The screening of multi-drug resistance ( MDR ) susceptibilities of Staphylococcus aureus and Staphylococcus epidermidis to methicillin and vancomycin in teaching hospitals in Nigeria Yah

Background: In Nigeria, the widespread use of antibiotics had led to high levels of resistance among bacterial isolates from patients with nosocomial infections. This had led to prolonged hospital stay and antibiotic therapy, especially β-lactam antibiotics that predispose patients to acquisition of methicillin -resistant Staph. aureus (MRSA) and coagulase negative resistant staphylococci. Objective: to evaluate the resistant pattern of multi-drug resistant strains of 80 clinical Staph. aureus, 22 environmental Staph. aureus, 30 clinical Staph. epidermidis and 12 environmental Staph. epidermidis to methicillin and vancomycin from teaching hospitals in Nigeria. Material and Methods: The Staphylococcus species were identified and confirmed by gram-positive cocci morphology, catalasepositive reaction, tested for mannitol salt fermentation and DNase production. The organisms were confirmed to be Staph. aureus and Staph. epidermidis by the tube coagulase test. The antibiotics susceptibility patterns were determined both by overnight broth-microdilution and agar disk diffusion methods. Results: The isolates were resistant to ampicillin, followed by penicillin, tetracycline, erythromycin and gentamicin but to a lesser extent were sensitive to ciprofloxacin. All the multi-drug resistant (MDR) Staphylococcus species were 100% sensitive to vancomycin and methicillin with a minimum inhibition concentration (MIC) breakpoint < 4μg/ml to vancomycin and MIC < 5μg/ml to methicillin on Mueller Hinton agar supplemented with 2%NaCl. Conclusion: The results indicated that methicillin and vancomycin are still very potent antibiotics against staphylococcal infections in Nigeria.


Introduction
In Nigeria, the widespread use of antibiotics had led to high levels of resistance among bacterial isolates from patients with nosocomial infections [19][20]15 . Thi had led to prolonged hospital stay and antibiotic therapy, especially βlactam antibiotics that predispose patients to acquisition of methicillin -resistant Staph.aureus (MRSA) and coagulase negative resistant staphylococci.Methicillin resistant strains that emerged by late 1980s have become increasingly present as nosocomial pathogens.The medical community was again relieved when vancomycin a glycoprotein was discovered that added effective therapy to all strains of methicillin resistant Staph.aureus.Nevertheless vancomycin resistant strains of coagulase-negative staphylococci were also a cause of concern 16,7,14,18 .Added to these concerns were observations that vancomycin resistant enterococci isolates or epidemics in some U.S. hospitals were becoming increasingly prevalent in critical care units 5,4 and high level vancomycin resistance were experimentally transferred from Enterococcus  4,7 .Strains of Staph.aureus and gram negative organisms resistant to vancomycin and other antimicrobial agents including quinolones are endemic already in numerous hospitals and health care institutions leaving only a few effective and costly antimicrobials for the treatment of patients infected with these pathogens 8 .In Nigeria, there has been a recent increase in resistant to gentamicin and variable susceptibility to other non-ß-lactam antibiotics, namely tetracycline, trimethoprim, erythromycin and ciprofloxacin 1,22,24 .In this study we investigated both the broth-micro-dilution and agar disk diffusion methods on multi-drug resistant on both hospital environment and long term clinical isolates of Staph.aureus and Staph.epidermidis from some selected teaching hospitals in Nigeria to ascertain their level of resistance to methicillin and vancomycin.
Methods Bacterial strains and selection of isolates for analysis: One hundred and forty four multidrug resistant Staph.aureus and Staph.epidermidis from some selected teaching hospitals in Nigeria were obtained and examined for their antibiotics susceptibility profiles to methicillin and vancomycin.The following antibiotics were used to screen for the resistance of the isolates; ampicillin (AM) 30µg, gentamicin (GN) 10µg, tetracycline (TE) 30µg, ciprofloxacin (CIP) 5µg, erythromycin (E) 10µg, Penicillin (PN) 30µg (Optun Laboratories Nig Ltd., Nigeria), methicillin 5µg (Bristol Meyers Squib) and vancomycin (VAN) 5µg (Mayne Pharma Warwickshire -UK).The inocula were prepared directly from an over night agar plate.Investigation of bactericidal activity were done by measuring the zone of inhibition with standard measuring procedures according to NCCLS, 17 after incubation at 30 -350C for 24 hours.Staphylococcus strains that showed resistance to three or more classes of antibiotics were titled as 10 multi drug resistant (MDR), and were further preserved for other analyses.The fully sensitive strains of the organisms were discarded.

Agar Dilution tests of methicillin and vancomycin:
The minimum inhibition concentration (MIC) of methicillin (MET) 500mg (Bristol Meyers Squib Hampshire-England) and vancomycin (VAN) 500mg (Mayne Pharma Warwickshire -UK) was determined by agar dilution method, according to the guidelines of NCCLS. 17Colonies of each strain from an over night growth were transferred to sterile saline.The suspension were adjusted to a 0.5 McFarland standard, diluted 1:10, and inoculated on Mueller Hinton agar (Difco Laboratories, Detroit, Mich) plates supplemented with 2% NaCl wt/vol.The plates were incubated at 30 -350C for 24 hours.

Results
The  3a to 3d.All the MDR isolates were sensitive to methicillin and vancomycin, but were resistant to ampicillin, followed by penicillin, tetracycline, erythromycin, gentamicin and ciprofloxacin.The results showed that both isolates were highly resistant to ampicillin and penicillin from all the teaching hospitals with resistant ranged of 40% to 71%.The isolates also had a resistant range of 00% to 17% to ciprofloxacin, although environmental Staph.epidermidis were 00% resistant to ciprofloxacin.Only Staph.epidermidis from UBTH as shown in Table 1 had a resistance of 17% to ciprofloxacin.The resistance pattern varied among the gentamicin, erythromycin and tetracycline as shown in Tables 3a to 3b.The minimum inhibitory concentration (MIC) range was 0.5µg/ml to 5µg/ml with a MIC break point of < 4µg/ml for both isolates as shown in Tables 3c and 3d.All the 144 MDR Staph.aureus and Staph.epidermidis were considered to be susceptible to methicillin and vancomycin according to published NCCLS guidelines.None of the isolates had a MIC > 5µg/ml

Discussion
The 144 MDR isolates in this report were described as sensitive isolates to methicillin and vancomycin.This contradicts other reports from Nigeria and some other African countries. 1,15 Since all the strains were sensitive to methicillin and vancomycin, the study therefore suggested that none of the strains contained vanA or mecA genes respectively.The rate of resistance continues to reduce from the earlier reports 15 , the isolates according to the present study were all sensitive to both methicillin and vancomycin.The current studies also found that methicillin, oxacillin and vancomycin were not at all among the commonly prescribed antibiotics in teaching hospital in Nigeria 25 .However, cloxacillin with similar mode of action was very rare in circulation as compared to commonly prescribed ampicillin, penicillin, aminoglycosides and quinolones 23 .
Despite the fact that a MIC = 4µg/mL was defined as susceptible by NCCLS standards 17 , it is still considered to be at the borderline of resistance.S. aureus strains that are methicillin or oxacillin resistant and have a MIC of vancomycin ≥4 µg/mL should be suspected for decreased susceptibility to vancomycin and should be considered for additional testing strategies because of the possible sub-population heterogeneity of S. aureus isolates with these MIC results 6,22 .Our results indicate that methicillin and vancomycin are still very potent antibiotics against Staph.aureus and Staph.epidermidis infections.According to Jan et al 10 , strains of MRSA with reduced susceptibility to vancomycin were isolated in Japan in 1997 and have since been described in the United States, France, Hong Kong, China, and Korea.Their findings as well had no strains of vancomycin intermediate Staph.aureus despite having three sites in Japan, consistent with the suggestion that these strains are still relatively rare.The isolation of these strains in an area of high endemicity indicates the need for continuous surveillance of antibiotic resistance of Staphylococcus species and the rationalization of antibiotic in clinical set up.

Conclusion
The results indicated that methicillin and vancomycin are still very potent antibiotics against Staph.aureus and Staph.epidermidis infections.Therefore the cry of methicillin-resistant Staph.aureus (MRSA) that was first identified in the United Kingdom in 1961 11 and since then assumed increasing importance internationally as a cause of both nosocomial and community-acquired infections should not be the case in Nigeria for now.
These isolates include 80 multidrug resistant clinical Staph.aureus strains, 22 multi-drug resistant environmental Staph.aureus strains and 30 multi-drug resistant clinical Staph.epidermidis strains and 12 multi-drug resistant environmental Staph.epidermidis strains.The clinical isolates were obtained randomly from routine specimens from different infected sites (urine, wounds, and diarrheal stool) of prolonged hospitalized patients between May 2003 and October 2004.The environmental isolates were obtained from the teaching hospitals wards (air) by the suck-let sampler method.The teaching hospitals were; University of Benin Teaching