Synergistic effect of ampicillin and gentamicin on beta-lactamase producing and methicillin resistant staphylococci

The antibacterial effect of ampicillin, gentamicin, vancomycin, methicillin and other drugs was tested on both beta-lactamase producing and methicillin resistant staphylococci. A total of 60 clinical isolates were collected from Irrua Specialist Teaching Hospital, Irrua. The results reveal that 32 (53.3%) of the staphylococci isolated produced beta-lactamase while 28 (47.7%) did not produce beta-lactamase. The difference in beta-lactamase production by the Staphylococcus aureus isolates was not statistically significant (P > 0.05). Out of the 32 beta-lactamase producing species, non was sensitive to methicillin while six of the 28 non beta-lactamase producing strains were sensitive to methicillin. The effects of ampicillin, gentamicin and the synergy of both drugs at different concentration (20, 15, 0.5 and 0.25 μg) did not show good sensitivity except for the 20 μg. A correlation (R < 1) was observed between betalactamase production and multi drug resistance, between beta-lactamase production and methicillin resistance and methicillin resistance and multi drug resistance.


INTRODUCTION
The three main Staphylococcus species of clinical importance are Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus saprophyticus (Cruickshank et al., 1975).Staphylococci causes infections either through their ability to multiply and spread on tissues or through their production of many extra-cellular substances like toxins and enzymes for example beta lactamase which are controlled by plasmids or chromosomes born genes (Ito et al., 2004).One of the reasons for the success of the staphylococci as human pathogen is its great variability, occurring at different periods and places with diverse clonal types and antibiotic resistance patterns within regions and countries.Although infections caused by antibiotic-resistant S. aureus bring about serious problems in the general population, such infec-tions can be particularly devastating for the very young, the elderly and the immuno-compromised (Adebayor and Lin, 2006).
The beta-lactamase antibiotics include the penicillins cephalosporins, carbapenems, clavams and monobactams (Singleton and Willey, 1999).Beta-lactam antibiotics act by disrupting synthesis of the cell envelope in growing cells.They do this by inactivating penicillin binding proteins thus inhibiting the synthesis of the peptideglycan.In many antibiotics, the beta-lactam ring is susceptible to cleavage by certain bacterial enzymes (Blactamase) (Edwards and Greenwood, 1992).
Such cleavage destroys the antibiotics and the organism which produces these enzymes show at least some degree of resistance to beta-lactam antibiotics.The production of beta-lactamase in S. aureus is specified by blaZi genes and is believed to be regulated by a repressor blaZi (Clarks and Duke, 2001).The production of beta-lactamase can be detected by the iodometric or acidometric method (Singleton and Wiley, 1999).
Over the past 20 years, there has been an increased interest in the development of resistance of pathogens against antibiotics caused by the indiscriminate use of modern antibiotic (Dash et al., 2011).Methicillin resistant S. aureus (MRSA) is a bacterium that developed antibiotic resistance first to penicillin in 1947 and later to methicillin and related "anti Staphylococcus drugs".It was first discovered in Britain in 1961 just one year after the launch of methicillin and is now widespread causing serious hospital and community acquired infections worldwide (Enright, 2003).Antibiotic resistance due to beta-lactamase production possesses a clinical problem which may be approached by the use of drug synergy.This study was aimed at studying the use of combination of drugs therapy staphylococcal infection.

Specimen collection/ isolation of organism
Specimens used in this study were collected from in and out patients at Irrua Specialist Teaching Hospital Irrua, Edo State, Nigeria.They included urine, ear swab, urethra swab, endocervical swab, sputum and pleural aspirate.A total of 60 clinical isolates was gotten.Samples were inoculated separately into Mannitol salt agar plate using the streak plate method and incubated aerobically at 37°C for 24 h.At the end of incubation, isolates were identified using various morphological and biochemical tests as described by Cheesbrough (2006).The acidometric method was used to identify beta-lactamase production.

Antibacterial susceptibility
The effect of ampicillin, gentamicin, and synergy of both drugs at different concentrations was tested on seven of the isolates and a standard S. aureus strain (NCTC 6571).Ampicillin (500 mg/2 ml) was diluted to 20 µg/ml.The isolates were suspended in nutrient broth before subjecting to different concentrations of ampicillin.Gentamicin (80 mg /ml) was diluted to 20 µg /ml.The synergistic effect was determined by diluting each drug to 40 mg/ml and equal volume was mixed to make up 20 µg /ml and the isolates were subjected to different concentrations of it and incubated at 37°C for 18 h.Antibacterial activity of conventional antibiotics was also tested on isolates using disc diffusion method and zones of inhibition were read according to the method of Stokes and Ridway (1980).

RESULTS AND DISCUSSION
The results show that 32 (53.3%) of the isolates were beta-lactamase producing while the remaining 28 (47.7%) were non beta-lactamase producing (Table 1).Of the 32 isolates positive for beta-lactamase, none was sensitive to methicillin, while six (21%) of the 28 isolates negative for beta-lactamase were sensitive to methicillin (Tables 2 and 3).The isolates also showed a multidrug resistant pattern to the antimicrobial agents tested except vancomycin (Tables 2 and 3).Tables 4 and 5 showed the effect of ampicillin, gentamicin and synergistic effect of both drugs on seven selected isolates and on S. aureus oxford strain (NCTC 6571) which was used as control.
Results from this study show a higher prevalence of beta lactamase producers than that recorded by Esumeh et al. (2004), were beta lactamase producers were 36% and non producers 63.3%.The production of beta lactamase is due to genes located on extrachromosomal plasmids.Livermore (2002) attributed this resistance to an unusual penicillin binding protein on the cell wall of such organism.The enzymes destroy the β-lactamase ring of penicillin (Gold and Moldering, 1996) and leads to an increasing resistance to the beta lactamase antibiotics.
the high resistance to the antibiotic (methicillin), which initially appeared not to be affected by the enzyme beta lactamase and thus became a salvaging agent in the treatment of many infections due to beta lactamase producing species of Staphylococcus.The emergence of methicillin resistant Staphylococcus led to the discovery of an antibiotic called vancomycin.This antibiotic has been observed to be very effective in the treatment of Staphylococci infections and this is proved from the results of this study.Of the 60 isolates, only seven (11.7%) were resistant to vancomycin and such vancomycin resistant S. aureus (VRSA) were not susceptible to any of the other antibiotics used.Thus, vancomycin could be used as a last resort in treating Staphylococcal infections.This result is in line with the work of Abbey (2004) who recorded only 15 (30%) of vancomycin resistant S. aureus of the 50 isolates examined.A general analysis of this study showed a trend in which there is serious antibiotic resistance from previously known antibiotics to the most recently developed ones.Also resistance could be attributed to the discovery by Sande et al. (1991) which explains that when the antimicrobial activity of a new drug is first tested, a pattern of sensitivity and resistant is usually defined.But the spectrum of activity can subsequently change because organisms have evolved the array of ingenious alterations that allow them to survive in the presence of antibiotics.The effect of ampicillin and gentamicin at different concentrations on selected isolates did not prove them to be good therapeutic agents for Staphylococcal infections.Table 4 showed that only the 20 ug/ml could inhibit the growth of Staphylococcus and this is twice the concentration in the disc.Gentamicin was a little more effective than ampicillin as the isolates showed total resistance to ampicillin.The synergistic effect did not give good susceptibility result either except for the 20 ug/ml as showed in Table 5.
This study shows a correlation coefficient (R<1) between beta lactamase production and multidrug resistance, between beta-lactamase production and methicillin resistance and between methicillin resistance and multidrug resistance.It was observed that betalactamase producing isolates have a lower susceptibility to antibiotics than non beta lactamase producers.It was  also seen that all beta-lactamase producers were not susceptible to methicillin.Those resistant to methicillin had a reduced susceptibility to other antibiotics when compared to the susceptibility of the non beta lactamase producers.
From this study, it can be concluded that Staphylococci are highly resistant to antibiotics mostly penicillin because of the enzyme beta-lactamase that they produce and antibiotic resistant genes in the plasmid.However resistance can be prevented by proper hygienic practices such as proper sanitary practices by hospital staff and patients, isolation of infected patients to avoid the easy spread of antibiotic resistant species of Staphylococcus and optimal and judicious selection of antimicrobial agents for the therapy of Staphylococcal infection.

Table 2 .
Antibacterial susceptibility of beta-lactamase producing stapylococci isolates no of isolates sensitive to antibacterial agents.

Table 3 .
Antimicrobial susceptibility of non b-lactamase producing isolates.

Table 4 .
Effects of ampicillin and gentamicin on selected isolates.

Table 5 .
Synergistic effect of ampicillin and gentamicin on selected isolates.