Sample Size Determination
The sample size for this study was calculated based on the equation for calculation of sample size for frequency in a population available at ‘OpenEpi, v.3, open source calculator-SSPropor’ (http://www.openepi.com/SampleSize/SSPropor.htm) last accessed on 3rd July 2018.
Sample size, n = [DEFF*Np (1-p)]/ [(d2/Z21-α/2*(N-1) + p*(1-p)].
Where,
N = Population size, which in our case is 121 frozen S. aureus isolates.
p = hypothesized % frequency of any of the virulence genes in the population (N): 50%+/-5
d = Confidence limits as % of 100 (absolute +/- %): 5% i.e. 95% confidence level.
DEFF = Design effect (for cluster surveys-DEFF): 1
By fitting in Confidence Level values, the sample size was calculated to be 93S. aureus isolates.
Sampling techniques. Consecutive sampling was used. All 121 isolates that had been phenotypically identified as S. aureus under the CHX study were consecutively retrieved from the − 800C freezer, thawed and sub-cultured on 7% sheep blood Agar (BA). Isolates were re-identified as S. aureus based on gram positive coccal morphology, positive catalase test, positive tube coagulase and DNAse tests. Of the 121 frozen isolates, 91 grew upon sub culturing, and 85 of these were re-confirmed as S. aureus based on all the four identification criteria set for this particular study.
PCR detection of S. aureus virulence genes
We conducted a multiplex PCR to determine the proportion of S. aureus possessing virulence factors and methicillin resistant Staphylococcus aureus (MRSA) genes. The PCR testing involved Nucleic acid (DNA) extraction, PCR reagent preparation, DNA amplification, gel electrophoresis and interpretation of results.
Nucleic acid extraction. DNA was extracted using thermo-lysing method. Briefly, 500ul of PCR water was added into sterile eppendorf tubes. A loop full of pure overnight grown culture of S. aureus on nutrient agar plates was emulsified in the PCR water and vortexed to re-suspend and wash off all media salts from the colonies. Tubes with bacterial cell suspension were centrifuge at 1300rpm for 10 minutes. The supernatant was aspirated off and the bacterial cell pellet re-suspended in 200ul of PCR water and this bacterial cell suspension was incubated at 1000C on a heat block for 30min. Tubes with bacterial lysates were cooled to room temperature and centrifuged at 1300rpm for 10 minutes. 50ul of the supernatant containing the extracted DNA was transferred into a new eppendorf tube and immediately used for PCR reactions or frozen at minus 80o until needed for PCR.
PCR reagent preparation: From the pre -amplification room, PCR reactions were prepared in a total volume of 35 µl consisting of the following reaction component 25ul of 2X taq PCR master mix (Qiagen cat# 1067520), 1µl (100ng/ul) of each of the forward/reverse primes (IDT) and 5ul of PCR water (Qiagen). The PCR reaction tubes were then transferred to the DNA extraction room and 2µl of extracted DNA added. The multiplex PCR-testing primers for the various genes was performed as follows: Set A: sea, seb, sec, sed and see genes; Set B: mecA, eta, etb and tst genes; Set C: hla, and hld genes; and Set D: 16S rRNA, pvl, mecA, and femA genes as detailed in table 1.
Amplification
The PCR tubes were loaded into the Gene Amp PCR system 9700 (Applied Biosystems, Inc. Forster City, CA). The PCR reaction tubes were incubated at 940C for 5 minutes followed by 37 cycles of denaturation at 940C for 45 seconds, annealing at 570C for 2 minutes and extension at 720C for 1 minute. The PCR reactions tubes were finally incubated at 720C for 10 minutes and the PCR products stored at 40C until agarose gel electrophoresis.
Agarose Gel Electrophoresis
A 2% agarose was prepared by weighing 2.0g of agarose powder in 100 mL of 1x Sodium Borate buffer (SB). The mixture was boiled in microwave oven for 5 minutes to allow thorough heating and mixing of the powder. The mixture was allowed to cool to 50ºC before adding Ethidium Bromide (EthBr). The dissolved agarose solution was then poured into an assembled gel tray with combs attached and allowed to set at room temperature for approximately one hour. Upon setting, the gel was placed into the electrophoretic tank and the combs vertically removed. 1ul of the loading dye was added into each PCR tube with amplicon, mixed well and then 10 ul loaded into the wells. For each electrophoretic run, a 1kb DNA ladder was included as molecular weight marker. DNA was electrophoresed at 120 Volts for 30 minutes. The gel was carefully transferred to a UV Trans-illuminator for visualization. Examples of the gel images for the different genes are shown in Fig. 1.
Quality Control Testing. For the phenotypic re-identification of S. aureus, a known S. aureus was used as positive control and known S. epidermidis used as a negative control. Each PCR batch had positive and negative controls for some of the genes under study as well as PCR water to check for reagent contamination possibilities. These positive controls were laboratory cocktails containing some of the genes under study as well and S. aureus 16s rRNA genes.
Data analysis. All study data was entered in Ms Excel 2013 and analyzed using SPSS v.20.
Table 1. Target gene, reagents and primer sequences for PCR Sets A, B, and C.