Easter Godwin Nwokah, Samuel Douglas Abbey and Confidence Kinikanwo Wachukwu.
Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria
All Correspondences to: firstname.lastname@example.org
The problem of Antimicrobial resistance has since become a global health challenge with renewed calls for global action, including surveillance, to contain the menace. Staphylococcus aureus strains are implicated in a wide range of diseases in humans and other animals. The aim of this study was to investigate the antimicrobial susceptibility pattern of Staphylococcus aureus isolates in Port Harcourt, Nigeria. Two hundred and five (205) isolates of Staphylococcus aureus from human sources were randomly collected from three health facilities- University of Port Harcourt Teaching Hospital, Braithwaite Memorial Specialist Hospital and De-Integrated Laboratories- all located in Port Harcourt. Isolates were grouped as Hospital-acquired (n=76) and Community-acquired (n=129) Staphylococcus aureus based on established criteria. The isolates collected were reconfirmed using standard laboratory protocols and thereafter, stored at +4°C. Using the disk diffusion method, the following antimicrobial agents (OXOID, UK) were tested- Cefoxitin(30µg) Vancomycin (30µg), Erythromycin (15µg), Fusidic acid (10µg), Penicillin G (10 Units), Tetracycline, Mupirocin (5 µg), Levofloxacin (30µg), Gentamicin (10 µg), Ceftazidime (30 µg), Cefuroxime (30µg), Clindamycin (2 µg), Amoxicillin/clavulanic acid (30µg), Tigecycline (15µg) and Linezolid (10µg), Quinupristine/dalfopristine (30µg), Ticarcillin/ clavulanic acid (85µg), Sulphamethoxazole/Trimethoprim (25µg), Piperacillin/Toxobactam (110µg). Organisms showed high levels of resistance to Cotrimoxazole (65.4%) and amoxicillin-clavulanic acid (44.9%) while only one isolate was resistant to Tegecycline. All isolates were susceptible to Vancomycin, Linezolid and Quinupristine/dalfopristine. MRSA detection was 12.2% and this, including the PCR methods for mecA status has been previously reported. Study further established the presence of multi-drug resistant (MDR) strains of Staphylococccus aureus (65.4%). Beta-lactamase production was detected in 94.1% of the isolates. There is need for sustained surveillance of antimicrobial resistance of S. aureus in this region to enhance guidance for treatment and also for infection control policies.
KEYWORDS: Antimicrobial Resistance, Staphylococcus aureus, MRSA,
The problem of Antimicrobial resistance has since become a global health challenge with renewed calls for global action, including surveillance, to contain the menace. Staphylococcus aureus strains are implicated in a wide range of diseases in humans and other animals such as boils, deep tissue abscesses, enterocolitis, bacteriuria, osteomyelitis, pneumonia, carditis, meningitis, septicemia and arthritis, with associated morbidities and mortalities and medical costs. Following the introduction of the ß-lactam antibiotic, penicillin in the early 1940s, which improved outcomes, penicillin-resistant strains of S. aureus was reported, and by 1946 it was estimated that 60% of hospital isolates in the UK were resistant to this antibiotic.  Since then, successive introduction of new antibiotics- streptomycin, tetracycline, chloramphenicol and erythromycin has, in each case, been rapidly accompanied by the emergence of resistant organisms. [2,3]
It is interesting that many strains acquiring resistance to the latest antimicrobials also usually expressed a ß-lactamase (penicillinase), providing resistance to penicillin while some are resistant to all of the other antibiotics. Introduction of the semisynthetic ß-lactamase-resistant penicillins, such as methicillin and oxacillin, during the early 1960s, led to a general decline in the prevalence of multiple-resistant S. aureus. . However, by the late 1960s to early 1970s, strains resistant to the ß-lactamase-resistant penicillins were isolated with increasing frequency.  Till date, there has been an increasing incidence of hospital-associated (nosocomial) and also community-acquired infections caused by multi-drug resistant strains of S. aureus, especially the methicillin-resistant S. aureus (MRSA), which term includes not only resistance to methicillin, but also to many other different antimicrobial compounds, including various biocides. Several genes have been found in strains of MRSA which confer on them high virulence and resistance to several antibiotic classes; these include mecA (that codes for penicillin resistance), lukS-lukF (responsible for widespread skin and soft tissue infections) and tetM (that codes for tetracycline resistance), erm (for macrolide resistance) among others. The acquisition of genes such as mecA that codes for penicillin binding protein (PBP2A) by the strains, confers almost complete resistance to all beta-lactam antibiotics, including the semi-synthetic penicillin. [6-8]
A number of studies have indicated that multi-drug resistant S. aureus is among the most frequently encountered microorganisms in microbiology laboratories in Nigeria.[9-15] It is common knowledge that there is indiscriminate use of antibiotics and poor hygienic practices in our locality, as well as poor infection prevention and control practices in our health care facilities. These are some of the reasons for the success of this pathogen, accounting for is its great variability, occurrence at different periods and places with diverse clonal types and antibiotic resistance patterns within regions and countries.
The ability to characterize S. aureus and monitor antimicrobial susceptibility patterns is important for clinicians selecting empirical antimicrobial therapy, rational formulation of public health care polices, and providing useful information on the global surveillance of this pathogen. However, data on the antimicrobial susceptibility patterns of this pathogen in Rivers State are inadequate, and in most cases, isolates are screened against commonly available first line antibiotics only, most of which have lately become unhelpful. This study was aimed to investigate the antimicrobial susceptibility pattern and Beta-lactamase production in Staphylococcus aureus isolates in Port Harcourt, Nigeria.
MATERIALS AND METHODS
Port Harcourt is a cosmopolitan city, located in Rivers State, one of the 36 States of Nigeria. There are two major tertiary healthcare facilities as well as other public and private health facilities. Clinical isolates were collected from the two tertiary health facilities- University of Port Harcourt Teaching Hospital and Braithwaite Memorial Specialist Hospital and De-Integrated Laboratories. Isolates were also grouped as Hospital in-patient (Nosocomial) or Out-patient isolates (Community-acquired) according to the criteria as prescribed by the US Centers for Disease Control and Prevention. 
Microbiological/ Identification Tests
Two hundred and five (205) non-duplicate human isolates of Staphylococcus aureus, were used for this study. Reconfirmation of isolates was done following standard microbiological protocols.  All confirmed isolates were stored at +4°C and later sub-cultured to carry out phenotypic characterizations.
Antimicrobial Susceptibility Testing
Antimicrobial Sensitivity Discs
The antibiotic sensitivity discs (Oxoid Ltd., Basingstoke, England) and their disc strengths are as follows: Oxacillin(1µg) Cefoxitin(30µg) Vancomycin(30µg), Erythromycin (15µg), Fusidic acid (10µg), Penicillin G (10 Units), Tetracycline (30µg), Levofloxacin (5µg),
Ciprofloxacin (5µg), Gentamicin (10µg), Ceftazidime (30µg), Cefuroxime (30µg), Clindamycin (2µg), Amoxicillin/clavulanic acid (Augmentin) (30µg), Tigecycline (15µg), Linezolid (30µg), Quinupristine/dalfopristine(30µg), Ticarcillin/clavulanic acid(85µg), Sulphamethoxazole/Trimethoprim (Bactrim) (25µg), Piperacillin/Toxobactam (110µg),
Disk Diffusion method
All isolates of Staphylococcus aureus were subjected to in-vitro antimicrobial susceptibility testing on Muller-Hinton agar (MH) as per the method described by Kirby and Bauer (1966) . Briefly, inocula of bacteria were prepared to 0.5 McFarland standards and tested against all the aforementioned antibiotics disks. Following incubation at 37°C for 24 hrs, the zones of inhibition around the discs were measured with ruler and interpreted using the interpretation chart as prescribed by CLSI (2009) . Multi-resistance was defined as resistance to at least three classes of antibiotics.
Test for Decreased Vancomycin Susceptibility
Isolates were screened further for vancomycin resistance using the vancomycin agar screening test whereby isolates were spot-inoculated onto Mueller Hinton agar supplemented with 6 µg/ml of vancomycin from a 0.5 McFarland standard suspension. The plates were incubated at 35°C for 24 h as recommended; and growth of two or more colonies on this agar would be considered as positive. 
Detection of Beta-lactamases production:
ß –lactamase test was also carried out on the S. aureus isolates to detect whether the organism would be able to produce the enzyme ß-lactamase- an enzyme that inactivates ß- lactam antibiotics. ß -lactamase production was detected by two different methods: Test tube iodometric technique and filter paper technique using 24 hour old culture and 10,000 units/ml of crystalline penicillin as per the method described by Sykes and Mathew (1979). 
Test tube method:
A loopful of heavy inoculum of 24 hours old culture from MH agar was mixed well with 1.0 ml Penicillin solution containing 10000 U per ml. The tubes were left for 60 minutes at room temperature with mixing at 15 minutes interval. Then 2 drops of 1% soluble starch solution was added, followed by one drop of Iodine solution. The tubes were mixed well and the results were recorded as follows- Instant discoloration: ++++ (Strong positive); Discoloration in 1-5 min: +++ (Average positive); Discoloration in 6 to 10 min: ++ (Moderately positive); Discoloration in 10-15 min: + (Weak positive) and No discoloration – (Negative). All test tubes showing discoloration within 10 minutes after adding iodine solution were taken as positive for beta-lactamase production.
Agar Plate method:
Isolates were inoculated on MH agar containing 1% soluble starch and incubated at 37°C for 48 hours. Then the plate was flooded with Penicillin solution containing 10000 U per ml and left at room temperature for 30 minutes. Then the penicillin solution was decanted completely and flooded with 1:5 dilution of iodine follows- More than 10 mm diameter discoloration around the culture: ++++ (Strong positive); 5-10 mm discoloration around culture: +++ (Average positive); 2-4 mm discoloration around culture: ++ (Moderately positive); 1 mm or discoloration below culture: + (Weakly positive) and No discoloration around or below culture: – (Negative).
Two hundred and five (205) non-duplicate isolates of Staphylococcus aureus collected from different clinical specimens were used in this study. Antimicrobial susceptibility pattern revealed varying degree of resistance to various types of antimicrobial agents tested (Table 1). Highest degree of antimicrobial resistance was recorded in cotrimoxazole- 134 (65.4%) out of 205 isolates of S. aureus and followed by amoxicillin-clavulanic acid (44.9%) while only one isolate was resistant to Tegecycline. All isolates were susceptible to vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid. The oxacillin disc susceptibility testing showed that 25 (12.2%) out of 205 isolates of S. aureus were resistant to oxacillin (Table 1).
Table2 shows comparison of antimicrobial
susceptibility between in-patient S.aureus isolates and out-patient S.aureus isolates. Resistance was significantly higher in in-patient S.aureus isolates (p<0.05).
Multi-drug Resistance (resistance to three or more classes of antimicrobial agents) was significantly high (p<0.05) as detected in 134 (65.4%) of the isolates
MRSA detection was significantly higher in in-patient isolates ((23.7% of 76) than out-patient (5.4% of 129) S. aureus (p = 0.000318) (Table 4).
Table 5 shows distribution of ß-lactamase producing S. aureus isolates. One hundred and ninety-three (94.1%) of the 205 isolates were positive. There was no significant difference (p > 0.05) in ß-lactamase production between MRSA and MSSA. There was also no significant difference (p > 0.05) in ß-lactamase production between in-patient and out-patient isolates.
Multi-Drug Resistance among pathogens is a significant challenge in both hospital and community settings that adds to the cost of medical care and the morbidity and mortality of patients. This becomes worrisome in the light of our local economic realities. Continual surveillance is essential to guide therapy and for the establishment of adequate infection control programmes.
This study revealed a multi-drug resistance rate of 65.4% (inclusive of all MRSA isolates) (Tables 3). MRSA detection was 12.2% and this, including the PCR methods for mecA status has been previously reported. The study further confirmed that MRSA are more resistant to various groups of antibiotics compared to MSSA (Table 3), which also agrees with other authors. [22-23] Furthermore, selective pressure on the organisms, occasioned by misuse of antibiotics, as well as the ease of transferability of genetic elements is instructive. Study confirmed that Hospital Acquired-MRSA (nosocomial) are more resistant to antimicrobial agents than the Community-Acquired-MRSA (out-patients) among S. aureus isolates from clinical sources in Port Harcourt.
None of the isolates in this study showed resistance to the glycoprotein- vancomycin (Table 1). This is in consonance with some other investigations in Nigeria. [24-26] However, certain reports have revealed the presence of VRSA in Nigeria. Akambi and Mbe, (2013), reported vancomycin resistance in 4 isolates out of 213 isolates of S. aureus in the University of Abuja Teaching Hospital, although only the disk diffusion method was employed. A VRSA prevalence rate of 57.7% has also been reported in Zaria, Northern Nigeria, 6.3% among MRSA. In another study, in non-clinical isolates, a prevalence rate of 89% was reported. The difference in rates of vancomycin resistance probably reflects differences in levels of over-prescription and abuse in different parts of the country. Vancomycin-resistant strains are a source of concern because until recently, vancomycin was the only uniformly effective treatment for staphylococcal infections, particularly MRSA. Resistance to vancomycin severely limits therapeutic options. It is therefore cheering that there was zero resistance for vancomycin in this study. Also, none of the isolates was resistant to teicoplanin, another glycoprotein.
Nasal mupirocin plays an important role in the eradication of MRSA carriage.  Overall, 13 isolates were resistant to mupirocin (Table 1). The 6.3% prevalence of mupirocin resistance in this study is comparable to the 7% reported by Shittu and Lin, (2006)  but higher than a study (2%) in South Africa and 1.5% in India. This trend suggests that mupirocin resistance in S. aureus is an emerging feature in this locality, and therefore, underscores the need for routine testing for early detection of resistant isolates and prompt institution of infection control measures.
Five of the isolates in this study showed resistance to fusidic acid (Table 1). Some other investigators have reported full susceptibility of S. aureus to fusidic acid,[32, 35] indicating that fusidic acid is a good and effective agent for the treatment of S. aureus infections. Monotherapy with fusidic acid has been associated with the emergence of resistance and therefore, in order to minimize the emergence of fusidic-acid resistant strains, Howden and Grayson, (2006)  advised that monotherapy with fusidic acid should be discouraged and a combination with another anti-staphylococcal agent (ß-lactams, rifampicin or glycopeptides) be recommended.
A number of reports have indicated an increase in the resistance of staphylococci to trimethoprim/ sulphametoxazole (cotrimoxazole) in Nigeria. In one study, resistance rate among MRSA was 92.1%. In the present study, 65.4% of S. aureus isolates was resistant to cotrimoxazole, accounting for the highest level of resistance observed. Twenty-one (84%) of MRSA were also resistance to cotrimoxazole. This antimicrobial has wide clinical application, inexpensive, orally administered and available over-the-counter in Nigeria, where they are sold with or without prescription. This could possibly explain the high level of staphylococcal resistance observed in this study.
Erythromycin is one of the commonest and affordable antimicrobial agents available in Nigeria. An erythromycin-resistance rate of 21.5% in this study is therefore a cause for concern. More worrisome is that 23 out of the 38 erythromycin-resistant S. aureus were inducibly resistant to clindamycin and this has been previously reported.
This study also detected resistance to the fluoroquinolones. 22.9% (30 MSSA and 17 MRSA) of the 205 isolates were resistant to levofloxacin while 21.0% (28 MSSA and 15 MRSA) were also resistant to ciprofloxacin. Fayomi et al., (2011), had reported 12.2% and 9.6% ciproxin resistance among MRSA and MSSA respectively in Ido-Ekiti, Nigeria. These rates, although lower than the findings from one study in Okigwe, Nigeria where resistance to various brands of quinolones ranged from 44% to 88%, constitute a growing concern for a therapeutic option in our setting, especially where the preferred vancomycin are largely unavailable. The reasons for the disparity in rates of quinolone resistance between MSSA and MRSA strains are uncertain but could include antibiotic selective pressure, especially in the hospital setting, resulting in the spread of the more antibiotic-resistant MRSA strains.
In this study, the enzyme, ß–lactamase was detected in 193 (94.1%) of the 205 isolates (Table 5), a rate higher than 64% earlier reported by Terry-Alli et al., (2011) but lower than the 100% previously reported by Olowe et al. (2007). Twenty-five (13.0%) of the 193 ß-lactamase-producing isolates in this study were MRSA. However, there was no significant difference (p>0.05) in ß-lactamase-production between MRSA and MSSA. A previous study reported majority of ß-lactamase producers as methicillin-resistant. The indiscriminate use of antibiotics and the over-the-counter availability of antibiotics without prescription have, to a large extent, contributed to the emergence of resistant strains. ß-lactamase production by S. aureus had been identified to be a risk factor for the prevalence of MRSA in South Western, Nigeria. The finding in this study is instructive for urgent surveillance, control and other intervention programmes.
In Nigeria, systematic reporting of infectious diseases is still rudimentary and most health institutions lack proper infection control programmes. Because there is no national policy or guidelines for screening, reporting and control of MRSA outbreaks, the tendency is either under-reporting or over-reporting of the true prevalence of MRSA and therefore infections/ outbreaks are largely undetected and their contributions to mortality, morbidity and cost of care, associated with hospital-acquired infections are unknown. This problem is expected to be overcome with the recent establishment of a reference laboratory in Nigeria.
The degree of Multidrug resistance among S. aureus and especially among MRSA as detected in this study in Port Harcourt, Nigeria is high enough to warrant the need for continuous antimicrobial resistance surveillance as well as molecular epidemiological typing. This will enhance guidance for treatment and also for infection control policies.
We are grateful for the technical support of all staff of the Medical Microbiology departments in UPTH, BMSH and De-Integrated Laboratories in Port Harcourt, Nigeria.
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