New York Science Journal 2013;6(9) http://www.sciencepub.net/newyork

The effect of p-nitrophenylglycerol on swarming and the production of some virulence factors in vulgaris

Mohammed Ghaidaa¹*, Wang Yanchang¹, and Hindi Abdallah²

¹Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida. ²Biology Department of Science College, Babylon University, Babil, Iraq *Email: [email protected]

Abstract: Proteus vulgaris is an opportunistic pathogen, commonly responsible for urinary and septic ; often nosocomial. Proteus vulagris has a number of putative virulence factors, including the secreted hemolytic, which has been suggested to contribute to host cell invasion and cytotoxicity, an inducible which, by generating ammonia, causes precipitation of bladder and kidney stones, fimbriae which promote bacterial adherence to the uroepithelium, a secreted protease able to digest immunoglobulins. In this study we have verified the ability of p-nitrophenylglycerol (anti-swarming agent of ) to inhibit Proteus vulgaris swarming and the expression of some virulence factor (haemolysin and urease).Swarming inhibition was determined on Luria Bertani agar with PNPG and then was harvested to assay cell length and the production of haemolysin and urease. P-nitrophenylglycerol significantly inhibited swarming and virulence factor expression but its effect on growth rate was not significant. [Mohammed Ghaidaa, Wang Yanchang, and Hindi Abdallah. The effect of p-nitrophenylglycerol on swarming and the production of some virulence factors in Proteus vulgaris. N Y Sci J 2013;6(9):8-14]. (ISSN: 1554-0200). 2

Keywords: P-nitrophenylglycerol, P.vulgaris, Swarming,virulence factors

Introduction and isolation of from specimens Proteus vulgaris is a rod-shaped Gram-negative contaminated with swarming strains of Proteus chemoheterotroph bacterium. The size of individual spp(Williams 1973).In addition, PNPG has little effect cells varies from 0.4~0.6μm by 1.2~2.5μm. P. on the results of a variety of identification tests vulgaris possesses peritrichous flagella, making it performed directly on colonies from media containing actively motile. It inhabits the soil, polluted water, raw PNPG( Lai 1994 and Jun et al. 2004). It is relatively meat, gastrointestinal tracts of animals, and dust. In cheap, nontoxic and doesn’t affect red blood cells; humans, Proteus species most frequently cause even fastidious pathogens will grow well and with urinary tract infections, but can also produce severe characteristic colony morphology in its presence abscesses; P. mirabilis produces 90 percent of cases, (Liaw et al. 2000).Its heat stability and long ‘shelf – and is encountered in the community, but P. vulgaris life’ make it convenient to use in the preparation of is associated with nosocomial (O'Hara et media(Liaw et al. 2000). al.2000 and Struble 2009). According to the ability of Proteus spp to The invader P. vulgaris has numerous factors express virulence factors and invade human urethelial including fimbriae, flagella, outer membrane proteins, cells is correlated with its swarming activity (Liaw et lipopolysaccharide, capsule antigen, urease, al. 2001 and 2004). So, the purpose of the present immunoglobulin A proteases, hemolysins, amino acid study was to investigate the activity of PNPG against deaminases, and, finally, the most characteristic uropathogenic P. vulgaris swarming and it’s attribute of Proteus, swarming growth, enabling them producing of some virulence factors (haemolysin and to colonize and survive in higher organisms (Rozalski urease) which are correlated with its ability to invade et al.1997 and Emody et al.2000). uroepithelial cells. P-nitrophenylglycerol or 1-(4-Nitrophenyl-β-D- Materials and methods glucuronicacid) (PNPG) is a chromogenic β- Chemicals: PNPG (Sigma) was sterilized by filtration glucuronidase substrate (Sartory and Watkins, 1999). through a 0.22um pore membrane. It has been found to has anti-swarm properties by Bacteria and growth conditions adding it to a solid culture medium.So, it used as one Proteus vulgaris was isolated from a patient with of methods which have been reported to prevent UTI and identified by biochemical tests (MacFaddin Proteus species from swarming over the surface of a 2000). P. vulgaris isolate was cultured overnight at solid culture medium(Hernandez et al.1999).The 37˚C in Luria Bertani (LB) broth and then used to antiswarming activity of p-nitrophenylglycerol inoculate LB broth and LB agar contained different (PNPG) has been found invaluable for the recognition concentration of PNPG (0, 10, 50, 100, 150 and

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New York Science Journal 2013;6(9) http://www.sciencepub.net/newyork

200µg/ml) to assay its growth rate, swarming activity hemolysis), whereas no change of zone referred to and ability to express virulence factors (haemolysin non-hemolysis (γ- hemolysis) (Buller 2004, Ray et al. and urease) in the presence of PNPG. 2004, and Al-Dulaimi 2009). Swarming behavior assay Urease production assay The swarming migration distance assay was Preparation of cells for urease assay was performed as described previously (Liaw et al. 2000 performed as described previously (Liaw et al. 2000 and 2004, Wang et al. 2006, and Al-Dulaimi 2009). and Echeverrigaray et al.2008).In this test we Briefly, an overnight bacterial culture (5 µl) was inoculated the urea slant from bacterial suspension by inoculated centrally onto the surface of dry LB streaking the entire slant surface, incubated the tubes swarming agar (1.5 % w/v) plates with or without with loosened caps at 37oC then color change of PNPG, which were then incubated at 37˚C. The medium was examined after 16h incubation. Urease swarming migration distance was assayed by production was indicated by changed medium color following swarm fronts of the bacterial cells and into pink (Winn et al. 2006 and Al-Dulaimi 2009). recording progress at 1h intervals. Statistical Analysis Bacterial growth assay All the results represent the average of three in independent experiments. The data were presented as ﹾP.vulgaris was cultured overnight at 37C Luria broth (LB) diluted 1:100 in LB containing mean and analyzed by one-way analysis of variance various concentrations of PNPG (0, 10, 50,100,150 with P <0.05 being significant, calculated using the and 200µg/ml) and the growth rate was monitored at GraphPad Prism 5 statistical software. 1h intervals (Liaw et al. 2000 and 2004, Wang et al. Results 2006, Echeverrigaray et al. 2008, Vanessa and Inhibition of P. vulgaris swarming by PNPG Zunino, 2009). In this study, we found that PNPG had the Measurement of cell length, haemolysin and urease ability to block the swarming migration of P.vulgaris activities in a dose-dependent manner (Fig. 1a). The swarming Sections of agar containing vegetative cells behavior was significantly inhibited at concentrations (colony center) and swarming cells (colony edge) 50µg/ml and was blocked completely at 150 and have been taken from plates with and without 200µg/ml (Fig. 1a, b). To test the inhibitory effect of appropriate concentration of PNPG. Bacteria were PNPG on bacterial growth in addition to swarming, an washed from agar segments with 5ml of phosphate overnight culture of P.vulgaris was inoculated into buffered saline (PBS) after that the cell length, LB containing various concentrations of PNPG and haemolysin and urease production was assayed (Liaw the growth rate of bacteria was monitored as shown in et al. 2000 and 2004, Wang et al. 2006, and Figure1c.The growth rate of P. vulgaris wasn’t Echeverrigaray et al.2008). inhibited by PNPG. At 16 h post-inoculation, the Measurement of cell length bacteria grew approximately to similar densities, Measurement of cell length was performed as regardless of the presence of PNPG. We concluded described by (Liaw et al. 2000 and Echeverrigaray et that the inhibitory effect of PNPG on swarming was al.2008]. Briefly, 150μl of stationary-phase LB unlikely to be due to inhibit of cell growth. cultures were spread onto LB agar plates without or Inhibition of swarming differentiation and with appropriate PNPG concentration and incubated at virulence factor expression of P. vulgaris by PNPG 37°C. After incubation, Cells from the entire surface Cell morphology was mointered after of agar plates were harvested by washing into 5 ml of inoculation of an overnight culture of P.vulgaris onto PBS. Bacterial cells were fixed and subjected to gram LB swarming plates containing various concentrations stain (Ward’s Science, USA), examined by light of PNPG. As shown in Figure 2a, in the absence of microscopy (Carl Zeiss, Germany) at a magnification PNPG, the swarming cells were longer than the of 100x, and digitalized using a digital camera. The bacterial cells in the presence of PNPG at the lengths of 100 cells in each sample were determined, concentration 200µg/ml, suggesting that swarming and the average was calculated. differentiation was inhibited. The inhibition of Haemolysin Production assay differentiation started to be observed at PNPG Haemolysin production was carried out by concentration of 50µg/ml. Very few elongated inoculating a blood agar medium containing swarming cells were observed at PNPG concentration 2%washed horse erythrocytes with bacterial cells of 100µg/ml. As PNPG concentration was increased taken after suspended in 5ml of PBS then incubated at to 150 and 200µg/ml, only short vegetative cells were 37◦C for 24h.The appearance of a clear zone around observed. These results indicate that swarming the colonies referred to a complete hemolysis (β- differentiation of P.vulgaris was indeed inhibited by hemolysis). The appearance of greenish zone around high concentrations of PNPG. the colonies referred to a partial hemolysis (α-

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New York Science Journal 2013;6(9) http://www.sciencepub.net/newyork

To investigate whether the production of of virulence factors was not affected significantly at virulence factors (haemolysin and urease) was also PNPG concentrations (0-100µg/ml) for urease and (0- influenced by PNPG, the production of haemolysin 50 µg/ml) for haemolysin but was inhibited and urease in P. vulgaris which taken from LB agar significantly in the presence of increasing plates containing different concentrations of PNPG concentrations (150 and 200µg/ml). was determined. As shown in Figure 3, the production

Figures

7 6 5 4 3 2 Distance(cm) 1 0 2 3 4 5 6 7 Time after inoculation(h)

0 10µg/ml 50µg/ml 100µg/ml 150µg/ml 200µg/ml

Fig.1.(a). Effect of PNPG on the swarming of P.vulgaris. The histogram shows the migration distance of P.vulgaris in the presence of various concentrations of PNPG (0, 10, 50, 100, 150, and 200µg/ml). The data represent the mean of three independent experiments and the differences are significant (P value <0.05).

0 10µg/ml 50µg/ml

100µg/ml 150µg/ml 200µg/ml

Fig.1.(b).Halo images of swarming plates containing different concentrations of PNPG (0, 10, 50, 100, 150, and 200 µg/ml) at 7h after inoculation.

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New York Science Journal 2013;6(9) http://www.sciencepub.net/newyork

0 10µg/ml 50µg/ml 100µg/ml 150µg/ml 200µg/ml 2

1.5

1

OD600 0.5

0 2 3 4 5 6 7 8 9 16 Time after inoculation (h)

Fig.1. (c). Effect of PNPG on the growth of P.vulgaris.OD600 was measured overtime in the presence of various concentrations of PNPG (0, 10, 50, 100, 150, and 200µg/ml). The data represent the mean of three independent experiments, there is no significant difference between concentrations (P value >0.05).

(0) (200µg/ml)

Fig.2 (a).Microscopic observation of P.vulgaris isolated from the LB plates without PNPG (0) and with (200 µg/ml) of PNPG.

140 120 100 80 60 40 20 Relative length(%) Relative 0 0 10 50 100 150 200

PNPG concentration(µg/ml)

Fig.2.(b). Effect of PNPG on the cell length of P.vulgaris. The histogram shows the cell length of P.vulgaris in the presence of various concentrations of PNPG (0, 10, 50, 100, 150, and 200µg/ml). The lengths of 100 cells in each sample were determined, and the average was calculated. The difference between concentrations is statistically significant (P value <0.05).

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New York Science Journal 2013;6(9) http://www.sciencepub.net/newyork

Heamolysin Urease

100 90 80 70 60 50 40

Percent(%) 30 20 10 0 0 10 50 100 150 200 PNPG concentration (µg/ml)

Fig.(3).The influence of PNPG on the expression of virulence factors in P.vulgaris. The histogram shows the production of haemolysin and urease at different concentrations of PNPG (0, 10, 50, 100, 150, and 200µg/ml). The data represent the mean of three independent experiments. The difference is statistically significant (P value<0.05).

Discussion It is now well known that many bacterial It has been well demonstrated that swarming functions including swarming, biofilm formation, and motility and virulence factor expression are secretion of virulence factors important in successful coordinately regulated in several pathogens including and recurrent establishment bacterial infections are Pr. mirabilis and Ps. aeruginosa which are often related to cell density-mediated gene expression which implicated in persistent UTI (Ronald 2002, Wang is termed ‘quorum sensing’ (QS). Quorum sensing 2006 and Nashikkar et al.2010). controls the virulence determinants in most P-Nitrophenylglycerol (PNPG) effectively . In this process bacteria communicate inhibits swarming and virulence factor production of with each other within their vicinity using chemical the enterobacterium Proteus mirabilis (Liaw et al. signaling molecules which are known as autoinducers. 2000 and 2001). The underlying mechanism of Quorum sensing (QS) relies on these autoinducers to inhibition is unclear. We have now found that PNPG control gene expression in response to changes in also inhibits swarming and virulence factor production bacterial cell density (Schauder et al.2001). Several in another enterobacterium, Proteus vulgaris. signaling molecules have been identified and the most In this study, we found that PNPG has the ability common type in most Gram-negative bacteria is the to inhibit P. vulgaris swarming significantly at a N-acylhomoserine lactones (AHLs) (Adonizio et al. concentration as low as 50 µg/ ml and inhibited 2008 and Yin et al.2012). swarming completely at 150 and 200µg/ml (Fig.1a QS regulates diverse bacterial functions and b). Also, it had the ability to suppress the including antibiotic formation, virulence factor production of virulence factor; hamolysin suppression expression, luminescence, biofilm formation, motility was at concentrations of (100,150 and 200 µg/ml) and pigment production (Fuqua and Greenberg, 2003, whereas urease suppressed at (150 and 200 µg/ml) Whitehead 2001 and Krishnan et al. 2012). Thus, (Fig.3). But it did not significantly affect the growth inhibiting QS or anti-QS is an important anti-infective of the bacteria even at high concentrations (200 measure that does not rely on antibiotics(Vattem et µg/ml) (Fig. 1c). This means PNPG have the ability to al.2007). Anti-QS agents will inhibit the QS inhibit swarming and virulence factor production mechanism and attenuate the virulence determinants without significant inhibition of P. vulgaris growth. and are unlikely to cause drug-resistance problems Based on this finding we concluded that the swarming (Adonizio et al.2008). ability of P.vulgaris is correlated with its ability to Antibiotics are commonly used for the treatment express virulence factors. So, this result corresponds of microbial infections with the widespread with results recorded by (Liaw et al. 2000, Ronald appearance of multi antibiotic-resistant bacteria; it is 2002, Wang 2006 and Nashikkar et al.2010). becoming increasingly more difficult to treat bacterial

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New York Science Journal 2013;6(9) http://www.sciencepub.net/newyork infections with conventional antibiotics. Thus, there is manual. Interpretation of biochemical an increasing need for new strategies to cope with identification test, 3: 121. infectious diseases. The discovery that many 4. -Echeverrigaray S, Michelim L, Delamare A L, pathogenic bacteria employ quorum sensing (QS) to Andrade, CP Costa SO and J Zacaria.2008. The regulate their pathogenicity and virulence factor Effect of Monoterpenes on Swarming production makes the QS system an attractive target Differentiation and Haemolysin Activity in for antimicrobial therapy. It has been suggested that Proteus mirabilis. Molecules, 13: 3107-3116. inactivating the QS system of a pathogen can result in 5. -Emody L, Pal T, Hacker, J and GB Ochler. 2000. a significant decrease in virulence factor production Genes and proteins underlying microbial (Schauder and Bassler, 2001, Lyon and Muir,2003, virulence. Advance in expiremental medicine and Mihalik et al.2008). So, the possible mechanism by biology, Vol.485.P.339. which PNPG could inhibit P. vulgaris swarming and 6. -Fuqua C and Greenberg EP.2002. Listening in on virulence factor expression may be due to its acting as bacteria: acyl-homoserine lactone signaling. Nat. an inhibitor compound for bacterial quorum sensing Rev. Mol. Cell Biol.3:685–695. (QS). Our results from this study indicate that PNPG 7. -Hernandez E, Ramisse, F and Jean-Didier has the potential to be an antimicrobial agent against C.1999. Abolition of Swarming of Proteus. J. P.vulgaris infection. Clin. Microbiol. 37(10):3435. 8. -Jun RW, Yu-Tze H, Hsin-chih L, Kwen-Tay L, Acknowledgments and Shen-Wu H.2004. Effect of PNPG on cell We are grateful to Biomedical Science growth cycle, motility machinery and quorum Department of the Medicine College at Florida State sensing in Serratia marcesccens. J Microbiol University especially the laboratory of Dr. Wang. We Immunol Infect.73:1-7. are so grateful to Dr. Wang Yanchang, Dr. Fengshan 9. -Krishnan T, Yin WF, and KG Chan.2012. Liang, Dr. Fengzhi Jin and Kelly McKnight for their Inhibition of Quorum Sensing Controlled assistance. Virulence Factor Production in PAO1 by Ayurveda Spice Clove Authors: (Syzygium Aromaticum) Bud Extract,12(4): Mohammed Ghaidaa¹*, Wang Yanchang¹, and Hindi 4016–4030. Abdallah² 10. -Lai HC.1994.Molecular studies on the swarming ¹Department of Biomedical Sciences, College of migration of Proteus mirabilis (PhD thesis).UK: Medicine, Florida State University, Department of pathology. Cambridge University. Tallahassee, Florida. 11. -Liaw SJ, Lai HC, Ho SW, Luh KT,and Wang ²Biology Department of Science College, Babylon WB. 2000. Inhibition of virulence factor University, Babil, Iraq expression and swarming differentiation in *Email: [email protected] Proteus mirabilis by p-nitrophenylglycerol. J Med Microbial. 49:725–731. FLORIDA STATE UNIVERSITY 12. -Liaw SJ, Lai HC, Ho SW, Luh KT, and WB College of Medicine Wang. 2001. Characterization of P- nitrophenylglycerol-resistant Proteus mirabilis super-swarming mutants. J Med Microbiol. 50:1039–1048. 13. -Liaw SJ, Lai HC,and Wang WB. 2004. Modulation of swarming and virulence by fatty acids through the RsbA protein in Proteus References mirabilis. Infect Immun. 72:6836–6845. 1. -Adonizio A, Kong, KF, and K Mathee.2008. 14. -Lyon GJ and Muir TW.2003. Chemical signaling Inhibition of Quorum Sensing-Controlled among bacteria and its inhibition.Chem Biol.10: Virulence Factor Production in Pseudomonas 1007-21. aeruginosa by South Florida Plant Extracts. 15. -MacFaddin J F. 2000. Biochemical tests for Antimicrob Agents Chemother. 52(1): 198–203. identification of medical bacteria. 1st Ed. 2. -Al-Dulaimi AA.2009.Inhibition of swarming and Williams and Wilkins. Baltimore, USA. some virulence factors expression in Proteus 16. -Mihalik K, Chung DW, Crixell SH, McLean mirabilis by amikacin in vitro. Diala Jour. RJC, and DA Vattem. 2008. Quorum sensing Volume, 32: 206. modulators of Pseudomonas aeruginosa 3. -Buller NB.2004. Bacteria from fish and other characterized in Camellia sinensis. Asian Journal aquatic animals: A practical identification of Traditional Medicines, 3 (1):12-23.

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