IDENTIFICATION OF TSST FROM METHICILLIN RESISTANCE
Aerobic, gram positive bacteria Staphylococcus aureus will be the causative agent of countless 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, together with 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 his or her skin (or) of their nose. (Fidalgo et al., 1990 – Roberts et al., 1991).
Staphylococcus aureus produces a selection of extracellular toxins and virulence factors that give rise to its pathogenic potential. S.aureus strains produce phyrogeneic exotoxins, including Staphylococcal enterotoxins (SES) and TSST-1 (Sharma et al., 2000). SES undoubtedly are a group of single – chain, low molecular mass protein which can be similar in composition and biological activity by differ in antigenicity (Fueyo et al., 2001). Enterotoxins of S.aureus may be detected by their biological activity, by immuno assays and also by PCR (MC Lauchlin et al., 2000) The prevalence of enterotoxigenic clinical S.aureus isolates are already reported in numerous 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 staph that’s resistant to certain antibiotics. These antibiotics include methicillin and other including cloxacillin, dicloxacillin, oxacillin and nafcillin and also a closely related class of medication known as cephalosporins. Methicillin resistant S.aureus strains are resistance against methicillin and essentially all the other beta-lactam antibiotics
MRSA was 1st reported in 1961, immediately after methicillin was introduced into human medicine to take care of penicillin resistant staphylococci. MRSA has since emerged for an important pathogen is human medicine (Lee JH – 2003) MRSA is often a major hospital associated together with community – associated pathogen causing a lots of disease, including endocarditis, Oesteomyelitis, TSS, Pneumonia, food poisoning and carbuncles (Oliveira D.C., et al., 2005).
In Indian hospital, MRSA is amongst the common reasons for hospital acquired infections and different hospitals have deported anywhere from around 30 to 80% Methicillin resistance determined by antibiotic sensitivity tests. Methicillin resistance is a result of the presence of A gene coding for penicillin – binding protein (PBP 2A) which has a low affinity for beta – Lactam antibiotics. This gene is persisted staphylococcal cassette chromosome (SCC) Mec, a distinctive mobile genetic element, included in the staphylococcal chromosome (Kalayama et al., 2001).
b) Coagulase Negative Staphylococci
There is definitely an increasing knowing of the abilities of coagulase – negative staphylococci to cause disease. Coagulase negative staphylococci happen to be recognized as the most common 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 list of organism and lots of new species happen to be proposed by Kloos and Schleifer (1976).
Since many staphylococci are penicillin resistant through manufacture of b-Lactamase, Methicillin and Other b-Lactamase-resistant penicillins are crucial 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) as well as associated with S.aureus were first explained by Todd et al., in 1978. S.aureus strains to make disease within clinical and food settings depends, among other determinants, on its ability to provide extracellular toxins. A number of staphylococcal enterotoxin (SES) classified as A, B, C1, C2, C3, D or might be produced by some strains (Pimbley and Patel 1998). Most staph aureus isolated from patient with TSS, an intense acute illness that rapidly causes multi organ system failure, create a Toxin called TSST-1. Although classically regarding tampon use,
TSS has also been related to a assortment 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 also the toxin is symptomatically in connection with the staphylococcal enterotoxin gang of toxins (Iandolo. J.J., 1989). (Blomster and Hantamaa D.A. et al., 1986).
d) Detection in the TSST-1 gene
Production of Coagulase, an item of coagulase (coa) gene, may be the principal criteria from the clinical microbiology laboratory for that identification of S.aureus. The coagulase ptn can be an important virulence factor for S.aureus. The coa gene has polymorphic repeat regions that is usually used for differentiating S.aureus isolates. The variable region of coa gene includes 81 bp tandem short sequence repeat (SSR S) (Van Belkum et al., 1998).
Amplification of coa and RFLP analysis of amplified products for that discrimination if S.aureus isolates formerly been reported (Goh et al., 1992; Shopsin et al., 2000; da silva & da silva, 2006).
TSST is really a potentially fatal multi system disease presenting with fever, hypotension, vomiting, diarrhoea, mucosal hyperaemia plus an erythematous rash. This is related to infection of mucosal or sequestered sites by TSST production S.aureus strains usually of bacteriophage group-I.
TSST type-1 (also referred to as enterotoxin type F (or) pyrogenic exotoxin C) is frequently responsible, through enterotoxins B (or) C might also cause the syndrome. TSS Strains produced TSST-1, the toxin accepted because the most likely root cause of TSS (MS Bergdoll et al., 1984). Coagulase negative strain produces TSST-1 and SEA. Since there’s been disagreement about the output 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 that studies. Pus samples were collected from wound-infected persons by utilizing sterile cotton dipped swab and samples collected from in around Namakkal area hospital. After assortment of samples immediately inoculated into peptone water and also the sample were taken to the laboratory within couple of hours.
b) ISOLATION OF Staphylococcus aureus
Loopful of culture from peptone water was streaked around the Mannital salt agar plate. The plates were incubated at 37oC for 24-48 hrs. The isolated colony from selective media was adopted 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 to get a minute, drained and rinsed with water; then Grams iodine solution for starters minute, drained and rinsed with water. Decolourised with ethyl alcohol for 30 Sec and later on counterstained with safranin for starters minute and observed under an oil immersion microscope.
ii) MOTILITY TEST
The hanging drop technique was followed to observe the motility from the organism. Observation is made under the microscope for that darting or cork screw movement with the organism.
iii) CATALASE TEST
A little culture was placed spanning a clean slide. A drop of 3% peroxide was placed within the culture and observed for effervescence. The creation of effervescence showed the ability to provide 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 without the need of colour change.
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 helpful to detect the creation 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, that has been 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 to 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 different sugars from the sugar media together 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 quarter-hour and made slants. Tubes were inoculated with test organisms stabbing on the agar and streaking within the slants. The tubes were incubated at 370c all day and night.
Positive result: Colour on the medium switch the signal from yellow, blackening on the medium, and break inside the medium of lifting in the medium indicates acid production, H2S production and gas production respectively.
C) ANTIBACTERIAL STABILITY TEST
The standard Kirby-Bauer disk diffusion method was adopted to determine the antimicrobial profiles in 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 on 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 in accordance with the recommendation on the disc manufacture.
D) COAGULATE TEST
The tube is packed with 0.5 ml of a single 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 nearly four hours. Positive result’s indicated by gelling from the plasma, which remains available 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 2 ml micro centrifuge tubes.
The tubes were centrifuged at 8000 rpm for 5 minutes.
After centrifugation, the supernatant was discarded and also 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 held 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 ten minutes to separate the phases.
The aqueous phase containing the DNA was carefully removed and moved to new tubes.
Equal number of 100% Isopropanol was included with the tubes containing the aqueous phase.
It was mixed by inverting the tubes three to four times.
The tubes were then centrifuged at 10000 rpm for 10-20 minutes to pellet the DNA.
The supernatant was discarded plus the pellet was collected.
To the pellet, 200μl of 70% ethanol was added and centrifuged at 10000 rpm for 10-20 minutes.
Then Ethanol was decanted completely along with the pellet was air-dried to offer 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 more work.
F) CONFIRMATION OF DNA BY AGAROSE GEL ELECTROPHORESIS:
Agarose gel electrophoresis is performed in a horizontal submarine electrophoresis unit. Thirty ml of just one % Agarose gel was prepared with 1X TBE buffer (don’t mix) and heated the information to get approximately clear solution for casting Agarose gel. After cooling the remedy, 7 µl of staining dye solution was added in the casting system.
The gel was in a position to solidify, and after that carefully disassembled on the casting system without disturbing the wells and put into 1X TBE buffer filled electrophoresis tank (the buffer level ought to be above gel). 5 µl of genomic sample DNA combined with 2 µl of gel loading dye after which loaded to gel and simultaneously loaded 3 µl of DNA marker provided from the nearby well.
The power card terminals was connected at respective positions, run the gel at 50 V, till the gel loading dye migrate over fifty percent the length of gel. Then powered down the unit and visualized the isolated DNA under UV Transilluminator.
I)SAMPLE LOADING DYE:
Sucrose 40% and bromophenol blue (0.25%) were combined with sterile double drinking water.
ii) ETHIDIUM BROMIDE:
Ethidium bromide (10mg) was blended with 10 ml of sterile sanitized water. (It is carcinogenic and must be treated or handled accordingly).
G) POLYMERASE CHAIN REACTION (Johnson & Tyler (1993)
The amplification method was carried by based on the Johnson & Tyler (1993) by modification.The sequences in 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 the Genei, India PCR system while using following procedure. The 20µl includes 2 μl of a single × PCR buffer, 1 mM of each in the primers (0.5 μl), 15 mM of each and every deoxynucleotide triphosphate (1 μl) and 5 U would be 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 afflicted by 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 with the reaction mixtures were analyzed by electrophoresis using a 1% agarose gel, containing ethidium bromide (0.2 mg/ml), in the use of an appropriate DNA molecular weight marker.
In the existing 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 make sure 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 additional studies.
The distribution of isolates in the different sorts of samples for example 5/10 (50%) isolates of burn, 3/4 (75%) from accident, 2/5 (40%) from skin samples
Among these types of samples highest occurrence extracted from accident (75%) and lowest occurrence in skin (40%)
b) COAGULASE TEST
Among the 10 degrees of S.aureus strains 8 (80%) isolates of coagulase negative S.aureus were obtained. There was a distribution of isolates from your different forms of samples for example 4/5 (80%) isolates from burn, 2/3 (66.6%) isolates from accident, 2/2 (100%) isolates from skin infection. Among the types of samples highest occurrence purchased 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) with the isolated Staphylococcus aureus showed sensitive or effectiveness methicillin antibiotic. Out off 10 isolates 8 (80%) was effectiveness Methicillin. Among the three types of samples the biggest resistances were noticed 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 based on the previous study. Among the 10 isolates the three 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 has not been observed.
5. SUMMARY AND CONCLUSION
A total of 19 samples were collected for the study. These samples were of three types namely Skin infection, Burn and Accident wound samples where Staphylococcus aureus was isolated by usage of selective media mannitol salt agar (MSA) and biochemical tests. Out from the 19 samples, 10 isolates were obtained. Among a few types of samples accident samples(75%) had highest prevalence of Staphylococcus aureus as well as burn wound samples (50%) and lastly skin infection samples (40%).
Among the 10 isolates of Staphylococcus aureus, 8 were found being coagulase negative by making use of coagulase make sure were distributed as, skin infection 100%, burn wound 80% and accident wound samples with all the least. (66.6%)
The 8 isolates of coagulase negative Staphylococcus aureus which were removed from above were subsequently tested for antibacterial drug resistance dependant on Kirby – Bauer disk diffusion method. They were all found to be resistant against Methicillin antibiotic.
In another, all isolates of coagulase negative staphylococcus aureus were exposed to the PCR assay based on previous studies completed. Among the 8 isolates, 3 isolates produced the TSST gene. The distribution of toxic gene from skin infection isolates was found to get 100% which of accident wound samples was 33.3% whereas there was clearly no toxic gene from the burn wound samples.