Introduction

The genus staphylococcus was identified in a purulent material by Robert Koch in 1878 and later cultivated by Louis Pasteur(NNISS,2002).They form the natural floral of human skin and mucous membrane(Iyer,2002).Staphylococcus is a member of the family micrococcacea in the order Eubacteriales(Mandell et al,1990 and 1995). They are gram positive,coccospherical in shape.The most widely encountered staphylococci are staphylococcus aureus,epidermidis and saprophyticus.Staphylococcus aureus is the most important medically because of its association with human diseases. Staphylococcus aureus is one  of the most versatile and dangerous human pathogen and have been implicated as a cause of urinary infections. (Lowy ,1998).Resistance to penicillin, which was the mainstay of anti-staphylococcal chemotherapy,  was identified among the β-lactamase producing staphylococcus aureus in the 1960s(Naimi et al 2003)hence ,the treatment of staphylococcus can be challenging and the associated mortality ranges from 20-30% despite the availability of highly active antimicrobial agents (Mourin et al,2001)

Based on this understanding,the idea of prescribing the penicillins alone may be reconsidered , since staphylococcus strains resistant to the β-lactam antibiotics have led to the use of several potent antibiotics like vancomycin which are largely unavailable in Nigeria and when obtainable prohibitively expensive or the quinolone which are availablebut expensive so difficult to obtain in rural hospitals. Some authors have reported good activity against staphylococci by the aminoglycoside, gentamicin.

This study  sought to determine the MIC and pattern of kinetics of ampicillin and gentamicin on both β-lactamase positive negative urinary isolates of staphylococci.

Materials and Methods

A total of two hundred and fifty clinical isolates of staphylococcus species from urine samples of patients with urinary symptoms were obtained from the medical microbiology laboratory of the university of Benin Teaching Hospital .

The isolates were identified using colony characteristics, gram stain, catalase test and the tube coagulase test in accordance with procedure described by Cowan and Steal (1985). (Stokes ,1980). Control experiments were set up using stock culture of Staphylococcus aureus and  Staphylococcus epidermidis ( coagulase negative Staphylococcus).  β-lactamase production by Staphylococci was detected using the iodometric method(Lennette et al,1980) and the β-positive isolates were separated from the negative isolates.

The MIC  was carried out on the different species and strains of staphylococci using a range of dilution of ampicillin of 1mg/ml made in nutrient broth to form concentrations of 5,10,15 20 and 25 g/ml for β-lactamase positive and negative staphylococci isolates. The same dilution was obtained for gentamycin 1mg/ml but at concentrations of 2.5,5,10,15 and 20µg/ml. The MIC defined as the least concentration of antibacterial agent that inhibits the visible growth of bacterial after incubation for 24hours was assesed (Hugo et al,1990). The nutrient agar plates were labelled with different concentrations of the ampicillin and vancomycin.The nutrient broth bottles containing  ampicillin, gentamicin and organisms were incubated for 24hours on nutrient agar plates. Within this time the plates were  withdrawn and colonies counted at intervals of 2,4,6,8 and 10hours respectively. The MIC was taken as the lowest drug concentration that inhibits the visible growth of the Staphylococci.

Results

 Table 1: The incidence of Staphylococcus aureus and coagulase negative staphylococci.

* CNS = Coagulase negative staphylococci

Table 2: Incidence of β-lactamase production amongst Staphylococcus aureus and coagulase negative staphylococci isolates.

 * CNS = Coagulase Negative Staphylococci

n = number tested

Table 3: The Kinetics of Ampicillin on β-lactamase negative staphylococci.

* N = Number

µg/ml = Microgram per milliliter

Table4: The kinetics of Ampicillin on β -lactamase producing staphylococci.

* N      = Numerous

µg/ml = Microgram per milliliter

Table 5: The kinetics of Gentamycin on β-lactamase negative staphylococci.

*  N = Numerous

µg/ml = Microgram per milliliter

Table 6: The kinetics of Gentamycin on β-lactamase positive staphylococci.

*  N     = Numerous

µg/ml = Microgram per millilitre

Discussion

The emergence of resistance to antibiotics of ,Staphylococci  have led to the emergence of new drugs and new approaches to chemotherapy. The increasing recognition of Staphylococcus aureus as a urinary pathogen especially in this part of the world makes the consideration of appriopriate chemotherapy important for the management of urinary tract infections. staphylococci isolates. As expected, the β-lactamase Staphylococci resisted ampicillin in this study, however at high doses ( equivalent of > 2 grammes per dose), there was significant reduction in bacterial count. The increasing resistance of β-lactamase producing staphylococci isolates to penicillins has rejuvenated the need to investigate alternatives which are cheap and effective. Cephalosporins, quinolones and vancomycin are excellent alternatives but rather expensive for example

1 ampoule of 500mg ampicillin cost—–40 naira

1 ampoule of 1000mg cefotaxime cost——800 naira

1 ampoule of 1000mg ceftriazone cost—–1800 naira

1 ampoule of 200mg ciprofloxacin cost—–500 naira

1 ampoule of 280mg gentamicin cost—–50 naira

Vancomycin is hardly available in Nigeria.

Scott et al demonstrated that β-lactamase producing organisms were susceptible to gentamycin.Nobert et al also reported the antibacterial action of gentamycin against certain gram positive and gram negative bacteria except bacteroids.The result of this present study  shows that gentamycin had an excellent antibacterial activity against staphylococci isolates irrespective of  β-lactamase production.This may occur because gentamycin is not a β-lactam antibiotics and its mode of action is by inhibition of protein synthesis of bacteria.Contrarily, Bint et al 1977 reported an outbreak of gentamycin resistant staphylococcus aureus.Recently,other researchers have reported good results with the use of gentamycin.The result of this study collaborates that of Linhua et al 2004 who revealed that staphylococcus aureus and epidermidis were susceptible to gentamycin and other antibiotics like vancomycin,ciprofloxacin,amikacin etc.This present study also agrees with the work of Lelievre et al who reported that the emergence and spread in French hospital of methicillin resistant staphylococcus aureus had an increasing susceptibility to gentamycin and other antibiotics.The MIC of gentamycin were 5µg/ml and 10µg/ml in the β-lactamase negative and positive staphylococci respectively.The lower MIC of gentamycin on the β-lactamase negative staphylococci shows that they were easily inhibited and more susceptible to gentamycin.The outcome of this study is in agreement with the work of Turano et al (1994) who emphasised that gentamycin was more active against coagulase negative than coagulase positive staphylococci isolates.Furthermore,Lemaitre et al (1998),reported an unusual heterogeneous staphylococci resistance to methicillin and tobramycin in the USA which were susceptible to gentamycin.In addition, the findings of Kalsoom et al (2006) shows that gentamycin was the second most effective drug against staphylococci isolates after vancomycin.she also noticed that the resistant pattern observed in gentamycin was less significant.In conclusion, gentamycin can be prescribed as a substitute for ampicillin in staphylococci infections that are resistant to the penicillins.We propose a case the synergistic combination of ampicillin and gentamycin in the management of methicillin or ampicillin resistant staphylococci isolates.

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