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Occurrence of ingression of Salmonella spp. in Betel leaf (Piper betle L.)

Occurrence of ingression of Salmonella spp. in Betel leaf (Piper betle L.) Background: Salmonella spp. is one of the most common pathogens associated with fresh produce related foodborne illness. This study aimed to determine Salmonella spp. contamination level in betel leaf, internalization potential and possible decontamination process. Results: A total of 77% betel leaf sample collected from local market was found to be contaminated with Salmonella spp. Of all the Salmonella spp. isolated and identified, 28.5% belong to Salmonella enterica subsp. enterica serovar Enteritidis, 19.5% belong to Salmonella Typhimurium, 15.6% to Salmonella Paratyphi, 10.4% to Salmonella Schottmuelleri, 9.1% to Salmonella Gallinarum, 10.4% to Salmonella Choleraesuis and 6.5% belong to Salmonella Bongori. Internalized Salmonella spp. showed moderate resistance to commonly used antibiotics. Treatment with common surface food disinfectants could not remove Salmonella spp. completely from betel leaf indicating the possibility that the bacteria may be in internal tissue of the leaf. Assessment of internalization potential showed that Salmonella spp. isolated from inner part of betel leaf pose better internalization potential (6.7–7.4 logCFU/gm) comparing with ATCC (American Type Culture Collection) strains (0.86–0.6 logCFU/gm). The isolates also showed better survivability in internalized condition, biofilm formation ability and motility than ATCC strains. Prevalence and expression of invasion (sefA and invA) and type 3 secretion system (TTSS) associated genes (hila, avrA and sopE) were high in internalized Salmonella isolates. Commercial disinfectants as well as H O were 2 2 found to have poor efficacy (log reduction around 2 CFU/gm) against internalized Salmonella. Ozonated water showed better decontamination efficiency (log reduction around 3 CFU/gm) whereas ethanolic extract of Terminalia arjuna stem bark showed higher decontamination (log reduction around 4.5 CFU/gm) of internalized Salmonella. Conclusion: Salmonella spp. can ingress into betel leaf and better decontamination treatment is needed to be established. Keywords: Internalization, Salmonella, Betel leaf, Disinfection Background part and subsequent translocation of bacteria to leaf and Foodborne illnesses with linkage to consumption of con- other aerial parts of the plant (Goldberg et al. 2011; taminated fresh products (such as betel leaf) is increas- Zhuang et al. 1995; Avila-Quezada et al. 2010; Hirneisen ing and becoming a significant food safety issue et al. 2012; Zheng et al. 2013; Bernstein et al. 2007a; worldwide nowadays (Gomes et al. 2009; Gorny 2006). Bernstein et al. 2007b). This event is alarming due to the Contamination can occur at any stage of production fact that internalization of pathogenic bacteria into fresh such as pre-harvest or post-harvest (Semenov et al. produce pose high risk to the consumers as they are sig- 2010; Goldberg et al. 2011; Bernstein 2011). Some recent nificantly resistant to external biocidal washing agents studies reported internalization of bacteria into the plant (Ibarra-Sanchez et al. 2004; Jablasone et al. 2005; Donkor et al. 2010). Though the mechanism of internalization of bacteria into plant parts are still poorly understood * Correspondence: fakruddinmurad@gmail.com Industrial Microbiology Laboratory, Institute of Food Science and (Auty et al. 2005), both plant and bacteria related factors Technology (IFST), Bangladesh Council of Scientific and Industrial Research contribute to its internalization (Erickson et al. 2010; Gu (BCSIR), Dr. Kudrat I Khuda Road, Dhanmondi, Dhaka 1205, Bangladesh et al. 2011). Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 2 of 10 Salmonella spp. is one of the most common pathogens Additionally, Salmonella spp. isolated from surface associated with fresh produce related foodborne illness (n = 25) and internalized condition (n = 17) during (Sivapalasingam et al. 2004; Lapidot et al. 2006). Recent this studywereused. reports clearly demonstrate that Salmonella not only sur- vive passively, but also infect plants actively (Wiedemann Sample collection et al., 2015; Schikora et al. 2012; Akhtyamova, 2013). In this experiment, a total of 100 samples was collected Moreover, infection of plants depends on the active sup- from different markets of Dhaka city, Bangladesh. Sterile pression of the host immune responses by Salmonella polybags were used to collect the samples aseptically. (Schikora et al. 2012). Most studies on Salmonella plant Samples were collected as number of leaves (not weight). interactions suggested an epiphytic lifestyle of Salmonella Twenty leaves were collected from each location. A total on plants (Berger et al. 2010). of ten sample was collected from each market. The Betel leaf (Piper betle L.) is a masticatory with import- sampling packs were marked properly and carried in ice ant socio-cultural and ceremonial use in south and box and transported to the laboratory within 3 h. Experi- southeast Asia. It is also an economically important ex- ments were carried out within 1–8 h after collecting the port commodity. Betel leaf is consumed raw through samples. All the samples were kept at 4 °C until these chewing mainly by South-Asian populations living were analyzed. worldwide. Betel leaf pose significant nutritional values and medicinal properties and is traditionally known to Identification and enumeration of Salmonella spp. be useful for the treatment of various diseases like bad Detection and identification of Salmonella spp. was done breath, boils and abscesses, conjunctivitis, constipation, according to ISO/DIS 6579-1. Briefly, 25 g leaf sample headache, hysteria, itches, mastitis, mastoiditis, was stomached with 225 ml 1% peptone water and leucorrhoea, otorrhoea, ringworm, swelling of gum, stomached sample was pre-enriched (Buffered peptone rheumatism, abrasion, cuts and injuries etc. as folk water) and enriched (tetrathionate broth and RV broth) medicine (Khanra 1997). Further, the essential oil and then selective media (XLD agar) was employed for contained in the leaves possesses antibacterial, anti- specific detection according to the method. Isolates were protozoan and antifungal properties (Hoque et al. 2011). further identified on the basis of biochemical tests (TSI Betel leaf is a product that is regularly consumed fresh agar/Urea agar/indole production/Voges-proskauer/Ly- and raw, but is difficult to decontaminate, as a result, sine decarboxylase). Enumeration of Salmonella spp. in like other fresh produces, can be a common vehicle of betel leaf was done according to Husna et al. (2015). transmission of enteropathogenic bacteria (Berger et al. Briefly, samples were stomached in peptone water and 2010). It is mainly produced in Bangladesh and India serially diluted and plate on XLD agar plate by spread and exported worldwide. Due to the contamination of plate technique and incubated at 37 °C for 24 h. Salmon- betel leaf with Salmonella spp., export of betel leaf has ella spp. were counted and expressed as logCFU/g. been reduced and sometimes suspended (Montanari 2015). Contamination of fresh produce like betel leaf Treatment of betel leaf with food disinfectants with Salmonella spp. pose great public health risk to the H O (10%, v/v), sodium hypochlorite (Sigma, USA) 2 2 consumer. Hence, research on contamination level and (10%, w/v) and commercial disinfectant (collected from pattern of Salmonella spp. and effective decontamin- local market) (1 & 2) were used to treat betel leaves (col- ation process is of great urgency. lected from local market) contaminated with Salmonella The present study aims to determine contamination spp. Briefly, the leaves (ten leave) were manually level and pattern and to evaluate potential decontamin- submerged into 200 ml solutions of the disinfectants for ation methods to neutralize Salmonella spp. contamin- 30 min and rinsed with sterile water for a couple of ation in betel leaf. times. Then the leaves were cut into pieces and Salmonella spp. load was determined as described above. Salmonella-free leaves were submerged for 5 min in Methods solution with ~10 cfu/ml of the bacteria and then Bacterial strains analyzed as the same procedure to serve as controls. Salmonella Enteritidis ATCC 13076, S. Typhimurium ATCC 13311 and S. Typhi ATCC 65154 were used as Detection of internalized Salmonella spp. positive culture in this study. Some environmental isolates Internalized Salmonella spp. has been detected and (n =15) of Salmonella were also included in the study to enumerated according to Franz et al. (Franz et al. 2007). observe differences in gene expression. All the isolates were Briefly, the surface of the collected whole leaf was disin- collected form culture collection pool of Industrial fected with serial washing using tap water, 80% ethanol Microbiology Laboratory, IFST, BCSIR, Dhaka, Bangladesh. for 10 s, 1%AgNO for 5 min, tap water and finally 3 Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 3 of 10 rinsing with deionized water. Then the leaves were cut in static condition. Diameter of motility halos was mea- into pieces and analyzed according to the method de- sured to determine motility. Biofi1m formation assays scribed earlier. Salmonella-free leaves were submerged were performed following the method of Fakruddin et for 5 min in solution with ~10 cfu/ml of the bacteria al. (2014). Bacterial suspension was inoculated into 96 and washed with water, ethanol and AgNO3 as described well polystyrene microtitre plates and incubated over- above and analyzed for both surface and internalized night at 30 °C. After incubation, wells were washed with Salmonella spp. This serves to eliminate the presence of PBS and stained with 0.1% safranin for 30 min at 30 °C. Salmonella as control to assure the presence of no Adhered safranin was solubilized with dimethyl sulfoxide Salmonella spp. in surface after washing. (DMSO) and absorbance of the wells were measured at Leaf internalization of ATCC strains was performed 490 nm wavelength. Specific biofilm formation was then according to Kroupitski et al. (2009). Briefly, ATCC calculated. Salmonella strains was grown overnight in Luria-Bertani (LB) medium and cells were collected and washed twice Virulence gene presence and expression with sterile distilled water and resuspended in sterile dis- Presence of six virulence associated gene in Salmonella tilled water. Intact betel leaves (which were previously spp. isolates has been determined. Isolates were grown tested to be Salmonella free) were submerged in 30 ml in LB broth overnight at 37 °C and total RNA was ex- sterile distilled water in 50 ml sterile tube (one piece per tracted. Description and primer sequence of the genes tube) for 30 min. After that, sterile distilled water of the are presented in Table 1. Virulence gene profile of leaf tube was replaced with 30 ml Salmonella suspension internalized Salmonella spp. was compared with that (~10 cfu/ml) and incubated for 2–3 h at 37 °C. After isolated from leaf surface and previously isolated envir- incubation, the leaves were washed twice with sterile onmental Salmonella strains. For determination of distilled water to remove unattached bacteria. Internal- expression level of the virulence genes, total RNA was ized Salmonella spp. was enumerated as described extracted from bacteria using Trizol reagent and cDNA earlier (Franz et al. 2007). was prepared using primescript RT reagent kit (TaKaRa Bio). Primers used for real time PCR are as follows: sefA Survivability of internalized Salmonella spp. (5’-GGCTTCGGTATCTGGTGGTGTG-3’ and 5’-GTCA Survivability of Salmonella spp. in internalized condition TTAATATTGGCTCCCTGAATA-3’), invA (5’-GCCTG was determined according to Gorbatsevich et al. (2013). CCGGAAGTATTGTTA-3’ and 5’-GGAGTTTCTCCCC Betel leaf samples were sanitized (as described above) and CTCTTCA-3’), hilA (5’-ATTAAGGCGACAGAGCTGG cut into small pieces aseptically and inoculated with bacter- A-3’ and 5’-GAATAGCAAACTCCCGACGA-3’), avrA ial suspension with known cell concentration (LogCFU/ml) (5’-GGAAACCGATCTCGAAATGA-3’ and 5’-TGCTGG and incubated at 37 °C for 14 days. A portion (10 g) of the TTCGAACAAAATCA-3’), sopE (5’-CAACACACTTT- sample was analyzed each day to enumerate internalized CACCGAGGAAG-3’ and 5’-GGTCTGGCTGGCGTAT Salmonella spp. as described earlier. GC-3’) and spvC (5’-AATGAACTACGAAGTGGGCG-3’ and 5’-TCAAACGATAAAACGGTTCCTC-3’), 16 s (5’- Antibiotic resistance of internalized Salmonella spp. TGTAGCGGTGAAATGCGTAG-3’ and 5’-CAAGGGC Antibiotic resistance pattern of the internalized Salmon- Table 1 Primer sequence of virulence genes detection by PCR ella spp. were determined according to the method de- Gene Virulence factor Primer sequence (5’–3’) Base pair scribed by Kirby-Bauer (1966) on Mueller Hinton agar (bp) using commercial discs (Oxoid, UK). The following anti- sefA Fimbria GATACTGCTGAACGTAGAAGG 488 biotics with the disc strength were used: Ciprofloxacin GCGTAAATCAGCATCTGCAGTAGC (CIP, 5 μg), chloramphenicol (C, 30 μg), penicillin (P, 5 unit), tetracycline (TE, 30 μg), doxycycline (DO, 30 μg), invA Invasion GTGAAATTATCGCCACGTTCGGGCAA 284 neomycin (N, 30 μg), gentamycin (GN, 200 μg), ampicil- TCATCGCACCGTCAAAGGAACC lin (AM, 10 μg), erythromycin (E, 15 μg) and nalidixic hilA Invasion CTGCCGCAGTGTTAAGGATA 497 acid (NA, 30 μg). A control strain of E. coli ATCC 25922 CTGTCGCCTTAATCGCATGT was included in each plate. Antimicrobial breakpoints avrA Effector protein GTTATGGACGGAACGACATCGG 385 and interpretation were taken from the CLSI standards. of TTSS ATTCTGCTTCCCGCCGCC Motility and biofilm formation of Salmonella spp. sopE Effector protein ACACACTTTCACCGAGGAAGCG 398 of TTSS Motility of the isolates was determined according to the GGATGCCTTCTGATGTTGACTGG method of Sperandio et al. (2002). Isolates were grown spvC Plasmid - CGGAAATACCATCTACAAATA 669 overnight in LB broth at 37 °C and was spot inoculated virulence CCCAAACCCATACTTACTCTG in center of 0.4% (w/v) LB agar and incubated at 37 °C Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 4 of 10 ACAACCTCCAAG-3’). Transcripts were quantified by LightCycler (Roche Diagnostics) using SYBR Premix Ex Taq (TaKaRa Bio) in accordance with the manufacturer’s instructions. The expression levels of each gene were normalized, with the 16S rRNA gene as an internal control. Decontamination of internalized Salmonella spp. Decontamination with two commercial fruit and vegetable disinfection agent (anshin-yasai & Yokosan; hereby denoted as commercial disinfectant 1 & 2 respectively) was performed according to manufacturer’s instruction. Information listed on the product revealed that active ingredient of disinfectant-1 was calcium oxide and of disinfectant-2 was calcium bicarbonate. Decon- tamination with H O , sodium hypochlorite and 2 2 ozonated water performed according to Bahreini et al. (2013). Ethanolic bark extract of Terminalia arjuna was prepared according to Mahbuba et al. (2012) and decon- tamination process was performed according to Orue et al. (2013). Briefly, selected chemicals and extracts were Fig. 1 Prevalence of Salmonella spp. in betel leaf. a depicts regional diluted with water at different concentrations. Artificially distribution of betel leaf samples infected with Salmonella spp. b inoculated betel leaf were immersed in the suspension shows serovar distribution of Salmonella spp. in infected betel leaf for 10 min. After that, the leaves were washed with ster- ile water, ethanol and AgNO3 (as described above) and presence and load of Salmonella spp. were determined reduction (2.37 log and 2.48 log respectively) (Table 2). as described previously. Control leaves (surface inoculated Salmonella-free leaves treated with same treatments) were found to be negative Statistical analysis for the presence of Salmonella spp. This result indicates All experiments were done twice and all samples per- that surface disinfection based treatment were not ef- formed at least in triplicate. Data were analyzed by SPSS fective to decontaminate Salmonella spp. from betel leaf. 17.0 (SPSS Inc., Chicago, Ill, USA). Occurrence of internalized Salmonella spp. in betel leaf was also determined (Table 3). Surface decontamination Result can reduce Salmonella loads by 1.97 log and treatment Prevalence of Salmonella spp. in betel leaf with disinfectants after surface decontamination reduce A total of seventy seven (77) out of 100 samples were Salmonella loads by 2.78 log, indicating that Salmonella found to be contaminated with Salmonella spp. From spp. may be internalized in inner parts of the leaf. the contaminated samples, 77 strains were isolated after pre-enrichment and enrichment and confirmed through biochemical and serological studies. The total percent- Internalization potential of isolate and ATCC strain ages of occurrence were 77%. Betel leaf collected from Comparison of internalization potential of two Salmon- all areas showed to be contaminated with Salmonella ella strains isolated from internalized condition in betel spp. (Fig. 1a). Prevalence observed in this study were leaf with three (3) ATCC Salmonella strains showed that 28.5%, 19.5%, 15.6%, 10.4%, 9.1%, 10.4% and 6.5% repre- the isolates pose better internalization potential than the senting Salmonella Enteritidis,S. Typhimurium,S. Para- ATCC strains (Table 4). Salmonella isolates from inter- typhi,S. Schottmuelleri,S. Gallinarum,S. Choleraesuis nalized condition showed considerable internalization and S. Bongori respectively (Fig. 1b). potential. Approx. 6.7 logCFU was found to be internal- ized in case of S. Typhimurium isolate and approx. 7.4 Treatment of betel leaf with food disinfectants logCFU was internalized in case of S. Typhi isolate Naturally contaminated betel leaves were washed with (Table 4). In contrast, ATCC isolates showed very little commercial food disinfectant 1 & 2, H O and Sodium internalization (approx. 1.2 logCFU for S. Enteritidis 2 2 hypochlorite. Commercial disinfectant 1 and 2 showed ATCC 13076 and 1.6 logCFU for S. Typhi ATCC 65154) 3.29 log and 3.87 log reduction respectively, while that (Table 3). Control leaves were found to be free of any of sodium hypochlorite and H O showed a lower log internalized Salmonella spp. 2 2 Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 5 of 10 Table 2 Disinfection of contaminated betel leaves with Table 4 Internalization potential of Salmonella spp. isolated different sanitizer from betel leaf Treatment Salmonella count Salmonella count Strain Inoculum Internalized Salmonella name (log cfu/g) (log cfu/g) (log cfu/g) (log cfu/g) before treatment after treatment S. Typhimurium (Isolated from 10.1 ± 0.33 7.7 ± 0.52 Commercial 10.75 ± 0.53 7.46 ± 0.37 internalized condition) disinfectant-1 (10%) S. Typhi (Isolated from internalized 10.1 ± 0.73 8.4 ± 0.73 Commercial disinfectant-2 (10%) 10.68 ± 0.09 6.81 ± 0.48 condition) H O (10%) 10.52 ± 0.32 8.15 ± 0.81 S. Enteritidis ATCC 13076 10.3 ± 0.19 2.2 ± 0.89 2 2 Sodium hypochlorite (10%) 10.31 ± 0.72 7.83 ± 0.19 S. Typhimurium ATCC 13311 10.0 ± 0.27 2.6 ± 0.12 S. Typhi ATCC 65154 10.1 ± 0.44 1.86 ± 0.21 Antibiotic susceptibility and Survivability comparison of internalized Salmonella Antibiotic susceptibility pattern on internalized Salmon- and 76.5% internalized Salmonella spp. isolate respect- ella isolates (n = 17) was shown in Fig. 2 and it shows ively in comparison to surface (72% & 60%) and environ- the isolates pose moderate antibiotic resistance. Com- mental (63.6% & 68.2%) Salmonella spp. isolates. Other parison of survivability of Salmonella Typhi isolate and virulence associated genes such as hilA, avrA and sopE ATCC strains in internalized condition in betel leaf is were present in 64.7%, 52.9% and 64.7% of internalized shown in Fig. 3. All the isolates were able to internalize isolate respectively. These three genes were present in and wild isolates internalized in a greater degree but sur- very low number of surface Salmonella isolates (4%, 4% vivality within the tissue seems to be the same. No sig- & 16%, respectively) and environmental Salmonella iso- nificant differences were observed. After 14 days lates (18.2%, 18.2% & 13.6% respectively). However, incubation, 6.72 logCFU Salmonella spp. were viable in prevalence of spvC gene in all type of isolate is very low internalized condition (initial count at day 0 was 8.31 (Fig. 4). The expression level of the virulence associated logCFU). In case of ATCC Salmonella strain, 5.23 genes in internalized Salmonella spp. were much higher logCFU Salmonella were viable after 14 days incubation than that in surface and environmental isolates (Fig. 5). (initial count 7.58 logCFU) (Fig. 3). Elevated expression of sefA and invA in internalized Sal- monella spp. suggest virulence potential of these isolates. Motility and biofilm forming capability of the isolated Increased expression of T3SS related genes (such as Salmonella hilA, avrA, sopE and sopC) indicates hyper-activation of Biofilm formation mediates survival of bacteria in inter- T3SS in internalized isolates contributing better survival nalized condition (Kroupitski et al. 2009) and motility in internalized conditions. mediates translocation in plant tissues (Warriner et al. 2003). Biofilm formation and motility of internalized Salmonella spp. isolated from betel leaf as well as of ATCC isolates was determined. Isolates showed better biofilm formation ability (SBF > 1) than the reference cultures (SBF < 1) (Table 5). Motility of the isolates were also higher (>40 mm) than that of reference cultures (<20 mm). These data coincide with the better surviv- ability of the isolates in internalized condition than the ATCC cultures. Virulence gene presence and expression sefA and invA are reported to impart virulence in Sal- monella spp. sefA and invA gene were present in 64.7% Table 3 Occurrence of internalized Salmonella spp. in betel leaf Betel leaf Salmonella count Fig. 2 Antibiotic resistance of Salmonella spp. isolated from Betel (logCFU/g) leaf (CIP = Ciprofloxacin; C = Chloramphenicol; P = Penicillin G; Without surface decontamination 9.45 (±0.33) TE = Tetracycline; AM = Ampicillin; DO = Doxycycline; N = Neomycin; With surface decontamination (Internalized) 7.48 (±1.03) GN = Gentamycin; E = Erythromycin; NA = Nalidixic acid). Salmonella strains (n = 17) isolated from internalized condition were used in Treated with disinfectants & without surface 6.67 (±0.11) this experiment decontamination Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 6 of 10 Fig. 4 Virulence gene profile of internalized Salmonella spp. (n =17) in comparison with surface (n = 25) and environmental isolates (n =15) Fig. 3 Survival potential of Salmonella spp. in internalized condition reported Salmonella spp. in betel leaf of Indian origin and Husna et al. (2015) reported Salmonella spp. in Decontamination treatment of internalized Salmonella betel leaf of Mymensingh region, Bangladesh and Decontamination method of internalized Salmonella Salmonella count was around 5 log in their samples. spp. has been performed and showed in Table 6. Com- Contaminated betel leaves were washed with four sur- mercial agents (1 & 2) have been found to have limited face disinfectants (anshin-yasai, yokosan, H O and 2 2 decontamination effect (around 1.2 log and 2 log reduc- sodium hypochlorite) and results showed that these tion respectively) on internalized Salmonella spp. with agents cannot fully decontaminate the betel leaf though increasing efficacy at elevated concentration. H O reduced Salmonella spp. level. From these results, the 2 2 showed almost similar efficacy compare with commer- possibility arises that Salmonella spp. were present not cial agents (1.9 log reduction at 10% concentration). only in surface, but also in the internal parts of the betel Washing leaves with internalized bacteria with ozonated leaf, hence imparting its resistance were to these surface water showed better neutralization effect (3 log reduc- disinfectants. Internalized Salmonella spp. in betel leaf tion) than both commercial agents and H O . Betel samples were enumerated and result showed that a sig- 2 2 leaves treated with Ethanolic bark extract of Terminalia nificant portion of total Salmonella spp. in the leaves are arjuna reduced internalized Salmonella spp. signifi- internalized. cantly. There was a log reduction of 3.8 at 5% concentra- Many previous researchers reported internalization of tion, 4.8 log at 10% concentration and 4.6 log reduction pathogenic bacteria, including Salmonella spp. into plant at 15% concentration (Table 6). leaves. Goldberg et al. (2011) reported internalization of Salmonella Typhimurium in detached leaves of seven Discussion A total of 100 betel leaf samples were collected from dif- ferent regions, 77% of the samples, were found to be contaminated with Salmonella spp. Regional distribution of contaminated betel leaf and species distribution of Salmonella spp. was shown in Fig. 1. Several studies has also reported presence of Salmonella spp. in betel leaf earlier. Singla et al. (2009) and Singh et al. (2006) have Table 5 Comparison of biofilm formation and motility of Internalized and ATCC Salmonella spp. Strain Specific biofilm Motility formation (SBF) (diameter in mm) Internalized S. Typhimurium 1.34 ± 0.14 42 ± 1.8 Internalized S. Typhi 1.53 ± 0.21 45 ± 2.2 S. Enteritidis ATCC 13076 0.76 ± 0.31 21 ± 1.2 S. Typhimurium ATCC 13311 0.81 ± 0.18 18 ± 1.2 Fig. 5 Expression of virulence gene in internalized Salmonella spp. (n = 17) in comparison with surface (n = 25) and environmental isolates (n=15) S. Typhi ATCC 65154 0.51 ± 0.08 20 ± 0.9 Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 7 of 10 Table 6 Decontamination of internalized Salmonella spp. from able to internalize and wild isolates internalized in a betel leaf greater degree but survivality within the tissue seems to Treatment Conc. Salmonella count Log be the same. Salmonella spp. isolates from betel leaf (log cfu/g) Reduction (approx.) showed better biofilm formation ability (SBF > 1) and Before After motility (>40 mm) than ATCC cultures (SBF < 1 and treatment treatment motility <20 mm). Results indicate that the isolated Commercial agent-1 5% 10.1 ± 0.94 8.9 ± 1.14 1.2 internalized Salmonella spp. have moderate resistance to 10% 10.6 ± 0.75 9.2 ± 1.21 1.4 commonly used antibiotics (Fig. 2) but antibiotic 15% 10.9 ± 0.89 8.2 ± 1.09 1.7 resistance were lower compared to the reports of other previous researchers who reported higher resist- Commercial agent-2 5% 10.2 ± 1.05 8.2 ± 0.97 2 ance in Salmonella spp. isolated from different food 10% 10.4 ± 1.12 7.9 ± 0.89 2.5 and poultry samples of Bangladesh (Nipa et al. 2011; 15% 10.1 ± 1.23 8.3 ± 0.76 1.8 Mahbuba et al. 2012). H O 5% 9.7 ± 0.88 8.9 ± 0.81 0.8 2 2 Internalized bacteria can evade disinfection, thus de- 10% 10.1 ± 0.69 8.2 ± 1.11 1.9 tailed study on the mechanism of internalization as well 15% 9.9 ± 1.31 8.1 ± 1.15 1.8 as on plant and environmental factors affecting internal- ization is needed to devise remediation methods ensur- TAEB 5% 10.7 ± 1.12 6.9 ± 1.07 3.8 ing safety of the fresh produces (Ge et al. 2013). 10% 11.2 ± 0.83 6.4 ± 0.89 4.8 Mechanism of internalization of bacteria in the plant 15% 10.9 ± 0.84 6.3 ± 0.96 4.6 has not been elucidated clearly till now. Generally, Ozonated water N/A 10.5 ± 0.95 7.5 ± 1.10 3 internalization is an active process dependent upon the (2 ppm) plant and the pathogen (Hora et al. 2005). Bacteria can (TAEB = Ethanolic bark extract of Terminalia arjuna) internalize through root during cultivation and through the stomata of leaf during pre-/post-harvest (Hoelzer et vegetables and fresh herbs. Hou et al. (2013) reported al. 2014). Bacterial internalization is influenced by the that bacteria such as Salmonella, E. coli O157:H7, Bacil- surface properties of the leaf, including morphology, lus, Enterobacter, Pseudomonas and Pantoea can chemical constituents and metabolic activities (Leveau internalize into lettuce leaves naturally through wounds 2009). Pathogenic bacteria can penetrate internal tissue or via roots and stomata. Avila-Quezada et al. (2010) of the plant through the roots (Solomon et al. 2002), and showed that Salmonella spp. can internalize and migrate seeds (Islam et al. 2004) for further translocation and into plant tissues such as seeds, fruits, leaves, roots and survival in the edible aerial plant tissues (Solomon et al. stems and survive for extended periods in internalized 2002). Some studies reported that bacterial strains varied condition. Guo et al. (2002) reported that Salmonella widely in their endophytic colonization abilities, which spp. can enter fruits and other plant parts through abra- could be related to the plant defense mechanisms that sions. Guo et al. (2001) also reported short distance targeted bacterial extracellular components (Dong et al. migration of Salmonella spp. into plant. Zheng et al. 2003; Iniguez et al. 2005). Water used for the washing of (2013) also reported that Salmonella spp. has the ability the fruits can be contaminated by the pathogens to internalize into tomato plants through roots, leaves while acting as the source of the internalization of and blossoms. Internalization of Salmonella spp. has also the pathogens through the lenticels, stomata and the been reported in mangoes (Penteado et al. 2004), toma- injured parts (Reina et al. 2002). toes (Buchanan et al. 1999), apple (Zhuang et al. 1995), It has been reported previously that many virulence lettuce (Reina et al. 2002), and sweet basil (Gorbatsevich associated genes mediate internalization and persistence et al. 2013). Internalization may occur naturally as of Salmonella spp. in plant. sefA gene is involved in at- reported in these studies, or may occur during washing tachment with plant tissue while invA gene is involved (due to immersion) at post-harvest stages (Gomez-Lopez in epithelial invasion of plant tissue. Of all the virulence et al. 2013). genes, type III secretion system (TTSS) associated genes Internalization efficiency of reference Salmonella cul- are most important for internalization of Salmonella ture (S. Typhimurium ATCC 13311, S. Enteritidis ATCC (Schikora et al. 2012). hilA gene product is a central 13076 and S. Typhi ATCC 65154) and S. Typhi and S. regulator of TTSS and also involved in epithelial Typhimurium isolated from betel leaf (leaf with internal- invasion. avrA gene product is an effector protein of ized Salmonella) has been compared and it was found TTSS and mediate Salmonella internalization and per- that the isolates pose better internalization potential sistence by suppressing the host inflammatory response. (6.7–7.4 logCFU/gm) when compared with the ATCC sopE gene product contributes invasion through gener- cultures (0.86–0.6 logCFU/gm). All the isolates were ation of membrane deformations. spvC gene (located on Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 8 of 10 virulence plasmid) product promotes rapid growth and as lack of fluorescence microscopy and efficient method of survival of Salmonella spp. within host cells (Borges et isolation of nucleic acids directly from leaf, this study al. 2013). Results showed that the prevalence of invasion results indicate occurrence of natural ingression of (sefA and invA) and TTSS associated genes (hila, avrA Salmonella spp. in betel leaf. Future research is needed to and sopE) are high in internalized Salmonella isolates further elucidate detailed mechanism of internalization of which indicates probable mechanisms of internalization Salmonella spp. in betel leaf as well as other plant. of the isolates. It can be postulated that TTSS play cen- tral role in the internalization and persistence of Sal- Conclusion monella spp. in betel leaf. Prevalence of hilA and avrA The present study provide indications of natural internal- gene was more in internalized S. Enteritidis, whereas ization of Salmonella spp. in betel leaf, though the mecha- prevalence of sopE gene is more prevalent in S. Typhi- nisms of internalization is yet to be elucidated. The murium and prevalence of spvC gene was prevalent in S. internalization of pathogenic bacteria like Salmonella spp. Paratyphi and S. Gallinarum. is a public health concern because a small number of Decontamination efficiency of different food disinfec- surviving cells can be potentially lethal. As Internalized tants at different concentrations against internalized Salmonella spp. in betel leaf evade surface disinfection, Salmonella spp. has been evaluated. Commercial disin- elucidation of internalization mechanisms and factors fectants (anshin-yasai and yokosan) as well as H O are (plant, bacterial and environmental) affecting internaliza- 2 2 found to have poor efficacy (log reduction around tion into betel leaf is needed to ensure the safety of this 2 CFU/gm). Ozonated water showed better decontamin- economically important fresh produce. Efficient decon- ation efficacy (log reduction around 3 CFU/gm). Singla tamination method has to be discovered to reduce the risk et al. (2009) reported that treatment of betel leaf with associated with internalized Salmonella in betel leaf. 2% acetic acid reduced artificially surface-contaminated Abbreviations Salmonella spp. by 4 log. Singla (2011) showed com- ATCC: American Type Culture Collection; CFU: Colony forming unit; bined treatment of 2% malic acid along with 2 ppm DMSO: Demethyl sulfoxide; LB: Luria Bertani; PCR: Polymerase chain reaction; RT: Reverse transcription; SBF: Specific biofilm formation; TSI: Triple sugar ozone significantly reduced Salmonella Typhimurium by iron; TTSS: Type 3 secretion system; XLD: Xylose lysine deoxycholate 7 log in turnip and reduced Cronobacter sakazakii by 6.8 log in betel leaf. Husna et al. (2015) showed treat- Acknowledgements The authors acknowledge the help of Ms. Sumaiya Islam, Scientific Officer, ment of betel leaf with sorbitol and sodium benzoate Industrial microbiology laboratory, IFST, BCSIR in conducting this study. can be effective to reduce Salmonella spp. in betel leaf though log reduction was very low compared to other Funding No external funding received. studies. Hadjok et al. (2008) reported UV light (254 nm) combined with H O can reduce contamination levels of 2 2 Author’s contributions human pathogens (Escherichia coli O157:H7, Pectobac- MF and MMA designed the study. MF, NH and RS performed all the experiments. MKI and MMA supervised the study. MF wrote the first draft of terium carotovora, Pseudomonas fluorescens and the manuscript and all authors read and approve the final manuscript. Salmonella) on or within (internalized) fresh produces but reduction of internalized pathogen was around half Competing interests The authors declare that they have no competing interests. of that for surface pathogens. Orue et al. (2013) reported that extracts from oregano and lime are as effective as chlorine based disinfectants for decontamination of Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Salmonella, Shigella and Escherichia coli O157:H7 on published maps and institutional affiliations. leafy vegetables. In our previous studies, we found etha- nolic extract of Terminalia arjuna stem bark has anti- Author details Industrial Microbiology Laboratory, Institute of Food Science and bacterial activity against Vibrio cholerae (Fakruddin et al. Technology (IFST), Bangladesh Council of Scientific and Industrial Research 2011) and Salmonella spp. (Mahbuba et al. 2012). In this (BCSIR), Dr. Kudrat I Khuda Road, Dhanmondi, Dhaka 1205, Bangladesh. study, ethanolic extract of Terminalia arjuna stem bark Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Sylhet 3114, Bangladesh. fared better reducing potential than the rest of the treat- ments (log reduction around 4.5 CFU/gm) (Table 6). Received: 8 September 2016 Accepted: 28 March 2017 Plant extract contains essential oil with antibacterial activity and could be useful for decontamination of References internalized Salmonella spp. Auty M, Duffy G, O’Beirne D, McGovern A, Gleeson E, Jordan K. In situ localization Many reports exist describing internalization of bacteria of Escherichia coli O157:H7 in food by confocal scanning laser microscopy. J Food Protect. 2005;68:482–6. in to plants through artificial inoculation experiments Avila-Quezada G, Sanchez E, Gardea-Bejar AA, Acedo-Felix E. Salmonella spp. and whereas report of incidence of natural internalization was Escherichia coli: survival and growth in plant tissue. New Zealand J Crop still very few. Despite some limitations of this study such Horticul Sci. 2010;38(2):47–55. Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 9 of 10 Bahreini M, Jahed E, Lotfian F, Khaneghah AM, Ghaderifarah M. The efficacy of Hadjok C, Mittal GS, Warriner K. Inactivation of human pathogens and spoilage ozonated water on reduction of four food borne pathogens and microbial bacteria on the surface and internalized within fresh produce by using a quality of lettuce. J Appl Sci Agricul. 2013;8(7):1425–30. combination of ultraviolet light and hydrogen peroxide. J Appl Microbiol. Bauer AW, Kirby WM, Sheris JC, Turck M. Antibiotic susceptibility testing by a 2008;104:1014–24. standardized single disc method. Am J Clin Path. 1996;45:149–58. Hirneisen KA, Sharma M, Kniel KE. Humanenteric pathogen internalization by root uptake into food crops. Foodborne Pathogen Dis. 2012;9(5): Berger CN, Sodha SV, Shaw RK, Griffin PM, Pink D, Hand P, Frankel G. Fresh fruit 395–405. and vegetables as vehicles for the transmission of human pathogens. Environ Microbiol. 2010;12(9):2385–97. Hoelzer K, Pouillot R, Van Doren JM, Dennis S. Reduction of Listeria monocytogenes contamination on produce—A quantitative analysis of common liquid fresh Bernstein N. Potential for contamination of crops by microbial human pathogens introduced to the soil by irrigation with treated-effluent. Israel J Plant Sci. produce wash compounds. Food Control. 2014;46:430–40. 2011;59:115–23. Hoque MM, Ratilla S, Shishir MA, Bari ML, Inatsu Y, Kawamoto S. Antibacterial Bernstein N, Sela S, Neder-Lavon S. Assessment of contamination potential of activity of ethanol extract of betel leaf (Piper betle L.) against some food lettuce by Salmonella enterica serovar Newport added to the plant growing borne pathogens. Bangladesh J Microbiol. 2011;28(2):58–63. medium. J Food Protect. 2007a;70:1717–22. Hora R, Warriner K, Shelp BJ, Griffiths MW. Internalization of Escherichia coli O157: Bernstein N, Sela S, Neder-Lavon S. Effect of irrigation regimes on persistence of H7 following biological and mechanical disruption of growing spinach Salmonella enterica serovar Newport in small experimental pots designed for plants. J Food Protect. 2005;68(12):2506–9. plant cultivation. Irrig Sci. 2007b;26:1–8. Hou Z, Fink RC, Radtke C, Sadowsky MJ, Diez-Gonzalez F. Incidence of naturally internalized bacteria in lettuce leaves. Intl J Food Microbiol. Borges KA, Furian TQ, Borsoi A, Moraes HLS, Salle CTP, Nascimento VP. Detection 2013;62:260–5. of virulence-associated genes in Salmonella enteritidis isolates from chicken in south of Brazil. Pesqui Vet Bras. 2013;33(12):1416–22. Husna AA, Islam MA, Rahman MT, Khatun MM. Efficacy of vinegar, sorbitol and Buchanan RL, Edelson SG, Miller RL, Sapers GM. Contamination of intact apples sodium benzoate in mitigation of Salmonella contamination in betel leaf. J after immersion in an aqueous environment containing Escherichia coli O157: Adv Vet Anim Res. 2015;2(2):190–4. H7. J Food Protect. 1999;62(5):444–50. Ibarra-Sanchez LS, Alvarado-Casillas S, Rodriguez-Garcia MO, Martinez-Gonzales Dong Y, Iniguez AL, Ahmer BM, Triplett EW. Kinetics and strain specificity of NE, Castillo A. Internalization of bacterial pathogens in tomatoes and their rhizosphere and endophytic colonization by enteric bacteria on seedlings of control by selected chemicals. J Food Protect. 2004;67(7):1353–8. Medicago sativa and Medicago truncatula. Appl Environ Microbiol. Iniguez AL, Dong Y, Carter HD, Ahmer BM, Stone JM, Triplett EW. Regulation of 2003;69(3):1783–90. enteric endophytic bacterial colonization by plant defenses. Mol Plant Donkor ES, Lanyo R, Kayang BB, Quaye J, Edoh DA. Internalizations of microbes in Microbe Interact J. 2005;18(2):169–78. vegetables: Microbial load of Ghanaian vegetables and the relationship with Islam M, Doyle MP, Phatak SC, Millner P, Jiang X. Persistence of different water sources of irrigation. Pakistan J Biol Sci. 2010;13(17):857–61. enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or Erickson MC, Webb CC, Diaz-Perez JC, Phatak SC, Silvoy JJ, Davey L, et al. Surface irrigation water. J Food Protect. 2004;67(7):1365–70. and internalized Escherichia coli O157:H7 on field-grown spinach and lettuce treated with spray-contaminated irrigation water. J Food Protect. ISO/DIS 6579-1. Microbiology of the food chain—Horizontal method for the 2010;73:1023–29. detection, enumeration and serotyping of Salmonella—Part 1: Horizontal Fakruddin M, Alam KMA, Mazumdar RM, Islam S, Nipa MN, Iqbal A, Bhuiyan HR. method for the detection of Salmonella spp. (http://www.iso.org/iso/home/ Anti-bacterial activity of the Extract of Terminalia arjuna against multi store/catalogue_tc/catalogue_detail.htm?csnumber=56712). antibiotic resistant Vibrio cholerae. J Sci Res. 2011;3(1):129–37. Jablasone J, Warriner K, Griffiths M. Interactions of Escherichia coli O157:H7, Fakruddin M, Mannan KSB, Mazumdar RM. Correlation between in vitro biofilm Salmonella typhimurium and Listeria monocytogenes plants cultivated in a formation and virulence properties of extra-intestinal pathogenic Escherichia gnotobiotic system. Intl J Food Microbiol. 2005;99:7–18. coli (EXPEC). OnLine J Biol Res. 2014;14(4):261–70. Khanra S. Paan Vittik Silpakendra (In Bengali). “Betel Leaf Based Industry”. Nabanna Bharati. 1997;30(2):169. Franz E, Visser AA, Van Diepeningen AD, Klerks MM, Termorshuizen AJ, Van Kroupitski Y, Golberg D, Belausov E, Pinto R, Swartzberg D, Granot D, Sela S. Bruggen AHC. Quantification of contamination of lettuce by GFP-expressing Internalization of Salmonella enterica in leaves is induced by light and Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Food Microbiol. 2007;24:106–12. involves chemotaxis and penetration through open stomata. Appl Environ Microbiol. 2009;75(19):6076–86. Ge C, Bohrerova Z, Lee J. Inactivation of internalized Salmonella Typhimurium in lettuce and green onion using ultraviolet C irradiation and chemical Lapidot A, Romling U, Yaron S. Biofilm formation and the survival of Salmonella sanitizers. J Appl Microbiol. 2013;114:1415–24. Typhimurium on parsley. Intl J Food Microbiol. 2006;109:229–33. Goldberg D, Kroupitski Y, Belausov E, Pinto R, Sela S. Salmonella Typhimurium Leveau JHJ. Microbiology: life on leaves. Nature. 2009;461:741. internalization is variable in leafy vegetables and fresh herbs. Intl J Food Mahbuba A, Islam S, Mazumdar RM, Fakruddin M, Bhuiyan HR, Sarker A. A Microbiol. 2011;145:250–7. biological tool to combat against multidrug-resistant Salmonella isolated Gomes C, Da Silva P, Moreira RG, Castell-Perez E, Ellis EA, Pendleton M. from poultry of Chittagong City, Bangladesh. Intl J Nat Sci. 2012;2(1):71–5. Understanding E. coli internalization in lettuce leaves for optimization of Montanari F. Managing Risks in Imports of Non-Animal Origin: The EU System of irradiation treatment. Intl J Food Microbiol. 2009;135:238–47. Reinforced Border Surveillance, Risk Regulation in Non-Animal Food Imports: The European Union Approach, 29–56. Cham: Springer International Gómez-López VM, Alicia M, Ana A, Beuchat LR, GIL MI. Postharvest. Handling Publishing; 2015. p. 39. http://dx.doi.org/10.1007/978-3-319-14014-8_2. Conditions Affect Internalization of Salmonella in Baby Spinach during Washing. J. Food Prot.. 2013;76(7):1145–51. Nailya A. Human Pathogens - The Plant And Useful Endophytes. J Med Microbiol Gorbatsevich E, Saldinger SS, Pinto R, Bernstein N. Root internalization, transport & Diagnosis 2013;2(03). and in-planta survival of Salmonella enterica serovar Newport in sweet basil. Nipa MN, Mazumdar RM, Hasan MM, Fakruddin M, Islam S, Bhuiyan HR, Iqbal A. Environ Microbiol Rep. 2013;5(1):151–9. Prevalence of Multi drug resistant bacteria on raw salad vegetables sold in Gorny J. Microbial contamination of fresh fruits and vegetables. In: Sapers GM, major markets of Chittagong city, Bangladesh. Middle-East J Sci Res. Gorny JR, Yousef AE, editors. Microbiology of Fresh Produce. New York: CRC 2011;10(1):70–7. Taylor and Francis; 2006. p. 3–32. Orue N, Garcia S, Feng P, Heredia N. Decontamination of Salmonella, Shigella and Gu G, Cevallos-Cevallos JM, van Bruggen AHC. Ingress of Salmonella enterica Escherichia coli O157:H7 from leafy green vegetables using edible plant Typhimurium into Tomato Leaves through Hydathodes. PLoS One. extracts. J Food Sci. 2013;78(2):M290–6. 2011;8(1):e53470. Penteado AL, Eblen BS, Miller AJ. Evidence of Salmonella internalization into fresh mangos during simulated post-harvest insect disinfection procedures. J Food GuoX,ChenJ,BrackettRE, BeuchatLR. Survival of Salmonella on and in tomato Protect. 2004;67(1):181–4. plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening. Appl Environ Microbiol. 2001;67(10):4760–4. Reina LD, Fleming HP, Bredit Jr F. Bacterial contamination of cucumber fruit Guo X, Van Iersel MW, Chen J, Brackett RE, Beuchat LR. Evidence of association of through adhesion. J Food Protect. 2002;65(12):1881–7. Salmonellae with tomato plant grown hydroponically in inoculated nutrient Schikora A, Garcia AV, Hirt H. Plants as alternative hosts for Salmonella. Trends solution. Appl Environ Microbiol. 2002;68(7):3639–43. Plant Sci. 2012;17(5):245–9. Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 10 of 10 Semenov AM, Kuprianov AA, van Bruggen AH. Transfer of enteric pathogens to successive habitats as part of microbial cycles. Microbial Ecol. 2010;601:239–49. Singh BR, Singh M, Babu N, Chandra M, Agarwal RK. Prevalence of multidrug- resistant Salmonella on ready-to-eat betel leaves (Paan) and in water used for soaking betel leaves in North Indian cities. J Food Protec. 2006;69(2):288–92. Singla R. Prevalence of high risk bacterial pathogens in Indian fresh vegetables and novel strategies for their inactivation. Diss. Thapar University, Patiala, 2011. Weblink: http://dspace.thapar.edu:8080/jspui/bitstream/10266/1741/3/1741.pdf. Singla R, Ganguli A, Ghosh M, Sohal S. Evaluation of sanitizing efficacy of acetic acid on Piper betle leaves and its effect on antioxidant properties. Intl J Food Sci Nutr. 2009;99999:1. doi:10.1080/09637480903114110. Sivapalasingam S, Friedman CR, Cohen L, Tauxe RV. Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J Food Protect. 2004;67:2342–53. Solomon EB, Yaron S, Matthews KR. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl Environ Microbiol. 2002;68(1):397–400. Sperandio V, Torres AG, Kaper JB. Quorum sensing Escherichia coli regulators B and C (QseBC): A novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in Escherichia coli. Mol Microbiol. 2002;43:809–21. Warriner K, Ibrahim F, Dickinson M, Wright C, Waites WM. Internalization of human pathogens within growing salad vegetables. Biotechnol Genetic Eng Rev. 2003;20:117–34. Wiedemann A, Virlogeux-Payant I, Chausse A-M, Schikora A, Velge P. Interactions of Salmonella with animals and plants. Front Microbiol. 2015; 5: 792. doi:10.3389/fmicb.2014.00791 Zheng J, Allard S, Reynolds S, Millner P, Arce G, Blodgett RJ, Brown EW. Colonization and Internalization of Salmonella enterica in Tomato Plants. Appl Environ Microbiol. 2013;79(8):2494–502. Zhuang RY, Beuchat LR, Angulo FJ. Fate of Salmonella Montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine. Appl Environ Microbiol. 1995;61:2127–31. 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Occurrence of ingression of Salmonella spp. in Betel leaf (Piper betle L.)

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Springer Journals
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Copyright © 2017 by The Author(s).
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Chemistry; Food Science
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2196-2804
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10.1186/s40550-017-0051-0
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Abstract

Background: Salmonella spp. is one of the most common pathogens associated with fresh produce related foodborne illness. This study aimed to determine Salmonella spp. contamination level in betel leaf, internalization potential and possible decontamination process. Results: A total of 77% betel leaf sample collected from local market was found to be contaminated with Salmonella spp. Of all the Salmonella spp. isolated and identified, 28.5% belong to Salmonella enterica subsp. enterica serovar Enteritidis, 19.5% belong to Salmonella Typhimurium, 15.6% to Salmonella Paratyphi, 10.4% to Salmonella Schottmuelleri, 9.1% to Salmonella Gallinarum, 10.4% to Salmonella Choleraesuis and 6.5% belong to Salmonella Bongori. Internalized Salmonella spp. showed moderate resistance to commonly used antibiotics. Treatment with common surface food disinfectants could not remove Salmonella spp. completely from betel leaf indicating the possibility that the bacteria may be in internal tissue of the leaf. Assessment of internalization potential showed that Salmonella spp. isolated from inner part of betel leaf pose better internalization potential (6.7–7.4 logCFU/gm) comparing with ATCC (American Type Culture Collection) strains (0.86–0.6 logCFU/gm). The isolates also showed better survivability in internalized condition, biofilm formation ability and motility than ATCC strains. Prevalence and expression of invasion (sefA and invA) and type 3 secretion system (TTSS) associated genes (hila, avrA and sopE) were high in internalized Salmonella isolates. Commercial disinfectants as well as H O were 2 2 found to have poor efficacy (log reduction around 2 CFU/gm) against internalized Salmonella. Ozonated water showed better decontamination efficiency (log reduction around 3 CFU/gm) whereas ethanolic extract of Terminalia arjuna stem bark showed higher decontamination (log reduction around 4.5 CFU/gm) of internalized Salmonella. Conclusion: Salmonella spp. can ingress into betel leaf and better decontamination treatment is needed to be established. Keywords: Internalization, Salmonella, Betel leaf, Disinfection Background part and subsequent translocation of bacteria to leaf and Foodborne illnesses with linkage to consumption of con- other aerial parts of the plant (Goldberg et al. 2011; taminated fresh products (such as betel leaf) is increas- Zhuang et al. 1995; Avila-Quezada et al. 2010; Hirneisen ing and becoming a significant food safety issue et al. 2012; Zheng et al. 2013; Bernstein et al. 2007a; worldwide nowadays (Gomes et al. 2009; Gorny 2006). Bernstein et al. 2007b). This event is alarming due to the Contamination can occur at any stage of production fact that internalization of pathogenic bacteria into fresh such as pre-harvest or post-harvest (Semenov et al. produce pose high risk to the consumers as they are sig- 2010; Goldberg et al. 2011; Bernstein 2011). Some recent nificantly resistant to external biocidal washing agents studies reported internalization of bacteria into the plant (Ibarra-Sanchez et al. 2004; Jablasone et al. 2005; Donkor et al. 2010). Though the mechanism of internalization of bacteria into plant parts are still poorly understood * Correspondence: fakruddinmurad@gmail.com Industrial Microbiology Laboratory, Institute of Food Science and (Auty et al. 2005), both plant and bacteria related factors Technology (IFST), Bangladesh Council of Scientific and Industrial Research contribute to its internalization (Erickson et al. 2010; Gu (BCSIR), Dr. Kudrat I Khuda Road, Dhanmondi, Dhaka 1205, Bangladesh et al. 2011). Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 2 of 10 Salmonella spp. is one of the most common pathogens Additionally, Salmonella spp. isolated from surface associated with fresh produce related foodborne illness (n = 25) and internalized condition (n = 17) during (Sivapalasingam et al. 2004; Lapidot et al. 2006). Recent this studywereused. reports clearly demonstrate that Salmonella not only sur- vive passively, but also infect plants actively (Wiedemann Sample collection et al., 2015; Schikora et al. 2012; Akhtyamova, 2013). In this experiment, a total of 100 samples was collected Moreover, infection of plants depends on the active sup- from different markets of Dhaka city, Bangladesh. Sterile pression of the host immune responses by Salmonella polybags were used to collect the samples aseptically. (Schikora et al. 2012). Most studies on Salmonella plant Samples were collected as number of leaves (not weight). interactions suggested an epiphytic lifestyle of Salmonella Twenty leaves were collected from each location. A total on plants (Berger et al. 2010). of ten sample was collected from each market. The Betel leaf (Piper betle L.) is a masticatory with import- sampling packs were marked properly and carried in ice ant socio-cultural and ceremonial use in south and box and transported to the laboratory within 3 h. Experi- southeast Asia. It is also an economically important ex- ments were carried out within 1–8 h after collecting the port commodity. Betel leaf is consumed raw through samples. All the samples were kept at 4 °C until these chewing mainly by South-Asian populations living were analyzed. worldwide. Betel leaf pose significant nutritional values and medicinal properties and is traditionally known to Identification and enumeration of Salmonella spp. be useful for the treatment of various diseases like bad Detection and identification of Salmonella spp. was done breath, boils and abscesses, conjunctivitis, constipation, according to ISO/DIS 6579-1. Briefly, 25 g leaf sample headache, hysteria, itches, mastitis, mastoiditis, was stomached with 225 ml 1% peptone water and leucorrhoea, otorrhoea, ringworm, swelling of gum, stomached sample was pre-enriched (Buffered peptone rheumatism, abrasion, cuts and injuries etc. as folk water) and enriched (tetrathionate broth and RV broth) medicine (Khanra 1997). Further, the essential oil and then selective media (XLD agar) was employed for contained in the leaves possesses antibacterial, anti- specific detection according to the method. Isolates were protozoan and antifungal properties (Hoque et al. 2011). further identified on the basis of biochemical tests (TSI Betel leaf is a product that is regularly consumed fresh agar/Urea agar/indole production/Voges-proskauer/Ly- and raw, but is difficult to decontaminate, as a result, sine decarboxylase). Enumeration of Salmonella spp. in like other fresh produces, can be a common vehicle of betel leaf was done according to Husna et al. (2015). transmission of enteropathogenic bacteria (Berger et al. Briefly, samples were stomached in peptone water and 2010). It is mainly produced in Bangladesh and India serially diluted and plate on XLD agar plate by spread and exported worldwide. Due to the contamination of plate technique and incubated at 37 °C for 24 h. Salmon- betel leaf with Salmonella spp., export of betel leaf has ella spp. were counted and expressed as logCFU/g. been reduced and sometimes suspended (Montanari 2015). Contamination of fresh produce like betel leaf Treatment of betel leaf with food disinfectants with Salmonella spp. pose great public health risk to the H O (10%, v/v), sodium hypochlorite (Sigma, USA) 2 2 consumer. Hence, research on contamination level and (10%, w/v) and commercial disinfectant (collected from pattern of Salmonella spp. and effective decontamin- local market) (1 & 2) were used to treat betel leaves (col- ation process is of great urgency. lected from local market) contaminated with Salmonella The present study aims to determine contamination spp. Briefly, the leaves (ten leave) were manually level and pattern and to evaluate potential decontamin- submerged into 200 ml solutions of the disinfectants for ation methods to neutralize Salmonella spp. contamin- 30 min and rinsed with sterile water for a couple of ation in betel leaf. times. Then the leaves were cut into pieces and Salmonella spp. load was determined as described above. Salmonella-free leaves were submerged for 5 min in Methods solution with ~10 cfu/ml of the bacteria and then Bacterial strains analyzed as the same procedure to serve as controls. Salmonella Enteritidis ATCC 13076, S. Typhimurium ATCC 13311 and S. Typhi ATCC 65154 were used as Detection of internalized Salmonella spp. positive culture in this study. Some environmental isolates Internalized Salmonella spp. has been detected and (n =15) of Salmonella were also included in the study to enumerated according to Franz et al. (Franz et al. 2007). observe differences in gene expression. All the isolates were Briefly, the surface of the collected whole leaf was disin- collected form culture collection pool of Industrial fected with serial washing using tap water, 80% ethanol Microbiology Laboratory, IFST, BCSIR, Dhaka, Bangladesh. for 10 s, 1%AgNO for 5 min, tap water and finally 3 Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 3 of 10 rinsing with deionized water. Then the leaves were cut in static condition. Diameter of motility halos was mea- into pieces and analyzed according to the method de- sured to determine motility. Biofi1m formation assays scribed earlier. Salmonella-free leaves were submerged were performed following the method of Fakruddin et for 5 min in solution with ~10 cfu/ml of the bacteria al. (2014). Bacterial suspension was inoculated into 96 and washed with water, ethanol and AgNO3 as described well polystyrene microtitre plates and incubated over- above and analyzed for both surface and internalized night at 30 °C. After incubation, wells were washed with Salmonella spp. This serves to eliminate the presence of PBS and stained with 0.1% safranin for 30 min at 30 °C. Salmonella as control to assure the presence of no Adhered safranin was solubilized with dimethyl sulfoxide Salmonella spp. in surface after washing. (DMSO) and absorbance of the wells were measured at Leaf internalization of ATCC strains was performed 490 nm wavelength. Specific biofilm formation was then according to Kroupitski et al. (2009). Briefly, ATCC calculated. Salmonella strains was grown overnight in Luria-Bertani (LB) medium and cells were collected and washed twice Virulence gene presence and expression with sterile distilled water and resuspended in sterile dis- Presence of six virulence associated gene in Salmonella tilled water. Intact betel leaves (which were previously spp. isolates has been determined. Isolates were grown tested to be Salmonella free) were submerged in 30 ml in LB broth overnight at 37 °C and total RNA was ex- sterile distilled water in 50 ml sterile tube (one piece per tracted. Description and primer sequence of the genes tube) for 30 min. After that, sterile distilled water of the are presented in Table 1. Virulence gene profile of leaf tube was replaced with 30 ml Salmonella suspension internalized Salmonella spp. was compared with that (~10 cfu/ml) and incubated for 2–3 h at 37 °C. After isolated from leaf surface and previously isolated envir- incubation, the leaves were washed twice with sterile onmental Salmonella strains. For determination of distilled water to remove unattached bacteria. Internal- expression level of the virulence genes, total RNA was ized Salmonella spp. was enumerated as described extracted from bacteria using Trizol reagent and cDNA earlier (Franz et al. 2007). was prepared using primescript RT reagent kit (TaKaRa Bio). Primers used for real time PCR are as follows: sefA Survivability of internalized Salmonella spp. (5’-GGCTTCGGTATCTGGTGGTGTG-3’ and 5’-GTCA Survivability of Salmonella spp. in internalized condition TTAATATTGGCTCCCTGAATA-3’), invA (5’-GCCTG was determined according to Gorbatsevich et al. (2013). CCGGAAGTATTGTTA-3’ and 5’-GGAGTTTCTCCCC Betel leaf samples were sanitized (as described above) and CTCTTCA-3’), hilA (5’-ATTAAGGCGACAGAGCTGG cut into small pieces aseptically and inoculated with bacter- A-3’ and 5’-GAATAGCAAACTCCCGACGA-3’), avrA ial suspension with known cell concentration (LogCFU/ml) (5’-GGAAACCGATCTCGAAATGA-3’ and 5’-TGCTGG and incubated at 37 °C for 14 days. A portion (10 g) of the TTCGAACAAAATCA-3’), sopE (5’-CAACACACTTT- sample was analyzed each day to enumerate internalized CACCGAGGAAG-3’ and 5’-GGTCTGGCTGGCGTAT Salmonella spp. as described earlier. GC-3’) and spvC (5’-AATGAACTACGAAGTGGGCG-3’ and 5’-TCAAACGATAAAACGGTTCCTC-3’), 16 s (5’- Antibiotic resistance of internalized Salmonella spp. TGTAGCGGTGAAATGCGTAG-3’ and 5’-CAAGGGC Antibiotic resistance pattern of the internalized Salmon- Table 1 Primer sequence of virulence genes detection by PCR ella spp. were determined according to the method de- Gene Virulence factor Primer sequence (5’–3’) Base pair scribed by Kirby-Bauer (1966) on Mueller Hinton agar (bp) using commercial discs (Oxoid, UK). The following anti- sefA Fimbria GATACTGCTGAACGTAGAAGG 488 biotics with the disc strength were used: Ciprofloxacin GCGTAAATCAGCATCTGCAGTAGC (CIP, 5 μg), chloramphenicol (C, 30 μg), penicillin (P, 5 unit), tetracycline (TE, 30 μg), doxycycline (DO, 30 μg), invA Invasion GTGAAATTATCGCCACGTTCGGGCAA 284 neomycin (N, 30 μg), gentamycin (GN, 200 μg), ampicil- TCATCGCACCGTCAAAGGAACC lin (AM, 10 μg), erythromycin (E, 15 μg) and nalidixic hilA Invasion CTGCCGCAGTGTTAAGGATA 497 acid (NA, 30 μg). A control strain of E. coli ATCC 25922 CTGTCGCCTTAATCGCATGT was included in each plate. Antimicrobial breakpoints avrA Effector protein GTTATGGACGGAACGACATCGG 385 and interpretation were taken from the CLSI standards. of TTSS ATTCTGCTTCCCGCCGCC Motility and biofilm formation of Salmonella spp. sopE Effector protein ACACACTTTCACCGAGGAAGCG 398 of TTSS Motility of the isolates was determined according to the GGATGCCTTCTGATGTTGACTGG method of Sperandio et al. (2002). Isolates were grown spvC Plasmid - CGGAAATACCATCTACAAATA 669 overnight in LB broth at 37 °C and was spot inoculated virulence CCCAAACCCATACTTACTCTG in center of 0.4% (w/v) LB agar and incubated at 37 °C Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 4 of 10 ACAACCTCCAAG-3’). Transcripts were quantified by LightCycler (Roche Diagnostics) using SYBR Premix Ex Taq (TaKaRa Bio) in accordance with the manufacturer’s instructions. The expression levels of each gene were normalized, with the 16S rRNA gene as an internal control. Decontamination of internalized Salmonella spp. Decontamination with two commercial fruit and vegetable disinfection agent (anshin-yasai & Yokosan; hereby denoted as commercial disinfectant 1 & 2 respectively) was performed according to manufacturer’s instruction. Information listed on the product revealed that active ingredient of disinfectant-1 was calcium oxide and of disinfectant-2 was calcium bicarbonate. Decon- tamination with H O , sodium hypochlorite and 2 2 ozonated water performed according to Bahreini et al. (2013). Ethanolic bark extract of Terminalia arjuna was prepared according to Mahbuba et al. (2012) and decon- tamination process was performed according to Orue et al. (2013). Briefly, selected chemicals and extracts were Fig. 1 Prevalence of Salmonella spp. in betel leaf. a depicts regional diluted with water at different concentrations. Artificially distribution of betel leaf samples infected with Salmonella spp. b inoculated betel leaf were immersed in the suspension shows serovar distribution of Salmonella spp. in infected betel leaf for 10 min. After that, the leaves were washed with ster- ile water, ethanol and AgNO3 (as described above) and presence and load of Salmonella spp. were determined reduction (2.37 log and 2.48 log respectively) (Table 2). as described previously. Control leaves (surface inoculated Salmonella-free leaves treated with same treatments) were found to be negative Statistical analysis for the presence of Salmonella spp. This result indicates All experiments were done twice and all samples per- that surface disinfection based treatment were not ef- formed at least in triplicate. Data were analyzed by SPSS fective to decontaminate Salmonella spp. from betel leaf. 17.0 (SPSS Inc., Chicago, Ill, USA). Occurrence of internalized Salmonella spp. in betel leaf was also determined (Table 3). Surface decontamination Result can reduce Salmonella loads by 1.97 log and treatment Prevalence of Salmonella spp. in betel leaf with disinfectants after surface decontamination reduce A total of seventy seven (77) out of 100 samples were Salmonella loads by 2.78 log, indicating that Salmonella found to be contaminated with Salmonella spp. From spp. may be internalized in inner parts of the leaf. the contaminated samples, 77 strains were isolated after pre-enrichment and enrichment and confirmed through biochemical and serological studies. The total percent- Internalization potential of isolate and ATCC strain ages of occurrence were 77%. Betel leaf collected from Comparison of internalization potential of two Salmon- all areas showed to be contaminated with Salmonella ella strains isolated from internalized condition in betel spp. (Fig. 1a). Prevalence observed in this study were leaf with three (3) ATCC Salmonella strains showed that 28.5%, 19.5%, 15.6%, 10.4%, 9.1%, 10.4% and 6.5% repre- the isolates pose better internalization potential than the senting Salmonella Enteritidis,S. Typhimurium,S. Para- ATCC strains (Table 4). Salmonella isolates from inter- typhi,S. Schottmuelleri,S. Gallinarum,S. Choleraesuis nalized condition showed considerable internalization and S. Bongori respectively (Fig. 1b). potential. Approx. 6.7 logCFU was found to be internal- ized in case of S. Typhimurium isolate and approx. 7.4 Treatment of betel leaf with food disinfectants logCFU was internalized in case of S. Typhi isolate Naturally contaminated betel leaves were washed with (Table 4). In contrast, ATCC isolates showed very little commercial food disinfectant 1 & 2, H O and Sodium internalization (approx. 1.2 logCFU for S. Enteritidis 2 2 hypochlorite. Commercial disinfectant 1 and 2 showed ATCC 13076 and 1.6 logCFU for S. Typhi ATCC 65154) 3.29 log and 3.87 log reduction respectively, while that (Table 3). Control leaves were found to be free of any of sodium hypochlorite and H O showed a lower log internalized Salmonella spp. 2 2 Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 5 of 10 Table 2 Disinfection of contaminated betel leaves with Table 4 Internalization potential of Salmonella spp. isolated different sanitizer from betel leaf Treatment Salmonella count Salmonella count Strain Inoculum Internalized Salmonella name (log cfu/g) (log cfu/g) (log cfu/g) (log cfu/g) before treatment after treatment S. Typhimurium (Isolated from 10.1 ± 0.33 7.7 ± 0.52 Commercial 10.75 ± 0.53 7.46 ± 0.37 internalized condition) disinfectant-1 (10%) S. Typhi (Isolated from internalized 10.1 ± 0.73 8.4 ± 0.73 Commercial disinfectant-2 (10%) 10.68 ± 0.09 6.81 ± 0.48 condition) H O (10%) 10.52 ± 0.32 8.15 ± 0.81 S. Enteritidis ATCC 13076 10.3 ± 0.19 2.2 ± 0.89 2 2 Sodium hypochlorite (10%) 10.31 ± 0.72 7.83 ± 0.19 S. Typhimurium ATCC 13311 10.0 ± 0.27 2.6 ± 0.12 S. Typhi ATCC 65154 10.1 ± 0.44 1.86 ± 0.21 Antibiotic susceptibility and Survivability comparison of internalized Salmonella Antibiotic susceptibility pattern on internalized Salmon- and 76.5% internalized Salmonella spp. isolate respect- ella isolates (n = 17) was shown in Fig. 2 and it shows ively in comparison to surface (72% & 60%) and environ- the isolates pose moderate antibiotic resistance. Com- mental (63.6% & 68.2%) Salmonella spp. isolates. Other parison of survivability of Salmonella Typhi isolate and virulence associated genes such as hilA, avrA and sopE ATCC strains in internalized condition in betel leaf is were present in 64.7%, 52.9% and 64.7% of internalized shown in Fig. 3. All the isolates were able to internalize isolate respectively. These three genes were present in and wild isolates internalized in a greater degree but sur- very low number of surface Salmonella isolates (4%, 4% vivality within the tissue seems to be the same. No sig- & 16%, respectively) and environmental Salmonella iso- nificant differences were observed. After 14 days lates (18.2%, 18.2% & 13.6% respectively). However, incubation, 6.72 logCFU Salmonella spp. were viable in prevalence of spvC gene in all type of isolate is very low internalized condition (initial count at day 0 was 8.31 (Fig. 4). The expression level of the virulence associated logCFU). In case of ATCC Salmonella strain, 5.23 genes in internalized Salmonella spp. were much higher logCFU Salmonella were viable after 14 days incubation than that in surface and environmental isolates (Fig. 5). (initial count 7.58 logCFU) (Fig. 3). Elevated expression of sefA and invA in internalized Sal- monella spp. suggest virulence potential of these isolates. Motility and biofilm forming capability of the isolated Increased expression of T3SS related genes (such as Salmonella hilA, avrA, sopE and sopC) indicates hyper-activation of Biofilm formation mediates survival of bacteria in inter- T3SS in internalized isolates contributing better survival nalized condition (Kroupitski et al. 2009) and motility in internalized conditions. mediates translocation in plant tissues (Warriner et al. 2003). Biofilm formation and motility of internalized Salmonella spp. isolated from betel leaf as well as of ATCC isolates was determined. Isolates showed better biofilm formation ability (SBF > 1) than the reference cultures (SBF < 1) (Table 5). Motility of the isolates were also higher (>40 mm) than that of reference cultures (<20 mm). These data coincide with the better surviv- ability of the isolates in internalized condition than the ATCC cultures. Virulence gene presence and expression sefA and invA are reported to impart virulence in Sal- monella spp. sefA and invA gene were present in 64.7% Table 3 Occurrence of internalized Salmonella spp. in betel leaf Betel leaf Salmonella count Fig. 2 Antibiotic resistance of Salmonella spp. isolated from Betel (logCFU/g) leaf (CIP = Ciprofloxacin; C = Chloramphenicol; P = Penicillin G; Without surface decontamination 9.45 (±0.33) TE = Tetracycline; AM = Ampicillin; DO = Doxycycline; N = Neomycin; With surface decontamination (Internalized) 7.48 (±1.03) GN = Gentamycin; E = Erythromycin; NA = Nalidixic acid). Salmonella strains (n = 17) isolated from internalized condition were used in Treated with disinfectants & without surface 6.67 (±0.11) this experiment decontamination Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 6 of 10 Fig. 4 Virulence gene profile of internalized Salmonella spp. (n =17) in comparison with surface (n = 25) and environmental isolates (n =15) Fig. 3 Survival potential of Salmonella spp. in internalized condition reported Salmonella spp. in betel leaf of Indian origin and Husna et al. (2015) reported Salmonella spp. in Decontamination treatment of internalized Salmonella betel leaf of Mymensingh region, Bangladesh and Decontamination method of internalized Salmonella Salmonella count was around 5 log in their samples. spp. has been performed and showed in Table 6. Com- Contaminated betel leaves were washed with four sur- mercial agents (1 & 2) have been found to have limited face disinfectants (anshin-yasai, yokosan, H O and 2 2 decontamination effect (around 1.2 log and 2 log reduc- sodium hypochlorite) and results showed that these tion respectively) on internalized Salmonella spp. with agents cannot fully decontaminate the betel leaf though increasing efficacy at elevated concentration. H O reduced Salmonella spp. level. From these results, the 2 2 showed almost similar efficacy compare with commer- possibility arises that Salmonella spp. were present not cial agents (1.9 log reduction at 10% concentration). only in surface, but also in the internal parts of the betel Washing leaves with internalized bacteria with ozonated leaf, hence imparting its resistance were to these surface water showed better neutralization effect (3 log reduc- disinfectants. Internalized Salmonella spp. in betel leaf tion) than both commercial agents and H O . Betel samples were enumerated and result showed that a sig- 2 2 leaves treated with Ethanolic bark extract of Terminalia nificant portion of total Salmonella spp. in the leaves are arjuna reduced internalized Salmonella spp. signifi- internalized. cantly. There was a log reduction of 3.8 at 5% concentra- Many previous researchers reported internalization of tion, 4.8 log at 10% concentration and 4.6 log reduction pathogenic bacteria, including Salmonella spp. into plant at 15% concentration (Table 6). leaves. Goldberg et al. (2011) reported internalization of Salmonella Typhimurium in detached leaves of seven Discussion A total of 100 betel leaf samples were collected from dif- ferent regions, 77% of the samples, were found to be contaminated with Salmonella spp. Regional distribution of contaminated betel leaf and species distribution of Salmonella spp. was shown in Fig. 1. Several studies has also reported presence of Salmonella spp. in betel leaf earlier. Singla et al. (2009) and Singh et al. (2006) have Table 5 Comparison of biofilm formation and motility of Internalized and ATCC Salmonella spp. Strain Specific biofilm Motility formation (SBF) (diameter in mm) Internalized S. Typhimurium 1.34 ± 0.14 42 ± 1.8 Internalized S. Typhi 1.53 ± 0.21 45 ± 2.2 S. Enteritidis ATCC 13076 0.76 ± 0.31 21 ± 1.2 S. Typhimurium ATCC 13311 0.81 ± 0.18 18 ± 1.2 Fig. 5 Expression of virulence gene in internalized Salmonella spp. (n = 17) in comparison with surface (n = 25) and environmental isolates (n=15) S. Typhi ATCC 65154 0.51 ± 0.08 20 ± 0.9 Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 7 of 10 Table 6 Decontamination of internalized Salmonella spp. from able to internalize and wild isolates internalized in a betel leaf greater degree but survivality within the tissue seems to Treatment Conc. Salmonella count Log be the same. Salmonella spp. isolates from betel leaf (log cfu/g) Reduction (approx.) showed better biofilm formation ability (SBF > 1) and Before After motility (>40 mm) than ATCC cultures (SBF < 1 and treatment treatment motility <20 mm). Results indicate that the isolated Commercial agent-1 5% 10.1 ± 0.94 8.9 ± 1.14 1.2 internalized Salmonella spp. have moderate resistance to 10% 10.6 ± 0.75 9.2 ± 1.21 1.4 commonly used antibiotics (Fig. 2) but antibiotic 15% 10.9 ± 0.89 8.2 ± 1.09 1.7 resistance were lower compared to the reports of other previous researchers who reported higher resist- Commercial agent-2 5% 10.2 ± 1.05 8.2 ± 0.97 2 ance in Salmonella spp. isolated from different food 10% 10.4 ± 1.12 7.9 ± 0.89 2.5 and poultry samples of Bangladesh (Nipa et al. 2011; 15% 10.1 ± 1.23 8.3 ± 0.76 1.8 Mahbuba et al. 2012). H O 5% 9.7 ± 0.88 8.9 ± 0.81 0.8 2 2 Internalized bacteria can evade disinfection, thus de- 10% 10.1 ± 0.69 8.2 ± 1.11 1.9 tailed study on the mechanism of internalization as well 15% 9.9 ± 1.31 8.1 ± 1.15 1.8 as on plant and environmental factors affecting internal- ization is needed to devise remediation methods ensur- TAEB 5% 10.7 ± 1.12 6.9 ± 1.07 3.8 ing safety of the fresh produces (Ge et al. 2013). 10% 11.2 ± 0.83 6.4 ± 0.89 4.8 Mechanism of internalization of bacteria in the plant 15% 10.9 ± 0.84 6.3 ± 0.96 4.6 has not been elucidated clearly till now. Generally, Ozonated water N/A 10.5 ± 0.95 7.5 ± 1.10 3 internalization is an active process dependent upon the (2 ppm) plant and the pathogen (Hora et al. 2005). Bacteria can (TAEB = Ethanolic bark extract of Terminalia arjuna) internalize through root during cultivation and through the stomata of leaf during pre-/post-harvest (Hoelzer et vegetables and fresh herbs. Hou et al. (2013) reported al. 2014). Bacterial internalization is influenced by the that bacteria such as Salmonella, E. coli O157:H7, Bacil- surface properties of the leaf, including morphology, lus, Enterobacter, Pseudomonas and Pantoea can chemical constituents and metabolic activities (Leveau internalize into lettuce leaves naturally through wounds 2009). Pathogenic bacteria can penetrate internal tissue or via roots and stomata. Avila-Quezada et al. (2010) of the plant through the roots (Solomon et al. 2002), and showed that Salmonella spp. can internalize and migrate seeds (Islam et al. 2004) for further translocation and into plant tissues such as seeds, fruits, leaves, roots and survival in the edible aerial plant tissues (Solomon et al. stems and survive for extended periods in internalized 2002). Some studies reported that bacterial strains varied condition. Guo et al. (2002) reported that Salmonella widely in their endophytic colonization abilities, which spp. can enter fruits and other plant parts through abra- could be related to the plant defense mechanisms that sions. Guo et al. (2001) also reported short distance targeted bacterial extracellular components (Dong et al. migration of Salmonella spp. into plant. Zheng et al. 2003; Iniguez et al. 2005). Water used for the washing of (2013) also reported that Salmonella spp. has the ability the fruits can be contaminated by the pathogens to internalize into tomato plants through roots, leaves while acting as the source of the internalization of and blossoms. Internalization of Salmonella spp. has also the pathogens through the lenticels, stomata and the been reported in mangoes (Penteado et al. 2004), toma- injured parts (Reina et al. 2002). toes (Buchanan et al. 1999), apple (Zhuang et al. 1995), It has been reported previously that many virulence lettuce (Reina et al. 2002), and sweet basil (Gorbatsevich associated genes mediate internalization and persistence et al. 2013). Internalization may occur naturally as of Salmonella spp. in plant. sefA gene is involved in at- reported in these studies, or may occur during washing tachment with plant tissue while invA gene is involved (due to immersion) at post-harvest stages (Gomez-Lopez in epithelial invasion of plant tissue. Of all the virulence et al. 2013). genes, type III secretion system (TTSS) associated genes Internalization efficiency of reference Salmonella cul- are most important for internalization of Salmonella ture (S. Typhimurium ATCC 13311, S. Enteritidis ATCC (Schikora et al. 2012). hilA gene product is a central 13076 and S. Typhi ATCC 65154) and S. Typhi and S. regulator of TTSS and also involved in epithelial Typhimurium isolated from betel leaf (leaf with internal- invasion. avrA gene product is an effector protein of ized Salmonella) has been compared and it was found TTSS and mediate Salmonella internalization and per- that the isolates pose better internalization potential sistence by suppressing the host inflammatory response. (6.7–7.4 logCFU/gm) when compared with the ATCC sopE gene product contributes invasion through gener- cultures (0.86–0.6 logCFU/gm). All the isolates were ation of membrane deformations. spvC gene (located on Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 8 of 10 virulence plasmid) product promotes rapid growth and as lack of fluorescence microscopy and efficient method of survival of Salmonella spp. within host cells (Borges et isolation of nucleic acids directly from leaf, this study al. 2013). Results showed that the prevalence of invasion results indicate occurrence of natural ingression of (sefA and invA) and TTSS associated genes (hila, avrA Salmonella spp. in betel leaf. Future research is needed to and sopE) are high in internalized Salmonella isolates further elucidate detailed mechanism of internalization of which indicates probable mechanisms of internalization Salmonella spp. in betel leaf as well as other plant. of the isolates. It can be postulated that TTSS play cen- tral role in the internalization and persistence of Sal- Conclusion monella spp. in betel leaf. Prevalence of hilA and avrA The present study provide indications of natural internal- gene was more in internalized S. Enteritidis, whereas ization of Salmonella spp. in betel leaf, though the mecha- prevalence of sopE gene is more prevalent in S. Typhi- nisms of internalization is yet to be elucidated. The murium and prevalence of spvC gene was prevalent in S. internalization of pathogenic bacteria like Salmonella spp. Paratyphi and S. Gallinarum. is a public health concern because a small number of Decontamination efficiency of different food disinfec- surviving cells can be potentially lethal. As Internalized tants at different concentrations against internalized Salmonella spp. in betel leaf evade surface disinfection, Salmonella spp. has been evaluated. Commercial disin- elucidation of internalization mechanisms and factors fectants (anshin-yasai and yokosan) as well as H O are (plant, bacterial and environmental) affecting internaliza- 2 2 found to have poor efficacy (log reduction around tion into betel leaf is needed to ensure the safety of this 2 CFU/gm). Ozonated water showed better decontamin- economically important fresh produce. Efficient decon- ation efficacy (log reduction around 3 CFU/gm). Singla tamination method has to be discovered to reduce the risk et al. (2009) reported that treatment of betel leaf with associated with internalized Salmonella in betel leaf. 2% acetic acid reduced artificially surface-contaminated Abbreviations Salmonella spp. by 4 log. Singla (2011) showed com- ATCC: American Type Culture Collection; CFU: Colony forming unit; bined treatment of 2% malic acid along with 2 ppm DMSO: Demethyl sulfoxide; LB: Luria Bertani; PCR: Polymerase chain reaction; RT: Reverse transcription; SBF: Specific biofilm formation; TSI: Triple sugar ozone significantly reduced Salmonella Typhimurium by iron; TTSS: Type 3 secretion system; XLD: Xylose lysine deoxycholate 7 log in turnip and reduced Cronobacter sakazakii by 6.8 log in betel leaf. Husna et al. (2015) showed treat- Acknowledgements The authors acknowledge the help of Ms. Sumaiya Islam, Scientific Officer, ment of betel leaf with sorbitol and sodium benzoate Industrial microbiology laboratory, IFST, BCSIR in conducting this study. can be effective to reduce Salmonella spp. in betel leaf though log reduction was very low compared to other Funding No external funding received. studies. Hadjok et al. (2008) reported UV light (254 nm) combined with H O can reduce contamination levels of 2 2 Author’s contributions human pathogens (Escherichia coli O157:H7, Pectobac- MF and MMA designed the study. MF, NH and RS performed all the experiments. MKI and MMA supervised the study. MF wrote the first draft of terium carotovora, Pseudomonas fluorescens and the manuscript and all authors read and approve the final manuscript. Salmonella) on or within (internalized) fresh produces but reduction of internalized pathogen was around half Competing interests The authors declare that they have no competing interests. of that for surface pathogens. Orue et al. (2013) reported that extracts from oregano and lime are as effective as chlorine based disinfectants for decontamination of Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Salmonella, Shigella and Escherichia coli O157:H7 on published maps and institutional affiliations. leafy vegetables. In our previous studies, we found etha- nolic extract of Terminalia arjuna stem bark has anti- Author details Industrial Microbiology Laboratory, Institute of Food Science and bacterial activity against Vibrio cholerae (Fakruddin et al. Technology (IFST), Bangladesh Council of Scientific and Industrial Research 2011) and Salmonella spp. (Mahbuba et al. 2012). In this (BCSIR), Dr. Kudrat I Khuda Road, Dhanmondi, Dhaka 1205, Bangladesh. study, ethanolic extract of Terminalia arjuna stem bark Department of Genetic Engineering & Biotechnology, Shahjalal University of Science & Technology, Sylhet 3114, Bangladesh. fared better reducing potential than the rest of the treat- ments (log reduction around 4.5 CFU/gm) (Table 6). Received: 8 September 2016 Accepted: 28 March 2017 Plant extract contains essential oil with antibacterial activity and could be useful for decontamination of References internalized Salmonella spp. Auty M, Duffy G, O’Beirne D, McGovern A, Gleeson E, Jordan K. In situ localization Many reports exist describing internalization of bacteria of Escherichia coli O157:H7 in food by confocal scanning laser microscopy. J Food Protect. 2005;68:482–6. in to plants through artificial inoculation experiments Avila-Quezada G, Sanchez E, Gardea-Bejar AA, Acedo-Felix E. Salmonella spp. and whereas report of incidence of natural internalization was Escherichia coli: survival and growth in plant tissue. New Zealand J Crop still very few. Despite some limitations of this study such Horticul Sci. 2010;38(2):47–55. Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 9 of 10 Bahreini M, Jahed E, Lotfian F, Khaneghah AM, Ghaderifarah M. The efficacy of Hadjok C, Mittal GS, Warriner K. Inactivation of human pathogens and spoilage ozonated water on reduction of four food borne pathogens and microbial bacteria on the surface and internalized within fresh produce by using a quality of lettuce. J Appl Sci Agricul. 2013;8(7):1425–30. combination of ultraviolet light and hydrogen peroxide. J Appl Microbiol. Bauer AW, Kirby WM, Sheris JC, Turck M. Antibiotic susceptibility testing by a 2008;104:1014–24. standardized single disc method. Am J Clin Path. 1996;45:149–58. Hirneisen KA, Sharma M, Kniel KE. Humanenteric pathogen internalization by root uptake into food crops. Foodborne Pathogen Dis. 2012;9(5): Berger CN, Sodha SV, Shaw RK, Griffin PM, Pink D, Hand P, Frankel G. Fresh fruit 395–405. and vegetables as vehicles for the transmission of human pathogens. Environ Microbiol. 2010;12(9):2385–97. Hoelzer K, Pouillot R, Van Doren JM, Dennis S. Reduction of Listeria monocytogenes contamination on produce—A quantitative analysis of common liquid fresh Bernstein N. Potential for contamination of crops by microbial human pathogens introduced to the soil by irrigation with treated-effluent. Israel J Plant Sci. produce wash compounds. Food Control. 2014;46:430–40. 2011;59:115–23. Hoque MM, Ratilla S, Shishir MA, Bari ML, Inatsu Y, Kawamoto S. Antibacterial Bernstein N, Sela S, Neder-Lavon S. Assessment of contamination potential of activity of ethanol extract of betel leaf (Piper betle L.) against some food lettuce by Salmonella enterica serovar Newport added to the plant growing borne pathogens. Bangladesh J Microbiol. 2011;28(2):58–63. medium. J Food Protect. 2007a;70:1717–22. Hora R, Warriner K, Shelp BJ, Griffiths MW. Internalization of Escherichia coli O157: Bernstein N, Sela S, Neder-Lavon S. Effect of irrigation regimes on persistence of H7 following biological and mechanical disruption of growing spinach Salmonella enterica serovar Newport in small experimental pots designed for plants. J Food Protect. 2005;68(12):2506–9. plant cultivation. Irrig Sci. 2007b;26:1–8. Hou Z, Fink RC, Radtke C, Sadowsky MJ, Diez-Gonzalez F. Incidence of naturally internalized bacteria in lettuce leaves. Intl J Food Microbiol. Borges KA, Furian TQ, Borsoi A, Moraes HLS, Salle CTP, Nascimento VP. Detection 2013;62:260–5. of virulence-associated genes in Salmonella enteritidis isolates from chicken in south of Brazil. Pesqui Vet Bras. 2013;33(12):1416–22. Husna AA, Islam MA, Rahman MT, Khatun MM. Efficacy of vinegar, sorbitol and Buchanan RL, Edelson SG, Miller RL, Sapers GM. Contamination of intact apples sodium benzoate in mitigation of Salmonella contamination in betel leaf. J after immersion in an aqueous environment containing Escherichia coli O157: Adv Vet Anim Res. 2015;2(2):190–4. H7. J Food Protect. 1999;62(5):444–50. Ibarra-Sanchez LS, Alvarado-Casillas S, Rodriguez-Garcia MO, Martinez-Gonzales Dong Y, Iniguez AL, Ahmer BM, Triplett EW. Kinetics and strain specificity of NE, Castillo A. Internalization of bacterial pathogens in tomatoes and their rhizosphere and endophytic colonization by enteric bacteria on seedlings of control by selected chemicals. J Food Protect. 2004;67(7):1353–8. Medicago sativa and Medicago truncatula. Appl Environ Microbiol. Iniguez AL, Dong Y, Carter HD, Ahmer BM, Stone JM, Triplett EW. Regulation of 2003;69(3):1783–90. enteric endophytic bacterial colonization by plant defenses. Mol Plant Donkor ES, Lanyo R, Kayang BB, Quaye J, Edoh DA. Internalizations of microbes in Microbe Interact J. 2005;18(2):169–78. vegetables: Microbial load of Ghanaian vegetables and the relationship with Islam M, Doyle MP, Phatak SC, Millner P, Jiang X. Persistence of different water sources of irrigation. Pakistan J Biol Sci. 2010;13(17):857–61. enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or Erickson MC, Webb CC, Diaz-Perez JC, Phatak SC, Silvoy JJ, Davey L, et al. Surface irrigation water. J Food Protect. 2004;67(7):1365–70. and internalized Escherichia coli O157:H7 on field-grown spinach and lettuce treated with spray-contaminated irrigation water. J Food Protect. ISO/DIS 6579-1. Microbiology of the food chain—Horizontal method for the 2010;73:1023–29. detection, enumeration and serotyping of Salmonella—Part 1: Horizontal Fakruddin M, Alam KMA, Mazumdar RM, Islam S, Nipa MN, Iqbal A, Bhuiyan HR. method for the detection of Salmonella spp. (http://www.iso.org/iso/home/ Anti-bacterial activity of the Extract of Terminalia arjuna against multi store/catalogue_tc/catalogue_detail.htm?csnumber=56712). antibiotic resistant Vibrio cholerae. J Sci Res. 2011;3(1):129–37. Jablasone J, Warriner K, Griffiths M. Interactions of Escherichia coli O157:H7, Fakruddin M, Mannan KSB, Mazumdar RM. Correlation between in vitro biofilm Salmonella typhimurium and Listeria monocytogenes plants cultivated in a formation and virulence properties of extra-intestinal pathogenic Escherichia gnotobiotic system. Intl J Food Microbiol. 2005;99:7–18. coli (EXPEC). OnLine J Biol Res. 2014;14(4):261–70. Khanra S. Paan Vittik Silpakendra (In Bengali). “Betel Leaf Based Industry”. Nabanna Bharati. 1997;30(2):169. Franz E, Visser AA, Van Diepeningen AD, Klerks MM, Termorshuizen AJ, Van Kroupitski Y, Golberg D, Belausov E, Pinto R, Swartzberg D, Granot D, Sela S. Bruggen AHC. Quantification of contamination of lettuce by GFP-expressing Internalization of Salmonella enterica in leaves is induced by light and Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Food Microbiol. 2007;24:106–12. involves chemotaxis and penetration through open stomata. Appl Environ Microbiol. 2009;75(19):6076–86. Ge C, Bohrerova Z, Lee J. Inactivation of internalized Salmonella Typhimurium in lettuce and green onion using ultraviolet C irradiation and chemical Lapidot A, Romling U, Yaron S. Biofilm formation and the survival of Salmonella sanitizers. J Appl Microbiol. 2013;114:1415–24. Typhimurium on parsley. Intl J Food Microbiol. 2006;109:229–33. Goldberg D, Kroupitski Y, Belausov E, Pinto R, Sela S. Salmonella Typhimurium Leveau JHJ. Microbiology: life on leaves. Nature. 2009;461:741. internalization is variable in leafy vegetables and fresh herbs. Intl J Food Mahbuba A, Islam S, Mazumdar RM, Fakruddin M, Bhuiyan HR, Sarker A. A Microbiol. 2011;145:250–7. biological tool to combat against multidrug-resistant Salmonella isolated Gomes C, Da Silva P, Moreira RG, Castell-Perez E, Ellis EA, Pendleton M. from poultry of Chittagong City, Bangladesh. Intl J Nat Sci. 2012;2(1):71–5. Understanding E. coli internalization in lettuce leaves for optimization of Montanari F. Managing Risks in Imports of Non-Animal Origin: The EU System of irradiation treatment. Intl J Food Microbiol. 2009;135:238–47. Reinforced Border Surveillance, Risk Regulation in Non-Animal Food Imports: The European Union Approach, 29–56. Cham: Springer International Gómez-López VM, Alicia M, Ana A, Beuchat LR, GIL MI. Postharvest. Handling Publishing; 2015. p. 39. http://dx.doi.org/10.1007/978-3-319-14014-8_2. Conditions Affect Internalization of Salmonella in Baby Spinach during Washing. J. Food Prot.. 2013;76(7):1145–51. Nailya A. Human Pathogens - The Plant And Useful Endophytes. J Med Microbiol Gorbatsevich E, Saldinger SS, Pinto R, Bernstein N. Root internalization, transport & Diagnosis 2013;2(03). and in-planta survival of Salmonella enterica serovar Newport in sweet basil. Nipa MN, Mazumdar RM, Hasan MM, Fakruddin M, Islam S, Bhuiyan HR, Iqbal A. Environ Microbiol Rep. 2013;5(1):151–9. Prevalence of Multi drug resistant bacteria on raw salad vegetables sold in Gorny J. Microbial contamination of fresh fruits and vegetables. In: Sapers GM, major markets of Chittagong city, Bangladesh. Middle-East J Sci Res. Gorny JR, Yousef AE, editors. Microbiology of Fresh Produce. New York: CRC 2011;10(1):70–7. Taylor and Francis; 2006. p. 3–32. Orue N, Garcia S, Feng P, Heredia N. Decontamination of Salmonella, Shigella and Gu G, Cevallos-Cevallos JM, van Bruggen AHC. Ingress of Salmonella enterica Escherichia coli O157:H7 from leafy green vegetables using edible plant Typhimurium into Tomato Leaves through Hydathodes. PLoS One. extracts. J Food Sci. 2013;78(2):M290–6. 2011;8(1):e53470. Penteado AL, Eblen BS, Miller AJ. Evidence of Salmonella internalization into fresh mangos during simulated post-harvest insect disinfection procedures. J Food GuoX,ChenJ,BrackettRE, BeuchatLR. Survival of Salmonella on and in tomato Protect. 2004;67(1):181–4. plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening. Appl Environ Microbiol. 2001;67(10):4760–4. Reina LD, Fleming HP, Bredit Jr F. Bacterial contamination of cucumber fruit Guo X, Van Iersel MW, Chen J, Brackett RE, Beuchat LR. Evidence of association of through adhesion. J Food Protect. 2002;65(12):1881–7. Salmonellae with tomato plant grown hydroponically in inoculated nutrient Schikora A, Garcia AV, Hirt H. Plants as alternative hosts for Salmonella. Trends solution. Appl Environ Microbiol. 2002;68(7):3639–43. Plant Sci. 2012;17(5):245–9. Fakruddin et al. International Journal of Food Contamination (2017) 4:6 Page 10 of 10 Semenov AM, Kuprianov AA, van Bruggen AH. Transfer of enteric pathogens to successive habitats as part of microbial cycles. Microbial Ecol. 2010;601:239–49. Singh BR, Singh M, Babu N, Chandra M, Agarwal RK. Prevalence of multidrug- resistant Salmonella on ready-to-eat betel leaves (Paan) and in water used for soaking betel leaves in North Indian cities. J Food Protec. 2006;69(2):288–92. Singla R. Prevalence of high risk bacterial pathogens in Indian fresh vegetables and novel strategies for their inactivation. Diss. Thapar University, Patiala, 2011. Weblink: http://dspace.thapar.edu:8080/jspui/bitstream/10266/1741/3/1741.pdf. Singla R, Ganguli A, Ghosh M, Sohal S. Evaluation of sanitizing efficacy of acetic acid on Piper betle leaves and its effect on antioxidant properties. Intl J Food Sci Nutr. 2009;99999:1. doi:10.1080/09637480903114110. Sivapalasingam S, Friedman CR, Cohen L, Tauxe RV. Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J Food Protect. 2004;67:2342–53. Solomon EB, Yaron S, Matthews KR. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl Environ Microbiol. 2002;68(1):397–400. Sperandio V, Torres AG, Kaper JB. Quorum sensing Escherichia coli regulators B and C (QseBC): A novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in Escherichia coli. Mol Microbiol. 2002;43:809–21. Warriner K, Ibrahim F, Dickinson M, Wright C, Waites WM. Internalization of human pathogens within growing salad vegetables. Biotechnol Genetic Eng Rev. 2003;20:117–34. Wiedemann A, Virlogeux-Payant I, Chausse A-M, Schikora A, Velge P. Interactions of Salmonella with animals and plants. Front Microbiol. 2015; 5: 792. doi:10.3389/fmicb.2014.00791 Zheng J, Allard S, Reynolds S, Millner P, Arce G, Blodgett RJ, Brown EW. Colonization and Internalization of Salmonella enterica in Tomato Plants. Appl Environ Microbiol. 2013;79(8):2494–502. Zhuang RY, Beuchat LR, Angulo FJ. Fate of Salmonella Montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine. Appl Environ Microbiol. 1995;61:2127–31. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com

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International Journal of Food ContaminationSpringer Journals

Published: Apr 4, 2017

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