Phenotypic Detection of Extended-Spectrum ß- lactamase Produced by Escherichia coli Isolates from Health facilities in Otukpo, Benue State Nigeria

Department of Microbiology, Federal College of Education, Kano, Nigeria Department of Microbiology, Federal University of Agriculture, Makurdi, Nigeria. Department of Medical Microbiology/Parasitology, Benue State University Teaching Hospital, Makurdi, Nigeria. Phenotypic Detection of Extended-Spectrum ßlactamase Produced by Escherichia coli Isolates from Health facilities in Otukpo, Benue State Nigeria Print ISSN: | Online ISSN: 2705-3385 2705-4004


INTRODUCTION
scherichia coli is a Gram-negative, facultatively anaerobic, rod-shaped, coliform bacterium of the E genius Escheria that is commonly found in the lower 1 intestine of warm-blooded organisms .Extended spectrum beta-lactamases (ESBLs) are enzymes produced by certain bacteria that are able to hydrolyze extended spectrum cephalosporins. ESBL was first reported in 1983 from Germany in isolates of 1 Klebsiella pneumonia . The frequency of ESBL production is high among E. coli and Klebsiella species. This has created a worldwide problem resulting in treatment failure. ESBLs significantly complicate therapeutic procedures when involved in infections and they lead to increased mortality. Any time ESBLs are detected, they always indicate the need for use of appropriate antibacterial agents. Failure to detect ESBL production by routine disk-diffusion tests has been 2, ,3 well documented. Many clinical laboratories are yet to be aware of the importance of ESBLs. Some that are aware lack the resources to detect them. This lack of understanding or resources is responsible for a continuing failure to respond appropriately to prevent the rapid worldwide dissemination of pathogens 4 possessing these beta-lactamases Some conditions have been observed to be risk factors for contracting infections from ESBL-producing pathogens. These include old age (> 65 years), gender, previous use of beta-lactam antibiotics and 5 fluoroquinolones . It has also been observed that health care interactions such as transfers from one hospital to another are identified risk factor for 6 acquiring infections due to ESBL producers . In Nigeria where antibiotic use is unregulated, and where extended spectrum cephalosporins and fluoroquinolones are widely prescribed as broads p e c t r u m a n t i b i o t i c s , E S B L -p r o d u c i n g microorganisms may be prevalent. Studies from various parts of Nigeria have reported 7 varying prevalences: reported a prevalence of 2.7% from studies conducted on blood stream infections in Benin-city, Southern Nigeria. A prevalence of 44.3% 8 was reported by another researcher from Benin-city . 9 reported 20.9% from South-West Nigeria. A very 10 recent study by reported a prevalence of 16.0% from studies conducted on isolates from various clinical specimens obtained from different health facilities in Makurdi, Benue State. Interactions with laboratory personnel from across Benue State reveal that bacteriological investigations for clinical specimens stop at the routine disk-diffusion antimicrobial susceptibility tests. Inability of this technique to detect resistance to antimicrobials by 3 ESBL-producers has since been established . Phenotypic detection of ESBL production by double disk synergy test is a simple technique that does not require high tech equipment and advanced expertise. The method is cheap and can be employed to meet local demands of resource-poor health facilities such as Otukpo and other cities in Benue State. Outcomes obtained from here will be compared with what has been gotten from other places and may reveal the extent of spread of this public health threat in this locality.
This study was carried in Otukpo local Government Area of Benue State, which is located in the middle belt region of Nigeria. It has a latitude of 6° 49' 0" N and longitude of 8° 40' 0" E with a population projection of 359,600 according to National Bureau of Statistics. The study examined four hundred clinical specimens involving 224 urine, 97 stool, 65 wound swabs and 14 blood samples collected from 222 female and 178 male patients (1:1.2 female to male ratio) that attended selected health facilities in Otukpo metropolis. The demographics of these patients, including age, sex and the health facility attended were reviewed and ethical clearance was obtained from the ethics review

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For Reprint Contact: jrbcs.org@gmail.com J Res Bas Clin Sci | Vol 2 | No 2 | 2021 committee of Benue State Hospitals Management Board (HMB). These specimens were inoculated on to various culture media to isolate Escherichia coli bacteria which were presumptively identified by standard microbiological and biochemical methods. Extended Spectrum beta (ESBL) production was tested for in Escherichia coli isolates by using combination disk method based on the inhibitory effect of clavulanic acid according to 11 Clinical Laboratory Standards Institute criteria. The clinical isolates were tested for their susceptibility to selected antimicrobials which included beta-lactam antibiotics like penicillin, ceftazidim, cefotaxime, cefixime and imipenem and non-beta-lactam antibiotics such as gentamycin and ciprofloxacin using Kirby Bauer disk diffusion method. The inoculated plates for the Double Disk Synergy Test (DDST) for the detection of ESBL and antibiotic o susceptibility test were incubated at 37 C for 24 hours. 4 Results were interpreted according to guidelines, where interpretive criteria for ESBL activity was based on an increase of greater or equal to 5mm in diameter of the inhibition zone around disk containing clavulanic acid as compared to the diameters of the inhibition zone around disks free of clavulanic acid. Data obtained from the study was analyzed using Statistical Package for Social Sciences (SPSS) for Windows version 16.0 (Armon. K. NY: IBM Corp). Simple frequencies and cross tabulations were -lactamase applied, and association between ESBL producing organisms and patient demographics was determined using Chi square test at significant level of p = 0.05.
The study revealed that sixty-eight (68) patients comprising 38 female (17.1%) and 30 male (16.9%) were infected with ESBL-producing Escherichia coli. This gave a prevalence of 17% for both sexes (table 1) Highest percentage of ESBLs were recovered from the isolates of stool specimens (22.7%), followed by isolates from blood specimens (21.4%). Isolates from urine specimens harboured the lowest percentage of ESBLs (13.8%), table 2. Table 3 showed that penicillin, one of the oldest antibiotics discovered was the most resisted (93.8%; n =375). This was followed closely by gentamycin 359(89.8%), which is a non-beta-lactam antibiotic. Table 3 further revealed that the ESBL-producing isolates of Escherichia coli were most susceptible to imipenem 321(80.1%), a carbapenem. Cefixime, a beta-lactam antibiotic came next in the susceptibility profile as the most potent against ESBL-producing Escherichia coli bacteria. Table 4 revealed that Otukpo General Hospital, 20(20.0%) had the highest prevalence of ESBLproducing E. coli, followed by Otia Hospital 18(18.0%). The least prevalence was found in Otukpo Comprehensive Health Centre 14(14.0%).

DISCUSSION
The spread of ESBL-producing organisms has defied geographical locations. The prevalence of 17.0 obtained in this study location which is a 10 semi-urban setting is even slightly higher than 16.0% reported by from studies conducted in Makurdi city, an urban setting. The finding is also at 12 variance with other previous works such as who reported 18.6% from Jos injection to treat all manner of infections ranging from sexually transmitted infections to systemic infections. Its easy accessibility and sale over the counters in patent medicine stores and pharmacy shops might have contributed to the high resistance. Non-human use of antibiotics, especially in poultry and other livestock production which eventually get consumed by man are also contributory factors to multidrug 16 resistance . Imipenem, (80.1%; n = 321) a carbapenem is the most active antibiotic against ESBL-producing Escherichia coli, followed by cefixime (49.8%; n = 199). Since only 19.8% of all isolates of ESBL-producing Escherichia coli are resistant to imipenem, this agent becomes the best treatment for serious infections by ESBL-producing enterobacteriaceae. This finding 17 agrees with who reported 100% susceptibility to imipenem by ESBL-producing Escherichia coli. Further research into the molecular determination of resistance genes by ESBL-producing isolates from these specimens will give a clearer picture of resistance patterns by the organism.
There is decreased susceptibility of pathogenic microorganisms to readily available drugs. With the slow pace in the development of new antimicrobials, there is need for proper utilization of available antibiotics and accurate detection and reporting of antibiotic resistance in order to keep multidrug resistant bacteria (MDR) under control. The use of carbapenems such as imipenem should be done with utmost caution as the study has shown that it remains the most potent antibiotic for treatment of infections caused by ESBL-producing organisms.
Findings from this study have revealed that antimicrobial surveillance has become imperative in all health facilities. Health professionals who prescribe antibiotics need to be properly trained on the art of CONCLUSION Recommendation prescription. Use of beta-lactam antibiotics as broad spectrum drugs needs to be checked.
Data for this study was obtained only from consenting patients that attended the selected health facilities during the period of the study. If data was gotten from the community and from more respondents, the prevalence may be higher. Also isolates were only subjected to phenotypic screening only. Molecular determination may yield higher prevalence.