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Enterobacteriaceae members are largely distributed in the environment and responsible for a wide range of bacterial infections in hospitalized patients. Pseudomonas aeruginosa (P. aeruginosa) causes severe nosocomial infections associated with severe inflammation due to its potent virulent factors including lipopolysaccharide (LPS). The aim of this study is to assess the bacterial LPS effect on Enterobacteriaceae biofilm and other virulence factors in vitro. The effect of P. aeruginosa LPS on biofilm formation of two other species of Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae) was assessed using a standard biofilm assay. PCR was performed on genes of biofilm and virulence factors. Expression of biofilm, type-1-fimbriae and serum resistance genes in treated and untreated cells was measured with RT-PCR. P. aeruginosa LPS has the ability to stimulate biofilm formation and stabilize the already formed biofilm significantly in all tested strains. In addition, LPS significantly increased the level of expression of Bss, FimH,and Iss genes when measured by RT-PCR. P. aeruginosa LPS has a direct stimulatory effect on the biofilm formation, type-1-fimbriae, and serum resistance in both E. coli and K. pneumoniae. So, the presence of P. aeruginosa in mixed infection with Enterobactereacea leads to increase their virulence. . . . . Keywords P. aeruginosa LPS E. coli K. pneumoniae Biofilm Virulence factors Introduction majority of human infections. They frequently comprise of either mono-species or multispecies of bacteria and fungi such Lipopolysaccharide (LPS), a Gram-negative bacterial endo- as Candida (Nobile and Mitchell 2007; Bandara et al. 2009; toxin, is an outer component of the cell wall that causes septic Bandara et al. 2010). Most studies focused on biofilm forma- shock in animals and humans by its immunomodulatory and tion due to the ability of sessile bacteria than planktonic bacteria proinflammatory properties (Aurell and Wistrom 1998). LPS in toleration of exogenous stress (Goncalves Mdos et al. 2014). also plays a role in endotoxic shock mechanism (Tracey et al. The biofilm is composed of an extracellular matrix; of 1987) and in the infections pathophysiology (Morrison and which 10% is extracellular polymeric substances (EPS) and Ryan 1987; Bandara et al. 2010). the rest is water (Flemming and Wingender 2010). EPS com- Most micro-organisms occur as biofilm that is complex ponents are important for the biofilm formation and communities of one or more microbial species attached to solid stabilization. surfaces or to one another rather than free-floating planktonic Enterobacteriaceae is a large family of Gram-negative bac- cells (Samaranayake 1990). On comparing biofilms and plank- teria, which comprises many harmless symbiont and patho- tonic cells, biofilms are resistant to antimicrobials (O’Toole genic microbes, such as Salmonella, Shigella, Escherichia et al. 2000;Mah andO’Toole 2001) and involved in the coli, and Klebsiella. E. coli is known to be the main causative agent of bacterimia, cholecystitis, urinary tract infections (UTIs), pneumonia, and meningitis. It has been reported that * Shaymaa H. Abdel-Rhman many isolates of E. coli produce biofilm structures that are shaymaahassan@mans.edu.eg; mabdelrhman@taibahu.edu.sa responsible for 90% of UTIs (Beloin et al. 2008; Spurbeck et al. 2011). Curli (coiled extracellular appendages on the Department of Pharmaceutics and Pharmaceutical Biotechnology, E. coli surface) are necessary for biofilm development Faculty of Pharmacy, Taibah University, AlMadinah Al Munawwarah, Kingdom of Saudi Arabia (Manu Chaudhary et al. 2013). E. coli is one of the normal flora in human intestinal tract Department of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt that can convert to pathogenic ones by lateral gene transfer 300 Ann Microbiol (2019) 69:299–305 through gene loss or gain (Kaper et al. 2004). E. coli can cause points (Montoro et al. 2005). Briefly, a suspension of tested extra-intestinal infections due to its different virulence factors strains (100 μl) was dispersed into wells of a 96-well plate (ex: adhesions, polysaccharide capsules, toxins, proteases, with different LPS concentration (10 ng, 50 ng, 100 ng, and lipopolysaccharides) (JR and TA. 2002; Mokady et al. 250 ng, 500 ng, 750 ng, and 1000 ng) and incubated for 2005; Abdelmegeed et al. 2015). 24 h. The assay was continued as described before by Klebsiella pneumoniae is a main cause of nosocomial Abdel-Rhman and Rizk (2016). pneumonia and UTIs, endophthalmitis, and pyogenic liver abscess. Virulence factors like fimbriae type 1 and type 3 gave Effect of P. aeruginosa LPS on biofilm formation new insights into K. pneumoniae pathogenic policies, which help attachment to both host mucosal surfaces and inert ones The effects of the P. aeruginosa LPS on biofilm formation and play a role in biofilm formation (O’Toole and Kolter were evaluated by biofilm formation assay that was carried 1998;Sutherland 2001; Lejeune 2003). out in a 96-well polystyrene microtiter plate (Nunc, New Virulence factors can be determined by genomics to iden- York, NY, USA) and measured using the crystal violet stain- tify specific genes responsible for virulence of the pathogens. ing procedure (Goncalves Mdos et al. 2014). Briefly, 100 μlof For an organism to be virulent, it needs a combination of the overnight bacterial suspensions diluted to (1 × 10 CFU/ different factors rather than one virulent factor (Dobrindt ml) was incubated with different LPS concentrations for 24 h 2005; Abdelmegeed et al. 2015). P. aeruginosa is an impor- at 37 °C; control assays were also performed and the proce- tant bacterial pathogen that causes nosocomial infection in dure was carried out as described by Abdel-Rhman and Rizk which P. aeruginosa can inhabit, infect, and intoxicate suscep- (2016). tible patients. LPS produced by it is a key virulence factor that also affects both innate and acquired host responses to infec- Effect of LPS on the formed mature biofilms tion. They are localized in the outer layer of the membrane contributing to its integrity. It protects the cell against the bile One hundred microliters of E. coli and K. pneumoniae sus- salts and lipophilic antibiotics and mediates the entry of bac- pensions (OD = 0.257) was inoculated into separate wells teria into the cell (Pier 2007). of polystyrene 96-well plates (flat bottom; Nunc). The plates P. aeruginosae, E. coli,and K. pneumoniae are widely were incubated at 37 °C for 24 h. After incubation, the super- distributed in Egypt’s hospitals as in references. So, the aim natants were aseptically aspirated, and the formed biofilms of this study was to assess the effect of P. aeruginosa LPS on were washed twice with PBS; 100 μl of fresh LB broth con- Enterobacteriaceae biofilm and virulence factors as these or- taining different LPS concentrations was added to each well. ganisms may be present in mixed infections. LB broth without LPS was added to control wells. The plates were then incubated for 24 h at 37 °C. Non-adherent cells were removed through washing twice with 200 μl sterile PBS saline. Cells adherent to wells were quantified using Methodology crystal violet assay (X et al. 2003). Experiments were carried out in triplicate. Micro-organisms The reference strains used were E. coli ATCC BAA196, E. coli ATCC 23716, E. coli ATCC 12435, Molecular screening of various virulence genes K. pneumoniae ATCC 33495, K. pneumoniae ATCC 51503, among E. coli and K. pneumoniae strains and K. pneumoniae ATCC 4352. In addition to two clinical isolates of each coliform. Before experiments, all isolates were The tested strains were investigated by PCR for the presence stored at − 80 °C. of nine genes; eight virulence genes: FimH (mannose-specific adhesion subunit of type 1 fimbriae), Iss (increased serum LPS LPS from P. aeruginosa ATCC 27316 (catalog no. resistance) (Galil et al. 2011), KapsMTII (group2 capsule syn- L9143) was obtained as lyophilized powder from Sigma- thesis), fyuA (ferric yersiniabactin uptake), Afa/draBC (Dr- Aldrich and stored at 2–8°C tilluse. binding a fimbrial adhesins), PapA (P fimbriae), PapC (outer membrane protein of P fimbriae), iutA (aerobactin receptor), Evaluation of P. aeruginosa LPS effect and Bss (biofilm formation) (Galil et al. 2011). Total DNA, on the metabolic activity reaction mixture, and PCR program were prepared and conducted as previously described by Galil et al. (2011)and To assess the effect of LPS on the vitality of E. coli and Abdelmegeed et al. (2015). K. pneumoniae strains, MTT (3-(4,5-dimethylthiazol-2-yl)- PCR products were analyzed on a 1.2% ethidium bromide- 2,5-diphenyltetrazolium bromide) reduction assay was done stained agarose gel and compared with 100 bp plus DNA on both LPS treated and control cells at predetermined time ladder and visualized under UV illumination. Ann Microbiol (2019) 69:299–305 301 Quantitative real-time PCR Effect of LPS on the already formed mature biofilms E. coli and K. pneumoniae strains were treated with LPS LPS had the ability to significantly stabilize and to increase the (10 ng/ml and 1000 ng/ml) and total RNA was extracted by already formed biofilm in all tested strains (1.40–7.20-fold in- TRI Reagent (T9424 Sigma-Aldrich). The purity and concen- crease). LPS (1000 ng/ml) can stabilize the already formed ma- tration for RNA were determined spectrophotometrically ture biofilm significantly than other used concentrations (Fig. 1) using NanoDrop (ND-1000 Spectrophotometer, NanoDrop especially in K. pneumoniae strains (2.50–7.20 times compared Technologies, Wilmington, USA). Complementary DNA to control untreated samples). All LPS concentrations caused (cDNA) was prepared from 1 μg of RNA using QuantiTect the same level of stabilization in E. coli ATCC BAA196 (around Reverse Transcription kit (QIAGEN, USA). 3.10 times) and E. coli ATCC 23716 (around 4.35 times). The level of virulence genes expression (Bss, FimH, Iss, FyuA,and iutA) was measured using the primers described Molecular screening of various virulence genes before. Amplification was done using 5x FIREPol among E. coli and K. pneumoniae strains EvaGreen, qPCR Mix, ROX Dye; Solis BioDyne; and Rotor Gene Q thermocycler (QIAGEN, Hilden, Germany). The re- Bss, FimH,and Iss genes were detected in all tested strains action mixture was prepared and RT-PCR program was done while fyuA gene was amplified in E. coli ATCC BAA196 only, as described previously by Abdelmegeed et al. (2015). The and iutA gene was present in all E. coli tested strains. The expressions of the virulence and biofilm genes in the treated other virulence genes (papA, papC, Afa/draBC, KapsMTII) samples were measured relative to untreated samples and were were not detected (Table 2). -ΔΔCt evaluated using the 2 method. Expression of biofilm and virulence genes by RT-PCR Statistical analysis Using RT-PCR, the expression level of (Bss, FimH,and iss) Statistical analysis was carried out using GraphPad Prism5. genes was significantly higher in the LPS treated cells com- One-way ANOVA followed by Dunnett posttest were carried pared to untreated ones. The other virulence genes (iutA and out to compare significant differences between the control and fyuA) were not expressed in the tested E. coli strains. The treated strains. A P value ≤ 0.05 was considered statistically effect of LPS on the gene expression was higher in significant. K. pneumoniae strains (relative expression = 7.4) than in E. coli strains (relative expression = 4.7) in the case of Bss gene. The high effect of LPS was also observed on the expres- Results sion of Bss gene than other genes (Fig. 2). Evaluation of P. aeruginosa LPS impact on Enterobacteriaceae Discussion The effect of different concentrations of P. aeruginosa LPS on E. coli and K. pneumoniae are Gram-negative commensals that Enterobacteriaceae metabolic activity was evaluated using live with its host in symbiosis by colonizing the gastrointestinal MTT reduction assay. All concentrations of LPS used had tracts (Kaper et al. 2004). Acquiring virulence traits by several turned the color of MTT from yellow to purple which indi- clones leads to intra- and extra-intestinal infections (Seaton cates that LPS had no effect on the metabolic activity of tested 2000; Witso et al. 2014). Previous studies reported that E. coli, strains. K. pneumoniae,and P. aeruginosa have been found as the most commonthreeuro-pathogenicbacteria(Behzadietal. 2010). Effect of P. aeruginosa LPS on biofilm formation Due to mixed infection caused by these three pathogens, the effect of P. aeruginosa LPS on E. coli and K. pneumoniae The biofilm formed by all tested (standard and clinical) strains biofilm and virulence factors was investigated. To our knowl- was significantly increased under different concentration of edge, this is the first study examining the effect of LPS compared to the control untreated samples (1.07–7.53- P. aeruginosa LPS on the biofilm and virulence factors in fold increase). The highest increase in biofilm formation Enterobacteriaceae. The present results showed that (7.53) was recorded in the presence of LPS (1000 ng/ml) as P. aeruginosa LPS had not any inhibitory effect on the growth compared with untreated cells. However, four strains (coli of E. coli or K. pneumoniae as evaluated by MTT reduction ATCC 12435, E. coli ATCC 23716, K. pneumoniae ATCC assay. This finding was in accordance with Bandara et al. 33495, and K1) showed marked increase in biofilm formation (Bandara et al. 2009) who reported similar results on with increasing concentrations of LPS (Table 1). Candida growth. 302 Ann Microbiol (2019) 69:299–305 Table 1 Effect of LPS on biofilm formation by tested strains using the crystal violet staining procedure Strain Fold increase in biofilm formation in presence of LPS 10 ng/ml 50 ng/ml 100 ng/ml 250 ng/ml 500 ng/ml 750 ng/ml 1000 ng/ml E. coli ATCC BAA196 2.08*** 2.10*** 2.11*** 2.31*** 2.49*** 2.51*** 2.44*** ATCC 12435 1.07* 1.09* 1.56*** 2.36*** 3.56*** 4.17*** 4.34*** ATCC 23716 1.07* 1.39*** 2.31*** 2.41*** 2.48*** 2.66*** 2.92*** E1 1.23*** 1.40*** 1.50*** 1.98*** 1.99*** 2.0*** 2.33*** E2 1.07* 1.08* 1.28*** 1.34*** 1.39*** 1.42*** 1.44*** K. pneumoniae ATCC 33495 1.27*** 1.30*** 1.32*** 2.7*** 2.83*** 2.93*** 3.31*** ATCC 4352 1.10* 1.05* 1.15*** 1.20*** 1.23*** 1.21*** 1.55*** ATCC 51503 1.86*** 1.87*** 2.08*** 2.13*** 2.25*** 2.43*** 2.46*** K1 1.08* 1.10* 3.27*** 3.89*** 4.68*** 5.81*** 7.53*** K2 1.2*** 1.4*** 1.75*** 1.77*** 1.89*** 2.76*** 2.9*** *Significant difference (P < 0.05), ***high significant difference (P < 0.0001) In this work, LPS was found to stimulate E. coli and stabilization of bacterial biofilm (Hori and Matsumoto K. pneumoniae biofilm formation at all concentrations used 2010). However, another study by Benitez et al. found that as it significantly increased its formation (P < 0.0001) (1.07– Vibrio cholera LPS can inhibit in vitro adhesion on colonic 7.53 times). The tested LPS has the ability not only to stimu- cell lines HT29-18N2 (Benitez et al. 1997). LPS can prevent late the biofilm formation but also to significantly stabilize the biofilm formation of competing strains. Bandara et al. studied already formed biofilm of both coliform, which was observed the impact of LPS from four different Gram-negative bacteria with all concentrations used (1.40–7.20 times). Although, the on biofilm formation by different Candida species. The au- phenotype of the reference isolates could vary from that of thors concluded that bacterial LPS effect on biofilm formation clinical isolates due to repeated subculture, and both by Candida was species-specific and this effect may be due to standard and clinical strains gave the same results as their alteration in cell number or activity in the formed biofilm biofilms were increased and stabilized in the presence of (Bandara et al. 2010; Rendueles et al. 2012). LPS. This finding was in accordance with Rendueles et al. Motility and swarming of E. coli are critical aspects for (2012) who reported that LPS of Gram-negative bacteria can biofilm formation (Pratt and Kolter 1998;O’Toole et al. affect biofilm formation, in addition to cohesion and 2000). According to Pratt and Kolter, flagella are responsible control Fig. 1 Effect of LPS on the 10 ng already formed mature biofilm by E. coli and K. pneumoniae strains. 50 ng EB, E. coli ATCC BAA196; E12, 100 ng E. coli ATCC 12435; E23, E. coli 250 ng ATCC 23716; K3, K. pneumoniae 500 ng ATCC 33495; K4, K. pneumoniae 750 ng ATCC 4352; and K5, 1000 ng K. pneumoniae ATCC 51503 EB E12 E23 K3 K4 K5 Isolate code % of Biofilm Stabilization Ann Microbiol (2019) 69:299–305 303 Table 2 PCR detection of various virulence genes among tested strains Strain PCR of biofilm and virulence genes Biofilm Serum resistance Adhesion Iron chelation bss iss kapsMTII fimH afa/draBC papC papA fyuA iutA E. coli ATCC BAA196 + + − + −− − ++ ATCC 12435 + + − + −− − − + ATCC 23716 + + − + −− − − + E1 + + − + −− − − + E2 + + − + −− − − + K. pneumoniae ATCC 33495 + + − + −− − − − ATCC 4352 + + − + −− − − − ATCC 51503 + + − + −− − − − K1 + + − + −− − − − K2 + + − + −− − − − Control Control Bss gene FimH gene LPS 10 LPS 10 LPS 1000 2.5 LPS 1000 *** *** *** *** *** *** *** *** 6 *** *** *** *** *** 1.5 *** 4 *** *** *** *** *** *** 3 1 *** *** 2 *** *** 1 0.5 EB E12 E23 K3 K4 K5 Isolate code EB E12 E23 K3 K4 K5 Isolate code Control Iss gene LPS 10 2.5 LPS 1000 2 *** *** *** *** *** *** *** *** 1.5 *** *** *** *** 0.5 EB E12 E23 K3 K4 K5 Isolate code Fig. 2 Relative expression levels of a Bss, b FimH,and c Iss calculated K. pneumoniae ATCC 4352; K5, K. pneumoniae ATCC 51503; LPS by the comparative (ΔΔct) method using 16srRNA gene as endogenous 10, lipoplysaccharide concentration 10 ng/ml; and LPS 1000, reference gene. EB, E. coli ATCC BAA196; E12, E. coli ATCC 12435; lipoplysaccharide concentration 1000 ng/ml E23, E. coli ATCC 23716; K3, K. pneumoniae ATCC 33495; K4, Relave RNA expression level Relave RNA expression level Relave RNA expression level 304 Ann Microbiol (2019) 69:299–305 Acknowledgements All thanks and appreciation to Biodiversity Institute for bacterial motility and it does exclusive roles, as they help of Ontario, University of Guelph, Guelph, Ontario, N1G 2W1, Canada, bacteria to swim towards signals or nutrients. This helps bac- for providing the standard strains used in this study. This work was per- teria to reach a surface and spread along it. In addition, it formed at the Microbiology Department, Faculty of Pharmacy, Mansoura provides the physical adherence to the surfaces (Pratt and University, Egypt and Department of Pharmaceutics and Pharmaceutical Biotechnology, Faculty of Pharmacy, Taibah University, AlMadinah Al Kolter 1998). Flagella also mediate swarming. A critical cell Munawwarah, Kingdom of Saudi Arabia. density is required to initiate this organized movement process of bacteria. So, swarming is usually associated with quorum Compliance with ethical standards sensing (Eberl et al. 1996; Sperandio et al. 2002; Verstraeten et al. 2008; Witso et al. 2014). Conflict of interest The author declares that there is no conflict of In this study, different virulence factors associated genes of interest. Enterobacteriacea were investigated by PCR: biofilm (Bss), adhesions (FimH, PapA, PapC,and afa/draBC), siderophore Publisher’sNote Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. (iutA and fyuA), and serum resistance (Iss and KapsMTII). Bss, FimH, and Iss genes were present in all tested E. coli and K. pneumoniae strains. fyuA gene was detected in E. coli References ATCC BAA196 and iutA were detected in all E. coli strains. However, PapA, PapC, afa/draBC,and KapsMTII were ab- Abdel-Rhman SH, Rizk DE (2016) Effect of tyrosol on Staphylococcus sent in all tested strains. aureus antimicrobial susceptibility, biofilm formation and virulence The effect of LPS on the expression levels of Bss, FimH, factors. Afr J Microbiol Res 10:687–693 Iss, fyuA,and iutA genes was evaluated using RT-PCR. LPS Abdelmegeed ES, Barwa R, Galil KHAE (2015) Comparative study on prevalence and association of some virulence factors with extended (concentrations 10 and 1000 ng/ml) was able to increase the spectrum betalactamases and AmpC producing Escherichia coli. Afr level of Bss, FimH,and Iss genes expression as compared to J Microbiol Res 9:1165–1174 control (in absence of LPS) (P < 0.0014). For both iutA and Aurell CA, Wistrom AO (1998) Critical aggregation concentrations of fyuA, both genes were detected but not expressed in the tested Gram-negative bacterial lipopolysaccharides (LPS). 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Annals of Microbiology – Springer Journals
Published: Dec 19, 2018
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