MOLECULAR EPIDEMIOLOGY OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN UNIVERSITY OF JOS COMMUNITY

ABSTRACT

Staphylococcus aureus is a virulent pathogen that is currently not only the most common cause of infections in hospitalized patients worldwide but increasingly also as a community associated pathogen. This study was aimed at determining the prevalence of methicillin resistant S. aureus among apparently healthy student population of the University of Jos, Jos Nigeria. Two hundred and seventeen (217) urine samples were screened for Staphylococcus aureus. Isolates were characterized by conventional cultural and biochemical methods including rapid test kits (MicrogenID test kit). Their susceptibility profiles were determined against a panel of eleven antibiotics including cefoxitin and oxacillin. The MRSA status were confirmed by the phenotypic test using disc diffusion test method and molecular analysis employing multiplex PCR for the detection of the mecA, nuc genes and spa typing. Of the 217 samples collected 171 (78.8%) were positive for Staphylococcal species while 73(54.1%) of 135 screened with the MicrogenD test kit were identified as S. aureus. The susceptibility test showed that the highest level of resistance was demonstrated against penicillin (91.8%) followed by cefotaxime (80.7%), ofloxacin (79.2%), tetracycline (64.9%) and oxacillin (60.8%) in that order. None of the 33 isolates subjected to E-test was Vancomycin resistant. Phenotypic detection of methicillin resistance with the use of cefoxitin and oxacillin discs gave a prevalence of 79.5% (136/171) of the Staphylococcal isolates and 47.9% of S. aureus. These isolates were resistant to the betalactam antibiotics and were multidrug resistant. They were, however largely susceptible to ciprofloxacin, vancomycin, erythromycin, gentamicin and cotrimoxazole. 88.30% had MAR index greater than 0.3. Sixteen of the S. aureus isolates had the mecA and nuc genes. The nucleotide sequence of 16S rRNA gene of the 16 isolates showed that 12 of them had 77-92% identity with S. aureus strain N315. Three isolates (5, 9 and 16) could not be typed as there was no similarity with GenBank data base. Four of the isolates have common lineage with 8 BWI (2), S1123 and 363LF (1) and CLRSA1 (1) strains. The study shows that mecA gene is present in the study population. mecB gene was also detected in the 16 isolates. This is the first time this gene is being reported in S. aureus and this may increase the level and magnitude of resistance by MRSA. The identification of mecA and mecB genes in community isolates implies that any outbreak of infections caused by these strains may pose a threat to public health as these strains are resistant to all antibiotics tested except ciprofloxacin, vancomycin and co-trimoxazole.

CHAPTER ONE

1.0 INTRODUCTION

The discovery of antimicrobial agents (antibiotics and related medicinal drugs) has substantially reduced the threat posed by infectious diseases. Their use coupled with improvements in sanitation, housing, nutrition, and the advent of widespread immunization programmes has led to a dramatic drop in deaths from these diseases which were previously widespread, untreatable, and frequently fatal, thereby contributing immensely to saving lives and eased the sufferings of millions of people (Gelband et al., 2015; WHO, 2015). Their role has expanded from treating serious infections to preventing infections in surgical patients, protecting cancer patients and people with compromised immune systems, and promoting growth and preventing disease in livestock and other food animals (Gelband et al., 2015).

However, the overuse and misuse of these antimicrobial agents and the emergence and re-emergence of some infectious diseases is hampering efforts towards effective control and eventual elimination of these most dangerous infectious diseases. These gains of antibiotic therapy are now seriously jeopardized by the emergence and spread of microbes that are resistant to cheap and effective first-choice or “first-line” drugs. Once-treatable infections are becoming difficult to cure, raising costs to healthcare facilities, and patient mortality is rising, with costs to both individuals and society. Decreasing antibiotic effectiveness has risen from being a minor problem to a broad threat, regardless of a country’s income or the sophistication of its healthcare system (Gelband et al., 2015).

The consequences are severe. Infections caused by resistant microbes fail to respond to treatment, leading to prolonged illness and greater risk of death; longer periods of infectivity which increase the numbers of infected people moving in the community and thus exposing the general population to the risk of contracting a resistant strain of infection (WHO 2017).

Resistance to “first-line” drugs results in switch to second- or third-line drugs which are generally much more expensive and sometimes more toxic as well (WHO, Fact sheet, 2015). Resistance to antimicrobials is a natural biological phenomenon due to “selective pressure”. The microbes which adapt and survive carry genes for resistance, which can be passed on. Bacteria are particularly efficient at enhancing the effects of resistance, not only because of their ability to multiply very rapidly but also because they can transfer their resistance genes, when they replicate or to related species through processes like conjugation, transduction, and transformation. Resistance to a single drug can thus spread rapidly through a bacterial population. Given the ease and frequency with which people now travel, antibiotic resistance is a global problem, requiring efforts from all nations and many sectors.

In the early to late twentieth century, medicine and science were able to stay ahead through the discovery of potent new classes of antimicrobials. This process has since slowed down to a virtual standstill, partly because of misplaced confidence that infectious diseases had been conquered (at least in the industrialized world), increase in the number of infections, and the dearth of new discoveries probably due to poor funding.

The WHO launched the first global strategy for combating the serious problems of the emergence and spread of antimicrobial resistance in 2001. The strategy builds on a number of activities which include monitoring the global emergence and spread of antimicrobial resistance by establishing laboratory-based networks for the surveillance of antimicrobial resistance by microorganisms. Also in May 2015, the World Health Assembly endorsed a global action plan aiming to ensure prevention and treatment of infectious diseases with safe and effective medicines (WHO, Fact sheet, 2016).

Evidence from around the world as reported by the WHO indicates an overall decline in the total stock of antibiotic effectiveness: resistance to all first-line and last-resort antibiotics is rising (WHO, 2016). The report noted that while there are some new antibiotics in development, none of them are expected to be effective against the most dangerous forms of antibiotic-resistant bacteria. The patterns of bacteria resistance to specific antibiotics differ regionally and by country, mirroring patterns of infectious disease and antibiotic use. The U.S. Centers for Disease Control and Prevention (CDC) estimates that antibiotic resistance is responsible for more than 2 million infections and 23,000 deaths each year in the United States, at a direct cost of $20 billion and additional productivity losses of $35 billion (CDC 2013). In Europe, an estimated 25,000 deaths are attributable to antibiotic-resistant infections, costing €1.5 billion annually in direct and indirect costs (EMA and ECDC 2009). Although reliable estimates of economic losses in the developing world are not available, it is estimated that 58,000 neonatal sepsis deaths are attributable to drug- resistant infections in India alone (Laxminarayan et al. 2013). Studies from Tanzania and Mozambique indicate that resistant infections result in increased mortality in neonates and children under five (Kayange et al. 2010; Roca et al. 2008).

The bacterial infections which contribute most to human diseases are also those in which emerging antimicrobial resistance is most evident: diarrhoeal diseases, respiratory tract infections, meningitis, sexually transmitted infections, and hospital acquired infections. Some important examples include penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, multi-resistant salmonellae, and multi-resistant Mycobacterium tuberculosis and HIV/AIDS virus (WHO, 2000).

Staphylococcus aureus has been known to be a major pathogen causing a wide spectrum of clinical manifestations, such as wound infections, pneumonia, septicaemia, and endocarditis with beta-lactam antibiotics being the drugs of choice for therapy (Grisold et al., 2002; Onanuga et al, 2006). Since the introduction of methicillin into clinical use in 1961, the occurrence of methicillin-resistant S. aureus (MRSA) has steadily increased and nosocomial infections caused by such isolates have become a serious problem worldwide (Benner and Kayser, 1968; Lowy 1998; Boucher et al., 2010). Considerable research efforts have been put forward to improve our understanding of its complex pathogenesis. In spite of these efforts, the burden of staphylococcal infections is still on the rise. The unresolved questions regarding this pathogen, include: (i) the nature of the driving forces behind the rise and decline of methicillin-resistant S.aureus (MRSA) clones; (ii) the mechanisms by which a commensal becomes a pathogen; (iii) the molecular underpinnings of toxin over-expression in hypervirulent MRSA clones such as USA300; and (iv) the repeated failures of anti-S.aureus vaccine approaches (Rasigade and Vandenesh, 2014).

1.1 Statement of Research Problem

Staphylococcus aureus is a virulent pathogen that is currently the most common cause of infections in hospitalized patients worldwide. S. aureus infection can involve any organ system. The core resistance phenotype that seems to be most associated with the persistence of S. aureus in the hospital is methicillin resistance. The increase in the resistance of this virulent pathogen to antibacterial agents, coupled with its increasing prevalence not only as a nosocomial pathogen but also in community infections without the usual risk factors associated with hospital environment is of major concern and documented in several reports (Archer, 1998; Bukharie et al., 2001; Rasigade and Vandenesh, 2014). The patterns of bacteria resistance to specific antibiotics differ regionally and by country, mirroring patterns of infectious disease and antibiotic use. Methicillin-resistant Staphylococcus aureus (MRSA) has declined in incidence in Europe, the United States and Canada over the past eight years, to 18 percent, 44 percent, and 16 percent, respectively. It also has begun to decline in South Africa (to 28 percent), where antibiotic stewardship is taking hold. In sub-Saharan Africa, India, Latin America, and Australia, it is still rising (Gelband et al., 2015). In Nigeria, studies on methicillin-resistant Staph aureus have been conducted, particularly in southwestern zone (Adetayo et al., 2014). In their review, Falagas et al. (2013) observed that the prevalence of hospital associated MRSA appears to be different in the northern compared with the southern part of the country. For the northern part of Nigeria, the prevalence of MRSA varied from 9% in north-central to 28% in north-east, while in the south-west part of the country it was between 20% and 41%. Despite this epidemiological data on MRSA in Nigeria, available data are still relatively limited when compared to information from developed countries which may be attributable to high level of awareness of MRSA infections and its clinical and societal consequences (Adetayo et al., 2014). These data though limited have been mainly on hospital associated MRSA. The few community associated MRSA studies documented in Nigeria are from the Southern part of the country (Owolabi and Olorioke, 2015; Ibe et al., 2013; Iroha et al., 2014; Ike et al., 2016; Akerele et al., 2015). No similar studies have been carried out in the Northern part of the country, of which Jos metropolis is part. University of Jos is located in the heart of Jos metropolis, with a large student population. Considering the fact that MRSA prevalence varies based on geographical location, type of hospital and study population, it becomes necessary to determine the prevalence of CA-MRSA in a section of Jos metropolis.

1.2 Justification for the Research

Among the strategies recommended by the WHO for curbing antibiotic resistance is promoting regional antibiotic resistance surveillance in addition to encouraging new product development (WHO 2016). S. aureus though not a threat to healthy individuals, causes serious infections in immune compromised individuals such (HIV positive, traumatized individuals, etc.). The SCCmec resistant island of the MRSA carries genes that codes for resistance to other antibiotics. A knowledge of the presence and prevalence of MRSA carrying the mecA gene in this environment especially in community isolates will promote the understanding of the resistance problem and the best approach to tackling it. Moreover, surveillance studies on the prevalence of MRSA in this environment are limited. This study will therefore contribute to the data of knowledge available on this subject.

1.3 Aim and objectives:

The aim of this work is to determine the prevalence of methicillin resistant Staphylococcus aureus (MRSA) in sample population of healthy individuals of the University of Jos community who are not on antibiotics, using molecular techniques.

1.3.1 Specific objectives:

1. Isolate, purify and characterise Staphylococci SPP. from urine specimens from the University of Jos community.

2. Determine the antibiotics susceptibility profiles of the isolates by agar diffusion method and the multiple antibiotic resistance (MAR) indexing.

3. Phenotypically identify methicillin resistant isolates from the diffusion test and by the oxacillin breakpoint test.

4. Determine the incidence of vancomycin resistance by the dilution and break-point test.

5. Determine the presence of beta-lactamase and plasmids among the resistant S. aureus isolates.

6. Molecularly confirm the MRSA using PCR (mecA, 16SrRNA, spa, nuc).

1.4 Hypothesis

1.4.1: Null Hypothesis: There is no incidence of Methicillin resistance Staphylococcus aureus in healthy individuals of the University of Jos community.

1.4.2: Alternate Hypothesis: Methicillin resistance Staphylococcus aureus is prevalent among healthy individuals of the University of Jos community.