Tropical Journal of Pharmaceutical Research Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria ISSN: 1596-5996 EISSN: 1596-9827 Vol. 7, Num. 1, 2008, pp. 929-934

Tropical Journal of Pharmaceutical Research, Vol. 7, No. 1, March 2008, pp. 929-934

Research Article

Adebola Onanuga^{1, 2} and Josiah A Onaolapo¹

¹Department of Pharmaceutics and Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria; ²Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Amasoma Bayelsa State, Nigeria.
*Corresponding Author: E-mail: adebolaonanuga@yahoo.co.uk

Code Number: pr08009

Abstract

Purpose: An investigation of the antimicrobial susceptibility patterns of Staphylococcus aureus isolated from healthy women to ten commonly used antimicrobial drugs was carried out as a basis for a guide for empirical antimicrobial treatment using urine samples.
Method: The samples collected from healthy women volunteers in Zaria were cultured and screened for S. aureus using standard microbiological procedures. The antimicrobial susceptibility of the isolates was investigated using disc diffusion technique.
Result: A total of 54(36%) S. aureus isolates were isolated from 150 urine samples collected. Of the 54 isolates, 16 (29.6%), 15 (27.8%) and 23 (42.6%) were from married but not pregnant, pregnant and single women respectively. The isolates were highly susceptible to ciprofloxacin, gentamicin, ofloxacin, sparfloxacin and pefloxacin in both groups (married and single). The differences observed in all the antimicrobial drugs tested for both groups were not statistically significant (p>0.05). A total of 34 (63%) of the isolates showed multi-drug resistance and only 6 (11%) were susceptible to all the antimicrobial drugs tested.
Conclusion: This observation calls for measures to reduce the reservoir of antimicrobial resistant organisms in healthy populations.

Key words: Antimicrobial drugs, community-associated, susceptibility, Staphylococcus aureus, healthy women.

INTRODUCTION

Staphylococcus aureus is a worldwide pathogen with its natural reservoir in human. It is one of the most common causes of severe community associated infections of skin and soft tissue^{1, 2}. Treatment of serious S. aureus infections can be challenging, and the associated mortality rate remains 20% to 25% despite the availability of highly active antimicrobial drugs ³.

S. aureus colonises the nares, axillae, vagina and damaged skin surfaces. About 30% to 50% of healthy adults are colonised with 10 to 20% persistently colonised ⁴. Approximately 60% of women harbour this organism intermittently at one or more body sites ⁵. Studies have shown that 7-25% of women harbour toxin-producing S. aureus ⁶. Persons colonised with S. aureus strains are at increased risk of becoming infected with these strains ^{1, 7}.

In the early 1950s, penicillinase-producing strains were universally present in hospital while community-associated isolates of S. aureus were considered to be largely penicillin susceptible. However, over the past few years, community-associated S. aureus infections are not only resistant to penicillin but to all other β- lactam antibiotics ^{8, 9}. More so, it is known that epidemic strains of S. aureus are commonly resistant to many antimicrobial drugs thereby making the choice of appropriate therapy difficult.

We hereby report the antimicrobial susceptibility pattern of community associated S. aureus isolated from healthy women in Zaria community as guide for empirical antimicrobial treatment and a basis for their reduction in healthy communities. This is relevant since resistance is believed to be a common phenomenon among strains of this organism, which is a likely result of indiscriminate use of antimicrobial drugs, a common occurrence in most Nigerian communities.

MATERIALS AND METHODS

First “clean catch” urine samples were collected randomly from 150 healthy women of three (3) categories (single, married but not pregnant, and pregnant women of ages between 20-40 years) over a period of two (2) months from Zaria community after informed consent had been obtained from each woman. All the volunteers were not on any antimicrobial drug at the point of sampling. Samples (Fifty from each group) were collected into labelled sterile bottles, kept in an iced-bag and transported to the laboratory.

Bacteriology

Within two (2) hours of collection, each urine sample was inoculated (in duplicates) into Mannitol salt agar plates on arrival at the laboratory. The plates were incubated aerobically at 37^OC for 24 hours. The characteristic isolates were identified using colonial, morphological and biochemical characteristics as described by Cheesbrough¹⁰ . Isolates that were Gram-positive cocci, catalase positive and coagulated human plasma were considered as S. aureus in this study.

Definition of Community-associated Isolates

For the purpose of this study, communityassociated isolates were defined as isolates from the samples of the healthy women who were not on any antimicrobial drug at the time of sampling and had not been admitted in hospital in the last one year.

Antimicrobial Susceptibility testing

Antimicrobial susceptibility pattern of all isolated S. aureus to the following ten (10) commonly used antimicrobial drugs in the community [ampicillin 10µg (Medreich sterilab, India), cephalexin 30µg (Fidson, India), ciprofloxacin (ciprotab^®) 5µg (Fidson, India), clindamycin (Dalacin C^®) 2µg (Pharmacia, Belgium), gentamicin (Hefogenta^®) 10µg (Wuham, china), methicillin 10µg (Oxoid, UK), ofloxacin (Fluxor^®) 5µg (Pathoteq Lab, India), pefloxacin (Peflotab^®) 5µg (Fidson, India), Sparfloxacin (Sparbact^®) 5µg (Pathoteq Lab. India) and vancomycin 30µg (Dumex-Alpharma, S. Demark)] were determined by the modified Kirby-Bauer diffusion technique ¹⁰. Standardised overweight culture of each isolates (containing about 10⁸ cfu/ml) was used to flood the surface of Mueller Hinton agar (MHA) plates; the excess was drained off and the surface was allowed to dry aseptically. The standard antimicrobial discs were then aseptically placed at reasonable equidistance on the inoculated MHA plates and allowed to stand for 1hour. The plates (prepared in duplicate for each isolate) were then incubated at 37^OC for 18 hours ⁷. The diameter of the zone of inhibition produced by each antimicrobial disc was measured, recorded and isolates were classified as “resistant”, “intermediate sensitive” or sensitive (susceptible) based on the standard interpretative chart updated according to the current the National Committee for Clinical Laboratory standards (NCCLS; now the Clinical and Laboratory Standards Institute [CLSI] guidelines ¹¹.

Statistical Analysis

Frequencies were obtained and percentages were calculated for study variables. Chisquare and two tailed Fisher’s exact test were used to calculate probabilities and determine significance. A p-value of less than or equal to 0.05 is considered to be statistically significant (p≤0.05).

RESULTS

Fifty-four (36%) out of 150 urine samples of healthy women volunteers screened yielded S. aureus isolates. The distribution of S. aureus isolates among the groups of women showed that it is more prevalent in the singles than the two groups of married women (Table 1).

The antimicrobial susceptibility test results in Table 2 show that the isolates from all the groups were generally highly susceptible to ciprofloxacin, gentamicin, ofloxacin, pefloxacin and sparfloxacin. They have generally very low susceptibility to ampicillin, cephalexin, clindamycin, methicillin and vancomycin. The observed differences in the susceptibility of the isolates from the two groups of women to the tested antimicrobial drugs is not statistically significant (p>0.05).

Multi-drug resistance in this study was taken as resistance to four or more of the ten antimicrobial drugs tested. The results showed 34 (63%) of the isolates as multi-drug resistant and were methicillin resistant Staphylococcus aureus (MRSA). Only 6 (11%) of the isolates were fully susceptible to all the tested antimicrobial drugs. The distribution of prevalence of multi-drug resistant S. aureus is shown in Figure 1.

DISCUSSION

S. aureus is a virulent organism that is renowned for its potential to acquire resistance to antimicrobial agents and it is one of the common cause of community-acquired and nosocomial infections ³.

Analysis of the healthy women urine in this study gave a total prevalence rate of 36%, which supported previous reports of studies carried out in Zaria ⁸ and Abuja ⁹. This result points to the increasing importance of this organism as a urinary pathogen and genital colonizers in our society. It may equally infer correspondingly high prevalence in healthy children and men because of the role of women (as mothers and wives) in our society. Further, broader based studies should be carried out to ascertain this postulation. The difference in the colonization rate of S. aureus in the married and single women was not significant (p>0.05) indicating that marital status is not a notable factor in colonization and there is no activity or behaviour of any of the groups, which predisposes them to S. aureus infection.

As expected the highest antimicrobial resistance was observed in ampicillin (77%) in both groups. This continuing upward trend has been noted in other studies ^{8, 9, 12}. Resistance to cephalexin and clindamycin is in conformity with previous observations that most isolates of S. aureus are resistant to large number of commonly prescribed antibiotics ¹³. The low vancomycin (30-40%) in the two groups support the previous reports in our communities ^{14, 15}. The resistance may be due to the acquisition of resistance determining genes, such as mecA (methicillin), van A, B, C responsible for vancomycin resistance in enterococci ^{16, 17} or as a result of the thickening of the cell wall as reported by some authors ^{17, 18}. These were however not determined in this work. A total of 70-96% of the isolates was highly susceptible to gentamicin, ofloxacin, ciprofloxacin, sparfloxacin and pefloxacin in both groups. This has been widely reported in most other studies ^{8, 9, 12}. Susceptibility to gentamicin (through a cheap drug) might be due to the route of administration which hinder its frequent misuse while the high susceptibility observed in the fluoroquinolones tested may be due to the fact that they are relatively expensive and newer antimicrobial drugs, therefore less available for abuse.

The community-associated S. aureus isolates tested exhibited a high level of multi-drug resistance, which calls for great concern. A total of 63% of the isolates were multiresistant, 89% were resistant to at least one antibiotic and only 11% were susceptible to all the antimicrobial drugs. These observations confirm the postulation that healthy members of the community are the highest reservoirs of antimicrobial resistant bacteria ^{19, 20}.

CONCLUSION

The enormous level of use of antimicrobial drugs indiscriminately or justifiably has great potential for selecting for or enhancing the growth of multi-resistant strains. The results from this study show the need to reassess policies on antimicrobial drugs use within and outside the hospital environment. There is also the need for regular monitoring of the antimicrobial susceptibility status of important pathogens so as to ensure the administration of an effective antibiotic whenever there is need to do so.

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