Plesiomonas shigelloides in stool samples of patients in the Venda Region : Possible considerations on pathogenicity and antibiogram profiles

This study determined the haemolytic, haemagglutinating and antibiotic susceptibility activities of Plesiomonas shigelloides isolated from stool samples of patients attending different health centers in the Venda region of South Africa. P.shigelloides was isolated and identified using the API 20E, API 20NE systems. Antibiotic susceptibility profiles of the isolates were determined using the disc diffusion method and analyzed according to NCCLS standards. The hemolytic and hemagglutination activities of the isolates on human, sheep, pig and chicken red blood cells were determined using the plate and slide methods. A total of 89 (13%) P. shigelloide were isolated from 660 samples. The hemolytic activities of the isolates were variable with no heamolysis on sheep red cells. 33 (37%) of isolates were beta lactamase producers. There was a high level of resistance to the penicilllins with 100% resistance to Penicillin G, Amoxicillin and Ampicillin. This study has demonstrated multiple resistance to different antibiotics and production of beta lactamase. Most of the isolates showed evidence of pathogenicity as demonstrated by hemolytic and haemagglutinating activities.


INTRODUCTION
Plesiomonas shigelloides is gram-negative rod which is found in freshwater, freshwater fish, shellfish and different types of animals such as cattle, goats, swine, cats, dogs, monkeys, vultures, snakes, and toads.P. shigelloides, though aquatic in origin, is extensively distributed in the environment and is reportedly an agent of gastroenteritis (Salerno et al., 2007).Several reports have implicated the organism as a cause of diarrhoeal disease in humans and animals (Farmer et al., 1992).Human infections due to this bacterium are mostly waterborne.The organism may be present in unsanitary water, which has been used as drinking water, recreational water, or water used to rinse foods that are consumed without cooking or heating.P. shigelloides infections occur in summer months and correlate with environmental con-*Corresponding author.E-mail: c355251@yahoo.com.tamination of freshwater (rivers, streams and ponds).Outbreaks of gastroenteritis linked to consumption of oysters contaminated with P. shigelloides have given impetus to the public health significance of the organism (Miller and Koburger, 1985).P. shigelloides gastroenteritis is usually a mild self-limiting disease with fever, chills, abdominal pain, nausea, diarrhea, or vomiting; symptoms may begin 20 -24 h after eating contaminated food or drinking contaminated water (Ueda et al., 1999).Diarrhea is usually watery, non-mucoid, and non-bloody and in severe cases may be greenish-yellow, foamy, and blood tinged.
Extra-intestinal complications such as septicemia (Nolte et al., 1988) may occur in people who are immunocompromised or seriously ill with cancer, blood disorders, or hepatobiliary disease.Inspite of the clinical significance of P. shigelloides, little is known about the genetic landscape, updated antibiograms, structure of the population and extent of infections caused by the organism (Salerno et al., 2007).As a prelude to unraveling the genetic diversity, which will impact on epidemiological control, a study on the prevalence, virulence potential and antibiograms of the organism is critical in order to gauge the extent of the problem, ascertain pathogenic factors and antibiograms of isolates for clinicoepidemiological relevance and for empiric management of infections requiring antibiotics.
In this first communication, we report on possible considerations of pathogenicity and antibiogram profiles of isolates as a prelude to an account of the genetic landscape of P. shigelloides in the Venda region of South Africa.

Study site and patients
The study was carried out in the Vhembe district, Limpopo Province of South Africa.Diarrheic stool samples were collected from patients with diarrhea attending different hospitals in the region including Donald Frazer (Vhufuli), Elim, Tshilidzini, Siloam and Makhado hospitals.Each patient was given a 50 ml sterile plastic container and advised on how to collect the stool samples which were then kept in a cooler box with ice and taken to the Microbiology laboratory, University of Venda for microbiological analysis.A total 660 stool samples were collected and demographic information such as age and sex of the patients were recorded.

Culture methods
Specimens were investigated for the presence of P. shigelloides.The specimens were cultured using the method previously described (Obi et al., 2007a(Obi et al., , 2007b)).Briefly, freshly collected stool specimens were plated onto MacConkey agar and xylose deoxycholate citrate agar (XDCA) and incubated at 37 o C for 24 h, the suspected colonies for P. shigeloides were tested for cytochrome oxidase (Kovac's method) as previously reported (Wong et al., 2003).The oxidase positive strains were further evaluated by triple sugar iron agar slants and API-20E and API-20NE commercial strips (Analytab products, Inc., plainview, N.Y).In addition, the sugars listed in Table 1 were also used in conjunction with commercial kit to identify P. shielloides.

Determination of beta-hemolytic activity
Beta-hemolytic activity was determination, using sheep blood agar, human blood agar, pig blood agar and chicken blood agar.Single colonies of each isolates were streaked across the various blood agar plates using sterile inoculating wire loops.The inoculated blood agar plates were incubated at 37ºC for 18 to 24 h.After incubation the hemolytic activities were determined by observing hemolysis on the blood agar (Obi et al., 2007a;Samie et al., 2007).

Beta-lactamase-production
The isolates were tested for the production of beta-lactamase.Two to three well-isolated colonies were picked and applied onto filter paper with applicator stick.A small quantity of Nitrocefin was drawn with a syringe and needle and a tiny drop of the fluid was applied onto each specimen (Samie et al., 2007).A change of colour of the filler paper from white to yellow indicated a positive reaction.

Determination of minimum inhibitory concentrations by microdilution
The minimum inhibitory concentrations (MICs) patterns of the isolates to 11 antibiotics were determined in microplates (NCCLS, 2003).

Reference strains
Escherichia coli ATCC 25922 and klebscella pneumoniae ATCC 700603 were used as the reference strains for antimicrobial susceptibility testing.

RESULTS AND DISCUSSION
Out of a total of 660 samples collected from 580 (88%) females and 80 (12%) males with ages ranging from 1 -60 years, only 89 (13%) were positive for P.shigelloides.Of 89 isolates of P. shigelloides isolated, 82 (92%) showed beta-haemolytsic activities on human red blood cells with 56 (63%) on chicken red blood cells whereas none (O%) showed haemolysis on sheep red blood cells and pig red blood cells.Only 33(37%) were betalactamase producers.Tables 2 and 3 show the MICs of selected antibiotics.The MICs were measured in µg/ml as well as antibiotic resistance of P. shigelloides to different antibiotics.
P. shigelloides is a bacterium of clinical significance but  Antimicrobial susceptibility testing was performed on 89 stool isolates of P. shigelloides isolated from patients with diarrhea in the Venda region of South Africa using a standard agar dilution technique.Results obtained showed that imipenem, meropenem, cephazoline, cefepime, ciprofloxacin, gentamicin and cefoxitin may be useful in the treatment of P. shigelloides infections.A study by Obi et al. (2007) demonstrated that the carbapenems and cephalosporins had the best in vitro activity against Aeromonas species, an organism related to P. shigelloides.
Results of our study are also in harmony with those of other researchers in terms of susceptibility of the organism to ciprofloxacin, gentamicin and imipenem (Kain and Kelly, 1989;Stock and Wiedemann, 2001;Wong et al., 2003).However, the organism showed resistance to multiple antibiotics such as penicillin, vancomycin amoxicillin, tetracycline and erythromycin.
Results also showed that majority of the isolates (92%) were beta-hemolytic and 63% showed haemolysis on human red blood cells.37% of the isolates were betalactamase producers.Haemolysis and beta-lactamase production are indices of pathogenicity and results on these pathogenic factors simulate previous findings on related organisms (Obi et al., 2007;Samie et al;2007;Wong et al., 2003).
In conclusion, meropenem and imipenem had good activity against P. shigelloides isolated from stool samples and may be useful in cases of gastrointestinal infection caused by P. shigelloides.However, further studies are needed to unravel genetic profiles of resistance genes as well as genes coding for virulence in P. shigelloides.

Table 1 .
Profiles used for identification of Plesiomonas shielloides.

Table 3 .
Antibiotic susceptibility of Plesiomonas shigelloides isolated from stool samples in the Venda region.