Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Antibacterial Activity and Characterization of Bacteria Isolated from Diverse Types of Greek Honey against Nosocomial and Foodborne Pathogens

Antibacterial Activity and Characterization of Bacteria Isolated from Diverse Types of Greek... applied sciences Article Antibacterial Activity and Characterization of Bacteria Isolated from Diverse Types of Greek Honey against Nosocomial and Foodborne Pathogens 1 2 1 1 Christina Tsadila , Marios Nikolaidis , Tilemachos G. Dimitriou , Ioannis Kafantaris , 2 3 1 , Grigoris D. Amoutzias , Spyros Pournaras and Dimitris Mossialos * Microbial Biotechnology-Molecular Bacteriology-Virology Laboratory, Department of Biochemistry & Biotechnology, University of Thessaly, 41500 Larissa, Greece; tsadila@bio.uth.gr (C.T.); tidimitr@bio.uth.gr (T.G.D.); kafantarisioannis@gmail.com (I.K.) Bioinformatics Laboratory, Department of Biochemistry & Biotechnology, University of Thessaly, 41500 Larissa, Greece; marionik23@gmail.com (M.N.); amoutzias@bio.uth.gr (G.D.A.) Clinical Microbiology Laboratory, Medical School, “Attikon” University General Hospital, National and Kapodistrian University of Athens, 12462 Athens, Greece; spournaras@med.uoa.gr * Correspondence: mosial@bio.uth.gr; Tel.: +30-2410565270 Abstract: It has been suggested that microorganisms present in honey are a potential source of antimicrobial compounds. This study aimed to isolate and characterize bacteria from 46 Greek honey samples of diverse botanical and geographical origin and to determine whether these bacteria Citation: Tsadila, C.; Nikolaidis, M.; demonstrate antibacterial activity against five important nosocomial and foodborne pathogens. In Dimitriou, T.G.; Kafantaris, I.; total, 2014 bacterial isolates were obtained and screened for antibacterial activity. Overall, 16% of Amoutzias, G.D.; Pournaras, S.; the isolates inhibited the growth of Staphylococcus aureus, 11.2% inhibited the growth of Pseudomonas Mossialos, D. Antibacterial Activity aeruginosa and Acinetobacter baumannii, 10.2% inhibited the growth of Salmonella Typhimurium and and Characterization of Bacteria 12.4% of the isolates affected the growth of Citrobacter freundii. In total, 316 isolates that inhibited Isolated from Diverse Types of Greek the growth of more than two of the tested pathogens were grouped by restriction fragment length Honey against Nosocomial and polymorphisms (RFLP) analysis of the 16S rRNA gene amplicon. Fifty of them were identified by Foodborne Pathogens. Appl. Sci. 2021, 16S rRNA gene sequencing. The majority, 62% of the isolates, belonged to the genus Bacillus. Only 11, 5801. https://doi.org/10.3390/ 10% of the isolates were identified as Gram-negative bacteria. Furthermore, in several bacterial app11135801 isolates, genes encoding polyketide synthases and nonribosomal peptide synthetases that catalyze Academic Editors: Daniel the biosynthesis of secondary metabolites which might contribute to the exerted antimicrobial activity, Severus Dezmirean and Adela were detected. This study demonstrates that honey microbiota exerts antimicrobial activity and is Ramona Moise a putative source of secondary metabolites against important nosocomial and food pathogens that warrants further investigation. Received: 19 May 2021 Accepted: 19 June 2021 Keywords: honey; microbiota; antimicrobial activity; 16S-rRNA gene; nonribosomal peptide syn- Published: 22 June 2021 thetase; polyketide synthase; secondary metabolites Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Honey is a natural substance produced by honeybees (Apis mellifera L.). Honeybees collect nectar (floral honey) or secretions of living parts of plants (honeydew honey) [1–3] and transform them by adding secretions from their own salivary glands [4]. It is a complex aqueous mixture, consisting of more than 200 compounds, mainly carbohydrates. More- Copyright: © 2021 by the authors. over, it contains small amounts of proteins, aminoacids, phytochemicals, and plant- and Licensee MDPI, Basel, Switzerland. bee-derived enzymes such as glucose oxidase and catalase [5,6]. It is characterized by low This article is an open access article water activity (aw = 0.50–0.60), low humidity (<18%), and high acidity (pH 3.4–6.1) [7,8]. distributed under the terms and The physicochemical and biological properties of honey depend on its botanical and conditions of the Creative Commons geographical origin [9–11]. Attribution (CC BY) license (https:// For centuries honey has been utilized not only as a food, but also as a medicine and creativecommons.org/licenses/by/ food preservative [12,13]. Recent studies confirmed its beneficial bioactivity on human 4.0/). Appl. Sci. 2021, 11, 5801. https://doi.org/10.3390/app11135801 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 5801 2 of 20 health [11,14,15]. Antibacterial activity is the most investigated biological property of honey [16,17] and it is attributed to different factors [2]. Honey is a hostile environment for the survival and growth of different microorganisms, due to its physicochemical proper- ties [8,12,18]. Hydrogen peroxide (H O ) is one of the major antimicrobial factors in honey. 2 2 It is generated by the bee-derived enzyme glucose oxidase (GOX) or via an alternative non-enzymatic pathway comprising polyphenolic compounds [6,17,19]. The presence of various phytochemical compounds is also important for the direct antimicrobial poten- tial of honey [20]. Other components responsible for the antibacterial activity exerted by honey are the peptide bee defensin-1 [11,16], methylglyoxal (MGO), which is the main antimicrobial substance of manuka honey [14,21], and other unknown components [22]. Despite the various antibacterial factors [17], several studies have reported the isola- tion of microorganisms from honeys produced in diverse geographical locations [4,7,12,18]. Microorganisms in honey might be derived from primary and secondary sources. Primary sources include pollen, flowers, nectar, honeydew secretions [5,22,23], the environment (dust, air, soil) [7], as well as the honeybees (gut microbiota) or other insects [24–26]. Furthermore, microorganisms can be inoculated to honey during its extraction and post- harvest processing [7,23]. They are often related to hygiene and storage conditions [3,27]. Usually, they are yeasts, molds, and bacterial species (especially spore-forming) [18,24,28]. Honey microbiome has been suggested as a potential source of antimicrobial com- pounds [19,28]. Honey bacterial isolates produce antimicrobial compounds in vitro, but there is no direct evidence of such compounds present in honey [29]. Many secondary metabolites of high pharmaceutical and biotechnological interest are synthesized by non- ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) [30,31]. These enzymes are large multifunctional complexes organized as modules and are implicated in the biosynthesis of nonribosomal peptides (NRPs) and polyketides (PKs) respectively. These two large classes of natural products exert various biological properties, such as antimicrobial, anticancer, and immunosuppressive activity [32,33]. The aim of this study was to isolate and characterize bacteria from honeys of diverse botanical origin and locations across Greece, exhibiting antibacterial activity against five important nosocomial and foodborne pathogens. Furthermore, the presence of genes encoding PKSs and NRPSs, that might be implicated in the synthesis of antimicrobial compounds, has been investigated. 2. Materials and Methods 2.1. Honey Samples A total of forty-six Greek honey samples from various geographical and botanical origins were provided by individual beekeepers and beekeeping associations. Each sample was assigned to a unique reference number. Details regarding botanical source, geographi- cal location, and harvest date were recorded (Table S1). The identification of the botanical source of honeys was based on the flora availability during the harvest season, the location of the apiary and, in several cases, palynological analysis. Honey samples were stored at room temperature in dark conditions. 2.2. Bacterial Isolation Each honey sample (500 L) was diluted into 1.5 mL sterile Mueller Hinton (MH) Broth (25%, v/v) (NEOGEN, Heywood, UK) and mixed well. Aerobic mesophilic bacteria: 100 L of each suspension were plated in triplicate onto standard plate count agar (PCA) (Condalab, Madrid, Spain) [7,8] or Luria Bertani (LB) agar (Lab M, Heywood, UK) [12] and then incubated aerobically at 30 C for 5 days. After incubation, in order to obtain pure bacterial cultures, single colonies were transferred onto new PCA Petri dishes using the successive subculturing method. Pure cultures were stored as glycerol stocks (MH broth +20% glycerol) in 96-well plates at 80 C, until further analysis. Appl. Sci. 2021, 11, 5801 3 of 20 Aerobic spore-forming bacteria: each diluted sample was subjected to heat activation at 80 C for 10 min and cooled at room temperature for another 15 min [8,18]. Then, 100 L of each sample were spread in triplicate onto the selective Bacillus cereus medium (BCM) (Lab M, Heywood, UK) and incubated under aerobic conditions at 30 C for 48 h. Pure cultures, obtained as described above, were stored as glycerol stocks (MH broth +20% glycerol) in 96-well plates at 80 C, until further analysis. Lactic acid bacteria (LAB): A 100 L aliquot of each diluted sample was spread in triplicate onto the selected media, Man Rogosa and Sharpe (MRS) agar (NEOGEN, Heywood, UK) and incubated anaerobically in an AnaeroJar AG25 with the AnaeroGen Atmosphere Generation system (Oxoid, Basingstoke, UK) at 30 C for approximately 4 days. Resazurin sodium salt (Alfa Aesar, Thermo FisherGmbH, Kandel, Germany) (30 mg/lt) was used as an indicator for anaerobic conditions (turns from blue to pink). Each colony was subcultured anaerobically onto MRS agar supplemented with 1% calcium carbonate (CaCO ) (Scharlau, Barcelona, Spain) [32]. A clear zone around a colony indicates hydrolysis of CaCO due to organic acid production [34]. Colonies that exhibited a clear zone were picked, cultured onto eppendorfs filled to the top with Thioglycolate Broth (Scharlau, Barcelona, Spain) +20% glycerol and stored at 80 C, until further analysis. 2.3. Pathogenic Bacterial Strains The antimicrobial activity of the bacterial isolates from different honeys was tested against methicillin-resistant Staphylococcus aureus (S. aureus) strain 1552, carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) strain 1773, Acinetobacter baumannii (A. baumannii), Citrobacter freundii (C. freundii), and Salmonella Typhimurium (S. Typhimurium) strains. All strains were isolated from clinical samples, identified, and characterized by standard labora- tory methods at Attikon University General Hospital [13,35]. All bacteria were routinely grown in MH Broth (NEOGEN, Heywood, UK) or MH agar (NEOGEN, Heywood, UK) at 37 C. 2.4. Dual Culture Overlay Assay The antibacterial activity was tested by dual culture overlay assay as previously described [28,36] with few modifications. A replicate of each 96-well plate was transferred onto PCA square Petri dishes (120  120 mm, Aptaca Spa, Canelli, Italy) by using a microplate replicator (Boekel Scientific, Pennsylvania, PA, USA) and incubated at 30 C for 24 h. Then, each pathogen (approximately 9  10 cfu/mL) was mixed into soft Nutrient Agar (Biolab, Budapest, Hungary) containing 0.75% (w/v) agar, keeping it liquid at 42 C. The mixture of soft agar and bacterial cells was poured as a thin overlayer on top of the plates with the overnight-cultivated replica of the bacterial isolates. The plates were incubated at 37 C for 24 h and inhibition zones were observed. Each bacterial isolate that exhibited a clear inhibition zone was tested again, in triplicate, in order to confirm the antibacterial activity. 2.5. Grouping and Identification of Bacteria Exerting Antibacterial Activity From the glycerol stock each isolate was plated on PCA agar and incubated aero- bically at 30 C for 24 h. A single colony was obtained and cultured in MH broth aero- bically at 30 C, overnight. Pure broth cultures were used for genomic DNA extraction with the ExtractMe Genomic DNA Kit (Blirt, Gdansk, ´ Poland). Universal primers 27F 0 0 0 (5 -AGAGTTTGATCMTGGCTCAG-3 ) [37] and 1492R (5 -GGTTACCTTGTTACGACTT- 3 ) [38] (Eurofins Genomics, Germany) were used to amplify the 16S rRNA gene by PCR. The reaction mixture contained: 1 U FastGene Taq DNA Polymerase (NIPPON Genetics, Tokyo, Japan), 1 PCR buffer A, 25 pmol of each primer, 1 mM dNTPs, 3 L DNA tem- plate, and deionized sterile water to a final volume of 50 L. The thermal cycler Primus 25 (PEQLAB Biotechnologie, Erlangen, Germany) was used in the following PCR conditions: initialization at 95 C for 3 min, followed by 35 cycles of denaturation at 95 C for 30 s, Appl. Sci. 2021, 11, 5801 4 of 20 annealing at 50 C for 30 s, and elongation at 72 C for 2 min. A final elongation step at 72 C for 2 min was added. Restriction fragment length polymorphisms analysis (RFLP) of 16S rRNA amplicons was conducted as a simple prescreening for dereplication and grouping of the bacterial isolates. Two L of each PCR product (approximately 1400 bp), were incubated at 37 C for 1 h, with 10 U of RsaI and HinfI endonucleases and 10  buffer K (Bioron, Römerberg, Germany) [5,18,25]. The analysis was performed by electrophoresis in a 3% agarose gel (Invitrogen, Paisley, UK) by standard electrophoresis. Following RFLP analysis, bacterial isolates from each group were phenotypically characterized by Gram staining [39]. Amplicons of representative isolates of each group demonstrating antibacterial ac- tivity were purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Düren, Germany) and then directly sequenced via Sanger dideoxy termination method by Macrogen Europe (Amsterdam, The Netherlands). Chromas version 2.6.6 software (Tech- nelysium Pty Ltd., South Brisbane, Australia, www.technelysium.com.au) (accessed on 21 June 2021) was used to check the quality of the obtained sequencing results. Sequences were assembled into a single sequence via MEGA X version 10.1.6 software [40] and Gene Runner version 6.5 software (www.generunner.net accessed on 21 June 2021) and subjected to a BlastN (Megablast) (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 21 June 2021, BLAST, NIH) search in the 16S rRNA Database-GENEBANK to identify sequences with the highest similarity [3,5,18]. Obtained sequences were deposited to GenBank under the following accession numbers: MW700012–MW700061. 2.6. NRPS and PKS Gene Screening Identified bacterial isolates were further screened for the presence of biosynthetic gene clusters of nonribosomal peptide synthetases (NRPS) and type-I polyketide synthases 0 0 (PKS-I). The oligonucleotide primer set NRPS-1F (5 -GCSTACSYSATSTACACSTCSGG-3 ) 0 0 and NRPS-1R (5 -SASGTCVCCSGTSCGGTAS-3 ) (Eurofins Genomics, Ebersberg, Ger- many) was used to amplify partially the adenylation (A) domain (approximately 750– 0 0 800 bp) and the primer set PKS-IF (5 -GTGCCGGTNCCRTGNGYYTC-3 ) and PKS-IR 0 0 (5 -CGATGGAYCCNCARCARYG-3 ) (Eurofins Genomics, Ebersberg, Germany) for the amplification of partially KS domain (approximately 600–750 bp) [32]. PCR reactions were carried out in 50 L final volume, using Primus 25 thermal cycler (PEQLAB Biotechnologie, Erlangen, Germany). The reaction mixture for NRPS A domain contained 1.5 U FastGene Taq DNA Polymerase (NIPPON Genetics, Tokyo, Japan), 1 PCR buffer B, 25 pmol of each primer, 1 mM dNTPs and 5 L DNA template. The amplification conditions were the following: initialization at 95 C for 3 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 59 C for 2 min and elongation at 72 C for 4 min with a final elon- gation step at 72 C for 1 min. For the KS domain the reaction mixture contained 1 U Taq DNA Polymerase (Invitrogene, UK), 1 PCR buffer, 25 pmol of each primer, 1 mM dNTPs, 1.5 mM MgCl , and 5 L DNA template. The initialization at 94 C for 3 min was followed by 40 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 1 min and elongation at 72 C for 30 s. The final elongation step was performed at 72 C for 5 min. Sequencing was performed via Sanger dideoxy termination method by CeMIA SA (Larissa, Greece). Sequencing data were subjected to a BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi, BLAST, NIH) (accessed on 21 June 2021) search and to BLASTX (E value: 1  10 ) in order to identify the closest known homologues and determine the level of homology of the amino acid sequence. 3. Results 3.1. Isolation of Bacteria from Honey In total, 2014 bacterial isolates were obtained from 46 Greek honey samples of various geographical and botanical origin, utilizing four different culture media (PCA, LB agar, BCM and MRS agar) (Table S1). Overall, 695 bacterial isolates were obtained on PCA, 851 on LB agar, 370 on BCM, and finally 98 isolates were obtained by using MRS agar. Bacterial Appl. Sci. 2021, 11, 5801 5 of 20 isolates were obtained from all honey samples no matter the botanical source, geographical origin, and the date of harvest. The only exception was honey sample 42, which was tested only for aerobic spore-forming bacteria on BCM. 3.2. Antimicrobial Activity of Bacterial Isolates All bacterial isolates (2014 in total) were screened by dual overlay assay, in order to determine whether they inhibited the growth of five important human and foodborne pathogens: S. aureus, P. aeruginosa, S. Typhimurium, A. baumannii, and C. freundii (Table 1). A clear inhibition zone (>5 mm) against a given tested pathogen was considered as pos- itive antibacterial activity. Overall, 323 (16%) isolates were found to inhibit the growth of S. aureus, 225 (11.2%) isolates inhibited the growth of P. aeruginosa, 206 (10.2%) isolates inhibited the growth of S. Typhimurium, while 225 (11.2%) isolates and 249 (12.4%) isolates affected the growth of A. baumannii, and C. freundii, respectively. Collectively, 296 (14.7%) isolates inhibited the growth of at least one of the tested pathogens, 167 (8.3%) isolates inhibited the growth of at least two of the five tested pathogens and 111 (5.5%) honey bacte- rial isolates affected the growth of at least three of the five tested pathogens. Forty (1.99%) isolates exerted antibacterial activity against at least four out of five tested pathogens, whereas 21 isolates (1.04%) inhibited the growth of all five pathogens. These results demon- strated that 31.5% (635/2014) of the obtained bacterial isolates exerted antibacterial activity against at least one pathogen. 3.3. Grouping and Identification of Bacterial Isolates Exerting Antibacterial Activity The bacterial isolates (316) that exerted antibacterial activity against at least two of the tested pathogens were selected for further analysis. The 16S rRNA gene of all these isolates was amplified by PCR and the amplicons were subjected to RFLP analysis. Overall, the 316 bacterial isolates were distributed in 19 groups (Table S2). Representative isolates from each group were further subjected to Gram staining. In total, 50 bacterial isolates were identified by 16S rRNA gene sequencing. Sequencing of the 16S rRNA gene revealed that 90% of the 50 bacterial isolates were classified as Gram-positive bacteria whereas only 10% as Gram-negative. Thirty-one out of 50 strains (62%) belonging to the genus Bacillus were classified into eight species (Table 2). B. safensis was the most frequently identified species (7 out of 31 isolates, 22.6%) followed by B. pumilus (5/31, 16.1%), B. subtilis (4/31, 12.9%), and B. cereus (2/31, 6.5%). Four isolates were identified as B. licheniformis, Priestia megaterium (former B. megaterium), B. paramycoides, and B. vallismortis respectively. Classification at species level within the Bacillus group was not possible for 9 isolates due to the high similarity of 16S rRNA gene sequence to more than one species. Other isolates were identified as Lysinibacillus fusiformis, Microbacterium imperiale, Micrococcus yunnanensis, Paenibacillus sp., Paenibacillus profundus, Terribacillus saccharophilus, and Terribacillus sp. (2% for each phylotype). Seven isolates were identified as Staphylococcus spp. (14%). Specifically, five isolates were identified as S. arlettae, S. epidermidis, S. hominis, S. pasteuri, and S. warneri respectively, and two isolates were identified as S. cohnii. Regarding the Gram-negative isolates, four (8%) were identified as Pseudomonas sp., P. coleopterorum, P. fulva, and P. stutzeri and one as Acinetobacter lwoffii (Table 2). Appl. Sci. 2021, 11, 5801 6 of 20 Table 1. Growth inhibition of selected human and foodborne pathogens exerted by bacterial isolates from various honeys. 1 1 1 1 1 2 2 2 2 2 Sample No Total % S. aureus P. aeruginosa S. Typhimurium A. baumannii C. freundii 1/5 2/5 3/5 4/5 5/5 1 25 23 26 6 25 23 17 12 3 - 55/127 43.3 2 8 7 5 7 8 11 6 4 - - 21/77 27.3 3 17 9 7 10 12 19 10 4 1 - 34/118 28.8 4 33 19 11 23 22 26 14 14 3 - 57/112 50.9 5 18 8 9 9 12 15 10 4 1 1 31/90 34.4 6 17 8 3 4 5 12 8 3 - - 23/70 32.9 7 15 15 8 11 18 24 12 5 1 - 42/117 35.9 8 5 5 7 8 7 7 9 1 1 - 18/62 29.0 9 8 3 1 4 4 12 4 - - - 16/81 19.8 10 13 6 11 5 5 17 6 2 - 1 26/83 31.3 11 8 5 4 10 6 9 6 1 1 1 18/56 32.1 12 7 4 4 3 6 6 2 2 2 - 12/41 29.3 13 18 7 9 11 14 22 5 6 1 1 35/112 31.3 14 35 15 19 25 27 51 22 6 2 - 81/190 42.6 15 4 3 5 5 4 - - 2 - 3 26.3 5/19 16 3 2 3 2 2 - 2 1 - 1 4/30 13.3 17 1 1 1 2 - - 1 1 - - 2/27 7.4 18 6 7 7 8 6 3 1 2 2 3 11/38 28.9 19 1 1 - 1 - - - 1 - - 1/7 14.3 20 2 3 2 3 1 - - 1 2 - 3/20 15.0 21 3 7 8 9 4 - 6 5 1 - 12/38 31.6 22 2 2 2 2 1 - 1 1 1 - 3/24 12.5 23 3 3 3 2 4 1 1 4 - - 6/9 66.7 24 3 3 3 1 3 4 1 1 1 - 7/36 19.4 25 - - - - - - - - - - 0/22 0,0 26 11 5 - 2 2 9 4 1 - - 14/40 35.0 27 2 2 1 1 2 5 - 1 - - 6/43 14.0 28 - 1 - - - 1 - - - - 1/28 3.6 29 17 17 15 22 13 2 4 7 7 5 25/57 43.9 30 5 2 1 1 2 2 3 1 - - 6/42 14.3 31 - - - 1 1 - 1 - - - 1/29 3.4 32 1 2 3 5 3 2 - 1 1 1 14.7 5/34 33 2 4 1 5 3 2 2 3 - - 7/37 18.9 34 1 1 1 1 1 - - - - 1 1/1 100 35 3 5 4 3 4 4 1 - 2 1 8/10 80.0 36 2 4 3 2 3 1 - 3 1 - 5/17 29.4 37 - - 1 - 1 - 1 - - - 1/1 100 38 1 1 1 1 - - - - 1 - 1/2 50.0 39 - 1 - - - 1 - - - - 1/4 25.0 40 1 - - - - 1 - - - - 1/2 50.0 41 10 10 8 6 9 1 5 5 3 1 15/17 88.2 42 - - - - - - - - - - 0/0 - 43 1 - - 2 - 3 - - - - 3/27 11.1 44 - - - - - - - - - - 0/1 0.0 45 11 4 9 2 9 - 2 6 2 1 11/14 78.6 46 - - - - - - - - - - 0/2 0.0 Total 323 225 206 225 249 296 167 111 40 21 635/2014 Total % 14.7 8.3 5.5 1.99 1.04 31.5 1 2 Columns demonstrate the number of bacterial isolates per honey sample that exerted activity against the corresponding pathogens. Total number of bacterial isolates that inhibited the growth of 1–5 pathogens. Appl. Sci. 2021, 11, 5801 7 of 20 Table 2. Molecular identification of bacterial isolates and molecular screening for the presence of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes. Group Strain bp % Identity Bacteria Accession Number NRPS PKS Antibacterial Activity CTA1 1381 100 1,2,3,4,5 B. pumilus MW700012 + * - CTA2 1395 100 1,2,3,4,5 B.pumilus MW700013 + - CTA15 1376 100 2,3,5 B.pumilus MW700019 + - CTA16 1389 99.6 2,3,5 B.vallismortis MW700020 + + * Bacillus sp. CTA31 1347 100 1,2,5 MW700025 + - (B. aerius/B. altitudinis/ B. aerophilus/B. stratosphericus) CTA57 1385 100 2,3,4 B.safensis MW700028 + + CTA146 1419 100 4,5 B.safensis MW700038 - + CTA163 1311 99.9 3,5 B.licheniformis MW700039 + * - CTB7 1400 100 1,2,3,4,5 B.safensis MW700041 - + CTB16 1403 100 1,2,3,4,5 B.safensis MW700043 + - CTB21 1300 100 1,2,3,4 B.pumilus MW700044 + - CTB26 1340 99.9 1,2,4,5 B.pumilus MW700045 - - Bacillus sp. CTB31 1094 99.9 1,3,4,5 MW700046 + + * (B. amyloliquefaciens/B. velezensis) CTB43 1403 100 2,3,4 B.safensis MW700049 + + CTB89 1132 100 1,5 B.safensis MW700053 + + CTB120 1369 100 3,4 B.safensis MW700057 + - CTA5 1396 99.9 1,2,3,5 B.subtilis MW700014 + + * CTA9 1384 100 2,3,4,5 B.subtilis MW700016 + + * CTA10 1373 100 2,3,4,5 B.subtilis MW700017 + * + * Bacillus sp. CTA14 681 99.6 2,3,5 MW700018 + * - (B. haynesii/B. piscis/ B. paralicheniformis/B. licheniformis) CTA20 1410 100 1,3,5 MW700021 + * + * B. subtilis Bacillus sp. CTA109 1414 99.9 2,5 MW700035 - + * (B. halotolerans/B. mojavensis) Bacillus sp. (B. subtilis subsp. Inaquosorum/ CTB11 1281 99.6 1,2,3,4,5 MW700042 - + * B. nakamurai/B. mojavensis/ B. halotolerans) Appl. Sci. 2021, 11, 5801 8 of 20 Table 2. Cont. Group Strain bp % Identity Bacteria Accession Number NRPS PKS Antibacterial Activity P. megaterium CTA7 1401 99.9 1,3,4,5 MW700015 + * - (B. megaterium) CTA28 1401 100 1,2,4 B.paramycoides MW700024 + - Bacillus sp. CTA46 1413 99.9 3,4,5 MW700027 + * + (B. proteolyticus/B. wiedmannii) CTB34 1414 99.9 1,2,3,5 B.cereus MW700048 - - CTB116 1405 100 2,5 B.cereus MW700056 - - CTA39 1349 99.9 3,4,5 MW700026 - - S. pasteuri CTA79 1370 99.9 1,5 MW700029 - - F S.cohnii CTB46 1411 99.9 1,2,4 MW700050 - + S.cohnii CTA107 897 100 2,5 P. stutzeri MW700034 - - CT110 1322 100 2,4 A. lwoffii MW700055 - - CTA23 1375 99.8 2,4,5 P. fulva MW700022 + * - Pseudomonas sp. (P. putida/P.fulva/P.parafulva/ H P. guariconensis/P. plecoglossicida/ CTA90 708 99.9 1,3 MW700031 + * + P. cremoricolorata/P. taiwanensis/ P. donghuensis/P.wadenswilerensis/ P. alkylphenolica) I CTA125 1436 98.9 1,2 Bacillus sp. MW700036 - + * Terribacillus sp. J CTA25 1371 99.9 2,4,5 MW700023 - - (T. goriensis/T. saccharophylus) K CTA84 1278 99.8 2,3 P.coleopterorum MW700030 - - L CTA95 1333 99.9 1,3 M. imperiale MW700032 + * - Bacillus sp. M CTA99 1349 100 1,3 MW700033 - - (B. proteolyticus/B. wiedmannii) Bacillus sp. CTA138 1405 99.9 2,4 MW700037 - - (B. proteolyticus/B. wiedmannii) CTB60 1404 99.9 3,4,5 L. fusiformis MW700051 - - S CTA169 496 99.8 3,5 P. profundus MW700040 + * + * T CTB32 1382 99.9 1,2,3,5 M. yunnanensis MW700047 - - Appl. Sci. 2021, 11, 5801 9 of 20 Table 2. Cont. Group Strain bp % Identity Bacteria Accession Number NRPS PKS Antibacterial Activity W CTB93 1379 99.8 1,2 T. saccharophilus MW700054 - - Paenibacillus sp. Y CTB69 1416 99.6 2,4,5 MW700052 + * + * (P. cucumis/P. polysaccharolyticus) CT2H 440 99.8 1,2,3,4 S. epidermidis MW700058 - - LAB1 CT7C 580 100 3,4,5 S. warneri MW700060 - - LAB2 CT6C 563 99.6 1,2,3,4,5 S.arlettae MW700059 + - LAB3 CT12B 432 99.8 1,2,5 S.hominis MW700061 - - * Amplicons were sequenced. (1) S. aureus, (2) P. aeruginosa, (3) S. Typhimurium, (4) A. baumani, (5) C. freundii. Appl. Sci. 2021, 11, 5801 10 of 20 Furthermore, all 50 isolates were screened for the presence of NRPS and PKS-I biosyn- thetic gene clusters. In fourteen isolates, NRPS genes were detected; six isolates were tested positive for PKS gene clusters whereas in 13 isolates both NRPS and PKS gene clusters were detected. Twenty-six out of 31 (83.9%) bacterial isolates identified as Bacillus spp. tested positive for NRPS or PKS or both. Specifically, 11 out of the 31 (35.5%) bacterial isolates identified as Bacillus spp. tested positive only for NRPS genes (78.6% of the isolates positive for NRPS, 11/14), 5 were positive only for PKS genes (83.3% of the isolates positive for PKS, 5/6) and 10 isolates were detected positive for both NRPS/PKS gene clusters (76.9% of all isolates positive for both NRPS and PKS, 10/13). Moreover, only NRPS genes were detected in 4 out of 5 identified B. pumilus strains (CTA1, CTA2, CTA15, CTB21), in 2 out of 9 Bacillus sp. (CTA31, CTA14), and in 2 out of 7 B. safensis strains (CTB16, CTB120). B. licheniformis (CTA163), P. megaterium (CTA7), and B. paramycoides (CTA28) were also positive just for NRPS genes. Two B. safensis (CTA146, CTB7) and 3 Bacillus sp. (CTA109, CTB11, CTA125) strains were positive just for PKS gene clusters. Both NRPS and PKS genes were detected in B. vallismortis (CTA16), in 4 B. subtilis strains (CTA5, CTA9, CTA10, CTA20), 3 B. safensis (CTA57, CTB43, CTB89), and 2 Bacillus spp. (CTB31, CTA46) strains. Two out of 4 (50%) bacterial isolates identified as Pseudomonas spp. tested positive for NRPS (P. fulva CTA23) or both NRPS/PKS (Pseudomonas sp. CTA90) gene clusters. In addition, 2 out of 7 isolates identified as Staphylococcus spp. tested positive for NRPS (S. arlettae CT6C) and PKS (S. cohnii CTB46) gene clusters. The strain M. imperiale (CTA95) was positive for NRPS encoding genes, while in P. profundus (CTA169) and Paenibacillus sp. (CTB69) both NPRS and PKS gene clusters were detected. Twelve NRPS amplicons and 11 PKS amplicons were sequenced, in order to validate the PCR result (Tables 3 and 4). Ten out of 12 amplicons were indeed identified as NRPS gene clusters. The identification of the other two amplicons was not possible due to low quality of sequencing data. In most cases there was an agreement regarding the genus classification and sequencing results, except for amplicons A1 and A7 that were amplified from bacterial isolates belonging to Bacillus spp., whereas the NPRS amplicons shared homology with NRPSs of P. moteilii. Amplicons A10, A20, and A46 from the strains B. safensis (CTA10), B. safensis (CTA20), and Bacillus sp. (CTA46) were identified as NRPSs that were implicated in surfactin biosynthesis, while B69 from the strain Paenibacillus sp. shared homology with the subunit B of gramicidin synthase. Furthermore, the sequencing data verified the PCR results for the 11 tested PKS ampli- cons. Six amplicons exhibited homology with ketosynthases, (A5, A9, A10, A20, A125, B11), 3 were identified as PKSs (A109, B31, B69), while amplicon A16 was identified as amino acid adenylation domain-containing protein. In all cases there was an agreement regarding the classification at genus level and the sequencing results. Interestingly, sequencing data regarding A9 amplicon of B. subtilis CTA9 strain suggest that bacillaene is biosynthesized by this strain. Appl. Sci. 2021, 11, 5801 11 of 20 Table 3. Sequencing results for the NRPS amplicons. Sample Species BLASTN % Identity BLASTX % Identity amino acid adenylation P. monteilii (AMA45849.1 A1 B. pumilus 84.4 domain-containing 78.7 hypothetical protein) protein (P. monteilii) P. monteilii (Gene APT63_12835, A7 P. megaterium 67.2 NRPS (P. monteilii) 41.7 AMA46431.1 hypothetical protein) B. subtilis strain JCL16 A10 B. subtilis chromosome (Gene srfAA), 84.2 Surfactin NRPS SrfAA (B. subtilis) 72.7 QKJ76470.1 surfactin NRPS SrfAA B. licheniformis strain P8_B2 D-alanine–poly(phosphoribitol) ligase A14 Bacillus sp. chromosome, QGI45530.1 95.9 91.1 subunit DltA (B. licheniformis) D-alanine–poly(phosphoribitol) ligase subunit DltA B. subtilis subsp. subtilis str. 168 chromosome (QJR44804.1 A20 B. subtilis 98.1 surfactin NRPS SrfAA (B. subtilis) 98.6 surfactin NRPS SrfAA amino acid adenylation P. monteilii strain USDA-ARS-USMARC-56711 A23 P. fulva 96.5 domain-containing 98.4 (Gene APT63_09540, AMA45849.1, hypothetical protein protein (P. monteilii) B. safensis strain PgKB20 A46 Bacillus sp. 96.5 NRPS (B. safensis) 99.49 chromosome Surfactin synthase subunit 2 (gene srfAB_2) amino acid adenylation P. monteilii strain USDA-ARS-USMARC-56711, gene A90.2 Pseudomonas sp. 91.8 domain-containing 87.7 APT63_09540, hypothetical protein protein (P. monteilii) A95 M. imperiale M. oleivorans strain I46 chromosome DNA gyrase subunit A 68.1 no no A163 B. licheniformis B. haynesii strain P19 chromosome 73.2 no no P. thiaminolyticus strain A169 P. profundus NRRL B-4156 amino acid 71.8 NRPS/PKS (P. apiarius) 56.5 adenylation domain-containing protein FLT43_25335 P. polymyxa E681 isolate type B chromosome Linear AMP-binding protein B69 83.5 96.6 Paenibacillus sp. gramicidin synthase subunit B, lgrB_2 (P. amylolyticus) Appl. Sci. 2021, 11, 5801 12 of 20 Table 4. Sequencing results for the PKS amplicons. Sample Species BLASTN % Identity BLASTX % Identity B. subtilis subsp. subtilis str. 168 chromosome amino acid A5 B. subtilis 81.8 ketosynthase, partial (Bacillus sp.) 82.7 adenylation domain-containing protein, HIR77_09320 B. subtilis subsp. subtilis strain UCMB5021 Malonyl CoA-acyl A9 B. subtilis carrier protein PksN transacylase involved in nonribosomal 77.1 ketosynthase, partial (Bacillus sp.) 76.1 synthesis of bacillaene, gene pksN B. subtilis strain JCL16 A10 B. subtilis chromosome, amino acid 78.9 ketosynthase, partial (Bacillus sp.) 78.2 adenylation domain-containing protein, gene HR084_09185 amino acid adenylation B. amyloliquefaciens strain R8-25 chromosome (or valezensis) A16 B. vallismortis amino acid adenylation domain-containing protein, gene 76.4 domain-containing protein 93.2 HT132_11940 (B. amyloliquefaciens) B. subtilis subsp. subtilis str. 168 chromosome, amino acid A20 B. subtilis 85.7 ketosynthase, partial (Bacillus sp.) 87.86 adenylation domain-containing protein, gene HIR77_09320 P. thiaminolyticus strain NRRL B-4156 chromosome acyltransferase domain-containing protein A109 Bacillus sp. 81.2 100 acyltransferase domain-containing protein, gene FLT43_06730 (P. apiaries) B. subtilis strain JCL16 A125 79.2 77.7 Bacillus sp. chromosome amino acid ketosynthase, partial (Bacillus sp.) adenylation domain-containing protein, gene HR084_09185 P. thiaminolyticus strain NRRL B-4156 chromosome acyltransferase domain-containing protein A169 81.1 100 P. profundus acyltransferase domain-containing protein, gene FLT43_06730 (P. apiarius) type I ketosynthase B. halotolerans strain KKD1 B11 Bacillus sp. 99 97.4 chromosome NRPS, gene HT135_09465 (Bacillus sp. LX-99) Bacillus sp. AM1(2019) B31 Bacillus sp. 72.9 PKS (B. velezensis) 60 chromosome zinc-binding dehydrogenase, gene GNE05_12430 PKS B69 Paenibacillus sp. Paenibacillus sp. B37 PKS gene, PKS 94.2 98.5 (Paenibacillus sp. B15) Appl. Sci. 2021, 11, 5801 13 of 20 4. Discussion The antimicrobial activity exerted by honey is one of its most well-established bioactiv- ities described in many studies [11]. Honey affects the growth and survival of microorgan- isms via multiple mechanisms [14,41]. Low pH, high sugar concentration, high osmolarity and the presence of antibacterial substances such as hydrogen peroxide and phytochemicals in honey provide a hostile environment for bacteria [42]. Consequently, honey is expected to contain a limited number and diversity of microorganisms [14]. Nevertheless, honey is not sterile since it is a natural product that is not processed [3,5,12,28]. Several studies have reported isolated microorganisms from honey samples produced in various geographical locations [4,7,12,18,43]. Interestingly, the number of microorganisms in honey ranged widely, from 0 to several thousand colony forming units (CFUs) per gram, depending on the sample and its freshness [8,9,18]. Sinacori et al. [18] reported low bacterial load in 33 out of 38 honey samples harvested in southern Italy. Similar results were reported by other researchers in Argentina [8], Morocco [15], Poland [12], Saudi Arabia [44], and Mexico [7]. Fernández et al. [27] studied the microbiological quality of honey from Argentina and their results were comparable to those reported in previous studies conducted in Argentina as well as other parts of the world [8,12,18,28,45]. Bacteria have also been isolated from stingless bee honey [4,23,42]. Moreover, there are few studies that have described the microbial communities of honey using culture-independent methods like next generation sequencing (NGS) [17,46–48]. Honey bacteria exerting antibacterial activity have been successfully isolated by sev- eral research groups [6,12,42,49–52]. There are also reports on the antibacterial activity exerted by various bacterial isolates from stingless bee (Heterotrigona itama) honey [34,42]. Lee et al. [28] screened more than two thousand bacterial strains isolated from six US domestic honeys and two manuka honeys against seven bacteria and one mold but identi- fication of bacterial isolates was not conducted. Recently, Pajor et al. [12] investigated the antimicrobial potential of 163 bacteria isolated from honey samples produced in Poland. A number of studies have demonstrated high antibacterial activity of Greek honeys against major nosocomial and foodborne pathogens [1,13,35,53]. However, this is the first study aiming to isolate and characterize bacteria from diverse types of Greek honeys exhibiting antibacterial potential. In total, 2014 bacterial isolates were screened against five important human and foodborne pathogens (S. aureus, P. aeruginosa, S. Typhimurium, A. baumannii, and C. freundii). Three of them belong to the “ESKAPE” pathogens group (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp.). The World Health Organization (WHO) included ESKAPE in a list of pathogens for which new antimicrobial compounds are urgently needed. A. baumannii and P. aeruginosa are listed in the critical priority list of pathogens, whereas S. aureus (MRSA) is in the high priority group [54–56]. Furthermore, multidrug-resistant C. freundii is of major concern, often implicated in nosocomial infections [57,58]. This is the first study regarding the antibacterial activity of honey bacterial isolates against C. freundii and A. baumanii. Moreover, in this study a higher number of bacterial isolates (2014) was screened against five pathogens, compared to relevant studies [4,42,51,52]. The most susceptible pathogen was S. aureus, followed by C. freundii, P. aeruginosa, and A. baumannii. In contrast, S. Typhimurium was the least affected tested pathogen. A possible explanation might be that Gram-negative bacteria possess an outer membrane which often contributes to resistance to a wide range of antibacterial factors (for instance, loss of porins located in outer membrane). In agreement with our study, Pajor et al. [12] reported that 20.3% of the honey isolated strains inhibited the growth of S. aureus ATCC 25923, while activity against P. aeruginosa ATCC 27857 was rarely observed. In previous studies, aerobic spore-forming bacteria belonging to Bacillus spp. and related genera (Brevibacillus, Lysinibacillus, Paenibacillus) have been identified as the most common bacteria present in honey [5,59]. Furthermore, bacteria belonging to Bacillus genus have been often isolated from the honeybee gut [60–62]. Similarly, in this study, 62% of the identified isolates belonged to the genus Bacillus. Malika et al. [15] detected Bacillus Appl. Sci. 2021, 11, 5801 14 of 20 spores in most of the honey samples from Morocco, while Pomastowski et al. [3] reported that regardless of the geographical and botanical origin, the main bacteria in honeys were spore-forming Bacillus spp., which is in agreement with our data. Bacteria belonging to B. subtilis or B. cereus groups are the most frequently detected in honey [3,25]. Indeed, B. safensis was the most frequently identified species in our study followed by B. pumilus, B. subtilis, and B. cereus. The species B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. safensis, B. subtilis, and B. vallismortis have been detected in honey samples from diverse geographical locations [3,4,9,22,25,42,44,60,63]. Nevertheless, to the best of our knowledge, this is the first time that B. paramycoides, L. fusiformis, M. imperiale, M. yunnanensis, P. profundus, and T. saccharophilus were isolated from honey. Paenibacillus strains isolated from various ecological niches synthesize a wide range of antibiotics (polymyxins, polypeptins, gavaserin) [51,64]. P. profundus strain Sl 79, a deep sediment bacterium, exerted a remarkable inhibitory activity against both Gram- positive and Gram-negative bacteria. It is known to produce isocoumarin and a new linear glyceryl acid-derived heptapeptide that exhibited antibacterial activity against S. aureus, S. epidermidis, B. subtilis, and E. faecium [64,65]. On the other hand, M. yunnanensis was previously detected in bee gut [61,66] but not in honey. Micrococcin was the first thiazolyl peptide isolated from a Micrococcus strain [67]. Sponge-derived M. yunnanensis F-256,44 was reported to produce kocurin, an antibiotic of the thiazolyl peptide family [67,68]. Moreover, Ravi Ranjan et al. [69] isolated M. yunnanensis strain rsk5, which produced a novel antibacterial compound. This compound exerted activity against antibiotic-resistant S. aureus and differs from other characterized secondary metabolites. T. saccharophilus, as its name suggests, is a sugar-loving bacterium [70]. A study reported that a T. saccharophillus strain isolated from the gut of an edible Indian freshwater fish could be applied as a probiotic in fish feed formulations [71]. Another study reported the production of copious amounts of exopolysaccharides by T. saccharophilus strain PS-47. Exopolysaccharides are secondary metabolites, used in the pharmaceutical and food industries [72]. Microbacterium spp. were previously isolated from the gastrointestinal tract of adult worker honeybees [62]. A study reported that M. imperiale (MAIIF2a) reduced fungal symptoms by Fusarium solani in cassava roots, indicating its antimicrobial potential [73]. The antibacterial activity of B. paramycoides and L. fusiformis against important human and food pathogens has been described for the first time in this study. However, these bacterial species are known for their bioremediation potential [74–76]. Moreover, B. paramy- coides was reported as a plant growth-promoting rhizobacterium [77], while L. fusiformis strain C250R produced a novel extracellular thermostable protease designated as SAPLF, which could be applied in pharmaceuticals, food, and biotechnology industries [78]. In this study, 14% of the characterized bacterial isolates were identified as Staphylo- coccus spp. Previous studies have reported the presence of S. epidermidis, S. pasteuri, and S. hominis in honey [3,22,79]. Moreover, all Staphylococcus spp. identified in this study, except S. arlettae, have been described as part of the honeybee gastrointestinal micro- biome [3,61,62,66,80,81]. S. arlettae is commonly found in the skin and nares of poultry and goats [82]. Surprisingly, none of the bacterial isolates grown on MRS agar that exerted antimicro- bial acitivity in this study was identified as lactic acid bacteria. However, all four isolates were identified as Staphylococcus spp., one of them (S. hominis) previously isolated using MRS agar [83]. Only 10% of the characterized bacterial isolates were identified as Gram-negative bacteria, namely Pseudomonas sp., P. coleopterorum, P. fulva, P. stutzeri, and A. lwoffii. Pseu- domonas spp. and Actinetobacter spp. are often present in soil [7]. However, Veress et al. [84] isolated for the first time the bacterium A. lwoffii from a Transylvanian honey sample, suggesting that it was derived from the honeybee microbiota. Pseudomonas spp. have been identified both in honey and the digestive tract of honeybees [9,10,17,22,62]. The Pseudomonas genus is very diverse and comprises 272 species, organized in several major taxonomic groups that are widely distributed in diverse niches [85]. Some of these strains Appl. Sci. 2021, 11, 5801 15 of 20 exert biotechnologically interesting metabolic potential (denitrification, degradation of aromatic compounds, nitrogen fixation) [86–89]. P. fulva is a widespread environmental species exhibiting high biodegrading potential. It is reported to degrade neonicotinoid in- secticides that contribute to honeybee colony collapse disorder [86], as well as a pyrethroid insecticide, reported to be accumulated in honeybee wax samples [87]. On the other hand, P. coleopterorum was detected in the gut microbiota of some plant-related insects [90]. Bee products have been suggested as a rich source of beneficial bacteria that might be potential biocontrol agents against bee pathogens [4,28,42]. Several Bacillus strains produce compounds against pathogenic and food-spoilage bacteria [2,51]. Moreover, B. subtilis, B. amyloliquefaciens, B. pumilus, and B. licheniformis are considered as probiotics and antibiotic producers. Indeed, B. subtilis has already been used as a probiotic supplement for human and animal diets [23,42]. LABs commonly isolated from honey produce secondary metabolites, like bacteri- ocins and lipopeptides [6,42,91,92]. However, we did not identify any LABs exhibiting antibacterial activity in the present study, presumably due to the relatively small number of isolates grown on MRS agar. Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are often implicated in the biosynthesis of medically useful secondary metabolites, such as antibiotics, antitumor agents, immunosuppressants, siderophores, and toxins [30,31,33]. In this study we report the detection of genes encoding NRPSs and PKSs in 14 and 6 out of 50 identified isolates, respectively. NRPS genes were detected more often in Bacillus spp. It is known that Bacillus and Paenibacillus genera produce non-ribosomally synthe- sized lipopetides with significant antimicrobial activity, such as iturin, fengycin, surfactin, and bacillomycin. These compounds are abundantly synthesized, particularly by B. amy- loliquefaciens, B. subtilis, B. licheniformis, and B. pumilus [92,93]. Furthermore, NRPs have been identified in Pseudomonas spp. by several studies [94–97]. Analysis of P. fluorescens In5 revealed the presence of two antifungal NRPs, nunamycin and nunapeptin [94]. An- other study showed that a monomodular NRPS from Pseudomonas fluorescens HKI0770 is implicated in biosynthesis of the antimicrobial pyreudiones [96]. Magdalena et al. [51] have targeted seven antibacterial peptide biosynthetic genes present in two B. velezensis honey isolates using specific primers. However, in our study the universal primer set NRPS-1F—NRPS-1R for the amplification of adenylation (A) domain was used, thus targeting a much broader range of NRPS biosynthetic clusters. PKS gene clusters were detected in B. safensis, Bacillus sp., and S. cohnii strains. Type I PKSs are large multifunctional enzymes, implicated in pharmacologically important metabolites, most often detected within prokaryotes [93,98]. PKs were detected in B. sub- tilis, B. amyloliquefaciens, B. methylotrophicus, B. atrophaeus, B. laterosporus, and Paenibacillus sp. [93]. Within the B. subtilis group, three types of antimicrobial PKs are produced, in- cluding bacillaene, difficidin, and macrolactin [98]. Moreover, Zhang et al. [99], reported the partial isolation of a gene cluster from Staphylococcus lentus encoding a PK exerting broad-spectrum activity against fungi and bacteria. To the best of our knowledge, this is the first time that PKS encoding genes have been detected in bacteria isolated from honey exerting antimicrobial potential. Interestingly, 13 isolates in this study contained both NRPS and PKS gene clusters. Often NRPSs and PKSs are synthesized as hybrid products by NRPS and PKS gene clus- ters [31,33,100], such as bacillaene, compactin, fusarin C, or salinosporamide [98]. Twelve NRPS amplicons and 11 PKS amplicons were sequenced, in order to validate our detection approach. Three out of the 12 NRPS amplicons were implicated in the synthesis of surfactin. Surfactin is a cyclic lipoheptapeptide produced by various strains of Bacillus subtilis, which alters membrane integrity and permeability acting against several microorganisms [51,92,101]. Furthermore, it has been reported that Paenibacillus strains, isolated from various environments, produce a wide range of antibiotics [64,92]. Three Paenibacillus species were detected in honey, P. alvei, P. polymyxa, and P. larvae [3,28,92,102]. Polymyxins produced by Appl. Sci. 2021, 11, 5801 16 of 20 P. polymyxa have been used as a last-resort treatment of multidrug-resistant Gram-negative bacteria infections. They also exhibit strong antifungal activity [93]. Finally, our sequencing results regarding the PKS amplicon from Bacillus subtilis CTA9 strain suggest the presence of a PKS implicated in bacillaene biosynthesis. Bacillaene was firstly isolated from B. subtilis strains, but since then has been reported to be produced by several other Bacillus spp. It is a hybrid PK synthesized by a PKS-I and NRPS operon and it has demonstrated a strong activity against various bacteria and fungi [92,93,98]. Taking together all these data, it is evident that most of the bacterial isolates in this study could be potent producers of secondary metabolites including antibiotics, bacteriocins, and enzymes that might lead to future biotechnological applications. 5. Conclusions In the present study, 2014 bacterial isolates were obtained from 46 Greek honey samples of diverse botanical and geographical origin. All bacterial isolates were tested against five important nosocomial and foodborne pathogens. Overall, 31.5% of the isolates exerted antibacterial activity against at least one pathogen. Gram-positive S. aureus was the most susceptible of the five tested pathogens, followed by C. freundii, P. aeruginosa, and A. baumanii. S. Typhimurium was found to be the least susceptible pathogenic bacterium. Fifty isolates that inhibited the growth of at least two tested pathogens were further characterized. Most of them (62%) were identified as Bacillus spp. Nevertheless, six identified species were isolated from honey samples for the very first time. Moreover, the detection of NRPS and PKS (PKS-I) biosynthetic gene clusters in several isolates is reported, underlining the high biosynthetic potential of honey bacteria. Collectively, the results presented in this study demonstrate that the honey microbiome is an untapped source of antimicrobial metabolites against multidrug-resistant nosocomial and food pathogens that warrants further investigation and bioprospecting. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/app11135801/s1, Table S1: Honey samples of diverse botanical, geographical origin, and harvest date. Number of bacterial isolates per honey sample and per culture medium, Table S2: Grouping of bacterial isolates by RFLP assay and Gram staining. Author Contributions: Conceptualization, D.M.; methodology, C.T., D.M.; investigation, C.T., I.K., T.G.D.; resources, S.P., D.M.; data curation, M.N., G.D.A.; formal analysis, M.N. writing—original draft preparation, C.T., D.M.; writing—review and editing, D.M., T.G.D., S.P., G.D.A.; supervision, D.M.; funding acquisition, D.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the General Secretariat for Research and Innovation (G.S.R.I) under the emblematic action “Honeybee Road”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The research work was supported by the Hellenic Foundation for Research and Innovation (HFRI) under the HFRI Ph.D. Fellowship grant (Fellowship Number: 545) for C.T. I.K. is recipient of a Postdoctoral Fellowship under the emblematic action “Honeybee Road”. Conflicts of Interest: The authors declare no conflict of interest. References 1. Stagos, D.; Soulitsiotis, N.; Tsadila, C.; Papaeconomou, S.; Arvanitis, C.; Ntontos, A.; Karkanta, F.; Adamou-Androulaki, S.; Petrotos, K.; Spandidos, D.; et al. Antibacterial and antioxidant activity of different types of honey derived from Mount Olympus in Greece. Int. J. Mol. Med. 2018, 42, 726–734. [CrossRef] 2. Ng, W.-J.; Sit, N.-W.; Ooi, P.A.-C.; Ee, K.-Y.; Lim, T.-M. The Antibacterial Potential of Honeydew Honey Produced by Stingless Bee (Heterotrigona itama) against Antibiotic Resistant Bacteria. Antibiotics 2020, 9, 871. [CrossRef] Appl. Sci. 2021, 11, 5801 17 of 20 3. Pomastowski, P.; Złoch, M.; Rodzik, A.; Ligor, M.; Kostrzewa, M.; Buszewski, B. Analysis of bacteria associated with honeys of different geographical and botanical origin using two different identification approaches: MALDI-TOF MS and 16S rDNA PCR technique. PLoS ONE 2019, 14, e0217078. [CrossRef] [PubMed] 4. Ngalimat, M.S.; Raja, A.; Rahman, R.N.Z.; Yusof, M.T.; Syahir, A.; Sabri, S. Characterisation of bacteria isolated from the stingless bee, Heterotrigona itama, honey, bee bread and propolis. PeerJ 2019, 7, e7478. [CrossRef] [PubMed] 5. López, A.C.; Alippi, A.M. Feasibility of using RFLP of PCR-amplified 16S rRNA gene(s) for rapid differentiation of isolates of aerobic spore-forming bacteria from honey. J. Microbiol. Methods 2019, 165, 105690. [CrossRef] [PubMed] 6. Aween, M.M.; Hassan, Z.; Muhialdin, B.J.; Eljamel, Y.A.; Al-Mabrok, A.S.W.; Lani, M.N. Antibacterial Activity of Lactobacillus acidophilus Strains Isolated from Honey Marketed in Malaysia against Selected Multiple Antibiotic Resistant (MAR) Gram- Positive Bacteria. J. Food Sci. 2012, 77, M364–M371. [CrossRef] 7. Vázquez-Quiñones, C.R.; Moreno-Terrazas, R.; Natividad-Bonifacio, I.; Quiñones-Ramírez, E.I.; Vázquez-Salinas, C. Microbiologi- cal assessment of honey in México. Rev. Argent. Microbiol. 2018, 50, 75–80. [CrossRef] 8. Iurlina, M.O.; Fritz, R. Characterization of microorganisms in Argentinean honeys from different sources. Int. J. Food Microbiol. 2005, 105, 297–304. [CrossRef] 9. Adadi, P.; Obeng, A.K. Assessment of bacterial quality of honey produced in Tamale metropolis (Ghana). J. Food Drug Anal. 2017, 25, 369–373. [CrossRef] 10. Kafantaris, I.; Amoutzias, G.D.; Mossialos, D. Foodomics in bee product research: A systematic literature review. Eur. Food Res. Technol. 2021, 247, 309–331. [CrossRef] 11. Albaridi, N.A. Antibacterial Potency of Honey. Int. J. Microbiol. 2019, 2019, 1–10. [CrossRef] 12. Pajor, M.; Worobo, R.; Milewski, S.; Szweda, P. The Antimicrobial Potential of Bacteria Isolated from Honey Samples Produced in the Apiaries Located in Pomeranian Voivodeship in Northern Poland. Int. J. Environ. Res. Public Health 2018, 15, 2002. [CrossRef] 13. Tsavea, E.; Mossialos, D. Antibacterial activity of honeys produced in Mount Olympus area against nosocomial and foodborne pathogens is mainly attributed to hydrogen peroxide and proteinaceous compounds. J. Apic. Res. 2019, 58, 756–763. [CrossRef] 14. Bucekova, M.; Bugarova, V.; Godocikova, J.; Majtan, J. Demanding New Honey Qualitative Standard Based on Antibacterial Activity. Foods 2020, 9, 1263. [CrossRef] [PubMed] 15. Malika, N.; And, M.; Adlouni, E.L.; Mohamed, F.; Chakib, E. Microbiological and Physico-chemical properties of Moroccan honey. Int. J. Agric. Biol. 2005, 50, 773–776. 16. Bucekova, M.; Jardekova, L.; Juricova, V.; Bugarova, V.; Di Marco, G.; Gismondi, A.; Leonardi, D.; Farkasovska, J.; Godocikova, J.; Laho, M.; et al. Antibacterial Activity of Different Blossom Honeys: New Findings. Molecules 2019, 24, 1573. [CrossRef] [PubMed] 17. Wen, Y.; Wang, L.; Jin, Y.; Zhang, J.; Su, L.; Zhang, X.; Zhou, J.; Li, Y. The Microbial Community Dynamics during the Vitex Honey Ripening Process in the Honeycomb. Front. Microbiol. 2017, 8, 1649. [CrossRef] [PubMed] 18. Sinacori, M.; Francesca, N.; Alfonzo, A.; Cruciata, M.; Sannino, C.; Settanni, L.; Moschetti, G. Cultivable microorganisms associated with honeys of different geographical and botanical origin. Food Microbiol. 2014, 38, 284–294. [CrossRef] 19. Grecka, K.; Kus, ´ P.; Worobo, R.; Szweda, P. Study of the Anti-Staphylococcal Potential of Honeys Produced in Northern Poland. Molecules 2018, 23, 260. [CrossRef] [PubMed] 20. Estevinho, L.; Pereira, A.P.; Moreira, L.; Dias, L.G.; Pereira, E. Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey. Food Chem. Toxicol. 2008, 46, 3774–3779. [CrossRef] 21. Lu, J.; Cokcetin, N.N.; Burke, C.M.; Turnbull, L.; Liu, M.; Carter, D.A.; Whitchurch, C.B.; Harry, E.J. Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa. Sci. Rep. 2019, 9, 18160. [CrossRef] 22. Grabowski, N.T.; Klein, G. Microbiology and Food-borne Pathogens in Honey. Crit. Rev. Food Sci. Nutr. 2015, 57. [CrossRef] [PubMed] 23. Rosli, F.N.; Hazemi, M.H.F.; Akbar, M.A.; Basir, S.; Kassim, H.; Bunawan, H. Stingless Bee Honey: Evaluating Its Antibacterial Activity and Bacterial Diversity. Insects 2020, 11, 500. [CrossRef] [PubMed] 24. Wirta, H.; Abrego, N.; Miller, K.; Roslin, T.; Vesterinen, E. DNA traces the origin of honey by identifying plants, bacteria and fungi. Sci. Rep. 2021, 11, 4798. [CrossRef] 25. López, A.C.; Alippi, A.M. Phenotypic and genotypic diversity of Bacillus cereus isolates recovered from honey. Int. J. Food Microbiol. 2007, 117, 175–184. [CrossRef] 26. McFrederick, Q.S.; Thomas, J.M.; Neff, J.L.; Vuong, H.Q.; Russell, K.A.; Hale, A.R.; Mueller, U.G. Flowers and Wild Megachilid Bees Share Microbes. Microb. Ecol. 2017, 73, 188–200. [CrossRef] [PubMed] 27. Fernández, L.A.; Ghilardi, C.; Hoffmann, B.; Busso, C.; Gallez, L.M. Microbiological quality of honey from the Pampas Region (Argentina) throughout the extraction process. Rev. Argent. Microbiol. 2017, 49, 55–61. [CrossRef] 28. Lee, H.; Churey, J.J.; Worobo, R.W. Antimicrobial activity of bacterial isolates from different floral sources of honey. Int. J. Food Microbiol. 2008, 126, 240–244. [CrossRef] 29. Kwakman, P.H.S.; Zaat, S.A.J. Antibacterial components of honey. IUBMB Life 2012, 64, 48–55. [CrossRef] 30. Santos-Gandelman, J.; Giambiagi-deMarval, M.; Oelemann, W.; Laport, M. Biotechnological Potential of Sponge-Associated Bacteria. Curr. Pharm. Biotechnol. 2014, 15, 143–155. [CrossRef] 31. Amoutzias, G.; Chaliotis, A.; Mossialos, D. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes. Mar. Drugs 2016, 14, 80. [CrossRef] Appl. Sci. 2021, 11, 5801 18 of 20 32. Chen, Y.-S.; Yanagida, F.; Hsu, J.-S. Isolation and characterization of lactic acid bacteria from dochi (fermented black beans), a traditional fermented food in Taiwan. Lett. Appl. Microbiol. 2006, 43, 229–235. [CrossRef] 33. Nikolouli, K.; Mossialos, D. Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics. Biotechnol. Lett. 2012, 34, 1393–1403. [CrossRef] [PubMed] 34. Syed Yaacob, S.N.; Huyop, F.; Kamarulzaman Raja Ibrahim, R.; Wahab, R.A. Identification of Lactobacillus spp. and Fructobacillus spp. isolated from fresh Heterotrigona itama honey and their antagonistic activities against clinical pathogenic bacteria. J. Apic. Res. 2018, 57, 395–405. [CrossRef] 35. Anthimidou, E.; Mossialos, D. Antibacterial Activity of Greek and Cypriot Honeys Against Staphylococcus aureus and Pseu- domonas aeruginosa in Comparison to Manuka Honey. J. Med. Food 2013, 16, 42–47. [CrossRef] [PubMed] 36. Olofsson, T.C.; Butler, È.; Markowicz, P.; Lindholm, C.; Larsson, L.; Vásquez, A. Lactic acid bacterial symbionts in honeybees—An unknown key to honey’s antimicrobial and therapeutic activities. Int. Wound J. 2016, 13, 668–679. [CrossRef] [PubMed] 37. Lane, D. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley and Sons: New York, NY, USA, 1991; pp. 115–175. 38. Turner, S.; Pryer, K.M.; Miao, V.P.W.; Palmer, J.D. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J. Eukaryot. Microbiol. 1999, 46, 327–338. [CrossRef] 39. Tripathi, N.; Sapra, A. Gram Staining; StatPearls Publishing: Treasure Island, FL, USA, 2021. 40. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [CrossRef] 41. Gobin, I.; Crnkovic, ´ G.; Magdalenic, ´ M.; Begic, ´ G.; Babic, ´ A.; Lušic, ´ D.; Vuckovi ˇ c, ´ D. Antibacterial potential of Croatian honey against antibiotic resistant pathogenic bacteria. Med. Glas. 2018, 15, 139–144. [CrossRef] 42. Zulkhairi Amin, F.A.; Sabri, S.; Ismail, M.; Chan, K.W.; Ismail, N.; Mohd Esa, N.; Mohd Lila, M.A.; Zawawi, N. Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia. Int. J. Environ. Res. Public Health 2019, 17, 278. [CrossRef] 43. Saha, S. Isolation, Detection and Characterization of Aerobic Bacteria from Honey Samples of Bangladesh. bioRxiv 2018. [CrossRef] 44. Sadik, M.W.; Al-Samie, M.A.; Ali, M.; Sadik, M.W.; Ali, M.A.M. Survey and identification of microorganisms in Bee Honey samples collected from different plant sources and regions in Saudi Arabia. Glob. Adv. Res. J. Microbiol. 2012, 1, 126–135. 45. Finola, M.S.; Lasagno, M.C.; Marioli, J.M. Microbiological and chemical characterization of honeys from central Argentina. Food Chem. 2007, 100, 1649–1653. [CrossRef] 46. Bovo, S.; Utzeri, V.J.; Ribani, A.; Cabbri, R.; Fontanesi, L. Shotgun sequencing of honey DNA can describe honey bee derived environmental signatures and the honey bee hologenome complexity. Sci. Rep. 2020, 10, 9279. [CrossRef] 47. Bovo, S.; Ribani, A.; Utzeri, V.J.; Schiavo, G.; Bertolini, F.; Fontanesi, L. Shotgun metagenomics of honey DNA: Evaluation of a methodological approach to describe a multi-kingdom honey bee derived environmental DNA signature. PLoS ONE 2018, 13, e0205575. [CrossRef] [PubMed] 48. Knazovick ˇ á, V.; Gábor, M.; Miluchová, M.; Bobko, M.; Medo, J. Diversity of bacteria in Slovak and foreign honey, with assessment of its physico-chemical quality and counts of cultivable microorganisms. J. Microbiol. Biotechnol. Food Sci. 2019, 9, 414–421. [CrossRef] 49. Lee, S.K.; Lee, H. Antimicrobial activity of solvent fractions and bacterial isolates of Korean domestic honey from different floral sources. Food Sci. Biotechnol. 2016, 25, 1507–1512. [CrossRef] 50. Abdel Wahab, W.A.; Saleh, S.A.A.; Karam, E.A.; Mansour, N.M.; Esawy, M.A. Possible correlation among osmophilic bacteria, levan yield, and the probiotic activity of three bacterial honey isolates. Biocatal. Agric. Biotechnol. 2018, 14, 386–394. [CrossRef] 51. Magdalena, S. Characterization of antibacterial activity produced by Bacillus spp. isolated from honey and bee-associated products against foodborne pathogens. Asian J. Trop. Biotechnol. 2020, 17. [CrossRef] 52. Lashani, E.; Davoodabadi, A.; Dallal, M.M.S. Some probiotic properties of lactobacillus species isolated from honey and their antimicrobial activity against foodborne pathogens. Vet. Res. Forum 2020, 11, 121–126. [CrossRef] [PubMed] 53. Voidarou, C.; Alexopoulos, A.; Plessas, S.; Karapanou, A.; Mantzourani, I.; Stavropoulou, E.; Fotou, K.; Tzora, A.; Skoufos, I.; Bezirtzoglou, E. Antibacterial activity of different honeys against pathogenic bacteria. Anaerobe 2011, 17, 375–379. [CrossRef] [PubMed] 54. De Oliveira, D.M.P.; Forde, B.M.; Kidd, T.J.; Harris, P.N.A.; Schembri, M.A.; Beatson, S.A.; Paterson, D.L.; Walker, M.J. Antimicro- bial Resistance in ESKAPE Pathogens. Clin. Microbiol. Rev. 2020, 33. [CrossRef] [PubMed] 55. Mulani, M.S.; Kamble, E.E.; Kumkar, S.N.; Tawre, M.S.; Pardesi, K.R. Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review. Front. Microbiol. 2019, 10, 539. [CrossRef] 56. Tacconelli, E.; Magrini, N. Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. Available online: http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant- bacteria/en/ (accessed on 13 May 2021). 57. Yang, L.; Li, P.; Liang, B.; Hu, X.; Li, J.; Xie, J.; Yang, C.; Hao, R.; Wang, L.; Jia, L.; et al. Multidrug-resistant Citrobacter freundii ST139 co-producing NDM-1 and CMY-152 from China. Sci. Rep. 2018, 8, 10653. [CrossRef] 58. Ranjan, K.P.; Ranjan, N. Citrobacter: An emerging health care associated urinary pathogen. Urol. Ann. 2013, 5, 313–314. 59. Alippi, A.M.; Abrahamovich, E. HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples. Int. J. Food Microbiol. 2019, 305, 108245. [CrossRef] [PubMed] Appl. Sci. 2021, 11, 5801 19 of 20 60. Gilliam, M.; Morton, H.L. Bacteria belonging to the genus Bacillus isolated from honey bees, Apis Mellifera, fed 2,4-D and antibiotics. Apidologie 1978, 9, 213–222. [CrossRef] 61. Gasper, J.; Terentjeva, M.; Kántor, A.; Ivanišová, E.; Kluz, M.; Kac ˇániová, M. Identification of Apis mellifera Gut Microbiota with MALDI-TOF MS Biotyper. Anim. Sci. Biotech. 2017, 50, 2344–4576. 62. Kac ˇániová, M.; Terentjeva, M.; Žiarovská, J.; Kowalczewski, P.Ł. In vitro antagonistic effect of gut bacteriota isolated from indigenous honey bees and essential oils against paenibacillus larvae. Int. J. Mol. Sci. 2020, 21, 6736. [CrossRef] 63. Alippi, A.M. Data associated with the characterization and presumptive identification of Bacillus and related species isolated from honey samples by using HiCrome Bacillus agar. Data Br. 2019, 25, 104206. [CrossRef] 64. Romanenko, L.A.; Tanaka, N.; Svetashev, V.I.; Kalinovskaya, N.I. Paenibacillus profundus sp. nov., a deep sediment bacterium that produces isocoumarin and peptide antibiotics. Arch. Microbiol. 2013, 195, 247–254. [CrossRef] 65. Kalinovskaya, N.I.; Romanenko, L.A.; Kalinovsky, A.I.; Dmitrenok, P.S.; Dyshlovoy, S.A. A New Antimicrobial and Anticancer Peptide Producing by the Marine Deep Sediment Strain “Paenibacillus profundus” sp. nov. Sl 79. Nat. Prod. Commun. 2013, 8. [CrossRef] 66. Aizenberg-Gershtein, Y.; Izhaki, I.; Halpern, M. Do Honeybees Shape the Bacterial Community Composition in Floral Nectar? PLoS ONE 2013, 8, e67556. [CrossRef] [PubMed] 67. Palomo, S.; González, I.; de la Cruz, M.; Martín, J.; Tormo, J.; Anderson, M.; Hill, R.; Vicente, F.; Reyes, F.; Genilloud, O. Sponge-Derived Kocuria and Micrococcus spp. as Sources of the New Thiazolyl Peptide Antibiotic Kocurin. Mar. Drugs 2013, 11, 1071–1086. [CrossRef] [PubMed] 68. Pham, T.M.; Wiese, J.; Wenzel-Storjohann, A.; Imhoff, J.F. Diversity and antimicrobial potential of bacterial isolates associated with the soft coral Alcyonium digitatum from the Baltic Sea. Antonie Van Leeuwenhoek 2016, 109, 105–119. [CrossRef] [PubMed] 69. Ranjan, R.; Jadeja, V. Isolation, characterization and chromatography based purification of antibacterial compound isolated from rare endophytic actinomycetes Micrococcus yunnanensis. J. Pharm. Anal. 2017, 7, 343–347. [CrossRef] [PubMed] 70. An, S.-Y.; Asahara, M.; Goto, K.; Kasai, H.; Yokota, A. Terribacillus saccharophilus gen. nov., sp. nov. and Terribacillus halophilus sp. nov., spore-forming bacteria isolated from field soil in Japan. Int. J. Syst. Evol. Microbiol. 2007, 57, 51–55. [CrossRef] 71. Kalarani, V.; Sumathi, V. Effect of Dietary Supplementation of Bacillus subtilis and Terribacillus saccharophillus on Innate Immune Responses of a Tropical Freshwater Fish, Labeo rohita. J. Clin. Cell. Immunol. 2016, 7, 1–9. [CrossRef] 72. Shukla, P.J.; Dave, B.P. Screening and molecular identification of potential exopolysaccharides (EPSs) producing marine bacteria from the Bhavnagar coast, Gujarat. Int. J. Pharm. Sci. Res. 2018, 9, 2973–2981. [CrossRef] 73. Freitas, M.A.; Medeiros, F.H.V.; Melo, I.S.; Pereira, P.F.; Peñaflor, M.F.G.V.; Bento, J.M.S.; Paré, P.W. Stem inoculation with bacterial strains Bacillus amyloliquefaciens (GB03) and Microbacterium imperiale (MAIIF2a) mitigates Fusarium root rot in cassava. Phytoparasitica 2019, 47, 135–142. [CrossRef] 74. Rashid, A.; Mirza, S.A.; Keating, C.; Ali, S.; Campos, L.C. Indigenous Bacillus paramycoides spp. and Alcaligenes faecalis: Sustainable solution for bioremediation of hospital wastewater. Environ. Technol. 2020, 1–14. [CrossRef] 75. He, M.; Li, X.; Liu, H.; Miller, S.J.; Wang, G.; Rensing, C. Characterization and genomic analysis of a highly chromate resistant and reducing bacterial strain Lysinibacillus fusiformis ZC1. J. Hazard. Mater. 2011, 185, 682–688. [CrossRef] [PubMed] 76. Ren, J.; Wang, C.; Huhetaoli, L.C.; Fan, B.; Niu, D. Biodegradation of acephate by Bacillus paramycoides NDZ and its degradation pathway. World J. Microbiol. Biotechnol. 2020, 36, 155. [CrossRef] 77. Noha, I.O.; Shixue, Y. Isolation and characterization of pea plant (Pisum sativum L.) growth-promoting Rhizobacteria. African J. Microbiol. Res. 2018, 12, 820–828. [CrossRef] 78. Mechri, S.; Kriaa, M.; Ben Elhoul Berrouina, M.; Omrane Benmrad, M.; Zaraî Jaouadi, N.; Rekik, H.; Bouacem, K.; Bouanane- Darenfed, A.; Chebbi, A.; Sayadi, S.; et al. Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R. Int. J. Biol. Macromol. 2017, 101, 383–397. [CrossRef] [PubMed] 79. Guran, H.S.; Durukan, G.; Mann, D. Assessment of the Microbiological Profile, Species Diversity and Antimicrobial Susceptibility of Recovered Bacteria from Retail Honeys in Turkey. J. Hell. Vet. Med. Soc. 2021, 72, 2765–2772. [CrossRef] 80. Anjum, S.I.; Shah, A.H.; Aurongzeb, M.; Kori, J.; Azim, M.K.; Ansari, M.J.; Bin, L. Characterization of gut bacterial flora of Apis mellifera from north-west Pakistan. Saudi J. Biol. Sci. 2018, 25, 388–392. [CrossRef] [PubMed] 81. Andere, C.I.; Monteavaro, C.; Palacio, M.A.; Catena, M.; Rodríguez, E.M.; Collins, A.M. Apis mellifera semen: Bacterial contamination and susceptibility to antibiotics. Apidologie 2011, 42, 551–559. [CrossRef] 82. Lavecchia, A.; Chiara, M.; De Virgilio, C.; Manzari, C.; Monno, R.; De Carlo, A.; Pazzani, C.; Horner, D.; Pesole, G.; Placido, A. Staphylococcus arlettae Genomics: Novel Insights on Candidate Antibiotic Resistance and Virulence Genes in an Emerging Opportunistic Pathogen. Microorganisms 2019, 7, 580. [CrossRef] 83. Sung, C.; Kim, B.-G.; Kim, S.; Joo, H.-S.; Kim, P.I. Probiotic potential of Staphylococcus hominis MBBL 2-9 as anti- Staphylococcus aureus agent isolated from the vaginal microbiota of a healthy woman. J. Appl. Microbiol. 2010, 108, 908–916. [CrossRef] 84. Veress, A.; Nagy, T.; Wilk, T.; Kömüves, J.; Olasz, F.; Kiss, J. Abundance of mobile genetic elements in an Acinetobacter lwoffii strain isolated from Transylvanian honey sample. Sci. Rep. 2020, 10, 2969. [CrossRef] [PubMed] 85. Nikolaidis, M.; Mossialos, D.; Oliver, S.G.; Amoutzias, G.D. Comparative Analysis of the Core Proteomes among the Pseudomonas Major Evolutionary Groups Reveals Species-Specific Adaptations for Pseudomonas aeruginosa and Pseudomonas chlororaphis. Diversity 2020, 12, 289. [CrossRef] Appl. Sci. 2021, 11, 5801 20 of 20 86. Zamule, S.M.; Dupre, C.E.; Mendola, M.L.; Widmer, J.; Shebert, J.A.; Roote, C.E.; Das, P. Bioremediation potential of select bacterial species for the neonicotinoid insecticides, thiamethoxam and imidacloprid. Ecotoxicol. Environ. Saf. 2021, 209, 111814. [CrossRef] [PubMed] 87. Yang, J.; Feng, Y.; Zhan, H.; Liu, J.; Yang, F.; Zhang, K.; Zhang, L.; Chen, S. Characterization of a Pyrethroid-Degrading Pseudomonas fulva Strain P31 and Biochemical Degradation Pathway of D-Phenothrin. Front. Microbiol. 2018, 9. [CrossRef] [PubMed] 88. Adeniji, A.A.; Aremu, O.S.; Loots, D.T.; Babalola, O.O. Pseudomonas fulva HARBPS9.1: Candidate anti-Fusarium agent in South Africa. Eur. J. Plant Pathol. 2020, 157, 767–781. [CrossRef] 89. Lalucat, J.; Bennasar, A.; Bosch, R.; García-Valdés, E.; Palleroni, N.J. Biology of Pseudomonas stutzeri. Microbiol. Mol. Biol. Rev. 2006, 70, 510–547. [CrossRef] 90. Menéndez, E.; Ramírez-Bahena, M.H.; Fabryová, A.; Igual, J.M.; Benada, O.; Mateos, P.F.; Peix, A.; Kolar ˇík, M.; García-Fraile, P. Pseudomonas coleopterorum sp. nov., a cellulase-producing bacterium isolated from the bark beetle Hylesinus fraxini. Int. J. Syst. Evol. Microbiol. 2015, 65, 2852–2858. [CrossRef] 91. Bulgasem, B.Y.; Lani, M.N.; Hassan, Z.; Yusoff, W.M.W.; Fnaish, S.G. Antifungal Activity of Lactic Acid Bacteria Strains Isolated from Natural Honey against Pathogenic Candida Species. Mycobiology 2016, 44, 302–309. [CrossRef] 92. Brudzynski, K. Honey as an Ecological Reservoir of Antibacterial Compounds Produced by Antagonistic Microbial Interactions in Plant Nectars, Honey and Honey Bee. Antibiotics 2021, 10, 551. [CrossRef] 93. Zhao, X.; Kuipers, O.P. Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species. BMC Genomics 2016, 17, 882. [CrossRef] 94. Michelsen, C.F.; Watrous, J.; Glaring, M.A.; Kersten, R.; Koyama, N.; Dorrestein, P.C.; Stougaard, P. Nonribosomal Peptides, Key Biocontrol Components for Pseudomonas fluorescens In5, Isolated from a Greenlandic Suppressive Soil. MBio 2015, 6. [CrossRef] [PubMed] 95. Pauwelyn, E.; Huang, C.-J.; Ongena, M.; Leclère, V.; Jacques, P.; Bleyaert, P.; Budzikiewicz, H.; Schäfer, M.; Höfte, M. New Linear Lipopeptides Produced by Pseudomonas cichorii SF1-54 Are Involved in Virulence, Swarming Motility, and Biofilm Formation. Mol. Plant Microbe Interact. 2013, 26, 585–598. [CrossRef] [PubMed] 96. Klapper, M.; Braga, D.; Lackner, G.; Herbst, R.; Stallforth, P. Bacterial Alkaloid Biosynthesis: Structural Diversity via a Minimalistic Nonribosomal Peptide Synthetase. Cell Chem. Biol. 2018, 25, 659–665.e9. [CrossRef] [PubMed] 97. Li, W.; Rokni-Zadeh, H.; De Vleeschouwer, M.; Ghequire, M.G.K.; Sinnaeve, D.; Xie, G.-L.; Rozenski, J.; Madder, A.; Martins, J.C.; De Mot, R. The Antimicrobial Compound Xantholysin Defines a New Group of Pseudomonas Cyclic Lipopeptides. PLoS ONE 2013, 8, e62946. [CrossRef] [PubMed] 98. Caulier, S.; Nannan, C.; Gillis, A.; Licciardi, F.; Bragard, C.; Mahillon, J. Overview of the Antimicrobial Compounds Produced by Members of the Bacillus subtilis Group. Front. Microbiol. 2019, 10, 302. [CrossRef] [PubMed] 99. Zhang, W.; Zhang, F.; Li, Z.; Miao, X.; Meng, Q.; Zhang, X. Investigation of bacteria with polyketide synthase genes and antimicrobial activity isolated from South China Sea sponges. J. Appl. Microbiol. 2009, 107, 567–575. [CrossRef] 100. Barrios-Llerena, M.E.; Burja, A.M.; Wright, P.C. Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites. J. Ind. Microbiol. Biotechnol. 2007, 34, 443–456. [CrossRef] 101. Carrillo, C.; Teruel, J.A.; Aranda, F.J.; Ortiz, A. Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochim. Biophys. Acta Biomembr. 2003, 1611, 91–97. [CrossRef] 102. Pajor, M.; Xiong, Z.R.; Worobo, R.W.; Szweda, P. Paenibacillus alvei MP1 as a Producer of the Proteinaceous Compound with Activity against Important Human Pathogens, Including Staphylococcus aureus and Listeria monocytogenes. Pathogens 2020, 9, 319. [CrossRef] http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Sciences Multidisciplinary Digital Publishing Institute

Antibacterial Activity and Characterization of Bacteria Isolated from Diverse Types of Greek Honey against Nosocomial and Foodborne Pathogens

Download PDF
20 pages

Loading next page...
 
/lp/multidisciplinary-digital-publishing-institute/antibacterial-activity-and-characterization-of-bacteria-isolated-from-RaFPzoODPa

References (102)

Publisher
Multidisciplinary Digital Publishing Institute
Copyright
© 1996-2021 MDPI (Basel, Switzerland) unless otherwise stated Disclaimer The statements, opinions and data contained in the journals are solely those of the individual authors and contributors and not of the publisher and the editor(s). MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Terms and Conditions Privacy Policy
ISSN
2076-3417
DOI
10.3390/app11135801
Publisher site
See Article on Publisher Site

Abstract

applied sciences Article Antibacterial Activity and Characterization of Bacteria Isolated from Diverse Types of Greek Honey against Nosocomial and Foodborne Pathogens 1 2 1 1 Christina Tsadila , Marios Nikolaidis , Tilemachos G. Dimitriou , Ioannis Kafantaris , 2 3 1 , Grigoris D. Amoutzias , Spyros Pournaras and Dimitris Mossialos * Microbial Biotechnology-Molecular Bacteriology-Virology Laboratory, Department of Biochemistry & Biotechnology, University of Thessaly, 41500 Larissa, Greece; tsadila@bio.uth.gr (C.T.); tidimitr@bio.uth.gr (T.G.D.); kafantarisioannis@gmail.com (I.K.) Bioinformatics Laboratory, Department of Biochemistry & Biotechnology, University of Thessaly, 41500 Larissa, Greece; marionik23@gmail.com (M.N.); amoutzias@bio.uth.gr (G.D.A.) Clinical Microbiology Laboratory, Medical School, “Attikon” University General Hospital, National and Kapodistrian University of Athens, 12462 Athens, Greece; spournaras@med.uoa.gr * Correspondence: mosial@bio.uth.gr; Tel.: +30-2410565270 Abstract: It has been suggested that microorganisms present in honey are a potential source of antimicrobial compounds. This study aimed to isolate and characterize bacteria from 46 Greek honey samples of diverse botanical and geographical origin and to determine whether these bacteria Citation: Tsadila, C.; Nikolaidis, M.; demonstrate antibacterial activity against five important nosocomial and foodborne pathogens. In Dimitriou, T.G.; Kafantaris, I.; total, 2014 bacterial isolates were obtained and screened for antibacterial activity. Overall, 16% of Amoutzias, G.D.; Pournaras, S.; the isolates inhibited the growth of Staphylococcus aureus, 11.2% inhibited the growth of Pseudomonas Mossialos, D. Antibacterial Activity aeruginosa and Acinetobacter baumannii, 10.2% inhibited the growth of Salmonella Typhimurium and and Characterization of Bacteria 12.4% of the isolates affected the growth of Citrobacter freundii. In total, 316 isolates that inhibited Isolated from Diverse Types of Greek the growth of more than two of the tested pathogens were grouped by restriction fragment length Honey against Nosocomial and polymorphisms (RFLP) analysis of the 16S rRNA gene amplicon. Fifty of them were identified by Foodborne Pathogens. Appl. Sci. 2021, 16S rRNA gene sequencing. The majority, 62% of the isolates, belonged to the genus Bacillus. Only 11, 5801. https://doi.org/10.3390/ 10% of the isolates were identified as Gram-negative bacteria. Furthermore, in several bacterial app11135801 isolates, genes encoding polyketide synthases and nonribosomal peptide synthetases that catalyze Academic Editors: Daniel the biosynthesis of secondary metabolites which might contribute to the exerted antimicrobial activity, Severus Dezmirean and Adela were detected. This study demonstrates that honey microbiota exerts antimicrobial activity and is Ramona Moise a putative source of secondary metabolites against important nosocomial and food pathogens that warrants further investigation. Received: 19 May 2021 Accepted: 19 June 2021 Keywords: honey; microbiota; antimicrobial activity; 16S-rRNA gene; nonribosomal peptide syn- Published: 22 June 2021 thetase; polyketide synthase; secondary metabolites Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Honey is a natural substance produced by honeybees (Apis mellifera L.). Honeybees collect nectar (floral honey) or secretions of living parts of plants (honeydew honey) [1–3] and transform them by adding secretions from their own salivary glands [4]. It is a complex aqueous mixture, consisting of more than 200 compounds, mainly carbohydrates. More- Copyright: © 2021 by the authors. over, it contains small amounts of proteins, aminoacids, phytochemicals, and plant- and Licensee MDPI, Basel, Switzerland. bee-derived enzymes such as glucose oxidase and catalase [5,6]. It is characterized by low This article is an open access article water activity (aw = 0.50–0.60), low humidity (<18%), and high acidity (pH 3.4–6.1) [7,8]. distributed under the terms and The physicochemical and biological properties of honey depend on its botanical and conditions of the Creative Commons geographical origin [9–11]. Attribution (CC BY) license (https:// For centuries honey has been utilized not only as a food, but also as a medicine and creativecommons.org/licenses/by/ food preservative [12,13]. Recent studies confirmed its beneficial bioactivity on human 4.0/). Appl. Sci. 2021, 11, 5801. https://doi.org/10.3390/app11135801 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 5801 2 of 20 health [11,14,15]. Antibacterial activity is the most investigated biological property of honey [16,17] and it is attributed to different factors [2]. Honey is a hostile environment for the survival and growth of different microorganisms, due to its physicochemical proper- ties [8,12,18]. Hydrogen peroxide (H O ) is one of the major antimicrobial factors in honey. 2 2 It is generated by the bee-derived enzyme glucose oxidase (GOX) or via an alternative non-enzymatic pathway comprising polyphenolic compounds [6,17,19]. The presence of various phytochemical compounds is also important for the direct antimicrobial poten- tial of honey [20]. Other components responsible for the antibacterial activity exerted by honey are the peptide bee defensin-1 [11,16], methylglyoxal (MGO), which is the main antimicrobial substance of manuka honey [14,21], and other unknown components [22]. Despite the various antibacterial factors [17], several studies have reported the isola- tion of microorganisms from honeys produced in diverse geographical locations [4,7,12,18]. Microorganisms in honey might be derived from primary and secondary sources. Primary sources include pollen, flowers, nectar, honeydew secretions [5,22,23], the environment (dust, air, soil) [7], as well as the honeybees (gut microbiota) or other insects [24–26]. Furthermore, microorganisms can be inoculated to honey during its extraction and post- harvest processing [7,23]. They are often related to hygiene and storage conditions [3,27]. Usually, they are yeasts, molds, and bacterial species (especially spore-forming) [18,24,28]. Honey microbiome has been suggested as a potential source of antimicrobial com- pounds [19,28]. Honey bacterial isolates produce antimicrobial compounds in vitro, but there is no direct evidence of such compounds present in honey [29]. Many secondary metabolites of high pharmaceutical and biotechnological interest are synthesized by non- ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) [30,31]. These enzymes are large multifunctional complexes organized as modules and are implicated in the biosynthesis of nonribosomal peptides (NRPs) and polyketides (PKs) respectively. These two large classes of natural products exert various biological properties, such as antimicrobial, anticancer, and immunosuppressive activity [32,33]. The aim of this study was to isolate and characterize bacteria from honeys of diverse botanical origin and locations across Greece, exhibiting antibacterial activity against five important nosocomial and foodborne pathogens. Furthermore, the presence of genes encoding PKSs and NRPSs, that might be implicated in the synthesis of antimicrobial compounds, has been investigated. 2. Materials and Methods 2.1. Honey Samples A total of forty-six Greek honey samples from various geographical and botanical origins were provided by individual beekeepers and beekeeping associations. Each sample was assigned to a unique reference number. Details regarding botanical source, geographi- cal location, and harvest date were recorded (Table S1). The identification of the botanical source of honeys was based on the flora availability during the harvest season, the location of the apiary and, in several cases, palynological analysis. Honey samples were stored at room temperature in dark conditions. 2.2. Bacterial Isolation Each honey sample (500 L) was diluted into 1.5 mL sterile Mueller Hinton (MH) Broth (25%, v/v) (NEOGEN, Heywood, UK) and mixed well. Aerobic mesophilic bacteria: 100 L of each suspension were plated in triplicate onto standard plate count agar (PCA) (Condalab, Madrid, Spain) [7,8] or Luria Bertani (LB) agar (Lab M, Heywood, UK) [12] and then incubated aerobically at 30 C for 5 days. After incubation, in order to obtain pure bacterial cultures, single colonies were transferred onto new PCA Petri dishes using the successive subculturing method. Pure cultures were stored as glycerol stocks (MH broth +20% glycerol) in 96-well plates at 80 C, until further analysis. Appl. Sci. 2021, 11, 5801 3 of 20 Aerobic spore-forming bacteria: each diluted sample was subjected to heat activation at 80 C for 10 min and cooled at room temperature for another 15 min [8,18]. Then, 100 L of each sample were spread in triplicate onto the selective Bacillus cereus medium (BCM) (Lab M, Heywood, UK) and incubated under aerobic conditions at 30 C for 48 h. Pure cultures, obtained as described above, were stored as glycerol stocks (MH broth +20% glycerol) in 96-well plates at 80 C, until further analysis. Lactic acid bacteria (LAB): A 100 L aliquot of each diluted sample was spread in triplicate onto the selected media, Man Rogosa and Sharpe (MRS) agar (NEOGEN, Heywood, UK) and incubated anaerobically in an AnaeroJar AG25 with the AnaeroGen Atmosphere Generation system (Oxoid, Basingstoke, UK) at 30 C for approximately 4 days. Resazurin sodium salt (Alfa Aesar, Thermo FisherGmbH, Kandel, Germany) (30 mg/lt) was used as an indicator for anaerobic conditions (turns from blue to pink). Each colony was subcultured anaerobically onto MRS agar supplemented with 1% calcium carbonate (CaCO ) (Scharlau, Barcelona, Spain) [32]. A clear zone around a colony indicates hydrolysis of CaCO due to organic acid production [34]. Colonies that exhibited a clear zone were picked, cultured onto eppendorfs filled to the top with Thioglycolate Broth (Scharlau, Barcelona, Spain) +20% glycerol and stored at 80 C, until further analysis. 2.3. Pathogenic Bacterial Strains The antimicrobial activity of the bacterial isolates from different honeys was tested against methicillin-resistant Staphylococcus aureus (S. aureus) strain 1552, carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) strain 1773, Acinetobacter baumannii (A. baumannii), Citrobacter freundii (C. freundii), and Salmonella Typhimurium (S. Typhimurium) strains. All strains were isolated from clinical samples, identified, and characterized by standard labora- tory methods at Attikon University General Hospital [13,35]. All bacteria were routinely grown in MH Broth (NEOGEN, Heywood, UK) or MH agar (NEOGEN, Heywood, UK) at 37 C. 2.4. Dual Culture Overlay Assay The antibacterial activity was tested by dual culture overlay assay as previously described [28,36] with few modifications. A replicate of each 96-well plate was transferred onto PCA square Petri dishes (120  120 mm, Aptaca Spa, Canelli, Italy) by using a microplate replicator (Boekel Scientific, Pennsylvania, PA, USA) and incubated at 30 C for 24 h. Then, each pathogen (approximately 9  10 cfu/mL) was mixed into soft Nutrient Agar (Biolab, Budapest, Hungary) containing 0.75% (w/v) agar, keeping it liquid at 42 C. The mixture of soft agar and bacterial cells was poured as a thin overlayer on top of the plates with the overnight-cultivated replica of the bacterial isolates. The plates were incubated at 37 C for 24 h and inhibition zones were observed. Each bacterial isolate that exhibited a clear inhibition zone was tested again, in triplicate, in order to confirm the antibacterial activity. 2.5. Grouping and Identification of Bacteria Exerting Antibacterial Activity From the glycerol stock each isolate was plated on PCA agar and incubated aero- bically at 30 C for 24 h. A single colony was obtained and cultured in MH broth aero- bically at 30 C, overnight. Pure broth cultures were used for genomic DNA extraction with the ExtractMe Genomic DNA Kit (Blirt, Gdansk, ´ Poland). Universal primers 27F 0 0 0 (5 -AGAGTTTGATCMTGGCTCAG-3 ) [37] and 1492R (5 -GGTTACCTTGTTACGACTT- 3 ) [38] (Eurofins Genomics, Germany) were used to amplify the 16S rRNA gene by PCR. The reaction mixture contained: 1 U FastGene Taq DNA Polymerase (NIPPON Genetics, Tokyo, Japan), 1 PCR buffer A, 25 pmol of each primer, 1 mM dNTPs, 3 L DNA tem- plate, and deionized sterile water to a final volume of 50 L. The thermal cycler Primus 25 (PEQLAB Biotechnologie, Erlangen, Germany) was used in the following PCR conditions: initialization at 95 C for 3 min, followed by 35 cycles of denaturation at 95 C for 30 s, Appl. Sci. 2021, 11, 5801 4 of 20 annealing at 50 C for 30 s, and elongation at 72 C for 2 min. A final elongation step at 72 C for 2 min was added. Restriction fragment length polymorphisms analysis (RFLP) of 16S rRNA amplicons was conducted as a simple prescreening for dereplication and grouping of the bacterial isolates. Two L of each PCR product (approximately 1400 bp), were incubated at 37 C for 1 h, with 10 U of RsaI and HinfI endonucleases and 10  buffer K (Bioron, Römerberg, Germany) [5,18,25]. The analysis was performed by electrophoresis in a 3% agarose gel (Invitrogen, Paisley, UK) by standard electrophoresis. Following RFLP analysis, bacterial isolates from each group were phenotypically characterized by Gram staining [39]. Amplicons of representative isolates of each group demonstrating antibacterial ac- tivity were purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Düren, Germany) and then directly sequenced via Sanger dideoxy termination method by Macrogen Europe (Amsterdam, The Netherlands). Chromas version 2.6.6 software (Tech- nelysium Pty Ltd., South Brisbane, Australia, www.technelysium.com.au) (accessed on 21 June 2021) was used to check the quality of the obtained sequencing results. Sequences were assembled into a single sequence via MEGA X version 10.1.6 software [40] and Gene Runner version 6.5 software (www.generunner.net accessed on 21 June 2021) and subjected to a BlastN (Megablast) (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 21 June 2021, BLAST, NIH) search in the 16S rRNA Database-GENEBANK to identify sequences with the highest similarity [3,5,18]. Obtained sequences were deposited to GenBank under the following accession numbers: MW700012–MW700061. 2.6. NRPS and PKS Gene Screening Identified bacterial isolates were further screened for the presence of biosynthetic gene clusters of nonribosomal peptide synthetases (NRPS) and type-I polyketide synthases 0 0 (PKS-I). The oligonucleotide primer set NRPS-1F (5 -GCSTACSYSATSTACACSTCSGG-3 ) 0 0 and NRPS-1R (5 -SASGTCVCCSGTSCGGTAS-3 ) (Eurofins Genomics, Ebersberg, Ger- many) was used to amplify partially the adenylation (A) domain (approximately 750– 0 0 800 bp) and the primer set PKS-IF (5 -GTGCCGGTNCCRTGNGYYTC-3 ) and PKS-IR 0 0 (5 -CGATGGAYCCNCARCARYG-3 ) (Eurofins Genomics, Ebersberg, Germany) for the amplification of partially KS domain (approximately 600–750 bp) [32]. PCR reactions were carried out in 50 L final volume, using Primus 25 thermal cycler (PEQLAB Biotechnologie, Erlangen, Germany). The reaction mixture for NRPS A domain contained 1.5 U FastGene Taq DNA Polymerase (NIPPON Genetics, Tokyo, Japan), 1 PCR buffer B, 25 pmol of each primer, 1 mM dNTPs and 5 L DNA template. The amplification conditions were the following: initialization at 95 C for 3 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 59 C for 2 min and elongation at 72 C for 4 min with a final elon- gation step at 72 C for 1 min. For the KS domain the reaction mixture contained 1 U Taq DNA Polymerase (Invitrogene, UK), 1 PCR buffer, 25 pmol of each primer, 1 mM dNTPs, 1.5 mM MgCl , and 5 L DNA template. The initialization at 94 C for 3 min was followed by 40 cycles of denaturation at 94 C for 30 s, annealing at 60 C for 1 min and elongation at 72 C for 30 s. The final elongation step was performed at 72 C for 5 min. Sequencing was performed via Sanger dideoxy termination method by CeMIA SA (Larissa, Greece). Sequencing data were subjected to a BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi, BLAST, NIH) (accessed on 21 June 2021) search and to BLASTX (E value: 1  10 ) in order to identify the closest known homologues and determine the level of homology of the amino acid sequence. 3. Results 3.1. Isolation of Bacteria from Honey In total, 2014 bacterial isolates were obtained from 46 Greek honey samples of various geographical and botanical origin, utilizing four different culture media (PCA, LB agar, BCM and MRS agar) (Table S1). Overall, 695 bacterial isolates were obtained on PCA, 851 on LB agar, 370 on BCM, and finally 98 isolates were obtained by using MRS agar. Bacterial Appl. Sci. 2021, 11, 5801 5 of 20 isolates were obtained from all honey samples no matter the botanical source, geographical origin, and the date of harvest. The only exception was honey sample 42, which was tested only for aerobic spore-forming bacteria on BCM. 3.2. Antimicrobial Activity of Bacterial Isolates All bacterial isolates (2014 in total) were screened by dual overlay assay, in order to determine whether they inhibited the growth of five important human and foodborne pathogens: S. aureus, P. aeruginosa, S. Typhimurium, A. baumannii, and C. freundii (Table 1). A clear inhibition zone (>5 mm) against a given tested pathogen was considered as pos- itive antibacterial activity. Overall, 323 (16%) isolates were found to inhibit the growth of S. aureus, 225 (11.2%) isolates inhibited the growth of P. aeruginosa, 206 (10.2%) isolates inhibited the growth of S. Typhimurium, while 225 (11.2%) isolates and 249 (12.4%) isolates affected the growth of A. baumannii, and C. freundii, respectively. Collectively, 296 (14.7%) isolates inhibited the growth of at least one of the tested pathogens, 167 (8.3%) isolates inhibited the growth of at least two of the five tested pathogens and 111 (5.5%) honey bacte- rial isolates affected the growth of at least three of the five tested pathogens. Forty (1.99%) isolates exerted antibacterial activity against at least four out of five tested pathogens, whereas 21 isolates (1.04%) inhibited the growth of all five pathogens. These results demon- strated that 31.5% (635/2014) of the obtained bacterial isolates exerted antibacterial activity against at least one pathogen. 3.3. Grouping and Identification of Bacterial Isolates Exerting Antibacterial Activity The bacterial isolates (316) that exerted antibacterial activity against at least two of the tested pathogens were selected for further analysis. The 16S rRNA gene of all these isolates was amplified by PCR and the amplicons were subjected to RFLP analysis. Overall, the 316 bacterial isolates were distributed in 19 groups (Table S2). Representative isolates from each group were further subjected to Gram staining. In total, 50 bacterial isolates were identified by 16S rRNA gene sequencing. Sequencing of the 16S rRNA gene revealed that 90% of the 50 bacterial isolates were classified as Gram-positive bacteria whereas only 10% as Gram-negative. Thirty-one out of 50 strains (62%) belonging to the genus Bacillus were classified into eight species (Table 2). B. safensis was the most frequently identified species (7 out of 31 isolates, 22.6%) followed by B. pumilus (5/31, 16.1%), B. subtilis (4/31, 12.9%), and B. cereus (2/31, 6.5%). Four isolates were identified as B. licheniformis, Priestia megaterium (former B. megaterium), B. paramycoides, and B. vallismortis respectively. Classification at species level within the Bacillus group was not possible for 9 isolates due to the high similarity of 16S rRNA gene sequence to more than one species. Other isolates were identified as Lysinibacillus fusiformis, Microbacterium imperiale, Micrococcus yunnanensis, Paenibacillus sp., Paenibacillus profundus, Terribacillus saccharophilus, and Terribacillus sp. (2% for each phylotype). Seven isolates were identified as Staphylococcus spp. (14%). Specifically, five isolates were identified as S. arlettae, S. epidermidis, S. hominis, S. pasteuri, and S. warneri respectively, and two isolates were identified as S. cohnii. Regarding the Gram-negative isolates, four (8%) were identified as Pseudomonas sp., P. coleopterorum, P. fulva, and P. stutzeri and one as Acinetobacter lwoffii (Table 2). Appl. Sci. 2021, 11, 5801 6 of 20 Table 1. Growth inhibition of selected human and foodborne pathogens exerted by bacterial isolates from various honeys. 1 1 1 1 1 2 2 2 2 2 Sample No Total % S. aureus P. aeruginosa S. Typhimurium A. baumannii C. freundii 1/5 2/5 3/5 4/5 5/5 1 25 23 26 6 25 23 17 12 3 - 55/127 43.3 2 8 7 5 7 8 11 6 4 - - 21/77 27.3 3 17 9 7 10 12 19 10 4 1 - 34/118 28.8 4 33 19 11 23 22 26 14 14 3 - 57/112 50.9 5 18 8 9 9 12 15 10 4 1 1 31/90 34.4 6 17 8 3 4 5 12 8 3 - - 23/70 32.9 7 15 15 8 11 18 24 12 5 1 - 42/117 35.9 8 5 5 7 8 7 7 9 1 1 - 18/62 29.0 9 8 3 1 4 4 12 4 - - - 16/81 19.8 10 13 6 11 5 5 17 6 2 - 1 26/83 31.3 11 8 5 4 10 6 9 6 1 1 1 18/56 32.1 12 7 4 4 3 6 6 2 2 2 - 12/41 29.3 13 18 7 9 11 14 22 5 6 1 1 35/112 31.3 14 35 15 19 25 27 51 22 6 2 - 81/190 42.6 15 4 3 5 5 4 - - 2 - 3 26.3 5/19 16 3 2 3 2 2 - 2 1 - 1 4/30 13.3 17 1 1 1 2 - - 1 1 - - 2/27 7.4 18 6 7 7 8 6 3 1 2 2 3 11/38 28.9 19 1 1 - 1 - - - 1 - - 1/7 14.3 20 2 3 2 3 1 - - 1 2 - 3/20 15.0 21 3 7 8 9 4 - 6 5 1 - 12/38 31.6 22 2 2 2 2 1 - 1 1 1 - 3/24 12.5 23 3 3 3 2 4 1 1 4 - - 6/9 66.7 24 3 3 3 1 3 4 1 1 1 - 7/36 19.4 25 - - - - - - - - - - 0/22 0,0 26 11 5 - 2 2 9 4 1 - - 14/40 35.0 27 2 2 1 1 2 5 - 1 - - 6/43 14.0 28 - 1 - - - 1 - - - - 1/28 3.6 29 17 17 15 22 13 2 4 7 7 5 25/57 43.9 30 5 2 1 1 2 2 3 1 - - 6/42 14.3 31 - - - 1 1 - 1 - - - 1/29 3.4 32 1 2 3 5 3 2 - 1 1 1 14.7 5/34 33 2 4 1 5 3 2 2 3 - - 7/37 18.9 34 1 1 1 1 1 - - - - 1 1/1 100 35 3 5 4 3 4 4 1 - 2 1 8/10 80.0 36 2 4 3 2 3 1 - 3 1 - 5/17 29.4 37 - - 1 - 1 - 1 - - - 1/1 100 38 1 1 1 1 - - - - 1 - 1/2 50.0 39 - 1 - - - 1 - - - - 1/4 25.0 40 1 - - - - 1 - - - - 1/2 50.0 41 10 10 8 6 9 1 5 5 3 1 15/17 88.2 42 - - - - - - - - - - 0/0 - 43 1 - - 2 - 3 - - - - 3/27 11.1 44 - - - - - - - - - - 0/1 0.0 45 11 4 9 2 9 - 2 6 2 1 11/14 78.6 46 - - - - - - - - - - 0/2 0.0 Total 323 225 206 225 249 296 167 111 40 21 635/2014 Total % 14.7 8.3 5.5 1.99 1.04 31.5 1 2 Columns demonstrate the number of bacterial isolates per honey sample that exerted activity against the corresponding pathogens. Total number of bacterial isolates that inhibited the growth of 1–5 pathogens. Appl. Sci. 2021, 11, 5801 7 of 20 Table 2. Molecular identification of bacterial isolates and molecular screening for the presence of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes. Group Strain bp % Identity Bacteria Accession Number NRPS PKS Antibacterial Activity CTA1 1381 100 1,2,3,4,5 B. pumilus MW700012 + * - CTA2 1395 100 1,2,3,4,5 B.pumilus MW700013 + - CTA15 1376 100 2,3,5 B.pumilus MW700019 + - CTA16 1389 99.6 2,3,5 B.vallismortis MW700020 + + * Bacillus sp. CTA31 1347 100 1,2,5 MW700025 + - (B. aerius/B. altitudinis/ B. aerophilus/B. stratosphericus) CTA57 1385 100 2,3,4 B.safensis MW700028 + + CTA146 1419 100 4,5 B.safensis MW700038 - + CTA163 1311 99.9 3,5 B.licheniformis MW700039 + * - CTB7 1400 100 1,2,3,4,5 B.safensis MW700041 - + CTB16 1403 100 1,2,3,4,5 B.safensis MW700043 + - CTB21 1300 100 1,2,3,4 B.pumilus MW700044 + - CTB26 1340 99.9 1,2,4,5 B.pumilus MW700045 - - Bacillus sp. CTB31 1094 99.9 1,3,4,5 MW700046 + + * (B. amyloliquefaciens/B. velezensis) CTB43 1403 100 2,3,4 B.safensis MW700049 + + CTB89 1132 100 1,5 B.safensis MW700053 + + CTB120 1369 100 3,4 B.safensis MW700057 + - CTA5 1396 99.9 1,2,3,5 B.subtilis MW700014 + + * CTA9 1384 100 2,3,4,5 B.subtilis MW700016 + + * CTA10 1373 100 2,3,4,5 B.subtilis MW700017 + * + * Bacillus sp. CTA14 681 99.6 2,3,5 MW700018 + * - (B. haynesii/B. piscis/ B. paralicheniformis/B. licheniformis) CTA20 1410 100 1,3,5 MW700021 + * + * B. subtilis Bacillus sp. CTA109 1414 99.9 2,5 MW700035 - + * (B. halotolerans/B. mojavensis) Bacillus sp. (B. subtilis subsp. Inaquosorum/ CTB11 1281 99.6 1,2,3,4,5 MW700042 - + * B. nakamurai/B. mojavensis/ B. halotolerans) Appl. Sci. 2021, 11, 5801 8 of 20 Table 2. Cont. Group Strain bp % Identity Bacteria Accession Number NRPS PKS Antibacterial Activity P. megaterium CTA7 1401 99.9 1,3,4,5 MW700015 + * - (B. megaterium) CTA28 1401 100 1,2,4 B.paramycoides MW700024 + - Bacillus sp. CTA46 1413 99.9 3,4,5 MW700027 + * + (B. proteolyticus/B. wiedmannii) CTB34 1414 99.9 1,2,3,5 B.cereus MW700048 - - CTB116 1405 100 2,5 B.cereus MW700056 - - CTA39 1349 99.9 3,4,5 MW700026 - - S. pasteuri CTA79 1370 99.9 1,5 MW700029 - - F S.cohnii CTB46 1411 99.9 1,2,4 MW700050 - + S.cohnii CTA107 897 100 2,5 P. stutzeri MW700034 - - CT110 1322 100 2,4 A. lwoffii MW700055 - - CTA23 1375 99.8 2,4,5 P. fulva MW700022 + * - Pseudomonas sp. (P. putida/P.fulva/P.parafulva/ H P. guariconensis/P. plecoglossicida/ CTA90 708 99.9 1,3 MW700031 + * + P. cremoricolorata/P. taiwanensis/ P. donghuensis/P.wadenswilerensis/ P. alkylphenolica) I CTA125 1436 98.9 1,2 Bacillus sp. MW700036 - + * Terribacillus sp. J CTA25 1371 99.9 2,4,5 MW700023 - - (T. goriensis/T. saccharophylus) K CTA84 1278 99.8 2,3 P.coleopterorum MW700030 - - L CTA95 1333 99.9 1,3 M. imperiale MW700032 + * - Bacillus sp. M CTA99 1349 100 1,3 MW700033 - - (B. proteolyticus/B. wiedmannii) Bacillus sp. CTA138 1405 99.9 2,4 MW700037 - - (B. proteolyticus/B. wiedmannii) CTB60 1404 99.9 3,4,5 L. fusiformis MW700051 - - S CTA169 496 99.8 3,5 P. profundus MW700040 + * + * T CTB32 1382 99.9 1,2,3,5 M. yunnanensis MW700047 - - Appl. Sci. 2021, 11, 5801 9 of 20 Table 2. Cont. Group Strain bp % Identity Bacteria Accession Number NRPS PKS Antibacterial Activity W CTB93 1379 99.8 1,2 T. saccharophilus MW700054 - - Paenibacillus sp. Y CTB69 1416 99.6 2,4,5 MW700052 + * + * (P. cucumis/P. polysaccharolyticus) CT2H 440 99.8 1,2,3,4 S. epidermidis MW700058 - - LAB1 CT7C 580 100 3,4,5 S. warneri MW700060 - - LAB2 CT6C 563 99.6 1,2,3,4,5 S.arlettae MW700059 + - LAB3 CT12B 432 99.8 1,2,5 S.hominis MW700061 - - * Amplicons were sequenced. (1) S. aureus, (2) P. aeruginosa, (3) S. Typhimurium, (4) A. baumani, (5) C. freundii. Appl. Sci. 2021, 11, 5801 10 of 20 Furthermore, all 50 isolates were screened for the presence of NRPS and PKS-I biosyn- thetic gene clusters. In fourteen isolates, NRPS genes were detected; six isolates were tested positive for PKS gene clusters whereas in 13 isolates both NRPS and PKS gene clusters were detected. Twenty-six out of 31 (83.9%) bacterial isolates identified as Bacillus spp. tested positive for NRPS or PKS or both. Specifically, 11 out of the 31 (35.5%) bacterial isolates identified as Bacillus spp. tested positive only for NRPS genes (78.6% of the isolates positive for NRPS, 11/14), 5 were positive only for PKS genes (83.3% of the isolates positive for PKS, 5/6) and 10 isolates were detected positive for both NRPS/PKS gene clusters (76.9% of all isolates positive for both NRPS and PKS, 10/13). Moreover, only NRPS genes were detected in 4 out of 5 identified B. pumilus strains (CTA1, CTA2, CTA15, CTB21), in 2 out of 9 Bacillus sp. (CTA31, CTA14), and in 2 out of 7 B. safensis strains (CTB16, CTB120). B. licheniformis (CTA163), P. megaterium (CTA7), and B. paramycoides (CTA28) were also positive just for NRPS genes. Two B. safensis (CTA146, CTB7) and 3 Bacillus sp. (CTA109, CTB11, CTA125) strains were positive just for PKS gene clusters. Both NRPS and PKS genes were detected in B. vallismortis (CTA16), in 4 B. subtilis strains (CTA5, CTA9, CTA10, CTA20), 3 B. safensis (CTA57, CTB43, CTB89), and 2 Bacillus spp. (CTB31, CTA46) strains. Two out of 4 (50%) bacterial isolates identified as Pseudomonas spp. tested positive for NRPS (P. fulva CTA23) or both NRPS/PKS (Pseudomonas sp. CTA90) gene clusters. In addition, 2 out of 7 isolates identified as Staphylococcus spp. tested positive for NRPS (S. arlettae CT6C) and PKS (S. cohnii CTB46) gene clusters. The strain M. imperiale (CTA95) was positive for NRPS encoding genes, while in P. profundus (CTA169) and Paenibacillus sp. (CTB69) both NPRS and PKS gene clusters were detected. Twelve NRPS amplicons and 11 PKS amplicons were sequenced, in order to validate the PCR result (Tables 3 and 4). Ten out of 12 amplicons were indeed identified as NRPS gene clusters. The identification of the other two amplicons was not possible due to low quality of sequencing data. In most cases there was an agreement regarding the genus classification and sequencing results, except for amplicons A1 and A7 that were amplified from bacterial isolates belonging to Bacillus spp., whereas the NPRS amplicons shared homology with NRPSs of P. moteilii. Amplicons A10, A20, and A46 from the strains B. safensis (CTA10), B. safensis (CTA20), and Bacillus sp. (CTA46) were identified as NRPSs that were implicated in surfactin biosynthesis, while B69 from the strain Paenibacillus sp. shared homology with the subunit B of gramicidin synthase. Furthermore, the sequencing data verified the PCR results for the 11 tested PKS ampli- cons. Six amplicons exhibited homology with ketosynthases, (A5, A9, A10, A20, A125, B11), 3 were identified as PKSs (A109, B31, B69), while amplicon A16 was identified as amino acid adenylation domain-containing protein. In all cases there was an agreement regarding the classification at genus level and the sequencing results. Interestingly, sequencing data regarding A9 amplicon of B. subtilis CTA9 strain suggest that bacillaene is biosynthesized by this strain. Appl. Sci. 2021, 11, 5801 11 of 20 Table 3. Sequencing results for the NRPS amplicons. Sample Species BLASTN % Identity BLASTX % Identity amino acid adenylation P. monteilii (AMA45849.1 A1 B. pumilus 84.4 domain-containing 78.7 hypothetical protein) protein (P. monteilii) P. monteilii (Gene APT63_12835, A7 P. megaterium 67.2 NRPS (P. monteilii) 41.7 AMA46431.1 hypothetical protein) B. subtilis strain JCL16 A10 B. subtilis chromosome (Gene srfAA), 84.2 Surfactin NRPS SrfAA (B. subtilis) 72.7 QKJ76470.1 surfactin NRPS SrfAA B. licheniformis strain P8_B2 D-alanine–poly(phosphoribitol) ligase A14 Bacillus sp. chromosome, QGI45530.1 95.9 91.1 subunit DltA (B. licheniformis) D-alanine–poly(phosphoribitol) ligase subunit DltA B. subtilis subsp. subtilis str. 168 chromosome (QJR44804.1 A20 B. subtilis 98.1 surfactin NRPS SrfAA (B. subtilis) 98.6 surfactin NRPS SrfAA amino acid adenylation P. monteilii strain USDA-ARS-USMARC-56711 A23 P. fulva 96.5 domain-containing 98.4 (Gene APT63_09540, AMA45849.1, hypothetical protein protein (P. monteilii) B. safensis strain PgKB20 A46 Bacillus sp. 96.5 NRPS (B. safensis) 99.49 chromosome Surfactin synthase subunit 2 (gene srfAB_2) amino acid adenylation P. monteilii strain USDA-ARS-USMARC-56711, gene A90.2 Pseudomonas sp. 91.8 domain-containing 87.7 APT63_09540, hypothetical protein protein (P. monteilii) A95 M. imperiale M. oleivorans strain I46 chromosome DNA gyrase subunit A 68.1 no no A163 B. licheniformis B. haynesii strain P19 chromosome 73.2 no no P. thiaminolyticus strain A169 P. profundus NRRL B-4156 amino acid 71.8 NRPS/PKS (P. apiarius) 56.5 adenylation domain-containing protein FLT43_25335 P. polymyxa E681 isolate type B chromosome Linear AMP-binding protein B69 83.5 96.6 Paenibacillus sp. gramicidin synthase subunit B, lgrB_2 (P. amylolyticus) Appl. Sci. 2021, 11, 5801 12 of 20 Table 4. Sequencing results for the PKS amplicons. Sample Species BLASTN % Identity BLASTX % Identity B. subtilis subsp. subtilis str. 168 chromosome amino acid A5 B. subtilis 81.8 ketosynthase, partial (Bacillus sp.) 82.7 adenylation domain-containing protein, HIR77_09320 B. subtilis subsp. subtilis strain UCMB5021 Malonyl CoA-acyl A9 B. subtilis carrier protein PksN transacylase involved in nonribosomal 77.1 ketosynthase, partial (Bacillus sp.) 76.1 synthesis of bacillaene, gene pksN B. subtilis strain JCL16 A10 B. subtilis chromosome, amino acid 78.9 ketosynthase, partial (Bacillus sp.) 78.2 adenylation domain-containing protein, gene HR084_09185 amino acid adenylation B. amyloliquefaciens strain R8-25 chromosome (or valezensis) A16 B. vallismortis amino acid adenylation domain-containing protein, gene 76.4 domain-containing protein 93.2 HT132_11940 (B. amyloliquefaciens) B. subtilis subsp. subtilis str. 168 chromosome, amino acid A20 B. subtilis 85.7 ketosynthase, partial (Bacillus sp.) 87.86 adenylation domain-containing protein, gene HIR77_09320 P. thiaminolyticus strain NRRL B-4156 chromosome acyltransferase domain-containing protein A109 Bacillus sp. 81.2 100 acyltransferase domain-containing protein, gene FLT43_06730 (P. apiaries) B. subtilis strain JCL16 A125 79.2 77.7 Bacillus sp. chromosome amino acid ketosynthase, partial (Bacillus sp.) adenylation domain-containing protein, gene HR084_09185 P. thiaminolyticus strain NRRL B-4156 chromosome acyltransferase domain-containing protein A169 81.1 100 P. profundus acyltransferase domain-containing protein, gene FLT43_06730 (P. apiarius) type I ketosynthase B. halotolerans strain KKD1 B11 Bacillus sp. 99 97.4 chromosome NRPS, gene HT135_09465 (Bacillus sp. LX-99) Bacillus sp. AM1(2019) B31 Bacillus sp. 72.9 PKS (B. velezensis) 60 chromosome zinc-binding dehydrogenase, gene GNE05_12430 PKS B69 Paenibacillus sp. Paenibacillus sp. B37 PKS gene, PKS 94.2 98.5 (Paenibacillus sp. B15) Appl. Sci. 2021, 11, 5801 13 of 20 4. Discussion The antimicrobial activity exerted by honey is one of its most well-established bioactiv- ities described in many studies [11]. Honey affects the growth and survival of microorgan- isms via multiple mechanisms [14,41]. Low pH, high sugar concentration, high osmolarity and the presence of antibacterial substances such as hydrogen peroxide and phytochemicals in honey provide a hostile environment for bacteria [42]. Consequently, honey is expected to contain a limited number and diversity of microorganisms [14]. Nevertheless, honey is not sterile since it is a natural product that is not processed [3,5,12,28]. Several studies have reported isolated microorganisms from honey samples produced in various geographical locations [4,7,12,18,43]. Interestingly, the number of microorganisms in honey ranged widely, from 0 to several thousand colony forming units (CFUs) per gram, depending on the sample and its freshness [8,9,18]. Sinacori et al. [18] reported low bacterial load in 33 out of 38 honey samples harvested in southern Italy. Similar results were reported by other researchers in Argentina [8], Morocco [15], Poland [12], Saudi Arabia [44], and Mexico [7]. Fernández et al. [27] studied the microbiological quality of honey from Argentina and their results were comparable to those reported in previous studies conducted in Argentina as well as other parts of the world [8,12,18,28,45]. Bacteria have also been isolated from stingless bee honey [4,23,42]. Moreover, there are few studies that have described the microbial communities of honey using culture-independent methods like next generation sequencing (NGS) [17,46–48]. Honey bacteria exerting antibacterial activity have been successfully isolated by sev- eral research groups [6,12,42,49–52]. There are also reports on the antibacterial activity exerted by various bacterial isolates from stingless bee (Heterotrigona itama) honey [34,42]. Lee et al. [28] screened more than two thousand bacterial strains isolated from six US domestic honeys and two manuka honeys against seven bacteria and one mold but identi- fication of bacterial isolates was not conducted. Recently, Pajor et al. [12] investigated the antimicrobial potential of 163 bacteria isolated from honey samples produced in Poland. A number of studies have demonstrated high antibacterial activity of Greek honeys against major nosocomial and foodborne pathogens [1,13,35,53]. However, this is the first study aiming to isolate and characterize bacteria from diverse types of Greek honeys exhibiting antibacterial potential. In total, 2014 bacterial isolates were screened against five important human and foodborne pathogens (S. aureus, P. aeruginosa, S. Typhimurium, A. baumannii, and C. freundii). Three of them belong to the “ESKAPE” pathogens group (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp.). The World Health Organization (WHO) included ESKAPE in a list of pathogens for which new antimicrobial compounds are urgently needed. A. baumannii and P. aeruginosa are listed in the critical priority list of pathogens, whereas S. aureus (MRSA) is in the high priority group [54–56]. Furthermore, multidrug-resistant C. freundii is of major concern, often implicated in nosocomial infections [57,58]. This is the first study regarding the antibacterial activity of honey bacterial isolates against C. freundii and A. baumanii. Moreover, in this study a higher number of bacterial isolates (2014) was screened against five pathogens, compared to relevant studies [4,42,51,52]. The most susceptible pathogen was S. aureus, followed by C. freundii, P. aeruginosa, and A. baumannii. In contrast, S. Typhimurium was the least affected tested pathogen. A possible explanation might be that Gram-negative bacteria possess an outer membrane which often contributes to resistance to a wide range of antibacterial factors (for instance, loss of porins located in outer membrane). In agreement with our study, Pajor et al. [12] reported that 20.3% of the honey isolated strains inhibited the growth of S. aureus ATCC 25923, while activity against P. aeruginosa ATCC 27857 was rarely observed. In previous studies, aerobic spore-forming bacteria belonging to Bacillus spp. and related genera (Brevibacillus, Lysinibacillus, Paenibacillus) have been identified as the most common bacteria present in honey [5,59]. Furthermore, bacteria belonging to Bacillus genus have been often isolated from the honeybee gut [60–62]. Similarly, in this study, 62% of the identified isolates belonged to the genus Bacillus. Malika et al. [15] detected Bacillus Appl. Sci. 2021, 11, 5801 14 of 20 spores in most of the honey samples from Morocco, while Pomastowski et al. [3] reported that regardless of the geographical and botanical origin, the main bacteria in honeys were spore-forming Bacillus spp., which is in agreement with our data. Bacteria belonging to B. subtilis or B. cereus groups are the most frequently detected in honey [3,25]. Indeed, B. safensis was the most frequently identified species in our study followed by B. pumilus, B. subtilis, and B. cereus. The species B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. safensis, B. subtilis, and B. vallismortis have been detected in honey samples from diverse geographical locations [3,4,9,22,25,42,44,60,63]. Nevertheless, to the best of our knowledge, this is the first time that B. paramycoides, L. fusiformis, M. imperiale, M. yunnanensis, P. profundus, and T. saccharophilus were isolated from honey. Paenibacillus strains isolated from various ecological niches synthesize a wide range of antibiotics (polymyxins, polypeptins, gavaserin) [51,64]. P. profundus strain Sl 79, a deep sediment bacterium, exerted a remarkable inhibitory activity against both Gram- positive and Gram-negative bacteria. It is known to produce isocoumarin and a new linear glyceryl acid-derived heptapeptide that exhibited antibacterial activity against S. aureus, S. epidermidis, B. subtilis, and E. faecium [64,65]. On the other hand, M. yunnanensis was previously detected in bee gut [61,66] but not in honey. Micrococcin was the first thiazolyl peptide isolated from a Micrococcus strain [67]. Sponge-derived M. yunnanensis F-256,44 was reported to produce kocurin, an antibiotic of the thiazolyl peptide family [67,68]. Moreover, Ravi Ranjan et al. [69] isolated M. yunnanensis strain rsk5, which produced a novel antibacterial compound. This compound exerted activity against antibiotic-resistant S. aureus and differs from other characterized secondary metabolites. T. saccharophilus, as its name suggests, is a sugar-loving bacterium [70]. A study reported that a T. saccharophillus strain isolated from the gut of an edible Indian freshwater fish could be applied as a probiotic in fish feed formulations [71]. Another study reported the production of copious amounts of exopolysaccharides by T. saccharophilus strain PS-47. Exopolysaccharides are secondary metabolites, used in the pharmaceutical and food industries [72]. Microbacterium spp. were previously isolated from the gastrointestinal tract of adult worker honeybees [62]. A study reported that M. imperiale (MAIIF2a) reduced fungal symptoms by Fusarium solani in cassava roots, indicating its antimicrobial potential [73]. The antibacterial activity of B. paramycoides and L. fusiformis against important human and food pathogens has been described for the first time in this study. However, these bacterial species are known for their bioremediation potential [74–76]. Moreover, B. paramy- coides was reported as a plant growth-promoting rhizobacterium [77], while L. fusiformis strain C250R produced a novel extracellular thermostable protease designated as SAPLF, which could be applied in pharmaceuticals, food, and biotechnology industries [78]. In this study, 14% of the characterized bacterial isolates were identified as Staphylo- coccus spp. Previous studies have reported the presence of S. epidermidis, S. pasteuri, and S. hominis in honey [3,22,79]. Moreover, all Staphylococcus spp. identified in this study, except S. arlettae, have been described as part of the honeybee gastrointestinal micro- biome [3,61,62,66,80,81]. S. arlettae is commonly found in the skin and nares of poultry and goats [82]. Surprisingly, none of the bacterial isolates grown on MRS agar that exerted antimicro- bial acitivity in this study was identified as lactic acid bacteria. However, all four isolates were identified as Staphylococcus spp., one of them (S. hominis) previously isolated using MRS agar [83]. Only 10% of the characterized bacterial isolates were identified as Gram-negative bacteria, namely Pseudomonas sp., P. coleopterorum, P. fulva, P. stutzeri, and A. lwoffii. Pseu- domonas spp. and Actinetobacter spp. are often present in soil [7]. However, Veress et al. [84] isolated for the first time the bacterium A. lwoffii from a Transylvanian honey sample, suggesting that it was derived from the honeybee microbiota. Pseudomonas spp. have been identified both in honey and the digestive tract of honeybees [9,10,17,22,62]. The Pseudomonas genus is very diverse and comprises 272 species, organized in several major taxonomic groups that are widely distributed in diverse niches [85]. Some of these strains Appl. Sci. 2021, 11, 5801 15 of 20 exert biotechnologically interesting metabolic potential (denitrification, degradation of aromatic compounds, nitrogen fixation) [86–89]. P. fulva is a widespread environmental species exhibiting high biodegrading potential. It is reported to degrade neonicotinoid in- secticides that contribute to honeybee colony collapse disorder [86], as well as a pyrethroid insecticide, reported to be accumulated in honeybee wax samples [87]. On the other hand, P. coleopterorum was detected in the gut microbiota of some plant-related insects [90]. Bee products have been suggested as a rich source of beneficial bacteria that might be potential biocontrol agents against bee pathogens [4,28,42]. Several Bacillus strains produce compounds against pathogenic and food-spoilage bacteria [2,51]. Moreover, B. subtilis, B. amyloliquefaciens, B. pumilus, and B. licheniformis are considered as probiotics and antibiotic producers. Indeed, B. subtilis has already been used as a probiotic supplement for human and animal diets [23,42]. LABs commonly isolated from honey produce secondary metabolites, like bacteri- ocins and lipopeptides [6,42,91,92]. However, we did not identify any LABs exhibiting antibacterial activity in the present study, presumably due to the relatively small number of isolates grown on MRS agar. Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are often implicated in the biosynthesis of medically useful secondary metabolites, such as antibiotics, antitumor agents, immunosuppressants, siderophores, and toxins [30,31,33]. In this study we report the detection of genes encoding NRPSs and PKSs in 14 and 6 out of 50 identified isolates, respectively. NRPS genes were detected more often in Bacillus spp. It is known that Bacillus and Paenibacillus genera produce non-ribosomally synthe- sized lipopetides with significant antimicrobial activity, such as iturin, fengycin, surfactin, and bacillomycin. These compounds are abundantly synthesized, particularly by B. amy- loliquefaciens, B. subtilis, B. licheniformis, and B. pumilus [92,93]. Furthermore, NRPs have been identified in Pseudomonas spp. by several studies [94–97]. Analysis of P. fluorescens In5 revealed the presence of two antifungal NRPs, nunamycin and nunapeptin [94]. An- other study showed that a monomodular NRPS from Pseudomonas fluorescens HKI0770 is implicated in biosynthesis of the antimicrobial pyreudiones [96]. Magdalena et al. [51] have targeted seven antibacterial peptide biosynthetic genes present in two B. velezensis honey isolates using specific primers. However, in our study the universal primer set NRPS-1F—NRPS-1R for the amplification of adenylation (A) domain was used, thus targeting a much broader range of NRPS biosynthetic clusters. PKS gene clusters were detected in B. safensis, Bacillus sp., and S. cohnii strains. Type I PKSs are large multifunctional enzymes, implicated in pharmacologically important metabolites, most often detected within prokaryotes [93,98]. PKs were detected in B. sub- tilis, B. amyloliquefaciens, B. methylotrophicus, B. atrophaeus, B. laterosporus, and Paenibacillus sp. [93]. Within the B. subtilis group, three types of antimicrobial PKs are produced, in- cluding bacillaene, difficidin, and macrolactin [98]. Moreover, Zhang et al. [99], reported the partial isolation of a gene cluster from Staphylococcus lentus encoding a PK exerting broad-spectrum activity against fungi and bacteria. To the best of our knowledge, this is the first time that PKS encoding genes have been detected in bacteria isolated from honey exerting antimicrobial potential. Interestingly, 13 isolates in this study contained both NRPS and PKS gene clusters. Often NRPSs and PKSs are synthesized as hybrid products by NRPS and PKS gene clus- ters [31,33,100], such as bacillaene, compactin, fusarin C, or salinosporamide [98]. Twelve NRPS amplicons and 11 PKS amplicons were sequenced, in order to validate our detection approach. Three out of the 12 NRPS amplicons were implicated in the synthesis of surfactin. Surfactin is a cyclic lipoheptapeptide produced by various strains of Bacillus subtilis, which alters membrane integrity and permeability acting against several microorganisms [51,92,101]. Furthermore, it has been reported that Paenibacillus strains, isolated from various environments, produce a wide range of antibiotics [64,92]. Three Paenibacillus species were detected in honey, P. alvei, P. polymyxa, and P. larvae [3,28,92,102]. Polymyxins produced by Appl. Sci. 2021, 11, 5801 16 of 20 P. polymyxa have been used as a last-resort treatment of multidrug-resistant Gram-negative bacteria infections. They also exhibit strong antifungal activity [93]. Finally, our sequencing results regarding the PKS amplicon from Bacillus subtilis CTA9 strain suggest the presence of a PKS implicated in bacillaene biosynthesis. Bacillaene was firstly isolated from B. subtilis strains, but since then has been reported to be produced by several other Bacillus spp. It is a hybrid PK synthesized by a PKS-I and NRPS operon and it has demonstrated a strong activity against various bacteria and fungi [92,93,98]. Taking together all these data, it is evident that most of the bacterial isolates in this study could be potent producers of secondary metabolites including antibiotics, bacteriocins, and enzymes that might lead to future biotechnological applications. 5. Conclusions In the present study, 2014 bacterial isolates were obtained from 46 Greek honey samples of diverse botanical and geographical origin. All bacterial isolates were tested against five important nosocomial and foodborne pathogens. Overall, 31.5% of the isolates exerted antibacterial activity against at least one pathogen. Gram-positive S. aureus was the most susceptible of the five tested pathogens, followed by C. freundii, P. aeruginosa, and A. baumanii. S. Typhimurium was found to be the least susceptible pathogenic bacterium. Fifty isolates that inhibited the growth of at least two tested pathogens were further characterized. Most of them (62%) were identified as Bacillus spp. Nevertheless, six identified species were isolated from honey samples for the very first time. Moreover, the detection of NRPS and PKS (PKS-I) biosynthetic gene clusters in several isolates is reported, underlining the high biosynthetic potential of honey bacteria. Collectively, the results presented in this study demonstrate that the honey microbiome is an untapped source of antimicrobial metabolites against multidrug-resistant nosocomial and food pathogens that warrants further investigation and bioprospecting. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/app11135801/s1, Table S1: Honey samples of diverse botanical, geographical origin, and harvest date. Number of bacterial isolates per honey sample and per culture medium, Table S2: Grouping of bacterial isolates by RFLP assay and Gram staining. Author Contributions: Conceptualization, D.M.; methodology, C.T., D.M.; investigation, C.T., I.K., T.G.D.; resources, S.P., D.M.; data curation, M.N., G.D.A.; formal analysis, M.N. writing—original draft preparation, C.T., D.M.; writing—review and editing, D.M., T.G.D., S.P., G.D.A.; supervision, D.M.; funding acquisition, D.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the General Secretariat for Research and Innovation (G.S.R.I) under the emblematic action “Honeybee Road”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The research work was supported by the Hellenic Foundation for Research and Innovation (HFRI) under the HFRI Ph.D. Fellowship grant (Fellowship Number: 545) for C.T. I.K. is recipient of a Postdoctoral Fellowship under the emblematic action “Honeybee Road”. Conflicts of Interest: The authors declare no conflict of interest. References 1. Stagos, D.; Soulitsiotis, N.; Tsadila, C.; Papaeconomou, S.; Arvanitis, C.; Ntontos, A.; Karkanta, F.; Adamou-Androulaki, S.; Petrotos, K.; Spandidos, D.; et al. Antibacterial and antioxidant activity of different types of honey derived from Mount Olympus in Greece. Int. J. Mol. Med. 2018, 42, 726–734. [CrossRef] 2. Ng, W.-J.; Sit, N.-W.; Ooi, P.A.-C.; Ee, K.-Y.; Lim, T.-M. The Antibacterial Potential of Honeydew Honey Produced by Stingless Bee (Heterotrigona itama) against Antibiotic Resistant Bacteria. Antibiotics 2020, 9, 871. [CrossRef] Appl. Sci. 2021, 11, 5801 17 of 20 3. Pomastowski, P.; Złoch, M.; Rodzik, A.; Ligor, M.; Kostrzewa, M.; Buszewski, B. Analysis of bacteria associated with honeys of different geographical and botanical origin using two different identification approaches: MALDI-TOF MS and 16S rDNA PCR technique. PLoS ONE 2019, 14, e0217078. [CrossRef] [PubMed] 4. Ngalimat, M.S.; Raja, A.; Rahman, R.N.Z.; Yusof, M.T.; Syahir, A.; Sabri, S. Characterisation of bacteria isolated from the stingless bee, Heterotrigona itama, honey, bee bread and propolis. PeerJ 2019, 7, e7478. [CrossRef] [PubMed] 5. López, A.C.; Alippi, A.M. Feasibility of using RFLP of PCR-amplified 16S rRNA gene(s) for rapid differentiation of isolates of aerobic spore-forming bacteria from honey. J. Microbiol. Methods 2019, 165, 105690. [CrossRef] [PubMed] 6. Aween, M.M.; Hassan, Z.; Muhialdin, B.J.; Eljamel, Y.A.; Al-Mabrok, A.S.W.; Lani, M.N. Antibacterial Activity of Lactobacillus acidophilus Strains Isolated from Honey Marketed in Malaysia against Selected Multiple Antibiotic Resistant (MAR) Gram- Positive Bacteria. J. Food Sci. 2012, 77, M364–M371. [CrossRef] 7. Vázquez-Quiñones, C.R.; Moreno-Terrazas, R.; Natividad-Bonifacio, I.; Quiñones-Ramírez, E.I.; Vázquez-Salinas, C. Microbiologi- cal assessment of honey in México. Rev. Argent. Microbiol. 2018, 50, 75–80. [CrossRef] 8. Iurlina, M.O.; Fritz, R. Characterization of microorganisms in Argentinean honeys from different sources. Int. J. Food Microbiol. 2005, 105, 297–304. [CrossRef] 9. Adadi, P.; Obeng, A.K. Assessment of bacterial quality of honey produced in Tamale metropolis (Ghana). J. Food Drug Anal. 2017, 25, 369–373. [CrossRef] 10. Kafantaris, I.; Amoutzias, G.D.; Mossialos, D. Foodomics in bee product research: A systematic literature review. Eur. Food Res. Technol. 2021, 247, 309–331. [CrossRef] 11. Albaridi, N.A. Antibacterial Potency of Honey. Int. J. Microbiol. 2019, 2019, 1–10. [CrossRef] 12. Pajor, M.; Worobo, R.; Milewski, S.; Szweda, P. The Antimicrobial Potential of Bacteria Isolated from Honey Samples Produced in the Apiaries Located in Pomeranian Voivodeship in Northern Poland. Int. J. Environ. Res. Public Health 2018, 15, 2002. [CrossRef] 13. Tsavea, E.; Mossialos, D. Antibacterial activity of honeys produced in Mount Olympus area against nosocomial and foodborne pathogens is mainly attributed to hydrogen peroxide and proteinaceous compounds. J. Apic. Res. 2019, 58, 756–763. [CrossRef] 14. Bucekova, M.; Bugarova, V.; Godocikova, J.; Majtan, J. Demanding New Honey Qualitative Standard Based on Antibacterial Activity. Foods 2020, 9, 1263. [CrossRef] [PubMed] 15. Malika, N.; And, M.; Adlouni, E.L.; Mohamed, F.; Chakib, E. Microbiological and Physico-chemical properties of Moroccan honey. Int. J. Agric. Biol. 2005, 50, 773–776. 16. Bucekova, M.; Jardekova, L.; Juricova, V.; Bugarova, V.; Di Marco, G.; Gismondi, A.; Leonardi, D.; Farkasovska, J.; Godocikova, J.; Laho, M.; et al. Antibacterial Activity of Different Blossom Honeys: New Findings. Molecules 2019, 24, 1573. [CrossRef] [PubMed] 17. Wen, Y.; Wang, L.; Jin, Y.; Zhang, J.; Su, L.; Zhang, X.; Zhou, J.; Li, Y. The Microbial Community Dynamics during the Vitex Honey Ripening Process in the Honeycomb. Front. Microbiol. 2017, 8, 1649. [CrossRef] [PubMed] 18. Sinacori, M.; Francesca, N.; Alfonzo, A.; Cruciata, M.; Sannino, C.; Settanni, L.; Moschetti, G. Cultivable microorganisms associated with honeys of different geographical and botanical origin. Food Microbiol. 2014, 38, 284–294. [CrossRef] 19. Grecka, K.; Kus, ´ P.; Worobo, R.; Szweda, P. Study of the Anti-Staphylococcal Potential of Honeys Produced in Northern Poland. Molecules 2018, 23, 260. [CrossRef] [PubMed] 20. Estevinho, L.; Pereira, A.P.; Moreira, L.; Dias, L.G.; Pereira, E. Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey. Food Chem. Toxicol. 2008, 46, 3774–3779. [CrossRef] 21. Lu, J.; Cokcetin, N.N.; Burke, C.M.; Turnbull, L.; Liu, M.; Carter, D.A.; Whitchurch, C.B.; Harry, E.J. Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa. Sci. Rep. 2019, 9, 18160. [CrossRef] 22. Grabowski, N.T.; Klein, G. Microbiology and Food-borne Pathogens in Honey. Crit. Rev. Food Sci. Nutr. 2015, 57. [CrossRef] [PubMed] 23. Rosli, F.N.; Hazemi, M.H.F.; Akbar, M.A.; Basir, S.; Kassim, H.; Bunawan, H. Stingless Bee Honey: Evaluating Its Antibacterial Activity and Bacterial Diversity. Insects 2020, 11, 500. [CrossRef] [PubMed] 24. Wirta, H.; Abrego, N.; Miller, K.; Roslin, T.; Vesterinen, E. DNA traces the origin of honey by identifying plants, bacteria and fungi. Sci. Rep. 2021, 11, 4798. [CrossRef] 25. López, A.C.; Alippi, A.M. Phenotypic and genotypic diversity of Bacillus cereus isolates recovered from honey. Int. J. Food Microbiol. 2007, 117, 175–184. [CrossRef] 26. McFrederick, Q.S.; Thomas, J.M.; Neff, J.L.; Vuong, H.Q.; Russell, K.A.; Hale, A.R.; Mueller, U.G. Flowers and Wild Megachilid Bees Share Microbes. Microb. Ecol. 2017, 73, 188–200. [CrossRef] [PubMed] 27. Fernández, L.A.; Ghilardi, C.; Hoffmann, B.; Busso, C.; Gallez, L.M. Microbiological quality of honey from the Pampas Region (Argentina) throughout the extraction process. Rev. Argent. Microbiol. 2017, 49, 55–61. [CrossRef] 28. Lee, H.; Churey, J.J.; Worobo, R.W. Antimicrobial activity of bacterial isolates from different floral sources of honey. Int. J. Food Microbiol. 2008, 126, 240–244. [CrossRef] 29. Kwakman, P.H.S.; Zaat, S.A.J. Antibacterial components of honey. IUBMB Life 2012, 64, 48–55. [CrossRef] 30. Santos-Gandelman, J.; Giambiagi-deMarval, M.; Oelemann, W.; Laport, M. Biotechnological Potential of Sponge-Associated Bacteria. Curr. Pharm. Biotechnol. 2014, 15, 143–155. [CrossRef] 31. Amoutzias, G.; Chaliotis, A.; Mossialos, D. Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes. Mar. Drugs 2016, 14, 80. [CrossRef] Appl. Sci. 2021, 11, 5801 18 of 20 32. Chen, Y.-S.; Yanagida, F.; Hsu, J.-S. Isolation and characterization of lactic acid bacteria from dochi (fermented black beans), a traditional fermented food in Taiwan. Lett. Appl. Microbiol. 2006, 43, 229–235. [CrossRef] 33. Nikolouli, K.; Mossialos, D. Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics. Biotechnol. Lett. 2012, 34, 1393–1403. [CrossRef] [PubMed] 34. Syed Yaacob, S.N.; Huyop, F.; Kamarulzaman Raja Ibrahim, R.; Wahab, R.A. Identification of Lactobacillus spp. and Fructobacillus spp. isolated from fresh Heterotrigona itama honey and their antagonistic activities against clinical pathogenic bacteria. J. Apic. Res. 2018, 57, 395–405. [CrossRef] 35. Anthimidou, E.; Mossialos, D. Antibacterial Activity of Greek and Cypriot Honeys Against Staphylococcus aureus and Pseu- domonas aeruginosa in Comparison to Manuka Honey. J. Med. Food 2013, 16, 42–47. [CrossRef] [PubMed] 36. Olofsson, T.C.; Butler, È.; Markowicz, P.; Lindholm, C.; Larsson, L.; Vásquez, A. Lactic acid bacterial symbionts in honeybees—An unknown key to honey’s antimicrobial and therapeutic activities. Int. Wound J. 2016, 13, 668–679. [CrossRef] [PubMed] 37. Lane, D. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley and Sons: New York, NY, USA, 1991; pp. 115–175. 38. Turner, S.; Pryer, K.M.; Miao, V.P.W.; Palmer, J.D. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J. Eukaryot. Microbiol. 1999, 46, 327–338. [CrossRef] 39. Tripathi, N.; Sapra, A. Gram Staining; StatPearls Publishing: Treasure Island, FL, USA, 2021. 40. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [CrossRef] 41. Gobin, I.; Crnkovic, ´ G.; Magdalenic, ´ M.; Begic, ´ G.; Babic, ´ A.; Lušic, ´ D.; Vuckovi ˇ c, ´ D. Antibacterial potential of Croatian honey against antibiotic resistant pathogenic bacteria. Med. Glas. 2018, 15, 139–144. [CrossRef] 42. Zulkhairi Amin, F.A.; Sabri, S.; Ismail, M.; Chan, K.W.; Ismail, N.; Mohd Esa, N.; Mohd Lila, M.A.; Zawawi, N. Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia. Int. J. Environ. Res. Public Health 2019, 17, 278. [CrossRef] 43. Saha, S. Isolation, Detection and Characterization of Aerobic Bacteria from Honey Samples of Bangladesh. bioRxiv 2018. [CrossRef] 44. Sadik, M.W.; Al-Samie, M.A.; Ali, M.; Sadik, M.W.; Ali, M.A.M. Survey and identification of microorganisms in Bee Honey samples collected from different plant sources and regions in Saudi Arabia. Glob. Adv. Res. J. Microbiol. 2012, 1, 126–135. 45. Finola, M.S.; Lasagno, M.C.; Marioli, J.M. Microbiological and chemical characterization of honeys from central Argentina. Food Chem. 2007, 100, 1649–1653. [CrossRef] 46. Bovo, S.; Utzeri, V.J.; Ribani, A.; Cabbri, R.; Fontanesi, L. Shotgun sequencing of honey DNA can describe honey bee derived environmental signatures and the honey bee hologenome complexity. Sci. Rep. 2020, 10, 9279. [CrossRef] 47. Bovo, S.; Ribani, A.; Utzeri, V.J.; Schiavo, G.; Bertolini, F.; Fontanesi, L. Shotgun metagenomics of honey DNA: Evaluation of a methodological approach to describe a multi-kingdom honey bee derived environmental DNA signature. PLoS ONE 2018, 13, e0205575. [CrossRef] [PubMed] 48. Knazovick ˇ á, V.; Gábor, M.; Miluchová, M.; Bobko, M.; Medo, J. Diversity of bacteria in Slovak and foreign honey, with assessment of its physico-chemical quality and counts of cultivable microorganisms. J. Microbiol. Biotechnol. Food Sci. 2019, 9, 414–421. [CrossRef] 49. Lee, S.K.; Lee, H. Antimicrobial activity of solvent fractions and bacterial isolates of Korean domestic honey from different floral sources. Food Sci. Biotechnol. 2016, 25, 1507–1512. [CrossRef] 50. Abdel Wahab, W.A.; Saleh, S.A.A.; Karam, E.A.; Mansour, N.M.; Esawy, M.A. Possible correlation among osmophilic bacteria, levan yield, and the probiotic activity of three bacterial honey isolates. Biocatal. Agric. Biotechnol. 2018, 14, 386–394. [CrossRef] 51. Magdalena, S. Characterization of antibacterial activity produced by Bacillus spp. isolated from honey and bee-associated products against foodborne pathogens. Asian J. Trop. Biotechnol. 2020, 17. [CrossRef] 52. Lashani, E.; Davoodabadi, A.; Dallal, M.M.S. Some probiotic properties of lactobacillus species isolated from honey and their antimicrobial activity against foodborne pathogens. Vet. Res. Forum 2020, 11, 121–126. [CrossRef] [PubMed] 53. Voidarou, C.; Alexopoulos, A.; Plessas, S.; Karapanou, A.; Mantzourani, I.; Stavropoulou, E.; Fotou, K.; Tzora, A.; Skoufos, I.; Bezirtzoglou, E. Antibacterial activity of different honeys against pathogenic bacteria. Anaerobe 2011, 17, 375–379. [CrossRef] [PubMed] 54. De Oliveira, D.M.P.; Forde, B.M.; Kidd, T.J.; Harris, P.N.A.; Schembri, M.A.; Beatson, S.A.; Paterson, D.L.; Walker, M.J. Antimicro- bial Resistance in ESKAPE Pathogens. Clin. Microbiol. Rev. 2020, 33. [CrossRef] [PubMed] 55. Mulani, M.S.; Kamble, E.E.; Kumkar, S.N.; Tawre, M.S.; Pardesi, K.R. Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review. Front. Microbiol. 2019, 10, 539. [CrossRef] 56. Tacconelli, E.; Magrini, N. Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. Available online: http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant- bacteria/en/ (accessed on 13 May 2021). 57. Yang, L.; Li, P.; Liang, B.; Hu, X.; Li, J.; Xie, J.; Yang, C.; Hao, R.; Wang, L.; Jia, L.; et al. Multidrug-resistant Citrobacter freundii ST139 co-producing NDM-1 and CMY-152 from China. Sci. Rep. 2018, 8, 10653. [CrossRef] 58. Ranjan, K.P.; Ranjan, N. Citrobacter: An emerging health care associated urinary pathogen. Urol. Ann. 2013, 5, 313–314. 59. Alippi, A.M.; Abrahamovich, E. HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples. Int. J. Food Microbiol. 2019, 305, 108245. [CrossRef] [PubMed] Appl. Sci. 2021, 11, 5801 19 of 20 60. Gilliam, M.; Morton, H.L. Bacteria belonging to the genus Bacillus isolated from honey bees, Apis Mellifera, fed 2,4-D and antibiotics. Apidologie 1978, 9, 213–222. [CrossRef] 61. Gasper, J.; Terentjeva, M.; Kántor, A.; Ivanišová, E.; Kluz, M.; Kac ˇániová, M. Identification of Apis mellifera Gut Microbiota with MALDI-TOF MS Biotyper. Anim. Sci. Biotech. 2017, 50, 2344–4576. 62. Kac ˇániová, M.; Terentjeva, M.; Žiarovská, J.; Kowalczewski, P.Ł. In vitro antagonistic effect of gut bacteriota isolated from indigenous honey bees and essential oils against paenibacillus larvae. Int. J. Mol. Sci. 2020, 21, 6736. [CrossRef] 63. Alippi, A.M. Data associated with the characterization and presumptive identification of Bacillus and related species isolated from honey samples by using HiCrome Bacillus agar. Data Br. 2019, 25, 104206. [CrossRef] 64. Romanenko, L.A.; Tanaka, N.; Svetashev, V.I.; Kalinovskaya, N.I. Paenibacillus profundus sp. nov., a deep sediment bacterium that produces isocoumarin and peptide antibiotics. Arch. Microbiol. 2013, 195, 247–254. [CrossRef] 65. Kalinovskaya, N.I.; Romanenko, L.A.; Kalinovsky, A.I.; Dmitrenok, P.S.; Dyshlovoy, S.A. A New Antimicrobial and Anticancer Peptide Producing by the Marine Deep Sediment Strain “Paenibacillus profundus” sp. nov. Sl 79. Nat. Prod. Commun. 2013, 8. [CrossRef] 66. Aizenberg-Gershtein, Y.; Izhaki, I.; Halpern, M. Do Honeybees Shape the Bacterial Community Composition in Floral Nectar? PLoS ONE 2013, 8, e67556. [CrossRef] [PubMed] 67. Palomo, S.; González, I.; de la Cruz, M.; Martín, J.; Tormo, J.; Anderson, M.; Hill, R.; Vicente, F.; Reyes, F.; Genilloud, O. Sponge-Derived Kocuria and Micrococcus spp. as Sources of the New Thiazolyl Peptide Antibiotic Kocurin. Mar. Drugs 2013, 11, 1071–1086. [CrossRef] [PubMed] 68. Pham, T.M.; Wiese, J.; Wenzel-Storjohann, A.; Imhoff, J.F. Diversity and antimicrobial potential of bacterial isolates associated with the soft coral Alcyonium digitatum from the Baltic Sea. Antonie Van Leeuwenhoek 2016, 109, 105–119. [CrossRef] [PubMed] 69. Ranjan, R.; Jadeja, V. Isolation, characterization and chromatography based purification of antibacterial compound isolated from rare endophytic actinomycetes Micrococcus yunnanensis. J. Pharm. Anal. 2017, 7, 343–347. [CrossRef] [PubMed] 70. An, S.-Y.; Asahara, M.; Goto, K.; Kasai, H.; Yokota, A. Terribacillus saccharophilus gen. nov., sp. nov. and Terribacillus halophilus sp. nov., spore-forming bacteria isolated from field soil in Japan. Int. J. Syst. Evol. Microbiol. 2007, 57, 51–55. [CrossRef] 71. Kalarani, V.; Sumathi, V. Effect of Dietary Supplementation of Bacillus subtilis and Terribacillus saccharophillus on Innate Immune Responses of a Tropical Freshwater Fish, Labeo rohita. J. Clin. Cell. Immunol. 2016, 7, 1–9. [CrossRef] 72. Shukla, P.J.; Dave, B.P. Screening and molecular identification of potential exopolysaccharides (EPSs) producing marine bacteria from the Bhavnagar coast, Gujarat. Int. J. Pharm. Sci. Res. 2018, 9, 2973–2981. [CrossRef] 73. Freitas, M.A.; Medeiros, F.H.V.; Melo, I.S.; Pereira, P.F.; Peñaflor, M.F.G.V.; Bento, J.M.S.; Paré, P.W. Stem inoculation with bacterial strains Bacillus amyloliquefaciens (GB03) and Microbacterium imperiale (MAIIF2a) mitigates Fusarium root rot in cassava. Phytoparasitica 2019, 47, 135–142. [CrossRef] 74. Rashid, A.; Mirza, S.A.; Keating, C.; Ali, S.; Campos, L.C. Indigenous Bacillus paramycoides spp. and Alcaligenes faecalis: Sustainable solution for bioremediation of hospital wastewater. Environ. Technol. 2020, 1–14. [CrossRef] 75. He, M.; Li, X.; Liu, H.; Miller, S.J.; Wang, G.; Rensing, C. Characterization and genomic analysis of a highly chromate resistant and reducing bacterial strain Lysinibacillus fusiformis ZC1. J. Hazard. Mater. 2011, 185, 682–688. [CrossRef] [PubMed] 76. Ren, J.; Wang, C.; Huhetaoli, L.C.; Fan, B.; Niu, D. Biodegradation of acephate by Bacillus paramycoides NDZ and its degradation pathway. World J. Microbiol. Biotechnol. 2020, 36, 155. [CrossRef] 77. Noha, I.O.; Shixue, Y. Isolation and characterization of pea plant (Pisum sativum L.) growth-promoting Rhizobacteria. African J. Microbiol. Res. 2018, 12, 820–828. [CrossRef] 78. Mechri, S.; Kriaa, M.; Ben Elhoul Berrouina, M.; Omrane Benmrad, M.; Zaraî Jaouadi, N.; Rekik, H.; Bouacem, K.; Bouanane- Darenfed, A.; Chebbi, A.; Sayadi, S.; et al. Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R. Int. J. Biol. Macromol. 2017, 101, 383–397. [CrossRef] [PubMed] 79. Guran, H.S.; Durukan, G.; Mann, D. Assessment of the Microbiological Profile, Species Diversity and Antimicrobial Susceptibility of Recovered Bacteria from Retail Honeys in Turkey. J. Hell. Vet. Med. Soc. 2021, 72, 2765–2772. [CrossRef] 80. Anjum, S.I.; Shah, A.H.; Aurongzeb, M.; Kori, J.; Azim, M.K.; Ansari, M.J.; Bin, L. Characterization of gut bacterial flora of Apis mellifera from north-west Pakistan. Saudi J. Biol. Sci. 2018, 25, 388–392. [CrossRef] [PubMed] 81. Andere, C.I.; Monteavaro, C.; Palacio, M.A.; Catena, M.; Rodríguez, E.M.; Collins, A.M. Apis mellifera semen: Bacterial contamination and susceptibility to antibiotics. Apidologie 2011, 42, 551–559. [CrossRef] 82. Lavecchia, A.; Chiara, M.; De Virgilio, C.; Manzari, C.; Monno, R.; De Carlo, A.; Pazzani, C.; Horner, D.; Pesole, G.; Placido, A. Staphylococcus arlettae Genomics: Novel Insights on Candidate Antibiotic Resistance and Virulence Genes in an Emerging Opportunistic Pathogen. Microorganisms 2019, 7, 580. [CrossRef] 83. Sung, C.; Kim, B.-G.; Kim, S.; Joo, H.-S.; Kim, P.I. Probiotic potential of Staphylococcus hominis MBBL 2-9 as anti- Staphylococcus aureus agent isolated from the vaginal microbiota of a healthy woman. J. Appl. Microbiol. 2010, 108, 908–916. [CrossRef] 84. Veress, A.; Nagy, T.; Wilk, T.; Kömüves, J.; Olasz, F.; Kiss, J. Abundance of mobile genetic elements in an Acinetobacter lwoffii strain isolated from Transylvanian honey sample. Sci. Rep. 2020, 10, 2969. [CrossRef] [PubMed] 85. Nikolaidis, M.; Mossialos, D.; Oliver, S.G.; Amoutzias, G.D. Comparative Analysis of the Core Proteomes among the Pseudomonas Major Evolutionary Groups Reveals Species-Specific Adaptations for Pseudomonas aeruginosa and Pseudomonas chlororaphis. Diversity 2020, 12, 289. [CrossRef] Appl. Sci. 2021, 11, 5801 20 of 20 86. Zamule, S.M.; Dupre, C.E.; Mendola, M.L.; Widmer, J.; Shebert, J.A.; Roote, C.E.; Das, P. Bioremediation potential of select bacterial species for the neonicotinoid insecticides, thiamethoxam and imidacloprid. Ecotoxicol. Environ. Saf. 2021, 209, 111814. [CrossRef] [PubMed] 87. Yang, J.; Feng, Y.; Zhan, H.; Liu, J.; Yang, F.; Zhang, K.; Zhang, L.; Chen, S. Characterization of a Pyrethroid-Degrading Pseudomonas fulva Strain P31 and Biochemical Degradation Pathway of D-Phenothrin. Front. Microbiol. 2018, 9. [CrossRef] [PubMed] 88. Adeniji, A.A.; Aremu, O.S.; Loots, D.T.; Babalola, O.O. Pseudomonas fulva HARBPS9.1: Candidate anti-Fusarium agent in South Africa. Eur. J. Plant Pathol. 2020, 157, 767–781. [CrossRef] 89. Lalucat, J.; Bennasar, A.; Bosch, R.; García-Valdés, E.; Palleroni, N.J. Biology of Pseudomonas stutzeri. Microbiol. Mol. Biol. Rev. 2006, 70, 510–547. [CrossRef] 90. Menéndez, E.; Ramírez-Bahena, M.H.; Fabryová, A.; Igual, J.M.; Benada, O.; Mateos, P.F.; Peix, A.; Kolar ˇík, M.; García-Fraile, P. Pseudomonas coleopterorum sp. nov., a cellulase-producing bacterium isolated from the bark beetle Hylesinus fraxini. Int. J. Syst. Evol. Microbiol. 2015, 65, 2852–2858. [CrossRef] 91. Bulgasem, B.Y.; Lani, M.N.; Hassan, Z.; Yusoff, W.M.W.; Fnaish, S.G. Antifungal Activity of Lactic Acid Bacteria Strains Isolated from Natural Honey against Pathogenic Candida Species. Mycobiology 2016, 44, 302–309. [CrossRef] 92. Brudzynski, K. Honey as an Ecological Reservoir of Antibacterial Compounds Produced by Antagonistic Microbial Interactions in Plant Nectars, Honey and Honey Bee. Antibiotics 2021, 10, 551. [CrossRef] 93. Zhao, X.; Kuipers, O.P. Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species. BMC Genomics 2016, 17, 882. [CrossRef] 94. Michelsen, C.F.; Watrous, J.; Glaring, M.A.; Kersten, R.; Koyama, N.; Dorrestein, P.C.; Stougaard, P. Nonribosomal Peptides, Key Biocontrol Components for Pseudomonas fluorescens In5, Isolated from a Greenlandic Suppressive Soil. MBio 2015, 6. [CrossRef] [PubMed] 95. Pauwelyn, E.; Huang, C.-J.; Ongena, M.; Leclère, V.; Jacques, P.; Bleyaert, P.; Budzikiewicz, H.; Schäfer, M.; Höfte, M. New Linear Lipopeptides Produced by Pseudomonas cichorii SF1-54 Are Involved in Virulence, Swarming Motility, and Biofilm Formation. Mol. Plant Microbe Interact. 2013, 26, 585–598. [CrossRef] [PubMed] 96. Klapper, M.; Braga, D.; Lackner, G.; Herbst, R.; Stallforth, P. Bacterial Alkaloid Biosynthesis: Structural Diversity via a Minimalistic Nonribosomal Peptide Synthetase. Cell Chem. Biol. 2018, 25, 659–665.e9. [CrossRef] [PubMed] 97. Li, W.; Rokni-Zadeh, H.; De Vleeschouwer, M.; Ghequire, M.G.K.; Sinnaeve, D.; Xie, G.-L.; Rozenski, J.; Madder, A.; Martins, J.C.; De Mot, R. The Antimicrobial Compound Xantholysin Defines a New Group of Pseudomonas Cyclic Lipopeptides. PLoS ONE 2013, 8, e62946. [CrossRef] [PubMed] 98. Caulier, S.; Nannan, C.; Gillis, A.; Licciardi, F.; Bragard, C.; Mahillon, J. Overview of the Antimicrobial Compounds Produced by Members of the Bacillus subtilis Group. Front. Microbiol. 2019, 10, 302. [CrossRef] [PubMed] 99. Zhang, W.; Zhang, F.; Li, Z.; Miao, X.; Meng, Q.; Zhang, X. Investigation of bacteria with polyketide synthase genes and antimicrobial activity isolated from South China Sea sponges. J. Appl. Microbiol. 2009, 107, 567–575. [CrossRef] 100. Barrios-Llerena, M.E.; Burja, A.M.; Wright, P.C. Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites. J. Ind. Microbiol. Biotechnol. 2007, 34, 443–456. [CrossRef] 101. Carrillo, C.; Teruel, J.A.; Aranda, F.J.; Ortiz, A. Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin. Biochim. Biophys. Acta Biomembr. 2003, 1611, 91–97. [CrossRef] 102. Pajor, M.; Xiong, Z.R.; Worobo, R.W.; Szweda, P. Paenibacillus alvei MP1 as a Producer of the Proteinaceous Compound with Activity against Important Human Pathogens, Including Staphylococcus aureus and Listeria monocytogenes. Pathogens 2020, 9, 319. [CrossRef]

Journal

Applied Sciences – Multidisciplinary Digital Publishing Institute

Published: Jun 22, 2021

Keywords: honey; microbiota; antimicrobial activity; 16S-rRNA gene; nonribosomal peptide synthetase; polyketide synthase; secondary metabolites

There are no references for this article.