Adherence of local and standard strains of Salmonella to human Uro-epithelial cells

This study include determination of the ability to adhere to human uroepithelial cells in three types of local isolates of Salmonella (S.typhi , S.typhimurium , S. montevideo) and compared this ability with standard strains (S.typhi 5535 , S.paratyphi B 5542 , S. paratyphi C a-55108). The results showed that all isolates were able to adhere to Human uroepithelial cells, and found that the standard strains adhered more efficiently than local isolates, but the statistical analysis revealed that there was no significant difference in adhesion rates between local and standard isolates. The results also showed that among the local isolates, the bacteria S. montevideo adhered in higher numbers than did other species, whereas the highest adherence among standard strains was by S. typhi 5535. Introduction Salmonella species are enteric pathogens that infects humans of all ages (Lindquist et al., 1987). At least four major groups of infection associated with Salmonellosis are recognized, these groups include gastroenteritis, septicemia with or with out focalized infection, and two syndromes (enteric fever and carrier state) traditionally associated with Salmonella typhi infection (Janda & Abbott, 1998). In addition to these four syndromes, anumber of less–well-described extraintestinal illnesses due to Salmonella have been reported, among these illnesses are Urinary tract infections (UTIs). (Cohen et al., 1987; Mathai et al., 1995 and Abbott et al., 1999). But its extremely rare are occurs in individuals with structural or functional abnormalities of the urinary tract, or immunosuppressive status. and may occur in conjunction with gastroenteritis .(Wilson & Feldman., 1982; Kappor et al., 1992; Ramos et al.,1996; Embil & Nicolle., 1997; Abbott et al.,1999; Richard.,2003; Mourani et al.,2005 and Gagnon et al.,2007). OGrady &Cattell (1966) reported that Salmonella enter the urinary tract either hematogenously or by direct invasion of the bladder via the urethra. In order to initiate all those infections mensioned above, Salmonella species must be able to attach to its host mucosal surfaces in the intestine or urinary tract and subsequent invasion of epithelial cells (ECs) (Baumler et Journal of Kirkuk University – Scientific Studies, vol.6, No.1, 2011 11 al .,1997) hence microbial attachment to mucosal surfaces is a first step in mucosal infection. Specific interaction between microbial surface ligands and host receptors influence the distribution of microbes in their sites of infection. Adhesion has often been regarded as a sufficient end point, explaining tissue tropism and bacterial persistence at mucosal sites. Adherence, however, is also a virulence factor through which microbes gain access to host tissues, upset the integrity of the mucosal barrier, and cause disease. (Deman et al., 1990; Connell et al., 1997). In gram negative bacteria adhesion is often mediated by fimbriae, which are thin filamentous organelles expressed on the surface of the bacterial cells (Clegg et al., 1987). The aim of the present study was to determine the adherence ability to human uro-epithelial cells by local and standard strain of Salmonella and hence comparing between them.


Introduction
Salmonella species are enteric pathogens that infects humans of all ages (Lindquist et al., 1987). At least four major groups of infection associated with Salmonellosis are recognized, these groups include gastroenteritis, septicemia with or with out focalized infection, and two syndromes (enteric fever and carrier state) traditionally associated with Salmonella typhi infection (Janda & Abbott, 1998). In addition to these four syndromes, anumber of less-well-described extraintestinal illnesses due to Salmonella have been reported, among these illnesses are Urinary tract infections (UTIs). (Cohen et al., 1987;Mathai et al., 1995 andAbbott et al., 1999). But its extremely rare are occurs in individuals with structural or functional abnormalities of the urinary tract, or immunosuppressive status. and may occur in conjunction with gastroenteritis . (Wilson & Feldman., 1982;Kappor et al., 1992;Ramos et al.,1996;Embil & Nicolle., 1997;Abbott et al.,1999;Richard.,2003;Mourani et al.,2005 andGagnon et al.,2007). OGrady &Cattell (1966) reported that Salmonella enter the urinary tract either hematogenously or by direct invasion of the bladder via the urethra.
In order to initiate all those infections mensioned above, Salmonella species must be able to attach to its host mucosal surfaces in the intestine or urinary tract and subsequent invasion of epithelial cells (ECs) (Baumler et al .,1997) hence microbial attachment to mucosal surfaces is a first step in mucosal infection. Specific interaction between microbial surface ligands and host receptors influence the distribution of microbes in their sites of infection. Adhesion has often been regarded as a sufficient end point, explaining tissue tropism and bacterial persistence at mucosal sites. Adherence, however, is also a virulence factor through which microbes gain access to host tissues, upset the integrity of the mucosal barrier, and cause disease. (Deman et al., 1990;Connell et al., 1997). In gram negative bacteria adhesion is often mediated by fimbriae, which are thin filamentous organelles expressed on the surface of the bacterial cells (Clegg et al., 1987). The aim of the present study was to determine the adherence ability to human uro-epithelial cells by local and standard strain of Salmonella and hence comparing between them.

Bacteria:-
Local isolates used in this study include :-(S.typhi, S.typhimurium , and S.montevideo) which belonged to groups D, B, and C respectively . These organisms were isolated from patients with typhoid and enteric fever, attending Azadi general hospital in kirkuk city. Diagnosis of the isolates were based on biochemical and serological tests (Andrews &Hammack., 2003) and then by API20E. Standard strains include: -(S. typhi 5535, S. paratyphi B 5542, S. paratyphi C a-55108) .These strains were obtained from University of Mosul-college of science-Dep of Biology.

Preparation of bacterial suspension:-
Cultures were grown at 37c‫ه‬ in Luria Bertani broth medium for 24h., after incubation period the broth cultures was centrifuged at 5000gx for 10 min. and the pellet was washed twice in phosphate buffered saline (PBS) and resuspended in the same buffer at a final concentration of (10 9 ) cells /ml by Mcfarland standard solution (Guzman et al.,1989)

Epithelial cells (ECs):-
Human uro-epithelial cells were collected by centrifugation of fresh morning urine from healthy woman. ECs were washed three times in phosphate buffered saline (PBS pH =7.2) and resuspend the pellet in the same buffer, the suspension passed through filter paper (Whattman No.1),and the epithelial cells retained on the filter placed on microscope slides ( Guzman et al.,1989).

Adherence assay: -
The slides that contain epithelial cells placed in a plate, and 0.5 ml of the bacterial suspension was added then the plate was incubated for 1hr. At 37c ‫ه‬ with shaking, after incubation the slides renced twice with PBS to eliminate unattached bacteria .then the cells on the slides were fixed with ethanol, and stained using Giemsa stain. ( Van-DenBosch et al., 1980;Guzman et al., 1989 andBories et al., 1989). The binding results were expressed as the number of bacteria adhered to each of 100 epithelial cells.

Statistical analysis:-
Adherence was expressed as the mean number of bound bacteria /epithelial cell ± the standard deviation. Comparisons were analyzed by student t-test.

Results and Discussion
The ability of bacteria to adhere to epithelial cells (ECs) has been shown to be an essential step in the pathogenesis of bacterial infections (Guzman et al., 1989). Furthermore, it has been shown that the propensity of certain bacteria to infect specific tissues is often related to the ability of these bacteria to adhere to the respective target cell in vitro (Beachey, 1981;Klemm &Schembri., 2000 andMikcha et al., 2004).
In this report, adhereing by all Salmonella (local and standard strains) were clearly seen on the surface of uro-epithelial cells (Fig 1). The results summarized in table (1) shows few differences in the dgree of attachment between local and standard strains, in fact standard strains shows higher rates of adherence than local isolates did, but no significant differences were observed in the adherence rates between them. These results are in agreement with the results of experiments reported by Shareef et al. (2009) on the adherence of the same Salmonella species to epithelial cells of rat intestine. There is an evidence that Salmonella spp. possesses some virulence factors on their cell surface such as type 1 fimbriae that mediates binding of bacterial cells to specific receptors on the surface of cells in the intestine or the urinary tract. (Stocker & Makela.,1986;Vanderveldn et al.,1998;Althouse et al.,2003;White et al., 2003;Duncan et al.,2005 andShareef et al.,2009).