1. Introduction
Aerobic, gram positive bacteria Staphylococcus aureus is the causative agent of many opportunistic infection in human and animals (Kools and Bannerman, 1995). As a human pathogen, S.aureus causes superficial, deep-skin and soft tissue infections, endocarditis and bacteremia, as well as a variety of Toxic - mediated diseases including gastroenteritis, staphylococcal scalded-skin syndrome and Toxic shock syndrome 25-30% of healthy people carry this organism on their skin (or) in their nose. (Fidalgo et al., 1990 - Roberts et al., 1991).
Staphylococcus aureus produces a variety of extracellular toxins and virulence factors that contribute to its pathogenic potential. S.aureus strains produce phyrogeneic exotoxins, such as Staphylococcal enterotoxins (SES) and TSST-1 (Sharma et al., 2000). SES are a group of single - chain, low molecular mass protein that are similar in composition and biological activity by differ in antigenicity (Fueyo et al., 2001). Enterotoxins of S.aureus can be detected by their biological activity, by immuno assays and by PCR (MC Lauchlin et al., 2000) The prevalence of enterotoxigenic clinical S.aureus isolates have been reported in different countries by many investigators (Mehrotra et al., 2000; Fueyo et al., 2001; Becker et al., 2003).
a)Methicillin - Resistant Staphylococcus aureus
Methicillin - resistant staphylococcus aureus is a type of staph that is resistant to certain antibiotics. These antibiotics include methicillin and other such as cloxacillin, dicloxacillin, oxacillin and nafcillin as well as a closely related class of drugs known as cephalosporins. Methicillin resistant S.aureus strains are resistant to methicillin and essentially all other beta-lactam antibiotics
MRSA was 1st reported in 1961, soon after methicillin was introduced into human medicine to treat penicillin resistant staphylococci. MRSA has since emerged as an important pathogen is human medicine (Lee JH - 2003) MRSA is a major hospital associated as well as community - associated pathogen causing a wide range of disease, including endocarditis, Oesteomyelitis, TSS, Pneumonia, food poisoning and carbuncles (Oliveira D.C., et al., 2005).
In Indian hospital, MRSA is one of the common causes of hospital acquired infections and different hospitals have deported anywhere from 30 to 80% Methicillin resistance based on antibiotic sensitivity tests. Methicillin resistance is due to the presence of A gene coding for penicillin - binding protein (PBP 2A) with a low affinity for beta - Lactam antibiotics. This gene is carried on staphylococcal cassette chromosome (SCC) Mec, a unique mobile genetic element, integrated into the staphylococcal chromosome (Kalayama et al.,2001).
b) Coagulase Negative Staphylococci
There is an increasing awareness of the abilities of coagulase - negative staphylococci to cause disease. Coagulase negative staphylococci have been recognized as the most frequent cause of prosthetic valve, endocarditis, neurosurgical shunt infection and infection of prosthetic orthopedic devices (Siebert and W.T et al., 1978). In general coagulase negative staphylococci are reported as "Staphylococcus epidermidis". However coagulase negative staphylococci and Interogenous group of organism and several new species have been proposed by Kloos and Schleifer (1976).
Since many staphylococci are penicillin resistant through production of b-Lactamase, Methicillin and Other b-Lactamase-resistant penicillins are important therapeutic agents for treatment of staphylococcal infection. However significant % of S.epidermidis is Methicillin resistant (Sieberet et al., (1978) and Sabath et al., 1969).
c) TSST - Toxic Shock Syndrome Toxin
Toxic shock syndrome (TSS) and its associated with S.aureus were first described by Todd et al., in 1978. S.aureus strains to produce disease in both clinical and food settings depends, among other determinants, on its ability to produce extracellular toxins. A number of staphylococcal enterotoxin (SES) classified as A, B, C1, C2, C3, D or can be produced by some strains (Pimbley and Patel 1998). Most staph aureus isolated from patient with TSS, a severe acute illness that rapidly leads to multi organ system failure, produce a Toxin known as TSST-1. Although classically associated with tampon use,
TSS has also been associated with a variety of non-menstruation related conditions (Hertzer, 2001).
Staphylococcus aureus is carrying the tst gene encoding TSST-1. (See, R.H. & A.W Chow, 1989). The tst gene is chromosomal and the toxin is symptomatically related to the staphylococcal enterotoxin group of toxins (Iandolo. J.J., 1989). (Blomster and Hantamaa D.A. et al., 1986).
d) Detection of the TSST-1 gene
Production of Coagulase, a product of coagulase (coa) gene, is the principal criteria in the clinical microbiology laboratory for the identification of S.aureus. The coagulase ptn is an important virulence factor for S.aureus. The coa gene has polymorphic repeat regions that can be used for differentiating S.aureus isolates. The variable region of coa gene is comprised of 81 bp tandem short sequence repeat (SSR S) (Van Belkum et al., 1998).
Amplification of coa and RFLP analysis of amplified products for the discrimination if S.aureus isolates have previously been reported (Goh et al., 1992; Shopsin et al., 2000; da silva & da silva, 2006).
TSST is a potentially fatal multi system disease presenting with fever, hypotension, vomiting, diarrhoea, mucosal hyperaemia and an erythematous rash. This is associated with infection of mucosal or sequestered sites by TSST production S.aureus strains usually belonging to bacteriophage group-I.
TSST type-1 (also known as enterotoxin type F (or) pyrogenic exotoxin C) is most often responsible, through enterotoxins B (or) C may also cause the syndrome. TSS Strains produced TSST-1, the toxin accepted as the most likely cause of TSS (MS Bergdoll et al., 1984). Coagulase negative strain produces TSST-1 and SEA. Since there has been disagreement about the production of TSST-1 by coagulase negative strains (Crass et al., 1986).
2. MATERIALS AND METHODS
a) COLLECTION OF SAMPLES
A Totally 19 pus samples were collected for our studies. Pus samples were collected from wound-infected persons by using sterile cotton dipped swab and samples collected from in around Namakkal area hospital. After collection of samples immediately inoculated into peptone water and the sample were brought to the laboratory within 2 hours.
b) ISOLATION OF Staphylococcus aureus
Loopful of culture from peptone water was streaked on the Mannital salt agar plate. The plates were incubated at 37oC for 24-48 hrs. The isolated colony from selective media was used for further analysis.
c) IDENTIFICATION OF Staphylococcus aureus
I) GRAM STAINING ( Hans Christian Gram (1853-1938)
Bacterial smears of 16-18 hrs old cultures were made on clean grease free slides, heat fixed and stained as follows. The slide was flooded with crystal violet solution for a minute, drained and rinsed with water; followed by Grams iodine solution for one minute, drained and rinsed with water. Decolourised with ethyl alcohol for 30 Sec and later counterstained with safranin for one minute and observed under an oil immersion microscope.
ii) MOTILITY TEST
The hanging drop technique was followed to observe the motility of the organism. Observation was made under the microscope for the darting or cork screw movement of the organism.
iii) CATALASE TEST
A small amount of culture was placed over a clean slide. A drop of 3% hydrogen peroxide was placed over the culture and observed for effervescence. The production of effervescence showed the ability to produce the enzyme catalase.
iv) OXIDASE TEST
The organism spotted on oxidase disc (HiMedia) the blue or purple colour change was observed within 10 seconds. Purple colour being positive while negative is denoted with no colour change.
(B)BIOCHEMICAL TEST
I) INDOLE TEST
A loopful of culture was inoculated into peptone water and incubated at 370 C for overnight. After incubation 0.5ml of Kovac's reagent was added.
Positive reaction -Cherry red ring
Negative reaction -Yellow colour ring
ii) METHYL RED TEST
The colony from nutrient agar slant was inoculated into MRVP medium and was then incubated at 370 C for overnight. The test employed to detect the production of acid during fermentation. In addition 0.5 ml of methyl red was added.
Positive reaction -Red colour
Negative reaction -Yellow colour
iii) VOGES PROSKAUER TEST
A loopful of culture was inoculated into MRVP medium, which was incubated at 370C for overnight, to detect acetyl methylcarbinol from pyruvic acid. In addition of VP reagent (0.5ml of 5% alpha-naphthol and 0.5ml of 40% KOH) was added.
Positive reaction -Red colour
Negative reaction -Remains colourless
iv) CITRATE UTILIZATION TEST:
The culture was streaked into the Simmon's citrate agar slant and incubated at 370C for overnight.
Positive reaction -Deep Purssian blue colour
Negative reaction -Green colour
v) UREASE TEST:
A loopful of culture was inoculated into Christenson's urea broth tube. Production of urease was indicated.
Positive reaction -Pink colour
Negative reaction -No colour change
vi) SUGAR FERMENTATION:
A loopful of culture was inoculated in to different sugars in the sugar media along with Durham's tube and Andrade's indicator.
Positive reaction -Pink colour
No fermentation -colourless
vii) TRIPLE SUGAR IRON:
Triple sugar iron agar was prepared and dispensed into tubes and sterilized at 1210C for 15 minutes and made slants. Tubes were inoculated with test organisms stabbing down the agar and streaking on the slants. The tubes were incubated at 370c for 24 hours.
Positive result: Colour of the medium change to yellow, blackening of the medium, and break in the medium of lifting of the medium indicates acid production, H2S production and gas production respectively.
C) ANTIBACTERIAL STABILITY TEST
The standard Kirby-Bauer disk diffusion method was used to determine the antimicrobial profiles of the Staphylococcus aureus isolates against Methicillin. The nutrient broth was prepared and sterilized at 121°C and inoculated the isolates then incubated at 37°C for 24 hrs. After incubation period the broth culture were inoculated into surface of the Mueller-Hinton agar plates and antibiotic discs were placed then plates were incubated at 37°C for 18 to 20 h. The zone of inhibition and resistance was measured, recorded and interpreted according to the recommendation of the disc manufacture.
D) COAGULATE TEST
The tube is filled with 0.5 ml of 1 in 10 diluted rabbit plasma. To the tube, 0.1 ml of overnight broth culture of test bacteria is added. All the tubes are incubated at 37ºC and observed up to four hours. Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube.
E) AMPLIFICATION OF TOXIC SHOCK SYNDROME GENE FROM Staphylococcus aureus
I) ISOLATION OF GENOMIC DNA
Took 1.5 ml of overnight broth culture in to 2 ml micro centrifuge tubes.
The tubes were centrifuged at 8000 rpm for 5 minutes.
After centrifugation, the supernatant was discarded and the pellet was collected. The pellet was suspended in 200μl of 1X TE buffer + 100μl of 10% SDS and mixed by vortexing.
The tubes were kept in water bath at 60°C for 20 minutes.
Then added with 300μl of Phenol: Chloroform: Isoamyl alcohol mixture (24:25:1) to extract the DNA and mixed completely by vortexing.
The tubes were then centrifuged at 10000 rpm for 10 minutes to separate the phases.
The aqueous phase containing the DNA was carefully removed and transferred to new tubes.
Equal volume of 100% Isopropanol was added to the tubes containing the aqueous phase.
It was mixed by inverting the tubes 3 to 4 times.
The tubes were then centrifuged at 10000 rpm for 10 minutes to pellet the DNA.
The supernatant was discarded and the pellet was collected.
To the pellet, 200μl of 70% ethanol was added and centrifuged at 10000 rpm for 10 minutes.
Then Ethanol was decanted completely and the pellet was air-dried to give purified DNA.
Re-suspended the dried DNA pellet in 20 μl of TE buffer and dissolved by tapping.
DNA solutions were stored at 4°C for further work.
F) CONFIRMATION OF DNA BY AGAROSE GEL ELECTROPHORESIS:
Agarose gel electrophoresis is carried out in a horizontal submarine electrophoresis unit. Thirty ml of 1 % Agarose gel was prepared with 1X TBE buffer (do not mix) and heated the content to get up to clear solution for casting Agarose gel. After cooling the solution, 7 µl of staining dye solution was added into the casting system.
The gel was allowed to solidify, and then carefully disassembled from the casting system without disturbing the wells and placed in 1X TBE buffer filled electrophoresis tank (the buffer level should be above gel). 5 µl of genomic sample DNA mixed with 2 µl of gel loading dye and then loaded to gel and simultaneously loaded 3 µl of DNA marker provided in the nearby well.
The power card terminals was connected at respective positions, run the gel at 50 V, till the gel loading dye migrate more than half the length of gel. Then switched off the unit and visualized the isolated DNA under UV Transilluminator.
I)SAMPLE LOADING DYE:
Sucrose 40% and bromophenol blue (0.25%) were mixed with sterile double distilled water.
ii) ETHIDIUM BROMIDE:
Ethidium bromide (10mg) was mixed with 10 ml of sterile distilled water. (It is carcinogenic and should be treated or handled accordingly).
G) POLYMERASE CHAIN REACTION (Johnson & Tyler (1993)
The amplification method was carried by according to the Johnson & Tyler (1993) with some modification.The sequences of the primers used were 5-ATGGCAGCATCAGCTTGATA-3 and 5- TTTCCAATAACCACCCGTTT-39 (Sigma, India). Thus yielding an amplicon 533 bp. PCR conditions were as described previously (Johnson & Tyler, 1993). Amplification was carried out in a Genei, India PCR system using the following procedure. The 20µl consists of 2 μl of 1 × PCR buffer, 1 mM of each of the primers (0.5 μl), 15 mM of each deoxynucleotide triphosphate (1 μl) and 5 U is the final concentration of Taq DNA polymerase (0.5 μl) Template 1 μl and 14.5 μl of molecular grade water. After initial denaturation at 94°C for 4 min, the samples were subjected to 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min. A final extension was performed at 72°C for 7 min. Following PCR, 20 μl of the reaction mixtures were analyzed by electrophoresis on a 1% agarose gel, containing ethidium bromide (0.2 mg/ml), in the presence of an appropriate DNA molecular weight marker.
4. RESULT
In the present study 19 wound swab samples were collected for Occurrence of Methicillin resistance Staphylococcal contamination and coagulase negative isolates. Among the 19 wound swab 10 (52.6%) isolates of Staphylococcus were obtained by standard test and selective media.
a) CONFIRMATION AND MAINTENANCE OF Staphylococcus ISOLATES.
The selected isolates from mannitol salt sugar are large circular, convex, smooth, shiny, opaque and golden yellow color colonies were obtained. The cells are Gram-positive, non- motile and grape like cluster cocci arrangement. The catalase resulted as positive. In biochemical test result MR showed positive. The Staphylococcus isolates were maintained on nutrient agar plate for further studies.
The distribution of isolates from the different types of samples such as 5/10 (50%) isolates of burn, 3/4 (75%) from accident, 2/5 (40%) from skin samples
Among the 3 types of samples highest occurrence obtained from accident (75%) and lowest occurrence in skin (40%)
b) COAGULASE TEST
Among the 10 samples of S.aureus strains 8 (80%) isolates of coagulase negative S.aureus were obtained. There was a distribution of isolates from the different types of samples such as 4/5 (80%) isolates from burn, 2/3 (66.6%) isolates from accident, 2/2 (100%) isolates from skin infection. Among the 3 types of samples highest occurrence obtained from skin infection and lowest occurrence in accident.
c) METHICILLIN STABILITY TEST
Coagulase negative isolates were subjected into Methicillin stability test by agar disc diffusion method (Kirby Bauer method). All (8) of the isolated Staphylococcus aureus showed sensitive or resistance to methicillin antibiotic. Out off 10 isolates 8 (80%) was resistance to Methicillin. Among the 3 types of samples the highest resistances were observed in Burn infection and skin infection wound samples (100%). In accident wound samples 33.3% of resistances isolates was obtained.
d) AMPLIFICATION OF TSST GENE FROM Staphylococcus aureus
In this PCR assay all isolates of Staphylococcus aureus was subjected according to the previous study. Among the 10 isolates the 3 isolates produce the TSST gene. The distributation of toxic gene from skin infection isolates has 100% and 33.3% from accident infection. In burn samples, TSST gene was not observed.
5. SUMMARY AND CONCLUSION
A total of 19 samples were collected for our study. These samples were of three types namely Skin infection, Burn and Accident wound samples from which Staphylococcus aureus was isolated by use of selective media mannitol salt agar (MSA) and biochemical tests. Out of the 19 samples, 10 isolates were obtained. Among the 3 types of samples accident samples(75%) had highest prevalence of Staphylococcus aureus followed by burn wound samples (50%) and finally skin infection samples (40%).
Among the 10 isolates of Staphylococcus aureus, 8were found to be coagulase negative by using coagulase test and were distributed as, skin infection 100%, burn wound 80% and accident wound samples with the least. (66.6%)
The 8 isolates of coagulase negative Staphylococcus aureus which were obtained from above were subsequently tested for antibacterial drug resistance based on Kirby - Bauer disk diffusion method. They were all found to be resistant to Methicillin antibiotic.
In the next, all isolates of coagulase negative staphylococcus aureus were subjected to the PCR assay according to previous studies carried out. Among the 8 isolates, 3 isolates produced the TSST gene. The distribution of toxic gene from skin infection isolates was found to be 100% and that of accident wound samples was 33.3% whereas there was no toxic gene in the burn wound samples.
In conclusion, the TSST developed in this study is a convenient and reliable method for the detection of tst in Staphylococci, which could be useful for both research and clinical purposes. Also, TSST demonstrated that the distribution of tst was more prevalent in MRSA. This is the first time that PCR detection of the TSST-1 gene in S. aureus has been reported from Namakkal, and further studies are needed on the occurrence of TSS in the community and the role of TSST-1-producing S. aureus in this disease in Namakkal.
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