Antibiotic Susceptibility Pattern of Extended Spectrum Betalactamase ( ESBL ) Producers and other Bacterial Pathogens in Kano , Nigeria

Purpose: To evaluate the antibiotic susceptibility pattern of various bacterial pathogens including extended spectrum betalactamase (ESBL) producers in Kano, Nigeria. Method: A total of 604 consecutive clinical samples obtained from Aminu Kano Teaching Hospital (AKTH), Kano between January and July 2010 were analyzed for bacterial pathogens using standard microbiological techniques for the isolation and identification of pathogens. Antibiotics susceptibility tests including, ESBL screening and confirmation, were carried out by disc diffusion technique using Clinical Laboratory Standard Institute (CLSI) criteria. Results: Ten different types of bacteria genera were observed from nine different clinical samples. E. coli was the most frequently isolated bacteria (30.5 %) followed by Staphylococcus aureus (21.3 %). ESBL producers showed high-level resistance against the quinolones, aminoglycoside and cotrimoxazole but were sensitive to carbapenems and levofloxaccin. Non-ESBL organisms showed increased resistance to amoxicillin-clavulanate, ceftazidime, cotrimoxazole, tetracycline and amoxycillin. The prevalence of ESBL producers was 12.8 %. E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa showed ESBL production of 17.3, 14.9 and 10.0 %, respectively. Conclusion: The findings of this study suggest that regular surveys should be carried out in this locality to provide baseline data that would always be of clinical relevance in the treatment of patients and to detect the emergence of multiple antibiotic resistance strains.


INTRODUCTION
Before the discovery of penicillin, which initiated the antibiotic era, the prognosis for people with infectious diseases, such as bacterial pneumonia, tuberculosis and staphylococcal infections, was poor [1].Today antibiotics are widely prescribed for various bacterial infections with a high degree of success.However, the unfortunate outcome of the wide-scale use of antibiotics is the development of antimicrobial resistance, an adaptive response in which microorganisms begin to tolerate an amount of drug to which it was previously susceptible.The development of mechanisms for circumventing or inactivating antibiotic drugs is due largely to the genetic versatility and adaptability of microbial populations [2].
Beta-lactam antimicrobial agents are the most common drugs for the treatment of bacterial infections and account for over 50 % of global antibiotic consumption [3].Bacterial resistance to B-lactam antibiotics has significantly increased in recent years and has been attributed to the spread of plasmid mediated B-lactamases.Some of these organisms have produced new forms of the older enzymes such as the extendedspectrum B-lactamases (ESBLs) that can hydrolyze newer celphalosporins and aztreonam [4].
Extended-spectrum b-lactamases (ESBLs) represent an important mechanism of resistance in Enterobacteriacaeae, because they inactivate penicillin, narrow-and extended-spectrum cephalosporins, and aztreonam [5].This study is focused on the prevalence and antibiotic susceptibility pattern of ESBL producers in bacterial isolates of urogenital infections and other bacterial pathogens in Kano, Nigeria.

Samples
A total of 604 clinical samples as listed above obtained from patients at Amino Kano teaching Hospital from various service points between January and July 2010 were analyzed for bacterial pathogens by standard microbiological procedures [6].
The samples were inoculated on blood and MacConkey agar.Salmonella Shigella agar was included for enteric pathogens.All were incubated aerobically at 37 o C for 18 -24 h.Anaerobic culture was not carried out.Gram stain, motility and biochemical tests were used to identify the various isolates.

Antibiotic susceptibility test
This was carried out by disc diffusion technique, (WHO modified) in accordance with the CLSI criteria and interpreted accordingly [7].Mueller Hinton culture plates were inoculated by dipping a sterile cotton wool swab into a suspension of the overnight growth of the organism prepared to the density of a Mc Farland no 0.5 opacity standard; excess liquid from the swab was expressed before inoculation by spread plate method.

ESBL producers-screening and confirmation
The isolates were tested against third generation cephalosporins (cefodoxime, cefotaxime and ceftriaxone) using Clinical Laboratory Standard Istitute (CLSI) recommended, WHO modified Kirby Bauer disc diffusion method [8].Zone diameters were interpreted using the revised National Committee on Clinical Laboratory (NCCL) Standard document [14].Isolates with reduced susceptibility to cefpodoxime (≤ 17 mm) cefotaxime (≤ 27 mm) and ceftriaxone (≤ 25 mm) were considered to be possible ESBL producers.
Phenotypic confirmation test was carried-out using Double Disc Synergy test.Disc containing the standard 10 ug of cefpodoxime and 30 ug of ceftazidime/ceftriaxone, are placed 15 mm apart (edge to edge); with amoxicillin-clavulanic acid disc containing 10 ug of the latter compound mounted exactly at their center.After 16 -20 h of incubation at 35 o C, any enhancement of the zone of inhibition between a beta-lactam disk and that containing the beta-lactamase inhibitor is indicative of the presence of an ESBL [9].

Statistical analysis
The data were computed as mean and standard deviation as well as in percentage.Chi-square analysis was carried out by Epi info Version 6 software.P < 0.05 was accepted as statistically significant.

RESULTS
The age group prevalence of infected patients is presented on Table 1.The age group that had the highest number of isolates was 0 -10 years (27.8 %) while > 70 years (0.9 %) had the least.
The difference was statistically significant X 2 = 356.9,df = 7, p < 0.001.The antibiotic susceptibility data for the bacterial pathogens, which shows high level of resistance against various antibiotics, are presented in Table 3.
Most pathogens were resistant to commonly used antibiotics such as tetracycline, cotrimoxazole and amoxycillin.E. coli and Klebsiella pneumonia showed 14 and 16 % susceptibility respectively against amoxicillin/clavulanate and 43 and 21 % respectively against ceftazidime.However, ceftriaxone, gentamicin and levofloxacin showed over 60 % susceptibility against most bacterial pathogens.
Table 4 shows the percentage prevalence of extended spectrum beta-lactamase producers among urogenital pathogens.E. coli had the highest prevalence of 17.3 % followed by Klebsiella pneumonia 14.9 % while the least was Proteus vulgaris 2.4 %.The difference however, was not statistically significant (X 2 = 9.22, df = 4, p = 0.05).
The antibiotic susceptibility pattern of ESBLs against six antibiotics is presented in Table 5.Besides exhibiting high level resistance against the cephalosporins, these pathogens also show resistance to other groups of antibiotics such as the aminoglycoside, fluoroquinolones and cotrimoxazole.

DISCUSSION
The prevalence of resistance to antibiotics varies greatly from one geographical area to another as well as between hospitals within community, mainly because of the differences in antimicrobial usage and infection control practices [10].
The age group prevalence of high infectious rate observed in infants and older children when exposed to bacterial pathogens could be attributed to their low level of immunity, other researchers made similar observations in their reports [11].The majority of the bacterial isolates 75.2 %, observed in the study were Gram negative bacilli mainly of the Enterobacteriaceae.604 Key: UR = urine, CT = catheter tip, ES = ear swab, WS = wound swab, ST = stool, ECS = endocervical swab, TS = throat swab, SM = semen, UBC = umbilical cord swab This is in agreement with the reports of other researchers [12].E. coli (30.5 %) was the most frequently isolated.It also accounted for most of all urinary isolates giving 45.0 %.This observation is in agreement with the findings of other research workers [13].The preponderance of this organism may be due to the fact that it constitutes a large proportion of the intestinal flora and with low hygienic practices, it will be expected to be isolated in diseases such as urinary tract infection, wound infections and other bacterial infections.
The prevalence of ESBL producers in this study was 12.8 %, which is higher than an earlier study in Kano [14] 50) Key: CN = gentamicin, COT = cotrimoxazole, CIP = ciprofloxacin, IMP = imipenem, MEP = meropenem, LEV = levofloxacin that are completely losing their efficacy against increasing wave of bacterial resistance.
Many clinical laboratories (as well as the wider medical community) are not fully aware of the importance of ESBLs and how to detect them; laboratories may also lack the resources to curb the spread of these resistance mechanisms [23].This ignorance and possible financial constraints may be responsible for the continuous dissemination of ESBLs worldwide.

Despite
the rapidity with which new chemotherapeutic agents are introduced, bacteria have shown a remarkable ability to develop resistance to these agents.In view of the steady rise in bacteria resistance to antibiotics, it has become very important to carry out in vitro antimicrobial susceptibility testing before prescription.

CONCLUSION
Regular studies of the antibiotic susceptibility pattern of isolates commonly observed in a locality will guide therapeutic judgment and enhance antibiotic prescribing.Such surveys will yield the first clue when multi drug resistant isolates are encountered to prevent therapeutic failures that could be fatal in the management of patients.

Table 1 :
Prevalence by age of infected patients

Table 2 :
Distribution of bacterial pathogens by clinical samples and Gram-staining reactions

Table 5 :
Antibiotic susceptibility pattern of ESBL producers