Isolation and molecular identification of methicillin resistant staphylococcus aureus (MRSA) from different type of wound (Cuts, burn and chemical)

Specific objectives of the study

Study area

The study was conducted in Murtala Muhammad Specialists Hospital (MMSH), Muhammad Abdullahi Wase Road, Kano municipal, Kano State. It was the largest state government hospital in northern Nigeria and has the highest patients’ attendance in the region. The hospital was commissioned in 1926, presently with an official bed capacity of 826 and has total number of 30 wards and units and 9 operating theatre and 14 clinics. It was also has the staff strength of 1656 and it is an NHIS accredited hospital. An ethical approval was obtained from Murtala Muhammad Specialist Hospital (MMSH), Kano state based on the consent of the Hospital Ethical Committee, under the consideration of Ministry of Health Kano.

Sample size

The sample size was calculated using 28% prevalence of MRSA in Kano. 3 The following formula was used to calculate the sample size as described by. 4

N = Z² P (1-P) / d

N=300

Inclusion criteria

All consented patients admitted in the surgical ward with cuts, burn and chemical wound for at least 14 days were included in the study.

Collection of clinical wound samples

Samples (Burn, cuts and chemical wound) were collected among the inpatients in Surgery Departmen at the 14 days interval of collection in Murtala Muhammad Specialist Hospital (MMSH), Kano State using swab technique as described by. 5

Isolation and Identification of staphylococcus aureus

Five milliter of prepared nutrient broth was added to test tubes and sterilized by autoclaved at 121⁰C for 15 minutes then allowed to cool. Swabbed sample was inoculated into sterilized 5ml of nutrient broth and uninoculated test tube was served as control. The test tubes were then incubated at 37⁰C for 24 hours. After the incubation, sterile swab stick was dipped into each test tube with reference turbidity and swabbed gently on to the prepared nutrient agar (NA) plate and incubated at 37⁰ for 24 hours. After 24hours of incubation, the examined colonies on a nutrient agar (NA) plate was then picked and inoculated on to the prepared mannitol salt agar (MSA) plate using sterile wire loop and incubated at 37⁰ for 24-48hours. Typical Staphylococcus colonies were examined after two days of the incubation. 6, 7

Gram stain

Gram stain was achieved through the standard procedure as described by 8

Standardization of the bacterial inoculums

A loopful of bacterial colony of the test isolates was picked and inoculated into test tube containing 5ml of distilled water using sterile wire loop, turbidity was adjusted to equivalent 0.5 McFarland standards.

Detection of methicillin-resistant staphylococcus aureus using cefoxitin disc diffusion

All Staphylococcus aureus isolates were subjected to cefoxitin disc diffusion test using 30ug of cefoxitin (Oxoid CT0019B). Prepared Mueller-Hinton agar plates were swabbed with standardized bacterial inoculums. Thirty microgram of cefoxitin disc were placed on the centre of MHA plates using sterile forceps and incubated at 37 ⁰C for 24 hours. Positive methicillin resistant strain of Staphylococcus aureus and methicillin susceptible strain of Staphylococcus aureus were served as positive and negative plates control respectively obtained from Murtala Muhammad specialist hospital, Kano. Zone of diameter inhibition was measured. 13, 14

DNA extraction

Pure colonies of Staphylococcus aureus from those that were cefoxitin resistance were cultured in nutrient broth for 24 hours at 37^oC with frequent agitation using micro centrifuge at 100rpm for 5minutes. Cell suspension were transferred into another test tube and centrifuged at 4500rpm for 5minutes. DNA was extracted using boiling lysis method, in which 1g of cell pellets were resuspended in 40ul of water and boiled at 100⁰C in water bath for 10minutes. After it was cooled on ice, and then centrifuged at 15000 x g for 10seconds. Cold 95% of ethanol was added to the supernatant fluid and stored at -20⁰C until polymerases chain reaction assay (PCR). 15

Polymerase chain reaction (PCR) assay

PCR mixture was prepared in thin walled 0.2ml tube, which contains 50ul of distilled water; Buffer 1x, MgCl₂ 0.5mM, Taq polymerase 0.05Units/ul, dNTP 200uM and mecA primer 0.5uM (forward) and 0.5uM (Reverse). 10ul of DNA sample was added to 50ul of PCR mixture, the mixture was placed inside the Bio-Rad thermal cycler for PCR amplification. Initial denaturation was carried out at 92⁰C for 30minutes followed denaturation at 92⁰C for 10minutes, annealing at 56⁰C for 10minutes and extension at 72⁰C for 10minutes. The final extension was performed at 72⁰c for 30minutes; the amplified product was detected by 1% of agarose gel electrophoresis with (0.5ug/ml) ethidium bromide staining and was observed using Ultraviolet light. 16

Occurrences of Methicillin-resistant Staphylococcus aureus (MRSA) from different type of wound

Occurrence of S.aureus and MRSA were presented in table 1 below; out of 100 samples from each sites of wound (cuts, burn and chemical) has positive isolates of 31(3), 56(8) and 24(2) respectively.

Percentage occurrences’ of Methicillin-resistant Staphylococcus aureus (MRSA)

Percentage occurrence of MRSA was presented in table 2 below, in which out of the 101 total number of Staphylococcus aureus isolates, 13(12.90%) isolates were positive MRSA and 88(87.10%) were negative MRSA.