Abstract
Purpose: Enterobacteriaceae members are largely distributed in the environment, and responsible for 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 objective of this study is to assess the effect of the bacterial LPS on Enterobacteriaceae biofilm formation and other virulence factors in vitro.
Methodology: The effect of P.aeruginosa LPS on biofilm formation of two other species of Enterobacteriaceae (E. coli and K. 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 with LPS was measured with RT-PCR.
Results: 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.
Conclusion: 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.

Key words: P. aeruginosa LPS, E. coli, K. pneumoniae, biofilm, virulence factors.

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Introduction
LPS, a Gram-negative bacterial endotoxin, is an outer component of the cell wall that causes septic shock in animals and humans by its immunomodulatory and proinflammatory properties (Rietschel et al. 1982; Morrison and Ryan 1987; Aurell and Wistrom 1998). LPS also plays a role in the mechanism of endotoxic shock (Michalek et al. 1980; Tracey et al. 1987) and in the pathophysiology of infections (Morrison and Ryan 1987; Bandara et al. 2010).
Most micro-organisms exist in nature as biofilm that is complex communities of one or more microbial species attached to solid surfaces or to one another rather than free-floating planktonic cells. (Samaranayake 1990). On comparing biofilms and planktonic counterparts, biofilms are resistant to antimicrobials (O’Toole et al. 2000; Mah and O’Toole 2001) and involved in the majority of human infections. They frequently comprise of either mono-species or multispecies of bacteria and fungi such as Candida (Nobile and Mitchell 2007; Bandara et al. 2009; Bandara et al. 2010). Most studies focused on biofilm formation due to the ability of sessile bacteria than planktonic bacteria in toleration of exogenous stress (Goncalves Mdos et al. 2014).
The nature of biofilm formation is the production of an extracellular matrix consist of 90% water and10% extracellular polymeric substances (EPS) (Flemming and Wingender 2010). Components of EPS mediate most of interactions that are important for the biofilm formation and stabilization.
Enterobacteriaceae is a large family of Gram-negative bacteria, which comprises many harmless symbiont and pathogenic microbes, such as Salmonella, Shigella, E. 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 many isolates of E. coli produce biofilm structures that is responsible for 90% of UTIs (Beloin et al. 2008; Spurbeck et al. 2011). Curli (coiled extracellular appendages on the E. coli surface) are necessary for biofilm development(Manu Chaudhary et al. 2013).
E. coli is one of the commensal bacteria in human intestinal tract that can convert to pathogenic ones by lateral gene transfer through gene loss or gain (Kaper et al. 2004). E. coli can cause extra intestinal infections due to its different virulence factors (ex: adhesions, polysaccharide capsules, toxins, proteases and lipopolysaccharides) (JR and TA. 2002; Mokady et al. 2005; Abdelmegeed et al. 2015).
Klebsiella pneumoniae is a leading cause of nosocomial pneumonia and UTIs, endophthalmitis, pyogenic liver abscess. Virulence factors like fimbriae type 1 and type 3 provided new insights into K. pneumoniae pathogenic strategies, , which mediate attachment to the host mucosal surfaces and inert surfaces and play a role in biofilm formation (O’Toole and Kolter 1998; Sutherland 2001; Lejeune 2003).
Virulence factors can be determined by genomics to identify specific genes responsible for virulence of the pathogens. For an organism to be virulent, it needs a combination of different factors rather than one virulent factor (Dobrindt 2005; Abdelmegeed et al. 2015). P. aeruginosa is important bacterial pathogens that causes nosocomial infection in which P. aeruginosa can colonize; infect and intoxicate susceptible patients.LPS produced by this organism is a key virulence factor that also affects both innate and acquired host responses to infection. They are localized in the outer layer of the membrane contributing to its integrity. It protects the cell against the bile salts and lipophilic antibiotics and mediate the entry of bacteria into the cell (Pier 2007).
P. aeruginosae, E. coli and K. pneumoniae are widely distributed in Egypt’s hospitals as in references. So, the aim of this study was to evaluate the effect of P. aeruginosa LPS on Enterobacteriaceae biofilm and virulence factors as these organisms may be present in mixed infections.

Methodology
Micro-organisms. The following reference laboratory strains were used: E. coli ATCC BAA196, E. coli ATCC 23716, E. coli ATCC 12435, K. pneumoniae ATCC 33495, K. pneumoniae ATCC 51503 and K. pneumoniae ATCC 4352. In addition to 2 clinical isolates of each coliform. Prior to experiments, all isolates were stored at -80 ºC.
LPS. LPS from P. aeruginosa ATCC 27316 (catalogue No. L9143) was purchased as lyophilized powder from Sigma Aldrich and stored at 2–8 ºC till use.
Evaluation of P. aeruginosa LPS effect on the metabolic activity.
To evaluate the effect of LPS on the vitality of E. coli and K. pneumoniae strains, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay was done on both LPS treated and control cells at a predetermined time points (Montoro et al. 2005). P. aeruginosa LPS (1 mg/ ml) was serially diluted with LB broth (10 ng, 50 ng, 100 ng, 250 ng, 500 ng, 750 ng and 1000 ng). The effect of LPS on the tested strains was evaluated as described below. Briefly, a suspension of E. coli and K. pneumoniae strains (100 µl) was dispersed into selected wells of a 96-well plate with LPS (100 µl of tested concentrations) and incubated for 24 h. A growth control containing sterile LB instead of LPS and a negative control without inoculums were also included for each isolate. Subsequently, 10µL of the MTT solution (5 g/L) was added to each well and the plate was re-incubated for another 4 h followed by addition of 50 µL of the DMS solution to each well. A change in color from yellow to violet (which indicates growth of bacteria) was measured with a microtitre plate reader (Spectra Max 340 tunable microplate reader; Molecular Devices Ltd, Sunnyvale, CA) at 540 nm. All assays were carried out in triplicate on three different occasions.
Effect of P. aeruginosa LPS on biofilm formation
The effects of the P. aeruginosa LPS on biofilm formation was evaluated by biofilm formation assay that was carried out in 96-well polystyrene microtiter plate (Nunc, New York, NY, USA) and measured using the crystal violet staining procedure (Goncalves Mdos et al. 2014). Briefly, overnight culture of each tested strain was diluted to 1×106 CFU/ml with fresh LB broth. Each well was inoculated with 100 µl of the bacterial suspensions with different LPS concentrations; control assays were also performed. The microtiter plates were incubated at 37ºC for 18 h without shaking, and non-adherent bacteria were removed by three washing steps with PBS. Biofilm was stained by 0.5% (w/v) crystal violet solution for 10 min. Then, the plates were rinsed with distilled water, air-dried, dissolved in 33% glacial acetic acid and the OD570 was determined.
Effect of LPS on the formed mature biofilms
One hundred ?l of E. coli and K. pneumoniae suspensions (OD600=0.257) was inoculated into separate wells of polystyrene 96-well plates (flat bottom; Nunc). The plates were incubated at 37°C for 24 h. After incubation, the supernatants were aseptically aspirated, and the wells were washed twice with PBS without disturbing the formed biofilms, 100 ?l of fresh LB broth containing different LPS concentrations was added to each well. LB broth without LPS was added to control wells. The plates were then incubated at 37°C for 24 h. Non-adherent cells were discarded through two rounds of washing with 200 ?l sterile PBS saline. Cells adherent to the plastic surfaces were quantified using crystal violet assay (X et al. 2003). Experiment was performed in triplicate.
Molecular screening of various virulence genes among E. coli and K. pneumoniae standard strains.
The tested strains were analyzed by PCR for the presence of nine genes; eight virulence genes: FimH (mannose specific adhesion subunit of type 1 fimbriae), Iss (increased serum resistance)(Galil et al. 2011), KapsMTII (group2 capsule synthesis), fyuA (ferric yersiniabactin uptake), Afa/draBC (Dr-binding a fimbrial adhesins), PapA (P fimbriae), PapC (outer membrane protein of P fimbrae), iutA (aerobactin receptor) and Bss (biofilm formation)( Galil et al. 2011). Total DNA was prepared as previously described (Abdelmegeed et al. 2015). A reaction mixture containing 0.5 ?M of each primer, 1.5 Mm MgCl2, 0.2 Mm dNTPs, 1 U Taq polymerase (promega), 5 ?l of DNA and nuclease free water was added for a total volume of 25 ?l per reaction.
The PCR program started with an initial denaturation step at 94 ºC for 3 min, then 40 cycles of (DNA denaturation at 94 ºC for 30 s, annealing for 40 s, then extension at 72 ºC for 1 min), followed by a final extension step at 72 ºC for 5 min.
PCR products were analyzed on a 1.2% ethidium bromide stained agarose gel and compared with 100 bp plus DNA ladder and visualized under UV illumination.
Quantitative real time-PCR
E. coli and K. pneumoniae strains were treated with LPS (10 ng/ml and 1000 ng/ml) and total RNA was extracted by TRI Reagent (T9424 Sigma-Aldrich). The purity and concentration for RNA were determined spectrophotometrically at 260 and 260/280 nm ratio respectively using NanoDrop (ND-1000 Spectrophotometer, NanoDrop Technologies, Wilmington, Delaware, USA). Complementary DNA (cDNA) was prepared from 1µg of RNA using QuantiTect Reverse Transcription kit (QIAGEN, USA).
The level of virulence genes expression (Bss, FimH, Iss, FyuA and iutA) were measured using the primers described before. Amplification and expression were performed using 5x FIREPol EvaGreen, qPCR Mix, ROX Dye; Solis BioDyne and Rotor Gene Q thermocycler (QIAGEN, Hilden, Germany). The reaction mixture was prepared and RT-PCR program were performed as previously described(Abdelmegeed et al. 2015). The expressions of the virulence and biofilm genes in the treated samples were measured relative to untreated samples and were analyzed using the 2-??Ct method.
Statistical analysis. Statistical analysis was performed using GraphPad Prism5. One-way ANOVA followed by Dunnett posttest were performed to compare significant differences between the control and treated strains. A P value ? 0.05 was considered statistically significant.

Results
Evaluation of P. aeruginosa LPS impact on Enterobacteriaceae
The effect of different concentrations of P. aeruginosa LPS on Enterobacteriaceae metabolic activity was evaluated using MTT reduction assay. All concentrations of LPS used had turned the color of MTT from yellow to purple which indicates that LPS had no effect on the metabolic activity of tested strains.
Effect of P. aeruginosa LPS on biofilm formation
The biofilm formed by all tested strains significantly increased under different concentration of LPS compared to the control untreated samples (1.07-7.53 fold increase). However, 4 strains (coli ATCC 12435, E. coli ATCC 23716, K. pneumoniae ATCC 33495 and K1) showed high increase in biofilm formation with increasing concentrations of LPS (table 1).
Effect of LPS on the already formed mature biofilms
LPS had the ability to significantly stabilize and to increase the already formed biofilm in all tested strains (1.40- 7.20 fold increase). LPS (1000 ng/ml) can stabilize the already formed mature biofilm significantly than other used concentrations (fig 1) especially in K. pneumoniae strains (2.50-7.20 times compared to control untreated samples). all LPS concentrations caused the same level of stabilization in E. coli ATCC BAA196 (around 3.10 times) and E. coli ATCC 23716 (around 4.35 times).
Molecular screening of various virulence genes among E. coli and K. pneumoniae strains.
Bss, FimH and Iss genes were detected in all tested strains while fyuA gene was amplified in E. coli ATCC BAA196 only, iutA gene was present in all E. coli tested strains. The other virulence genes (papA, papC, Afa/draBC, KapsMTII,) were not detected (table 2).
Expression of biofilm and virulence genes by RT-PCR
Using RT-PCR, the expression level of (Bss, FimH and iss) genes was significantly higher in the LPS treated cells compared to untreated ones. The other virulence genes (iutA and fyuA) were not expressed in the tested E. coli strains. The effect of LPS on the gene expression was higher in K. pneumoniae strains (relative expression=7.4) than in E. coli strains (relative expression=4.7) in case of Bss gene. The high effect of LPS was also observed on the expression of Bss gene than other genes (fig 2).
Discussion
E. coli and K. pneumoniae are Gram-negative commensals that live with its host in symbiosis by colonizing the gastrointestinal tracts (Kaper et al. 2004). Acquiring virulence traits by several clones leads to intra- and extra-intestinal infections (Seaton 2000; Witso et al. 2014). Previous studies reported that E. coli, K. pneumoniae and P. aeruginosa have been found as the most common three uro-pathogenic bacteria (Behzadi et al. 2010).
Due to mixed infection caused by these three pathogens, the effect of P. aeruginosa LPS on E. coli and K. pneumoniae biofilm and virulence factors was investigated. To our knowledge this is the first study exploring the effect of P. aeruginosa LPS on the biofilm and virulence factors in Enterobacteriaceae. The present results showed that P. aeruginosa LPS had no inhibitory effect on the growth of E. coli or K. pneumoniae as evaluated by MTT reduction assay. This finding was in accordance with Bandara et al. (Bandara et al. 2009) who reported similar results on Candida growth.
In this work, LPS was found to stimulate E. coli and K. pneumoniae biofilm formation at all concentrations used as it significantly increased its formation (P