Identification, Characterization and Antibiotic Susceptibility of Salmonella and Shigella species Isolated from Blood and Stool Samples of Patients Visiting N. I. H, Islamabad

MATERIALS AND METHODS

The study was conducted at the Bacteriology Laboratory, Public Health Division, NIH, Islamabad, between January and June 2001. One hundred and fifty samples, blood (110) and stool (40), were collected from diarrhea patients, residing in Rawalpindi Islamabad, visiting the Bacteriology laboratory at the NIH. These samples were streaked on blood, SS, MacConkey and chocolate agar and stool samples were inoculated in Selenite broth for sub-culturing, while Tryptic Soya Broth is used for sub-culturing blood samples and incubated for 24 to 72 hrs, respectively. The pathogens were identified by colony morphology as they form colorless colonies on all agars. Confirmatory tests included biochemical tests and serotyping (Annexure 1) (Ellen et al., 1994).

Serotyping: All the pathogenic forms of Salmonella and Shigella spp were confirmed up to species level by serotyping. For serological testing O (somatic), H (Flagellar) and Vi (capsular) antigens were used for confirmation. Shigella were serotyped by their O (somatic) antigens using polyvalent S. dysenteriae, S. flexneri, S. boydi and S. soneii antisera. Salmonella serotypes were done using O, H and Vi antigens. The groups were designated as A to Z, 51 to 61, 64 B 66. Medically important Salmonella belonged to groups A to G. The Salmonella was serotyped by their H antigen. They occurred in two antigenic forms referred as phase I and phase II. Vi antigen was mostly used for S. typhi and S. paratyphi C. (Cheesbrough, 1991). The procedure followed was in accordance with Edward and Ewing (1972).

Antibiogram pattern: Antibiogram pattern of isolated strains were determined on Mueller B Hinton agar. A sterile cotton swab was dipped into a sample from well-mixed colonies in peptone water and applied onto a Mueller B Hinton agar plate.

Annexure 1:	:Salmonella and Shigella isolation and identification

Commercially available anti-microbial disks were placed on the plate by means of a multi-disk dispenser and pressed firmly onto the agar with sterile forceps and incubated at 37^oC for 24 hours, before measuring the zones of inhibition to determine the susceptibility of the isolates. For comparison, the anti-microbial susceptibilities of isolates from the standard cultures were determined by a standard disk method according to the guidelines of the National Committee for Clinical Laboratory Standard (NCCLS). E. coli ATCC 25922 was included for quality control (Johnson et al., 1995).

Zones of inhibition were determined with the help of list of break points of antibiotics. If the zone of inhibition was less than the minimum value of break point then it was assumed to be ineffective and if the zone of inhibition was equal to the maximum value or greater than maximum value of break point, then the antibiotics were assumed to be effective against E. coli (Cheesbrough, 1991).

RESULTS AND DISCUSSION

Of the total blood and stool samples subjected to analysis for Salmonella and Shigella species, our study showed that 6 Salmonella isolates were identified all from the blood samples, whereas the 2 Shigella strains isolated were from stool samples (Table 1). Salmonella species isolated from blood culture included 2 isolates of S. typhi, 1 of S. paratyphi A, 1 of S. paratyphi B and 2 isolates of other Salmonella spp. of the 2 strains isolated from stool samples both were Shigella flexneri (Table 2).

Table 1:	Incidence of Salmonella and Shigella spp.

Table 2:	Incidence of Salmonella and Shigella species/serotypes isolated from clinical samples

Table 3:	Sex-wise distribution of Salmonella and Shigella species

Table 4:	Antibiotic susceptibility of Shigella species

Table 5:	Antimicrobial susceptibility of Salmonella species

This low percentage of isolates may be due to the fact that the samples were collected during the cold dry season, whereas Salmonella and Shigella infections are more common during the rainy season and often occur as outbreaks (Yamashiro et al., 1998).

Sex-wise distribution showed that the 2 Shigella flexneri isolates were evenly distributed, i.e., one male and one female case. Whereas, the 6 Salmonella species showed a distinct distribution pattern in that I. e., 4 were from males and 2 from females (Table 3). These findings are in agreement with the reports of other researchers (Saqib et al., 2000).

Drug susceptibility of S. flexneri isolates showed that they were resistant to vibramycin, cotrimoxazole, tetracycline and ampicillin, but sensitive to nalidixic acid, norfloxacin, ciprofloxacin, aztreonam, chloramphenicol, cefotaxime and amikacin (Table 4). Of the total 17 antibiotics used against Salmonella spp., all the S. paratyphi A strains showed intermediate resistance to tetracycline, and sensitivity to amikacin, tobramycin, gentamicin, ciprofloxacin, cefuroxime, nalidixic acid and ofloxacin. Whereas, S. typhi was resistant against cefuroxime, tetracycline, chloramphenicol, and cefamendole, while sensitive to amikacin, tobramycin, gentamicin, ofloxacin, cefotaxime, cefaclor, sulpha metoxazole-trimethoprim and cefoperazone. S. paratyphi B showed resistance against gentamicin and ampicillin, intermediate resistance against cefotaxime, but complete sensitivity against orelox, enoxacin, and cefamendole and intermediate against cefotaxime (Table 5). Similar findings have been reported by Binsztein et al. (1999) and Cruchaga et al. (2001).

Furthermore, the results of this study shows that Shigella flexneri resistant to tetracycline, ampicilline and cotrimoxazole, and sensitivity to amikacin, nalidixic acid, norfloxacin, aztreonam and chloramphenicol, which have also been reported by other researchers (Poiata et al., 1996; Urio et al., 2001; Yamashiro et al., 1998).

The study under discussion also revealed that S. typhi strains showed greater resistance agaisnt drugs, which is in agreement with the findings of other workers (Farooqi et al., 1990), who reported that S. typhi and S. paratyphi showed resistance against ampicilline and chloramphenicol, as indeed reflected in this study.

As for the sensitivity pattern, our study showed that cefotaxime, cefaclor, SXT, amikacin and ofloxacin were effective against S. typhi, which is in conformity with other findings (Binsztein et al., 1999, Butt et al., 2000) and the overall resistance pattern for Salmonella spp showed that chloramphenicol, ampicilline, gentamicin and cefamendole were ineffective, which has also been reported by Saqib et al. (2001).

The findings of this study do reflect that there is Shigella infections, although few, were equally in both males and females, while in Salmonella infections, especially those caused by S. typhi were more common in men.

However, despite the low prevalence of Salmonella and Shigella infections, the mere outbreak of six and two incidences, over a period of a couple of months, does reflect the poor level of hygiene and water treatment being practiced today in Pakistan. This issue of environmental health is very important and should be addressed by the concerned authorities so as to minimize such outbreaks. Drug susceptibility tests are also essential to ensure effective therapy. In addition, the clinicians must stress patient compliance and over-the-counter sales of antibiotics should be curtailed. Only then will the spread of resistance strains be brought under control.