Antibiotics Susceptibility Pattern of Methicillin Resistant Staphylococcus aureus (MRSA) In Enugu State, South-East Region of Nigeria

Development of antimicrobial resistance by bacteria is now a worldwide health issue, as infection is one of the leading causes of death in the world today. The aim of this study was to evaluate the prevalence and antimicrobials susceptibility pattern of Methicilin-Resistant Staphylococcus aureus in 3 different hospitals in South-East geopolitical region of Nigeria. The identification and confirmation of the S. aureus were done using selective and differential medium (Mannitol salt agar) for S. aureus and by coagulase/staphylase test using Oxoid ® reagents kits (DR0595A). The method used for antibiotics susceptibility pattern of the characterised S. aureus isolates was discs diffusion method, as recommended by the Clinical Laboratory Standards Institute (CLSI), discs containing oxacillin (5 μg/disc), vancomycin (30 μg/disc), cephalexin (30 μg/disc), levofloxacin (5 μg/disc), ciprofloxacin (5 μg/disc), tetracycline (30 μg/disc), cotrimoxazole (25 μg/disc), gentamicin Original Research Article Agboke and Attama; BMRJ, 10(1): 1-7, 2015; Article no.BMRJ.18219 2 (30 μg/disc), clindamycin (2 μg/disc) and rifampicin (5 μg/disc). MRSA confirmation was done using Oxoid ® DR0900 penicillin binding protein (pbp2 ’ ) latex agglutination test kits. The results showed that out of 218 characterized clinical isolates, 39 of it were confirmed MRSA with varying percentages of resistance to various antibiotics thus: oxacillin (62.07%), vancomycin (60.35%), cephalexin (55.18%), levofloxacin (56.90%), ciprofloxacin (65.52%), tetracycline (68.97%), cotrimoxazole (67.25%), gentamicin (62.07%), clindamycin (63.79%) and rifampicin (62.07%). The S. aureus are more sensitive to Levofloxacin and less sensitive to tetracycline, clindamycin and rimfapicin. Latex agglutination test confirmed 39 strains of the clinical isolates to be MRSA. The results shows open wound as a source with highest prevalence and sputum with lowest prevalence of the MRSA with no significant change (P > 0.05).


Methicillin-Resistant Staphylococcus aureus (MRSA)
Staphylococcus aureus is responsible for a broad range of clinical infections, most notable of which are cases of bacteremia and endocarditis [1]. Staphylococcus aureus is an important cause of serious infections in both hospitals and the community. Methicillin-resistant Staphylococcus aureus (MRSA) were first reported in 1961 and the first hospital outbreak of MRSA was reported in 1963 [2]. When MRSA strains first appeared, they occurred predominantly in the healthcare setting. Cases of community-associated MRSA (CA-MRSA) infections were first reported in the late 1980s and early 1990s [3]. Health careassociated MRSA (HA-MRSA) is particularly efficient at developing resistance to antimicrobial agents. Methicillin resistance among staphylococci has steadily increased worldwide, especially among cases acquired in hospitals. It is associated with longer hospital stay and more infections in intensive care units and leads to more antibiotic administration [1]. Asymptomatically colonized patients and health care workers are the major sources of Methicillin-Resistant Staphylococcus aureus (MRSA) in the hospital environment [3].
MRSA-infected patients in burns units are particularly problematic because the big surface area of denuded skin can produce a large inoculum of organisms that can be easily transmitted to other patients via the hands of health care workers. Extensive skin lesions also result in heavy shedders of MRSA [2]. The commonest site of MRSA carriage is the anterior nares. A significant risk factor for acquisition of MRSA is the duration of hospital stay. Prolonged stay in the hospital is likely with patients in orthopedics and dermatology wards, which may result in high rates of carriage observed in these patients [3].
HACO (Health care associated MRSA with a community onset) refers to community onset for a person associated with a hospital environment, e.g., a person living in a residential home, a health care worker, a dialysis patient, or an individual with a history of hospitalization within the previous year [4,5]. Risk factors associated with MRSA bacteremia include the following: residence in an extended care facility, prior antibiotic exposure, insulin dependent diabetes, prolonged hospitalization, urinary catheterization, nasogastric tube placement, prior surgery, and having an underlying disease [6]. The elderly population (≥ 65 years old) is at a significantly higher risk of death due to MRSA bacteremia than are younger populations.
The accurate and early determination of methicillin resistance is of key importance in the prognosis of infections caused by S. aureus. Strains that possess mecA gene are either heterogeneous or homogeneous in their expression of resistance [7,8].
The study aims to evaluate the prevalence and antimicrobials susceptibility pattern of Methicilin-Resistant Staphylococcus aureus in 3 different hospitals in South-East geopolitical region of Nigeria.

Specimens Collection
This study was conducted from September 2012 to June 2013 at 3 different hospitals in the South-

Characterisation of Clinical Isolates
The characterisation of the 2,372 isolates was done in sequence to eliminate the unwanted isolates and to confirm the S. aureus isolates. The viable clinical isolates used for this research was 218 isolates. The clinical isolates were characterised as cocci and non-cocci isolates by cultural and microscopic characters with gram staining reaction.
The catalase reaction test, mannitol salt agar test and slide coagulase test confirmed 58 (26.6%) clinical isolates to be S. aureus. The isolates were distributed as follows, 7 from sputum, 10 from skin swab, 11 from abscess, 19 from open wound and 11 from ear/nasal swab.
A direct colony method by CLSI [9] was used, pure isolated colonies were taken from Mueller Hinton agar into sterile broth tubes and a suspension equivalent to a 0.5 McFarland standard was prepared and used to inoculate the test media. The discs were applied to the plate within 15 minutes of inoculation and incubated for 24 hours at 35°C.

Penicilin-binding Protein (PBP2 ʹ`) Latex Agglutination Test for MRSA Confirmation
The  Table 1 shows the antibiotic sensitivity and resistance pattern of Staphylococcus aureus to various antibiotics, 67.24% (39/58) of the S. aureus isolates shows resistance in varying degree to the antibiotics (MRSA), while 32.75% (19/58) of the isolates were sensitive to all the antibiotics (MSSA). The highest frequency of sensitivity of the isolates to the antibiotics was observed with cephalexin 44.82% (26/58) followed by levofloxacin 43.10% (25/58). The least was observed with tetracycline 31% (18/58). However, there was no significant difference (p > 0.05) in susceptibility between cephalexin and levofloxacin and also in the resistance pattern. There was significant difference (p < 0.05) among the various antimicrobials resistance, with tetracycline and cotrimoxazole as the most resisted antibiotics by the S. aureus isolates.

Penicillin-binding Protein (PBP2 Latex Agglutination Test Results
The 39 clinical isolates of S. aureus percentages of resistance to the 10 antibiotics used for the susceptibility study were subjected to PBP2 1 latex agglutination test and result gave different types of resistance strains by latex agglutination test into VISA and MRSA. The results confirmed 39 strains of the isolates as MRSA, while some are suspected to be VISA as shown in Table 2.

Specimen Prevalence of Clinical Isolates of MSSA and MRSA
The prevalence of the S. aureus isolates of the different specimen are presented in Table 3. The

Specimen Prevalence of Clinical Isolates of MSSA and MRSA
isolates of the in Table 3. The   table shows the number of staphylase

DISCUSSION
A study at Ilorin [8], Nigeria reported wound infections of 38 % as the highest frequency of S. aureus isolates. This agrees with the result of the present study where the order of the Coagulase positive S. aureus, MSSA and MRSA prevalence is as follow: Open wound 19 (32.75%), greater than abscess 11 (18.96%), greater than Ear/Nasal swab 11 (18.96%), greater than Skin swab 10 (17.24%), greater than Sputum 7 (12.06%) except the little difference between the abscess and ear/nasal in the staphylase positive S. aureus and MRSA prevalence.
The association of MRSA with therapeutic challenges, complications, deaths and cost related to longer hospital stay compared with MSSA has been widely documented [10,11]. More importantly, the multidrug resistant hospital-acquired MRSA (HA-MRSA) strains and their intrinsic resistance to beta-lactam antibiotics made treatment difficult using the most available and less costly antibiotics in developing countries [11]. In agreement with the previous study by (Olowo O. A) [12] open wound has the highest prevalence of S.aureus and MRSA.

Antibiotics Susceptibility Pattern of the Clinical Isolates
The susceptibility and resistance pattern of the 58 clinical isolates S. aureus were as seen in the Table 1. Susceptibility test profile revealed a high level of resistance amongst the S. aureus (39 isolates) to all the commonly used antimicrobials, ciprofloxacin, gentamicin, clindamycin, levfloxacin, cephalexcin, tetracycline, rifampicin cotrimoxazole, oxacillin and vancomycin. The results were comparable to those in a previous study carried out by Nwankwo and Nasiru [13], but not as high as in this study. Vancomycin was the drug of choice for MRSA infections for many years, until the first S. aureus isolate with reduced sensitivity to vancomycin (vancomycin intermediate Staphylococcus aureus (VISA) was reported in Japan in 1997 [14].
The susceptibility and resistance pattern of these clinical isolates in oxacillin and vancomycin was not different, almost no significance difference (P > 0.05). Hence, most of the MRSA among these isolates were also VISA and VIRA as it is revealed in Table 2. It was discouraging to note that vancomycin resistance was highly observed among the isolates.
In this study levofloxacin and cephalexcin are the least resisted antibiotics, while tetracycline and cotrimoxazole are the highest resisted antibiotics [12,13].

CONCLUSION
In this study the highest prevalence of S. aureus isolates and MRSA strains were detected in wound samples. The S. aureus isolates were resistant to tetracycline (68.97%) and cotrimoxazole (67.25%), and sensitive to levofloxacin (43.10%) and cephalexin (44.82%)." The MRSA strains resisted all the antimicrobials in varying percentages including vancomycin (60.35%), levofloxacin (56.90%) and cephalexin (55.18%) respectively. This shows that the MRSA isolates are multiple drug resistant strains and a threat to public health and healthcare workers [15].