International Journal of Research in Pharmacy and Biosciences Volume 2, Issue 4, May 2015, PP 5-9 ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online) Antibiogram of Staphylococcus Aureus from Healthy School Pupils in Agulu, Southeastern Nigeria Ugwu MC 1*, Mokwe NM 1, Ejikeugwu PC 1, Enemor EC 1, Eze CO 1,2, Ugwu BC 1, Gugu TH 1 1Department of Pharmaceutical Microbiology and Biotechnology, Nnamdi Azikiwe University, Awka, Anambra Nigeria 2,1 Department of Pharmaceutical Microbiology, Faculty of Pharmacy Delta state University, Delta State ABSTRACT Studies have indicated that Staphylococcus aureus is a significant etiological agent of many infections in Nigerian Children. Adequate information is needed for treatment of these infections and for the purpose of formulating effective infection control measures as well as sound antibiotic-use policies. Thus this study was carried out to observe the biochemical characters and antibiotic susceptibility/ resistance profile of isolated S. aureus to conventional antibiotics among primary school pupils. The study population comprises of children that were between 4-8 years. A total of 100 nasal swabs were collected from primary school children from March to May 2013 following standards microbiological methods. Bacterial culture was done in Mueller-Hinton broth at 37 0 C. Characters of these strains were determined with standard biochemical tests. Antibiotic resistance pattern analysis was performed using the standard Kirby Bauer disk diffusion method. A total of 68 S. aureus isolates were recovered and characterized. Sixty nine per cent (69%) tested positive to catalase, while 32% tested positive to coagulase. Lactose fermenters were 19%, while those that tested positive to starch hydrolysis were 17.6%. The isolates were found to be very resistant to the penicillins, but very sensitive to ceftriaxone, gentamicin and the fluoroquinolones. The present study revealed that there is high prevalence of S. aureus among the healthy primary school pupils. The outcome of this form of surveillance exercises would find usefulness in shaping the existing antibiotic-use policies in order to achieve therapeutic endpoint while helping to slow down/ prevent the emergence of multiple drug-resistant strains. Keywords: Staphylococcus aureus, antibiotic, resistance, school pupi INTRODUCTION Staphylococcus aureus is a Gram positive bacterium commonly found on the skin and in the nose of most healthy individuals.globally, it is a leading cause of human bacterial infections[1,2]. S. aureus has been found to be the most frequently implicated pathogen in bloodstream infections, skin and soft tissue infections, and pneumonia. Infection rate from S. aureus is high. Recently, the increased recognition of its involvement in community acquired infections has some levels of clinical and pharmacological implications for the health care providers [2-3]. Colonization is an important step in the chain of events that leads to S. aureus infections. First, individuals are likely to become colonized, invaded and infected from this source[4]. Thus, colonization with S. aureus is a major risk factor for staphylococcal infections [5]. One of the most frequent bacterial infections in the pediatric population is caused by S. aureus. In Turkey, it was reported that 17.3% of the children had nasal carriage of multi-drug resistant Staphylococcus aureus [ 6]. Similarly in Ethiopia a study conducted in Jimma Town, Southwestern Ethiopia showed 47.74% carriage of staphylococcal species among primary school children [12]. In related study in Pokhara, Rijal, et al. [13], recorded isolation rate of 31% of S. aureus from school children. In southeastern Nigeria, infections in pediatrics are recognized to be an important public health problem and in a study by Elo-Ilo et al [7] S.aureus was reported to be the most common bacterial isolates in the children. However, the prevention as well as the reduction in the incidence of its infection requires that the population have a good perception of the disease as a health problem. The characterization of pathogenic organisms for epidemiological purposes is important in combating outbreaks of infection caused by these organisms. This is of particular interest *Address for correspondence mc.ugwu@unizik.edu.ng International Journal of Research in pharmacy and Biosciences V2 I4 May 2015 5
in developing countries where morbidity and mortality are prominently caused by microbial infections. It is desirable to characterize S. aureus isolates because of the ubiquity of this organism and its capacity to cause various types of infections which may occur in epidemic form [4]. Knowledge of the characteristics of Staphylococcus within a given locality may serve as a means of identifying strains that are more likely to lead to outbreaks of staphylococcal infection in that locality[4].thus data on susceptibility/ resistance patterns and characterization are of great importance as these data may be used to devise mechanisms to stem the emergence and subsequent spread of infections and drug resistance by the organism. MATERIALS AND METHODS Isolation and Identification of Bacterial Isolates Strains of S. aureus were isolated from nostrils of 100 healthy human subjects using a sterile swab stick for each individual. Informed consent was obtained from the school administration. The swabs were aseptically inoculated on mannitol- salt agar immediately and were incubated at 37 C for 24hrs under aerobic conditions. Colonies, which caused fermentation of mannitol, were isolated, sub cultured, Gram stained and examined microscopically. Thereafter, all Gram-positive cocci in clusters were stored in an agar slant at 4 0 C for further identification / characterization. Biochemical Characterization All Gram-positive cocci isolates that were in clusters and that fermented mannitol were subjected to the following standard characterization tests for Staphylococcus aureus: Catalase test: Coagulase test, Lactose fermentation and Starch hydrolysis tests as described by Cheesbrough [8]. Antibiotic sensitivity Antibiotic sensitivity of the isolates was determined using Kirby Bauer disc diffusion method following the guidelines of Clinical Laboratory Standard Institute (CLSI) as described elsewhere [9-10]. The antibiotic sensitivity disc (JIREH DISC) containing Augmentin (AG) 30µg, Amoxicillincloxacillin (APX) 30µg, Co-trimoxazole (SXT) 25µg, Ceftriazone (CRO) 30µg, Lincomycin (L) 30µg, Streptomycin (S) 10µg, Gentamicin (CN) 10µg, Ofloxacin (OFX) 10µg, Erythromycin (E) 5µg and Ciprofloxacin (CPX) 10µg were used for the antibiogram study. RESULTS AND DISCUSSIONS For proper understanding of the clinical and pharmacological implications of Staphylococcal species in infections and in order to provide data for the control and epidemiological measures, reliable identification of these organisms is crucial. A total of 100 samples were investigated bacteriologically to detect the occurrence of staphylococci among the primary school pupils. The isolation rate was observed to be 68 %. From table 1, out of 68 samples that were Gram positive 69% tested positive to catalase, while 32% tested positive to coagulase. Catalase test differentiates staphylococci from the streptococci while coagulase test differentiates Staphylococcus aureus from other species of staphylococci. Coagulase is an enzyme commonly used by S.aureus to induce coagulation and thus convert soluble fibrinogen into fibrin. This will protect the bacteria from the immune system of the host [ 8]. Those that were lactose fermenters were 19%, while those that tested positive to starch hydrolysis were 17.6%. The Staphylococcus aureus isolates were selected for sensitivity testing against some conventional used antibiotics. Figure 1 shows the percentage resistance of the isolates. Their pattern of resistance is in the order: Ampicillin-cloxacillin > Amoxicillin-clavulanic > Cotrimoxazole > Streptomycin> Ceftriaxone> Erythromycin~ Ciprofloxacin ~Lincomycin >Gentamicin~ Ofloxacin. The isolates were found to be very resistant to the penicillins, but very sensitive to the injectables (ceftriaxone and gentamicin) and the fluoroquinolones (ciprofloxacin and ofloxacin). The Ampicillin-cloxacillin and amoxicillin-clavulanic are commonly used antibiotics in the studied community being freely available in open markets and other un-registered drug out-lets. This observed resistance pattern is reflective of the rate of antibiotic misuse and abuse in the area. Several works have equally reported resistance to commonly used antibiotics in community settings [3]. Also, high susceptibility (low resistance) to gentamicin and the floroquinolones is also a pointer to the fact that antibiotic misuse encourages the development of resistance as these agents are rarely used in children. Fluoroquinolones are generally avoided in patients younger than 16 years because of concerns about the medical conditions known as arthropathy [11]. Gentamicin comes in parenteral forms, thus it is not misused or abused owing to difficulty in its administration and its invasive nature. Our results are comparable to a related study in 6 International Journal of Research in pharmacy and Biosciences V2 I5 May 2015
Ile Ife south western Nigeria where 99% isolates were found to be resistant to penicillin and only 2 % of isolates were resistant to gentamicin [4]. Similar high resistance to Penicillin (100%,) and high sensitivity to Gentamicin (95.3%) was observed when antibiotic susceptibility tests were conducted on 169 S. aureus isolates among primary school children and prisoners in Jimma Town, Southwest Ethiopia[ 12]. Also worthy of note from the percentage resistance profile results, is that the best agents showing relatively good susceptibility profile (Erythromycin,Ciprofloxacin,Lincomycin,Gentamicin and Ofloxacin) are agents that produce their antimicrobial effect through inhibition of protein synthesis and DNA gyrase. The increasing frequency of antimicrobial resistance among infectious organisms is of great concern to both healthcare providers and the general public, as the incidence of multidrug resistance S. aureus in a community, will make treatment of common infections much more difficult [12-13]. Table 2 shows the multiple antibiotic resistance index (MARI) index of the isolates. A total of 43% of the isolates had MAR indices less than 20%. The rest of the isolates (57%) had MAR indices greater than 20%. When the MAR index is greater than 20%, it shows that the organisms were isolated in an environment where antibiotics are abused widely [10]. It has been shown that resistance in Staphylococcus is frequently a consequence of wide use of the antibiotics in farming, hospitals and community [14]. Conclusively, the this study have presumptively shown remarkable antibiotic resistance rates amongst pathogenic S. aureus isolates and it is therefore critical for our health institutions to imbibe good antibiotic stewardship. Health institutions should also base their treatment on proper antimicrobial susceptibility test result(s) and avoid blind treatment as much as possible since the singular use of antibiotics cause resistant microbes to develop and spread via selective pressure. This is vital in order to preserve the efficacy of available drugs and thus contain the emergence of drug resistant pathogenic organisms. Table1. Biochemical characterization of sample isolates S/N Isolates Catalase test Coagulase test Lactose fermentation test Starch hydrolysis test 1 2 + - - - 2 3 + + + + 3 4 + - - - 4 7 - - - - 5 8 + + + + 6 11 + - - - 7 12 - - - - 8 13 + - - - 9 14 + + + - 10 15 + + + + 11 16 + + - - 12 17 - - - - 13 18 - - - - 14 19 - + + + 15 20 - + - - 16 22 - - - - 17 24 - - - - 18 26 + + - - 19 27 + - - - 20 28 + + - - 21 29 + - - - 22 30 + - - - 23 31 - - - - 24 32 - - - - 25 33 + + - - 26 34 - - - - 27 37 + + - - 28 38 + + + + 29 41 + + - - 30 43 + + + + 31 44 + - - - 32 45 + - - - 33 47 - - - - 34 49 + - - - 35 50 + - - - 36 52 - - - - 37 53 + - - - International Journal of Research in pharmacy and Biosciences V2 I4 May 2015 7
38 55 + - - - 39 56 + - - - 40 57 + - - - 41 58 + + - - 42 59 + - - - 43 62 + - - - 44 63 - - - - 45 64 - - - - 46 66 - - - - 47 67 + - - - 48 70 + - - - 49 71 + + + + 50 72 + - - - 51 73 + + + + 52 75 - - - - 53 77 - - - - 54 78 + - - - 55 79 + - - - 56 80 + - - - 57 81 + + + + 58 82 + - - - 59 83 + + + + 60 85 + + + + 61 87 - - - - 66 88 + + - - 63 90 + - - - 64 93 + - - - 65 95 - - - - 66 97 + - - - 67 98 + + + + 68 100 - - - - Total 68 47(69%) 22(32%) 13(19%) 12(17.6%) Table2. Multiple antibiotic resistance indices of the S.aureus isolates Isolates MARI (%) Isolates MARI (%) 3 14 41 14 8 43 43 14 14 43 58 29 15 14 62 14 16 29 71 0 20 29 81 43 26 29 83 14 28 14 85 43 31 43 88 14 33 14 98 29 36 29 Figure1. Percentage resistance of the S.aureus isolates 8 International Journal of Research in pharmacy and Biosciences V2 I5 May 2015
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