CHARACTERIZATION OF METHICILLIN – RESISTANT STAPHYLOCOCCUS AUREUS FROM ORTHOPAEDIC PATIENTS IN AHMADU BELLO UNIVERSITY TEACHING HOSPITAL, ZARIA, NIGERIA

ABSTRACT

Methicillin resistant Staphylococcus aureus (MRSA) is now a threat to both the hospitalized patients and community globally. This work was aimed at detecting molecularly, methicillin resistant Staphylococcus aureus from the orthopaedic patients in Ahmadu Bello University Teaching Hospital, Zaria, Nigeria. Conventional biochemical methods were used to identify the isolates while API STAPH identification test kit further characterized the isolates to species level. The susceptibility test was carried out using disc agar diffusion method while beta – lactamase production was tested for using nitrocefin. Methicillin resistance was detected phenotypically using cefoxitin 30 µg disc and oxacillin agar screen test. Multiplex polymerase chain reaction (PCR) was used to detect mecA gene, the gene coding methicillin resistance and blaZ gene, the gene coding for beta- lactamase production with 16SrRNA gene being the internal control. Sequencing was carried out for the amplified isolates. A total number of 126 samples were collected from wound, skin and bed of orthopaedic patients for 5 months. With the conventional biochemical method of identification, 100(79.4%) isolates were identified as S. aureus while with the use of API STAPH identification test kit 39(39%) of the 100 were characterized as S. aureus. The susceptibility test of the S. aureus isolates showed that gentamicin had the greatest activity: 100%, 100% and 93.8% in wound, bed and skin respectively, followed by ciprofloxacin (100%, 94.1% and 93.8%) and pefloxacin (100%, 88.2% and 75%) respectively. However, the greatest level of resistance was observed with ampicillin: 100%, 100% and 87.5% in wound, bed and skin respectively followed by ceftriaxone: 100%, 76.5% and 75%; and amoxicillin – clavulanate: 66.7%, 58.8%, 56.2%. Phenotypic detection of MRSA with the use of cefoxitin disc diffusion gave a MRSA prevalence of 83.3%, 64.5% and 56.3% from wound, bed and skin respectively. These MRSA isolates were generally resistant to the beta lactam antibiotics used, while 11/25 (44%) were multi-drug resistant. However, vancomycin, gentamicin and ciprofloxacin were most active against the MRSA isolates, 15% of these phenotypic MRSA isolates were hyper-producers of beta-lactamase. The gold standard for detecting MRSA using polymerase chain reaction is detection of mecA gene and only 2 (5.1%) S. aureus isolates were positive. Fifteen (78.9%) of the phenotypic MRSA tested carried plasmid with molecular weight ranging from 9.23 to 13.27 kilobase pairs. The presence of plasmid and hyper- production of beta-lactamase can be suggested to be responsible for the phenotypic detection of MRSA observed in this study; 33.3% of the S. aureus isolates amplified blaZ gene. The nucleotide sequence of 16SrRNA gene of isolate S41 in comparison with those from GenBank database showed that the S. aureus isolate has 99% identity with Staphylococcus aureus strain KIBGE-MB01 with sequence ID (accession) number HM061132.1.

CHAPTER ONE

INTRODUCTION

1.0 INTRODUCTION

Staphylococcus aureus is commonly carried on the skin or in the nose of healthy individuals. It is an important pathogen in human infections causing illness ranging from minor skin infections and abscesses to life – threatening diseases such as pneumonia, meningitis, endocarditis, toxic shock syndrome and septicaemia which may be rapidly fatal (Holmes et al., 2005).

Bacterial resistance to antibiotics has been recognized since the first drugs were introduced for clinical use. Penicillin was first introduced in 1941, when less than 1% of Staphylococcus aureus strains were resistant to its action. By 1947, 38% of hospital strains had acquired resistance and currently over 90% of Staphylococcus aureus isolates are resistant to penicillin. Increasing resistance to antibiotics is a consequence of selective pressure (Power, 1998).

Methicillin was the first penicillinase – resistant semisynthetic penicillin to be derived from the penicillin nucleus, 6- aminopenicillanic acid (6-APA) (Figure 1) (Knudsen and Rolinson, 1960). Initially, it was used widely, but because of its toxicity it was gradually replaced with other penicillinase-resistant penicillins such as nafallin, oxacillin etc.

Figure 1: Structure of Methicillin

Ever since the beginning of the use of antibiotics, bacteria have become very adept at becoming resistant to different antibiotics. Methicillin- resistant S. aureus (MRSA) was first discovered in 1961; they are isolates of S. aureus which have acquired genes encoding antibiotic resistance to all penicillins including methicillin and other narrow spectrum β lactamase resistant penicillin antibiotics. Since then hospitals worldwide have reported varying proportion of MRSA among S. aureus isolates (Foster, 1996). Thus MRSA has become a real clinical and therapeutic problem.

MRSA infections can be classified into two major groups: Hospital-acquired MRSA (HA-MRSA) and Community-acquired MRSA (CA-MRSA). HA-MRSA is responsible for post-operative wound infections, or infections resulting from implanted devices such as catheters, that are acquired within the healthcare setting. Typically, patients infected with HA-MRSA are immune-compromised and the resulting infections are generally more invasive. CA-MRSA typically manifests itself as skin infections, such as pimples or boils, and is classified as being acquired outside of any type of healthcare setting. These infections are typically more serious than minor skin irritation and affect otherwise healthy individuals (Raygada and Levine, 2009).

MRSA becomes resistant by acquiring a mecA gene, usually carried on a larger piece of DNA called a staphylococcal cassette chromosome (SCC) mec. Expression of mecA yields PBP 2a, a penicillin binding protein with reduced affinity for β-lactam antibiotic binding (Guiguard et al., 2005).

1.1 Statement of the Problem

In orthopaedics, MRSA has been implicated in surgical site infection, post operative infection, implant devices, infection following trauma, chronic osteomyelitis subsequent to an open fracture, meningitis following skull fracture (Nixon et al., 2006). Both hip joint surgery especially emergency procedures for femoral neck fractures and the presence of a wound present a high risk of infection. The morbidity and mortality of MRSA can be severe (Power, 1998). MRSA infection colonisation contributes to an increased length of hospital stay; 88 days compared to 11days on average for non MRSA patients (Ho et al., 2008). Diagnosis of MRSA in orthopaedic surgery and the understanding of its epidemiology are therefore crucial to ensure a decrease in the incidence of MRSA. There is need for the early detection of MRSA in orthopaedic patients and the use of appropriate antimicrobial agents to control its spread.

1.2 Brief history of Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria

The Ahmadu Bello University Teaching Hospitals complex started as Institute of health in 1967 in accordance with status 15 of Ahmadu Bello University law (Amendment Act schedule 16) by the then Interim Common Services Agency (ICSA) of the former Northern Nigerian government with the objective of providing health care services, training and to conduct research.

In 1976, the Federal Government took over all the Teaching Hospitals in the country, so the control of ABUTHs passed from Ahmadu Bello University (A.B.U.) to Federal Ministry of Health, although close relationship with the University continued. Thus, 1976-1985 was the period of gradual disengagement from the university with the accompanying handing over of facilities like Nursing Home in Kaduna, Orthopaedic Hospital, Dala-Kano (1980), School of Hygiene, Kano (1984) and dispensaries in Zaria and Kaduna to the relevant State Governments.

With the promulgation of the Decree No. 10 of January 1985, ABUTHs became legally and operationally separated from ABU and the hospitals were located in Kaduna and Zaria in Kaduna State and Malumfashi in Katsina State until the hospitals were moved to the permanent site, a 547- bedded Teaching hospital at Shika-Zaria which was commissioned on 11 November, 2005.

Various Departments in the hospital include Pharmaceutical services, Nursing Services, Anaesthesia, Catering services, Opthalmology, Dental Surgery, Radiology, Physiotherapy, Haematology and Blood Transfusion, Chemical Pathology, Medical Microbiology, Pathology, Paediatrics, Surgery, Medicine, Immunology, Radiology and Community Medicine.

In the Orthopaedics department, the commonly prescribed antibiotics are gentamicin, ciprofloxacin and ampicillin – cloxacillin combination. Even though there has not been a reported case of MRSA in this department, there is need to investigate the prevalence of MRSA among the in-patients haven known the clinical implications of MRSA and ABUTH being a major teaching hospital in the north-western part of Nigeria.

1.3 Justification of Research

Detection of MRSA is important for patient care and appropriate utilization of infection control resources. MRSA is a significant pathogen that has emerged over the last 4-decades causing both nosocomial and community – acquired infection. There had been reported cases of MRSA in Nigeria: Adesida et al., (2005) reported a detection of mecA gene in 1.4% of hospital isolates collected from hospitals in the South Western region of Nigeria. Likewise Shittu et al., (2006) reported a detection of mecA gene in 1.5% of S. aureus isolates from clinical samples in South, Western Nigeria. It should be noted that there has not been any report to the best of our knowledge, on the detection of mecA gene in the North-western region of Nigeria where this study was carried out, even though Ikeh (2003) and Taiwo et al., (2004) had reported 43% and 34.7% phenotypic expression of MRSA respectively from clinical isolates in this region. Also, Olonitola et al, (2007) and Onanuga et al., (2005) had reported phenotypic expression of MRSA among non-hospital isolates in Zaria, (in North-Western region) and Federal capital territory (Abuja) respectively.

In a retrospective study on the cost of MRSA infections in the elective and trauma orthopaedic population in a General Hospital in the United Kingdom, the cost of treating MRSA infection over 12 months was £384,000. This included blood tests, x-rays, days in hospital, minutes in theatre, theatre equipment, Electrocardiograms (ECGs), swabs, dressings and all drugs excluding staff cost (Hassan et al., 2007).

It has been identified that the implications for an orthopaedic patient who contract MRSA in hospital include: extended length of stay, infection and wound breakdown; loss of alignment of fractured bone, failure of internal fixation, delay or non-union of bone, loss of earnings, pain, anxiety and depression (John and David 1991, Makoni 2002). For the hospital staff, the effects include increased workload, disruption to ward routine and may even result in temporary ward closures. However, managing and controlling MRSA outbreaks can have less of a financial impact than if the outbreak is uncontrolled. Rapid and accurate detection of methicillin resistance in S. aureus is important for the use of appropriate antimicrobial therapy and for the control of nosocomial spread of MRSA strains.

1.4 Aim

The aim of this study is to isolate S. aureus from orthopaedic patients in Ahmadu Bello University Teaching Hospital, Zaria, detect molecularly the MRSA isolates from there and to determine their antibiotic susceptibility pattern.

1.5 Specific Research Objectives

The objectives of this work are to:

1. Isolate, purify and characterize staphylococci from wounds, skin and beds of orthopaedic patients using conventional and API Staph kit methods.

2. Determine the antibiotic susceptibility of the isolates by disc agar diffusion (DAD) using antibiotics commonly prescribed in the orthopaedic wards

3. Identify the methicillin – resistant isolates.

4. Test for beta – lactamase production and hyper production using nitrocefin.

5. Detect blaZ gene detection methods.

6. Test the isolates for carriage of mecA gene and sequencing of the amplified gene products.

7. Determine plasmid carriage by the resistant isolates.

1.6 Null Hypothesis

There are no cases of mecA – mediated MRSA isolates from clinical specimens in the orthopaedic wards of Ahmadu Bello University Teaching Hospital, Zaria Nigeria.