Prevalence and antibiotic susceptibility profile of methicillin resistant Staphylococcus aureus in Accra , Ghana

Over the last four decades, methicillin-resistant Staphylococcus aureus (MRSA) has spread throughout the world and become highly endemic in many geographical areas. This pathogen causes severe morbidity and mortality in hospitals worldwide. MRSA is also considered a major community acquired pathogen throughout the world. MRSA is implicated in serious clinical conditions such as bacteremia, pneumonia, and intra-abdominal infection. The objective of this study was to determine the prevalence of MRSA in Accra, Ghana, and also to determine its antibiotic susceptibility profile. Two hundred and fifty Staphylococcus aureus isolates from routine microbiological specimens were collected from five hospitals in Accra. MRSA screening assay was used to screen for MRSA. Disc diffusion method (KirbyBauer) was used to determine the susceptibility of the MRSA. The MRSA screening assay, which is very close to the polymerase chain reaction in terms of specificity and sensitivity, showed that 84 of the 250 isolates were MRSA, giving a prevalence rate of 33.6%. MRSA strains were susceptible to erythromycin; 63 out of the 84 MRSA isolates were susceptible representing 75%. This was followed by gentamicin 46 (54.7%), cotrimoxazole 35 (49%), cefuroxime 33 (38%), flucloxacillin 24 (28.6%), and ampicillin 13 (15.5%). Penicillin 4 (4.8%) and tetracycline 6 (7.1%) were the least susceptible. The findings from this study emphasize the need for continual surveillance of MRSA and of antibiotic resistance in general.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is considered a major nosocomial and community acquired pathogen throughout the world.MRSA is implicated in serious clinical conditions such as bacteremia, pneumonia, and intra-abdominal infection. 1Staphylococcus aureus strains resistant to methicillin have become a well-known etiological agent in a wide variety of infections.These infections have become a common problem in hospitals and in the community, and have been associated with prolonged hospital stay and increased hospital costs.
Moreover, few therapeutic options are available to treat infected patients. 1,20][11][12] Resistance to methicillin (and to all β-lactam antibiotics) in Staphylococcus aureus is primarily associated with the acquisition of the mecA gene coding for the penicillin-binding protein 2a (PBP 2a), involved in bacterial cell wall synthesis. 2A distinctive feature of methicillin resistance is its heterogeneous nature, 13,14 with the level of resistance varying according to the culture conditions and β-lactam antibiotic being used.The majority of cells in heterogeneous strains (typically 99.9% or more) are susceptible to low concentrations of β-lactam antibiotics, e.g. 1 to 5 mg/mL of methicillin. 14he detection of methicillin resistance, however, is complicated by the fact that its phenotypic expression in many strains is heterogeneous. 15This has resulted in the development of various laboratory techniques to enhance the expression of this resistance in vitro. 3,4Examples of these methods are slide latex agglutination test, rapid solid-phase immunoassay, and various disc diffusion methods.mecA gene detection tests based on polymerase chain reaction (PCR) or DNA hybridization have proved to be more specific and more sensitive than conventional tests, particularly in very heterogeneous strains. 5ittle work has been done on MRSA in Ghana.The objective of this study was to determine the prevalence and antibiogram of MRSA in Accra, Ghana.

Clinical specimen
Two hundred and fifty (250) Staphylococcus aureus isolates from routine microbiological specimens were collected from five hospitals in Accra.Categories of patients included those hospitalized with suspected clinical infections (in-patients) and those attending the outpa-tients' department for the first time without hospitalization (out-patients).Only one isolate was recovered from each patient, presenting with a particular case.The S. aureus isolates were obtained from cultures of different specimens including wounds, sputum, blood, aspirates, urine, and cerebrospinal fluids.These isolates were collected and sub-cultured on slopes of Mueller Hinton agar for storage.

Patient bio-data
Full patient bio-data such as age, sex, and types of infection was obtained from request forms.

Culture
Suspected colonies of S. aureus from primary culture plates of Blood agar, Chocolate agar and MacConkey agar were confirmed as S. aureus, by Gram Reaction, positive catalase, Tube coagulase and Deoxyribonucleases (DNAse) test.

Results
From Table 1, a total number of 84 isolates from 250 S. aureus isolates tested positive for MRSA.Out of this, 87 MRSA wound specimen produced the highest (46) of MRSA representing 54.7% of total MRSA detected.This is followed by isolates specimen from swabs representing 21.4% of MRSA isolated.Blood specimen is the third highest (14.3%) source of MRSA.This is followed by Aspirates (3.6%), Sputum (2.4%) and Cerebrospinal fluid (2.4%).Urine specimen was the least only with 1 specimen representing (1.2%) tested positive for MRSA.
Figure 1 shows the percentage of MRSA and susceptible Staphylococcus aureus (MSSA).The prevalence of MRSA from this study is therefore 33.6%.

Distribution of methicillin-resistant Staphylococcus aureus isolates
Table 2 shows the distribution of 84 MRSA isolates recovered in terms of out-patients and in-patients.In patients had the highest carriage of MRSA representing 69%, whiles out patients had a total of 26 isolates representing 31%.No MRSA (0%) was detected from sputum, aspirates, cerebrospinal fluids and urine specimen recovered from out patients.

Discussion
Undoubtedly the epidemiology of MRSA has continued to evolve since its first appearance more than three decades ago.Initially there were sporadic reports of methicillin resistance amongst nosocomial S. aureus isolates.But later MRSA became a well established hospital acquired pathogen with few reports of community acquired isolates.Recent studies report of increases of prevalence of MRSA.In this present study (Figure 1) 33.6% of S. aureus isolates  Personal communication).This implies that the incidence of MRSA keeps changing every year and is on a rise as compared to the last few years.
The MRSA screen assay detected 84 of the 250 S. aureus isolates as MRSA (Table 1).No agglutination occurred when the 250 isolates were tested with the negative-control latex.The MRSA PBP2a latex agglutination screen assay is latex particles sensitized with anticlonal antibody against PBP2a and react specifically with MRSA to cause agglutination.It is reported to have 97.6% sensitivity and it distinguishes between very low level MRSA from MSSA. 16,17Bowers et al., reported MRSAlatex agglutination test as a reliable and rapid detection test from both broth pure culture and selective media as well as being a reliable alternative to mec A PCR for the definite diagnosis of MRSA. 18Interestingly, Atoum et al. reported strains of negative mecA being methicillin resistant and positive mecA being methicillin sensitive.These observations are explained as being due to non functional mecA gene or non active PBP2a protein.Consequently, they recommended the use of a combination of both molecular and microbiological methods for detection of MRSA. 19However PBP2a latex agglutination has demonstrated 100% agreement for both mecA-positive and negative strains. 20t is well established that the major mechanism of resistance to methicillin and other antistaphyloccoccal penicilins in S. aureus involves the production of an additional membrane penicillin-biding protein.In adtion to the PBP2a production, most methicilin-resistant S. aureus also produce β-lactamases. 21here is however, limited information about the relationship between B-lactamase(s) of MRSA and the four B-lactamases (types A, B, C and D) produce by methicillin susceptible strains of S. aureus.
Most of the MRSA strains (34.8%) in this study came from wound and skin swabs (Table 2).This is similar to the study by Felten et al, in which it was reported that MSSA from skin lesions probably acquired the mecA gene by horizontal transfer from other skin Staphylococcus species. 1 MRSA is generally known to be resistant to β-lactam antibiotics (Figure 2).However penicillin, ampicillin and flucloxacillin (all β-lactam antibiotics) were included in the study to ascertain their antibiotic susceptibility patterns.From Table 3 penicillin had the highest resistance of 92.2% followed by ampicillin 84.5% and flucloxacillin 71.4%.These findings    agreed with the work done by Taiwo et al. 22 The difference in the resistance of these β-lactam antibiotics is explained by the fact that PBP 2abinding affinities differ among the various βlactam antibiotics; however, there is crossresistance to this general class of antibiotics because most, if not all, of the currently available agents bind too poorly to be active at clinically relevant concentrations. 23RSA isolates also showed resistance as follows: tetracycline 92.8%, and cotrimoxazole 58.3% (Table 3).High resistance of MRSA to gentamicin has also been reported. 24However, in this study only 45.2% resistance to gentamicin was found.Furthermore, in another study, conducted among the aminoglycosides, findings revealed that susceptibility was maximum to netilmicin 52.5% and minimum to gentamicin 10.3% (showing that MRSA was 90% resistant to gentamicin). 25However, because of various reasons, netilmicin cannot be recommended for empirical treatment of MRSA associated infections. 26Lyon et al. have also shown that the majority of MRSA strains are also resistant to erythromycin, clindamycin, and tetracycline. 27

Conclusions and Recommendations
First, the findings of this research have shown that there is an increasing prevalence rate of MRSA in Accra, Ghana.To reduce the prevalence of MRSA, regular surveillance of hospital associated infections, monitoring of antibiotic sensitivity patterns of MRSA and formulation of definite antibiotic policy may be helpful.There is therefore the need for health policy planners to be in the forefront in the development of comprehensive programs, policies and strategic measures as well as the development of research and surveillance units.It is recommended that molecular characterization is done in further studies.
MRSA screening assay was used to screen for Methicillin resistance.The MRSA-screen assay is a 5-minutes slide latex agglutination test based on detection of PBP2a.The method involves extraction of PBP2a from suspensions of colonies and detection by agglutination with latex particles coated with monoclonal antibodies to PBP2a.Inocula for susceptibility testing N o n -c o m m e r c i a l u s e o n l y were made using suspensions from over nightcultures of MRSA on Müller-Hinton agar.An inoculum equivalent to 0.5 McFarland (10 8 CFU/mL) turbidity standard was used for each test.Disc diffusion method (Kirby-Bauer method) was used to determine the susceptibility of the MRSA, to commonly used antimicrobial agents.These included: Penicillin (1 ug), Ampicillin (10 ug), Flucloxacillin (5 ug), Erythromycin (5 ug), Tetracycline (10 ug), Cotrimoxazole (25 ug), Cefuroxime (30 ug), Gentamicin (10 ug).The inoculum was compared to a 0.5 McFarland standard and the control was MRSA (ATCC 6571).

Figure 2 .
Figure 2. Multidrug resistance of methicillin-resistant Staphylococcus aureus.Multi drug resistance of methicillin-resistant Staphylococcus aureus.Generally, out of the 84 MRSA isolates, 25% were resistant to more than five antibiotics used, followed by 22.6%, 21.4%, 14.3%, 8.3% and 4.8% for six antibiotics, seven antibiotics, four antibiotics, three antibiotics and eight antibiotics respectively.Also 2.4% of MRSA were resistant to two antibiotics.Whiles 1.3% were resistant to one antibiotic.In general 96.3% of the isolates were resistant to three or more antibiotics.

Table 1 . Isolates of methicillin-resistant Staphylococcus aureus from different clinical specimen. Clinical No. of S. aureus No. of MRSA % MRSA Specimen n=250 n=84
total number of 84 isolates from 250 S. aureus isolates tested positive for methicillin-resistant Staphylococcus aureus (MRSA).Out of this, 87 MRSA wound specimen produced the highest (46) of MRSA representing 54.7 % of total MRSA detected.This is followed by specimen from swabs representing 21.4 % of MRSA isolated.Blood specimen is the third highest (14.3 %) source of MRSA.This is followed by Aspirates (3.6%), Sputum (2.4%) and Cerebrospinal fluid (2.4%).Urine specimen was the least only with 1speceimen representing (1.2 %) tested positive for MRSA. A

Table 2 . Distribution of methicillin-resistant Staphylococcus aureus isolates (outpatients and inpatient).
Distribution of 84 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from outpatients and inpatients.In patients had the highest carriage of MRSA representing 69%, whiles out patients had a total of 26 isolates representing 31%.