Prevalence of mecA gene among staphylococci from clinical samples of a tertiary hospital in Benin City, Nigeria

Background The staphylococci have increasingly been associated with infections worldwide and anti-microbial resistance has made these versatile pathogens more recalcitrant in the hospital setting. Objectives This study sought to investigate the occurrence and distribution of Staphylococcus species as well as determine the prevalence of methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase negative staphylococci (MRCoNS) among clinical samples from University of Benin Teaching Hospital (UBTH) in Benin City. Methods Ninety one (91) clinical isolates comprising S. aureus and Coagulase Negative staphylococci (CoNS) were recovered from routine clinical specimens and anti-microbial susceptibility tests were carried out. Polymerase Chain Reaction (PCR) was thereafter carried out on these isolates to detect mecA gene. Results Staphylococcus species had its highest prevalence from infected wounds of patients (28.8%) while urine samples showed the least (5.4%). The highest level of resistance was to ceftazidime (S. aureus - 68%, CoNS - 75.6%) while the least resistance was observed for meropenem (S. aureus- 26%, CoNS- 46.3%). Using phenotypic method (with 1µg oxacillin antibiotic disc), the distribution of MRSA and MRCoNS was 44.0% and 46.3% respectively. PCR analysis showed that 38.0% of S. aureus and 41.5% of the CoNS had mecA gene respectively; wound swabs showed the highest prevalence with 30.5% of staphylococcal isolates being mecA gene positive. There was also no significant association between the Staphylococcal isolates and their isolation rate, isolation site and mecA gene distribution (p > 0.05). Conclusion This study draws attention on the increase in the prevalence of mecA gene (39.6%) and an increase in multidrug resistant staphylococci when compared to previous studies in our country; it recommends laboratory guidance and periodic review to stem the tide of resistance.


Introduction
Staphylococcus aureus is a non-motile, aerobic or facultative anaerobic Gram positive coccus that inhabits the nasal membranes and skin of warm blooded animals and man, in whom it could cause a range of infections from mild, such as skin infections and food poisoning, to life threatening, such as pneumonia, sepsis, osteomyelitis and infectious endocarditis 1 . The CoNS are part of the normal flora of human skin 2 , these organisms have relatively low virulence but are increasingly recognized as agents of clinically significant infection of the bloodstream and other sites 3 . Risk factors for CoNS infection include foreign bodies (such as indwelling prosthetic devices or intravascular catheters) and immune compromise 3 . The introduction of methicillin in 1960 as an alternative antibiotic for the treatment of beta-lactamase (penicillinase) producing S. aureus was greeted with resistance al-African Health Sciences Vol 17 Issue 4, December, 2017 most immediately 4,5 . Since that time, strains of S. aureus resistant to methicillin emerged and became a major clinical problem within hospitals in Europe in the 1960s 6 , in the United States in the 1970s 7 , and in several countries of the world 8 . Resistance to methicillin and semisynthetic penicillins has been observed in more than 80% of coagulase-negative Staphylococcal isolates 9 . MRSAs and MRCoNS have therefore emerged as the most important cause of hospital-acquired infections (HAI) and community-acquired infections (CAI), resulting in increased morbidity and mortality in the hospital settings 10,11 .
The specific genetic mechanism of its resistance in MR-SAs has been identified as a mobile genetic element (staphylococcal cassette chromosome mec) integrated into the S. aureus chromosome, within which the mecA gene encodes a specific methicillin-resistant transpeptidase known as penicillin-binding protein 2a (PBP2a) 12 . This protein has a low affinity for beta-lactam antimicrobial drugs, thus bacteria expressing this protein are resistant to all types of these drugs 13 , and in recent times; aminoglycosides and quinolones 13 . In Nigeria, the occurrence of MRSA was first documented by researchers in 1987 with its prevalence pegged at 50.6% 14 . Subsequent studies have seen a variation in its prevalence 15,16,17 . Literature is growing on CoNS and its incrimination in clinical infections in Nigeria 18,19 ; several factors affect the reliability of the disk diffusion technique for the detection of methicilin resistance among the staphylococci 17,20 . Though molecular methods have emerged as the gold standard, there is still little data on the distribution of MRSA and MRCoNS in our locality. This study therefore aims at evaluating the prevalence and distribution of MRSAs and MRCoNS among staphylococci isolated from clinical samples by determining the distribution of mecA gene among these organisms in Benin City using PCR based method.

Isolation and identification
The isolates were identified to be S. aureus following Gram stain that showed Gram positive cocci and biochemical tests which showed positive results for catalase and coagulase (slide and/or tube) as described in standard Medical Microbiology laboratory manual 21 . Similarly isolates identified as CoNS were Gram positive cocci and catalase positive but were negative for both slide and tube coagulase tests. The isolates were thereafter stored at 4°C on Mueller Hinton agar slants for further work.

Antimicrobial susceptibility testing:
In vitro anti-bacterial susceptibility tests were performed on each isolate using anti-bacterial drugs. Plates were prepared with Mueller Hinton's agar for use in the Kirby-Bauer method. Broth cultures were adjusted to turbidity standard, equivalent to McFarland 0.5. This was then used for disc diffusion according to Clinical and Laboratory Standard Institute (CLSI) criteria, using the following discs; gentamicin (10 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), amoxicillin-clavulanate (30 µg), cefuroxime (30 µg), ceftriaxone (30 µg) ceftazidime (30 µg), cloxacillin (5 µg), meropenem (10 µg) (all from Abtek Biologicals Ltd,Liverpool, U.K). These were then incubated at 37˚C for 18 hrs. Sensitivity pattern was determined by measuring the zones of inhibition with a calibrated ruler and comparing with the control organism. Interpretative criteria for susceptibility tests were according to CLSI 22 . An oxacillin disc (1 µg) was used to detect methicillin resistance. Sensitivity was read after incubation for 24 hrs at 35° C. Isolates were regarded as sensitive or resistant according to CLSI criteria 23 . Multiple antibiotic resistance (MAR) index was calculated for each isolate using the formular; Chromosomal DNA Extraction: DNA extraction was carried out as previously described 24 ; Isolates were harvested into 1.0 ml of sterile water, vortexed to mix, and centrifuged at 10,000 r.p.m for 5 min. The supernatant was discarded and the pellets were washed again with sterile water. After this, 200µl of sterile water was added to the pellets, the pellets were vortexed to homogenize, and boiled in a dry bath at 100°C for 10 minutes. This was followed by vortexing and centrifugation at 12,000 r.p.m for 5 min. The supernatant containing the DNA were transferred to another tube and stored at -20°C. The concentration and purity of the extracted DNA was estimated using a nanodrop spectrophotometer.
PCR amplification of mecA gene. The mecA gene was amplified using the primer set me-cA1(AAAATCGATGGTAAAGGTTGGC) and mecA2 (5'AGTTCTGCAGTACCGGATTTTGC3') as described by Del Vechio et al (1995) 25 . PCR was performed in a 20 µl of a reaction mixture containing 1X PCR Buffer (Solis Biodyne), 1.5 mM MgCl 2 , 200 µM of each dNTP (Solis Biodyne), 20 pMol of each primer, 2.5 units of TaqD-NA polymerase (Solis Biodyne),10-200 ng of extracted DNA, and sterile distilled water was used to make up the reaction mixture. Thermal cycling was conducted in an Eppendorf Thermal Cycler Nexus series for an initial denaturation of 95°C for 5 min, followed by 30 consecutive cycles of 95ºC for 30 sec; 55ºC for 30 sec, and 72ºC for 1 min. This was followed by a final extension step of 72ºC for 10 min. The amplification product was separated on 1.5% agarose gel electrophoresis, visualized by ethidium bromide staining and photographed under Ultraviolet il-lumination. 100bp DNA ladder (Solis Biodyne) was used as DNA molecular weight standard. A single band with a molecular weight of 533bp signifies the presence of the mecA gene. S. aureus ATCC 43300 served as the positive control strain.

Calculation of sensitivity and specificity
Calculations of sensitivity were made by dividing the number of strains detected as resistant by the susceptibility test method by the total number of strains that were mecA positive. Specificity was calculated by dividing the number of strains detected as susceptible by the test method by the total number of strains that were mecA negative.

Statistical analysis
Statistical analysis was by the Chi (X 2 ) square test using INSTAT® software. A p value of < 0.05 was deemed statistically significant.

Result
A total of 91 clinical isolates of Staphylococcus species (S. aureus -50 (54.9%), CoNS -41 (45.1%)) were recovered from clinical samples during the period of study. Although the isolation rate of Staphylococcus species was higher from males (52.7%), gender was however not a risk factor for Staphylococcal infection (X 2 = 0.2637, P > 0.05). There was no significant association in the occurrence of Staphylococci and the isolation sites (X 2 = 0.5481, P> 0.05). The isolation rate of Staphylococcus species from clinical samples was highest from infected wounds of patients (28.8%) while urine samples showed the least isolation rate (5.4%) as seen in Table 1. The anti-microbial susceptibility pattern showed varying degrees of resistance. The highest level of resistance was toceftazidime (S. aureus -68%, CoNS -75.6%). The anti-biotic with the least resistance was meropenem (S. aureus-26%, CoNS-46.3%). The antibiogram is well detailed in Table 2.
Using phenotypic method (with 1µg oxacillin antibiotic disc), the distribution of MRSA and MRCoNS was 44.0% and 46.3% respectively. Multiple antibiotic resistance (MAR) index was calculated for each of the isolates. It was observed that majority of isolates had MAR indi-ces of above 0.2 (S. aureus -60%, CoNS -80.5%). PCR was carried out on all staphylococcal isolates to detect mecA gene, a gene that confers resistance to methicillin and most β-lactam antibiotics. Figure 1 shows the Agarose gel electrophoresis of PCR product amplified from mecA genes for S. aureus and CoNS.   On the whole, PCR analysis showed that 39.6% of Staphylococcus species recovered was mecA gene positive with 38.0% of S. aureus and 41.5% of the CoNS having mecA gene respectively. mecA gene was markedly distributed in wound swabs (30.5%) and was absent in high vaginal swabs and catheter tips (Table 4). However, there was no significant association between isolation site and the distribution of mecA gene among S. aureus (X 2 = 0.7735, P > 0.05), and CoNS (X 2 = 0.4259, P > 0.05). Seventeen (17)

Discussion
In this study, 28.8% of Staphylococci isolated were from wound swabs. In a study which had Staphylococcal isolates recovered from eight hospitals which cut across South-Western, North-central and North-Eastern Nigeria, more than 80% of the total number of S. aureus isolates recovered was from infected wounds 13 . It has been observed that the skin of 80-90% of people is colonized with S. epidermidis and that most CoNS infections are acquired from patients own flora 26 . The CoNS have also shown a higher frequency of isolation from wound swabs when compared to other samples 18 . In Nigeria, CoNS is one of the common causes of infections of open fractures in wounds and delay in wound debridement has been reported to be a major predisposing factor to wound infection 28 . Similarly, S. aureus is found on the skin, axilla, anterior nares and groins as normal flora 27 . It is therefore not hard to imagine that it would be implicated in wound infection as there is a breach in the structural integrity of the skin.
Resistance was observed to oxacillin in this study, 44.0% and 46.3% for S. aureus and the CoNS respectively. This is in contrast to an Iranian study, where resistance was 88% and 60.3% for S. aureus and CoNS respectively 29 . Oxacillin and methicilin are hardly used in our setting as compared with the Iranian study which noted the rampant use of oxacillin. Our report is however similar to observations by Olowe et al in Osogbo, Nigeria, where 40.4% of S. aureus clinical isolates were resistant to oxacillin. Similarly, demonstrable levels of resistance was observed for meropenem (S. aureus-26%, CoNS-46.3%), though the drug is expensive and one of last resort in our locality 30 .
The study also shows that majority of staphylococcal isolates were multidrug resistant, with MAR indices > 0.2 (S. aureus -60%, CoNS -80.5%). Worthy of note is the high prevalence of multidrug resistant CoNS. The finding is similar to an observation by Akinjogunla and Enabulele (2010). However, in their study on Staphylococcal isolates from ear swabs of patients with otitis media, they found that 19.2% of S. aureus and 9.2% of CoNS had MAR indices above 0.8 31 . An MAR index higher than 0.2 has been said to be indicative of isolates originating from an environment where antibiotics were often used 32 , the high indexes from this study therefore give credence to the observation of many researchers in Nigeria on the prevailing practice of indiscriminate use of antibiotics and lack of Laboratory guidance before institution of antimicrobial therapy 16,17 , as they may have induced resistance mechanisms over time in these versatile opportunists.
Using PCR method, 38.0% of S. aureus isolates and 41.5% of the CoNS had mecA gene respectively. Using phenotypic method (with oxacillin antibiotic disc) however, the distribution of MRSA and MRCoNS was 44.0% and 46.3% respectively. Differing rates in susceptibility pattern has been observed by researchers. Olowe et al in drawing comparisons between methicillin, oxacillin, cefoxitin and the gold standard PCR, observed that all the antibiotics mentioned slightly overestimated methicillin resistance in S. aureus 30 . Hyperproduction of β-lactamase enzyme has been observed among mecA negative MR-SAs in Nigeria 35 . Besides the challenge of heterogeneity of the strains, this could be a resistance mechanism for oxacillin-resistant mecA gene negative Staphylococci as observed in this study, more research is therefore needed.
There was no significant association between the distribution of Staphylococci and the distribution of mecA gene among clinical samples in this study (P > 0.05), as all samples showed some percentage distribution of the gene with the exception of catheter tips and high vaginal swabs. mecA gene was markedly distributed among the staphylococci recovered from wound swabs in this study (S. aureus-30.7%, CoNS-58.3%). This is comparable to a study in Osogbo (S. aureus-52.2%) 30 , and two multi-centre studies evaluating the prevalence of mecA gene among the staphylococci from clinical samples 13,17 . Noteworthy in this study is the rising prevalence of MRSA and MRCoNS from blood of patients showing signs of septicaemia, as 28.5% of S. aureus and 40.0% of the CoNS were mecA positive. This is in sharp contrast to some studies in Nigeria in which all S. aureus isolated from the blood of patients showing similar symptoms were mecA gene negative 17,30 , however, the gene was detected among CoNS notably S. haemolyticusca having septicemia in Lagos, Nigeria 13 . Some researchers have observed the rising prevalence of MRCoNS from patients with septicaemia in India; 12% and 54% respectively 33,34 . In our country, little attention has been accorded the CoNS to the extent of screening for mecA gene among them. Literature is however growing on its pathogenicity and increased isolation from clinical infections 18,19 . The marked distribution of mecA gene among these isolates (41.5%), as well as the insignificant difference in their isolation rate and distribution of mecA gene when compared with S. aureus recovered from clinical samples is a pointer to the fact that they should be accorded equal status as opportunistic pathogens (P > 0.05).

Conclusion
Summarily, this study notes a rising percentage in the distribution of mecA gene among the Staphylococci recovered from clinical samples in Benin when compared with previous studies in our country, it reports increasing level of multidrug resistant staphylococci with high MAR index, it reiterates the continued practice of indiscriminate use of antibiotics and emphasizes laboratory guidance before institution of anti-microbial therapy. Efforts should also be made to enact regulations on antibiotic usage. Periodic review of susceptibility pattern, molecular epidemiological surveys and surveillance is equally imperative. These when implemented promises to stem the tide of anti-mi crobial resistance.