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Prevalence of Enterotoxigenic and Toxic Shock Syndrome Toxin-1 Producing Coagulase Positive Staphylococcus Aureus in Human and Their Characterization

M. J. Islam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. S. Uddin
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. S. Nasrin
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

K. H. M. N. H. Nazir
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. T. Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. M. Alam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

The study was carried out with 73 human originated samples viz. surgical wound swab, pus, burn ulcer exudates, aural swab and diabetic ulcer exudates collected over a period of 5 months starting from September 2006 to identify and characterize enterotoxins and toxic shock syndrome toxin-1 (TSST-1) producing coagulase-positive S. aureus (CPSA) by Reverse Latex agglutination test, in which 30 (41.10%) were found as CPSA. Among the 30 CPSA isolates, 22 (73.33%), 6 (20%) and 2 (6.67%) were golden-yellow, yellow and whitish pigment producers, respectively and 29 (96.67%) isolates indicated βhemolysis on blood agar speculating their ability to produce β-hemolysin. A total of 30 CPSA were checked for enterotoxin and TSST-1 production of which 5 (16.67%) and 1 (3.33%) isolates produced enterotoxin-A and TSST-1, respectively. Other produced multiple toxins in which 2 (6.67%) produced both enterotoxins A and enterotoxin B, 2 (6.67%) produced both enterotoxin C and enterotoxin D and 2 (6.67%) produced both enterotoxin C and TSST-1. The antibiotic-resistant pattern of the CPSA indicated that 83.33% of isolates were resistant to penicillin-G and 70% to sulphamethoxazole. On the other hand, the results demonstrated that gentamicin, spiramicin, ciprofloxacin, oxacillin, oxytetracycline and streptomycin might be used for the treatment of S. aureus infection. Few multiple antibiotic-resistant CPSA were also identified. The prevalence of methicillin resistance S. aureus (MRSA) was 23.33%.

Keywords: CPSA, Reverse-latex-agglutination test, Enterotoxins, TSST-1, MRSA

Location: The Department of Microbiology and Hygiene and in the laboratory of the Department of Medicine, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh

Start Date: 00-09-2006

End Date: 00-04-2007

Program Area: Conservation and Biodiversity

Commodity: Genetic resources

Objectives:

To detect and characterize enterotoxigenic and TSST-1 producing S. aureus from human origin.

Methodology:

The study was conducted in the laboratory of the Department of Microbiology and Hygiene and in the laboratory of the Department of Medicine, Bangladesh Agricultural University (BAU), Mymensingh, during the period from September 2006 to April 2007. Sources of samples A total of 73 samples such as wound swab, pus and exudates from diabetic burn ulcers and aural swab of patients were collected from Mymensingh Medical College (MMC) with proper aseptic precautions. Media used for culture Both commercially available and laboratory-made media were used for the isolation, identification and characterization of S. aureus. The solid media used were blood agar (BA, HiMedia), nutrient agar (NA, HiMedia), mannitol salt agar (MSA, HiMedia) and Muller Hinton agar (MHA, Oxoid). The broths used were brain heart infusion broth (BHIB, HiMedia) and nutrient broth (NA, HiMedia). All the media were prepared according to the instructions of the manufacturer(s). Isolation of coagulase-positive S. aureus S. aureus were isolated based on their morphological, staining and cultural characteristics following the procedures of Cheesbrough (1985). Coagulase test was performed using human plasma to detect the CPSA as described by Carter (1979). Test for pigment production and hemolytic activity Pigment production and hemolytic activity of S. aureus were observed on NA and BA media, respectively, according to the procedures of Chatterjee et al. (1990). β-hemolysis was indicated by the complete clear zones of hemolysis around the colony on BA. Reverse latex agglutination test procedure Reverse latex agglutination test was performed according to the method of Pinto et al. (2004). In brief, the single pure colony of CPSA isolate was inoculated into test tube containing 5 ml BHIB and incubated aerobically at 370C for 24 h. After incubation, the broth culture was transferred into an Eppendorf tube and centrifuged at 10,000 rpm for 5 minutes. The supernatant containing the toxins were collected. The test was done in V–shaped 96 well micro-plates. An amount of 25 µl of PBS was poured in each well and 25 µl of supernatant fluid was then added into each well of first row and subsequently, a 2 fold dilution was made. Type-specific pink-colored antibody (25 µl) (Denka Seiken, Japan) was added to each well of a particular column. The micro-plates were kept on an electric shaker for few seconds and kept at room temperature for overnight. Finally, an agglutination reaction was observed. In positive cases, antigen-antibody bindings occurred and the pink color disappeared. Determination of antibiotic sensitivity pattern Antibiotic sensitivity test of the isolated CPSA was done by disc diffusion method using the Kirby-Bauser technique (Bauser et al., 1966) as per the recommendation of National Committee for Clinical Laboratory Standards (NCCLS, 1997). The entire test was performed on MHA (pH 7.2-7.4). The name of antibiotics (manufactured by Oxoid Ltd. Basingstoke, Hampshire, England) and their concentrations were: penicillin-G (10 µg/disc), spiramycin (100 µg/disc), amoxycillin (25 µg/disc), oxytetracycline (30 µg/disc), oxacillin (10 µg/disc), gentamicin (120 µg/disc), ciprofloxacin (5 µg/disc), sulphamethoxazole (25 µg/disc) and streptomycin (10 µg/disc). The MRSA were detected by their ability of being resistant against oxacillin as described by Walther et al. (2006).

Source of Information: Bangl. J. Vet. Med. (2007). 5 (1 & 2): 115–119

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

S. aureus is one of the important etiological agents responsible for pyogenic infection in man. Staphylococcal organisms are mainly associated with skin, gland and mucous membranes of man and animals. In the present study CPSA was isolated from pus, wound swab, aural swab, burn ulcers and diabetic burn ulcer exudates collected from human. Among the 73 samples, 30 (41.10%) were positive for the presence of CPSA. As the CPSA are usually pathogenic in nature hence, their presence indicates potential health risk for man. Aycicek et al. (2005) found 9.4% prevalence of CPSA by testing a total of 512 samples originating from human hands; however, Fabiano et al. (2005) reported 36% prevalence of CPSA in human skin samples. The increased number of resistant CPSA to penicillin G and sulphamethoxazole might be linked to the long and extensive use of those antibiotics in human. On the other hand, spiramycin, oxytetracycline, streptomycin and ciprofloxacin could be used for the treatment of staphylococcal infection in human in Bangladesh. Kadlubowska et al. (2006) found CPSA as resistant against spiramycin and ciprofloxacin in Poland. Several MRSA and multiple antibiotic-resistant CPSA were identified in present study. The prevalence of MRSA was 23.33%, which was in support of Loeffler et al. (2005) and Kadlubowska et al. (2006) who found 17.9% and 70% MRSA positive cases of human origin, respectively. Manian (2003) reported MRSA from wound infection of human. The results indicated MRSA as common nosocomial pathogen of human. Cloning and sequencing of mecA gene responsible for methicillin resistance may be done from the MRSA isolated here to understand their molecular epidemiology.

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