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ORIGINAL ARTICLE
Year : 2023 | Volume
: 22
| Issue : 4 | Page : 508-514
Antimicrobial susceptibility pattern of extended-spectrum beta-lactamase-producing uropathogens in aminu Kano teaching hospital, Northwestern Nigeria
Usman Yahya Umar1, Fatima Jummai Giwa2, Abdulrasul Ibrahim2, Farida Suleiman Gachi3
1 Department of Medical Microbiology, Aminu Kano Teaching Hospital, Kano; Nigeria Field Epidemiology and Laboratory Training Programme, Abuja, Nigeria
2 Department of Medical Microbiology, Faculty of Basic Clinical Sciences, College of Medical Sciences, Ahmadu Bello University, Zaria, Nigeria
3 Department of Medical Microbiology and Parasitology, Modibbo Adama University Teaching Hospital, Yola, Adamawa State, Nigeria
Correspondence Address:
Usman Yahya Umar
Department of Medical Microbiology, Aminu Kano Teaching Hospital, Kano
Nigeria
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_155_22
Abstract
Background: UTI is the most common nosocomial infection among hospitalized patients. Emerging increasing resistance has been observed among uropathogens of the family enterobacterales. Extended spectrum beta lactamase genes encode for multidrug resistance and unrestricted use of antibiotics in hospitals provides an environment for spread of infections with limited treatment options. This necessitates therapies based on culture and antimicrobial sensitivity to improve patients' outcomes We aimed to determine susceptibility pattern of ESBL uropathogens among hospitalized patients in Aminu Kano Teaching Hospital, Kano. Methodology: Three hundred and eighty-nine urine samples were obtained from in-patients with UTI between April 2020 and April 2021. Five samples were rejected and remaining analyzed. Susceptibility testing was done by modified Kirby Bauer technique. Clinical Laboratory Standards Institute guidelines 2019 (CLSI 2019) and the European Committee on Antimicrobial Susceptibility testing guidelines version 2 (EUCAST version 2) were used for screening and confirmation of ESBL production respectively. Results: Of the 384-urine processed, 105 (27.3%) were gram negatives and 81 were Enterobacterales, Isolation rates were E. coli-55.6%, K. pneumoniae-29.6%, Citrobacter spp.-12.3%, P. mirabilis-1.2% and Morganella spp.-1.2%. Among Enterobacterales, 32 (39.5%) were ESBL producers. Prevalence of ESBL were 62.5% for Escherichia coli, 28.1% for Klebsiella pneumoniae and 9.4% for Citrobacter species. Susceptibility showed that ESBL-producing Isolates were highly susceptible to amikacin (96.9%). Resistance to other antibiotics varied from 3.1% to 100%. Conclusion: We recommend strengthening laboratory capacity, antibiotics stewardship and Infection control to prevent spread of resistant pathogens including ESBLs.
Abstract in French Résumé
L'infection urinaire est l'infection nosocomiale la plus fréquente chez les patients hospitalisés. L'émergence d'une résistance croissante a été observée parmi les uropathogènes de la famille des entérobactéries. Les gènes de bêta-lactamase à spectre étendu codent pour la multirésistance et l'utilisation sans restriction d'antibiotiques dans les hôpitaux crée un environnement propice à la propagation des infections avec des options de traitement limitées. Nous avons cherché à déterminer le profil de sensibilité des uropathogènes BLSE chez les patients hospitalisés à l'hôpital universitaire Aminu Kano, à Kano. Méthodologie: Trois cent quatre-vingt-neuf échantillons d'urine ont été obtenus auprès de patients hospitalisés souffrant d'infections urinaires entre avril 2020 et avril 2021. Cinq échantillons ont été rejetés et les autres analysés. Les tests de sensibilité ont été effectués à l'aide de la technique Kirby Bauer modifiée. Les directives 2019 du Clinical Laboratory Standards Institute (CLSI 2019) et les directives de l'European Committee on Antimicrobial Susceptibility testing version 2 (EUCAST version 2) ont été utilisées respectivement pour le dépistage et la confirmation de la production de BLSE. Résultats: Sur les 384 urines traitées, 105 (27,3 %) étaient à Gram négatif et 81 étaient des Enterobacterales, les taux d'isolement étaient de 55,6 % pour E. coli, 29,6 % pour K. pneumoniae, 12,3 % pour Citrobacter spp, 1,2 % pour P. mirabilis et 1,2 % pour Morganella spp. Parmi les entérobactéries, 32 (39,5 %) étaient productrices de BLSE. La prévalence des BLSE était de 62,5 % pour Escherichia coli, de 28,1 % pour Klebsiella pneumoniae et de 9,4 % pour les espèces de Citrobacter. La sensibilité a montré que les isolats producteurs de BLSE étaient très sensibles à l'amikacine (96,9 %). La résistance aux autres antibiotiques variait de 3,1 % à 100 %. Conclusion: Nous recommandons de renforcer les capacités des laboratoires, la gestion des antibiotiques et le contrôle des infections afin de prévenir la propagation de pathogènes résistants, y compris les BLSE.
Mots-clés: Hôpital universitaire Aminu Kano, sensibilité aux antimicrobiens, bêta-lactamase à spectre étendu, Kano, uropathogènes
Keywords: Aminu Kano Teaching Hospital, antimicrobial susceptibility, extended spectrum beta lactamase, Kano, uropathogens
Introduction 
Urinary tract infection (UTI) is the most common nosocomial infection among hospitalized patients and is a major public health concern in developing countries. Emerging increasing resistance has been observed among uropathogens of the family Enterobacterales, especially Escherichia coli and Klebsiella spp.[1],[2],[3]
Increasing antibiotic resistance among bacterial pathogens escalates the financial burden associated with these infections. In the United States only, it was estimated that about 11 million cases of UTIs were reported annually.[4] Similarly, over 7 million consultations were due to symptoms of UTIs, leading to an estimated cost of 1.6 billion US dollars.[5] Extended-spectrum beta-lactamase (ESBL)-producing bacteria possess the ability to hydrolyze penicillins, oxyimino-cephalosporins, and aztreonam and are inhibited by clavulanic acid. They are reported from all over the globe including Africa.[6] Uropathogens of the family Enterobacterales are increasingly shown to contain ESBL-producing genes. This is not unconnected with the liberal and unrestricted use of antibiotics, especially third-generation cephalosporins in the hospitals coupled with the carriage of genes that encode the beta-lactamase on plasmids, which provides a favorable environment for the selection of antimicrobial resistance. More disturbing is the fact that the plasmids, apart from coding for ESBL, may also carry some other resistance genes which can make the bacteria resistant to other classes of antibiotics such as quinolones, aminoglycosides, and sulfonamides.[7] As such, these agents may not be very effective in the management of infections due to ESBL-producing Enterobacterales. Most clinicians resort to the use of carbapenems for the treatment of these infections. However, it is documented that resistance to carbapenems is evolving rapidly due to increased ESBLs-producing isolates globally, which led to the increased use of carbapenems that resulted in carbapenem resistance and emergence of carbapenems-resistant Enterobacterales.[2],[8],[9] This trend necessitates evidence-based targeted therapies based on patients culture, resistance testing, and antimicrobial sensitivity profile to improve clinical outcomes This study aimed to determine the antimicrobial susceptibility pattern of ESBL-producing uropathogens among patients in Aminu Kano Teaching Hospital (AKTH), Kano State, Nigeria.
Materials and Methods Study area
The study was conducted at AKTH, Kano State, Nigeria. Kano State is situated in the Northwestern part of Nigeria. The state has an estimated population of 20 million people (projected from 2006, National population commission (NPC)).[10] More than 60% of the population is rural with scanty or no school education. Similarly, the majority of them (61%) belong to the low socioeconomic status.[11]
AKTH is a tertiary health facility located in Kano state. It provides health-care services, training, and research to serve the needs of the community. It is also one of the major referral centers in the region offering services not only to the neighboring states but also neighboring countries such as Niger republic.[12] On a daily basis, more than 900 patients are attended to in the outpatients' department, which comprises consulting clinics, outpatients' service, and accident and emergency.[13] The hospital is a 700 bed-capacity with 60.2% bed occupancy and an average admission of 20,359 patients annually. They also provide laboratory services, with the Medical Microbiology Department processing approximately 35,000 specimens annually.[13] The urine bench has the highest turnover with an estimated 16,000 samples processed annually.[13] An audit conducted in 2018 for antimicrobial resistance profile of Enterobacterales isolated in the hospital revealed ESBL-producing isolates of up to 25%.
Study design and study duration
The study was a hospital-based, descriptive, cross-sectional study carried out from April 2020 to April 2021.
Sample size determination
The sample size was determined using the formula the Fisher's formula,[14]
Where n is the sample size, Z is the 95% confidence level at 1.96, d is the level of precision set at 5% (0.05), and p is the prevalence from a previous studies, using 34.4% (0.344).[15] A sample size of 347 was thereafter calculated.
Sampling technique
Patients were recruited consecutively as they presented to the hospital in the various wards until the sample size was obtained.
Study population
The study was conducted among inpatients in AKTH with suspected UTI irrespective of their age and sex (male and female medical wards, male surgical ward, accident and emergency, emergency pediatrics ward, pediatrics medical ward, and gynecology amenity ward).
Inclusion criteria
All patients admitted at AKTH for a period of at least 72 h irrespective of their age, sex, and clinical condition with the diagnosis of suspected UTI.
Exclusion criterion
Severely ill patients were excluded from the study.
Sample collection
Patients were given a sterile, leak proof wide-mouthed container, into which they supplied about 10ml of early morning, midstream, and clean-catch urine. For catheterized patients, 5–10 mL of urine specimen was obtained through the catheter tubing using a sterile needle and syringe after disinfection with 70% alcohol. Specimens were processed within 2 h of collection.
Bacterial identification
Urine microscopy was done using uncentrifuged urine and urine sediment.[16] The specimen was inoculated on cysteine lactose electrolyte deficient and blood media and incubated aerobically at 35–37°C for 18–24 h. Significant bacteriuria count was performed on blood agar using a calibrated wire loop. Secondary Gram stain was done using discrete colonies from the blood agar plate. Further identification was performed using conventional technique such as oxidase, motility, triple sugar iron agar, indole, urease, and citrate tests.[16] Analytical profile index Biomerieux was used for some of the difficult to identify isolates.
Antibiotic sensitivity testing
This test was conducted using modified Kirby–Bauer disc diffusion method as described in clinical laboratory standard institute 2019 guidelines. The interpretation was also conducted using the same guidelines.[17]
A saline suspension of the test organism was made using the direct colony suspension and its turbidity adjusted using the 0.5 McFarland standard. The suspension was inoculated on a Mueller–Hinton agar using a sterile swab stick dipped into the inoculum suspension. The whole surface of the medium was streaked three times by rotating the plate 60° every time to ascertain uniform distribution of the inoculum. The antibiotics selected were meropenem (10 μg), cefpodoxime (10 μg), cefotaxime (30 μg), amoxicillin-clavulanic acid (20/10 μg), gentamicin (10 μg), amikacin (30 μ), ciprofloxacin (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), ampicillin (30 μg), and nitrofurantoin (300 μg). The two plates, each containing five antibiotics were incubated aerobically at 35–37°C for 24 h, after which the diameter of zones of growth inhibition around the discs was measured. A similar procedure was conducted on Klebsiella pneumoniae ATCC 700603 and E. coli ATCC 25922 as positive and negative controls, respectively.
Extended spectrum beta-lactamase screening test
The test was conducted using the modified Kirby–Bauer technique (as described above). Cefpodoxime (10 μg) and cefotaxime (30 μg) discs were used to screen all the Gram-negative isolates. Any isolate with the zone of inhibition <17 mm for cefpodoxime and/or <27 mm for cefotaxime was subjected to confirmatory test.[18]
Double-disc synergy test
The test was conducted using the modified Kirby–Bauer method. Cefpodoxime (10 μg) and cefotaxime (30 μ) were placed each on either side of co-amoxiclav 15 mm apart center to center The plate was inverted and incubated overnight at 35–37°C. ESBL-positive strains showed the expansion of the zone of inhibition of cephalosporins toward the co-amoxiclav giving a dumb-bell appearance. This occurred because of the presence of the ESBL inhibitor clavulanic acid in the co-amoxiclav.[18],[19] Control strains of ATCC 700603 and ATCC 25922 were used (EUCAST, version 2).[20]
Ethical considerations
The study proposal was reviewed and approved by the Health Research Ethical Committee of AKTH, Kano. Consent was also obtained from the study participants, and confidentiality was maintained.
Data analysis
Data were cleaned and entered in Microsoft Excel version 2016, and analysis was conducted. Results were presented in the form of tables and charts as appropriate.
Results A total of 389 urine samples were collected from admitted patients with suspected UTI. Analysis was conducted on 384 specimens as five of them were rejected. Among those analyzed, 265 (69%) yielded no growth and 105 (27.3%) were found to be Gram negatives with 81 (21.1%) further identified as Enterobacterales. Furthermore, 14 (3.6%) were characterized as Gram positive [Figure 1]. The isolation rates for the Enterobacterales were 55.6%, 29.6, 12.3%, 1.2%, and 1.2% for E. coli, K. pneumoniae, Citrobacter spp., Proteus mirabilis, and Morganella spp., respectively. After initial screening, 67 (82.7%) of the isolates were found to be ESBL producers. Confirmation by double-disc synergy test revealed only 32 isolates to be ESBL producers, with 62.5%, 28.1%, and 9.4% for E. coli, K. pneumoniae, and Citrobacter specie, respectively. Resistance to multiple antibiotics was also observed among the ESBL-producing uropathogens [Figure 2], [Figure 3], [Figure 4], [Figure 5].
Figure 1: Distribution of uropathogens among in-patients in Aminu Kano Teaching Hospital, 2021 (n = 384)
Figure 2: Susceptibility pattern of extended spectrum beta-lactamase-positive isolates (n = 32)
Figure 3: Susceptibility pattern of extended spectrum beta-lactamase-positive strain of Escherichia coli (n = 20)
Figure 4: Susceptibility pattern of extended spectrum beta-lactamase-positive strain of Klebsiella pneumoniae (n = 9)
Figure 5: Susceptibility pattern of extended spectrum beta-lactamase-positive strain of Citrobacter species (n = 3) Discussion The present study has demonstrated that E. coli and K. pneumoniae were the most common uropathogens. Similar findings were reported by other researchers.[1],[15],[21],[22],[23] The study found a relatively significant ESBL prevalence of 39.5% among uropathogens. This is supported by the finding of Yusha'u et al. in Kano state, who documented an ESBL prevalence of 41.2%.[24] Higher prevalence of 61.5% and 57% was reported by workers in Southeastern Nigeria and India, respectively.[2],[25],[26] On the other hand, lower prevalence rates of 23% and 16% were reported by researchers in Maiduguri and Anambra states, respectively.[6],[13] Similarly, Sozen et al. reported a relatively low value of 26.6% in Sudan.[27] The findings of a relatively high prevalence of ESBL in this study are most likely due to the use and misuse of antibiotics, especially the third-generation cephalosporins which are workhorses for most physicians in our health-care facilities. The differences in ESBL prevalence reported can be explained by the methodologies used in the different studies as well as the geographical peculiarities of the various regions.
ESBLs were more prevalent in E. coli followed by K. pneumoniae, whereas Citrobacter spp. had the least. Similarly, resistance to multiple antibiotics was noted among the ESBL-producing uropathogens.[2],[22],[23],[25] This is likely from the fact that the plasmid carrying the ESBL gene can acquire other resistance genes that may confer resistance to other antibiotic classes, especially in hospital setup.[8],[24],[25],[26],[27] Other researchers have reported similar findings, where ESBL production was more frequent in E. coli followed by K. pneumoniae.[22],[28],[29],[30],[31],[32],[33],[34],[35] A study reported a very high prevalence of ESBL of 84% in E. coli among patients with UTI in India.[26]
The antimicrobial susceptibility pattern for the ESBL isolates in this study was 96.9%, 84.4%, and 78.1% for amikacin, nitrofurantoin, and meropenem, respectively, whereas susceptibility to gentamicin, ciprofloxacin, and co-amoxiclav was low at 46.9%, 28.1%, and 21.9%, respectively. While much lower values of 12.5% were observed for co-trimoxazole and a 100% resistance to ampicillin [Figure 2]. The high resistance observed with ampicillin may be related to the accessibility and unrestricted use of the antibiotic. Moreover, some of the enterobacterales including Citrobacter spp., Enterobacter spp., Klebsiella spp., Proteus spp., and Providentia spp. are intrinsically resistant to ampicillin.[36] Furthermore, multiple genes for resistance encoded by plasmids of ESBL producers and the known resistance of ESBL-producing pathogens to beta-lactams are additional factors.
In this study, ESBL producers showed a good susceptibility rate to meropenem [Figure 1]. Carbapenems are considered the mainstay and agents of choice in the treatment of infections caused by ESBL-producing pathogens. Due to their stability to the extended spectrum beta lactamase enzymes.[15],[33] However, higher susceptibility values of 100% and 98% for imipenem among uropathogens were documented by researchers in Northwestern Nigeria and India, respectively.[15],[26] In this study, however, 21% of the isolates showed resistance to meropenem. This was in consonant with findings documented in Ghana by Karikari et al., where 22% of the isolates demonstrated resistance to imipenem.[37] This observed phenomenon of increasing resistance to the antibiotic of last resort calls for an immediate implementation of antibiotic stewardship policies in our health-care institutions to reverse this precarious trend and preserve the use of carbapenems and the evolution of carbapenemase-producing isolates.
Susceptibility of 84.4% for nitrofurantoin was observed in this study [Figure 2]. This is corroborated by findings of researchers from Nigeria and Ghana who reported susceptibility of nitrofurantoin on ESBLs of 70%–75%, respectively.[15],[37] Low resistance to nitrofurantoin in various regions has been reported by researchers among Enterobacterales, including those not susceptible to other antibiotics.[15],[20],[38] This may be due to the ability of nitrofurantoin to attain a high urinary concentration. These findings suggest that the antibiotic can be used to treat uncomplicated UTI. On the other hand, a significant resistance of up to 40% was reported from Kampala, Uganda.[22] More research may be needed to further elucidate the reasons for this finding.
Similarly, studies have also revealed a high level of resistance among the ESBLs isolates to commonly prescribed antibiotics used in the treatment of UTIs. All ESBL producers showed complete (100%) resistance to ampicillin, followed by trimethoprim-sulfamethoxazole 87.5% and amoxicillin-clavulanic acid 78.1% [Figure 2], [Figure 3], [Figure 4], [Figure 5]. This is in consonant with trends reported from Nigeria, Ghana, and Kampala.[6],[22],[37] These findings simply imply that these antibiotics are no longer suitable and efficient to be used as empirical agents in the management of UTIs in our health facilities. As such, clinicians and other prescribers of these antibiotics should be guided by relevant culture and sensitivity results to avoid possible treatment failure that can result from their use in patients.
Variable susceptibility rates were observed for aminoglycosides with highest sensitivity to amikacin and lowest to gentamicin [Figure 2], [Figure 3], [Figure 4], [Figure 5]. Among the isolates tested, only few were found to be susceptible to ciprofloxacin [Figure 2], [Figure 3], [Figure 4], [Figure 5]. The excessive use of the quinolones such as ciprofloxacin has resulted in high selective pressure, leading to the increasing resistance observed in that class of antibiotics.[15],[22] This is corroborated by the findings of the study for monitoring antimicrobial resistance trends (SMART) study in the USA, where high resistance to fluoroquinolones was found among Gram negative pathogens causing UTIs.[39] Furthermore, resistance to third-generation cephalosporins among the ESBL-producing bacteria may coexist with resistance to other antibiotics including quinolones.[15],[22] A high susceptibility of 96.9% was recorded for amikacin among the ESBL-producing isolates in this study, [Figure 2] akin to these are findings reported by other researchers among ESBL-producing Enterobacterales.[3],[15],[19],[25] This property is attributed to the agent's ability to resist most of the aminoglycoside-modifying enzymes. As such, amikacin is employed to treat infections due to aminoglycoside-resistant organisms.[15],[33] This is further supported by the report of the SMART study in the USA that showed that amikacin is second only to carbapenem in the treatment of infections due to ESBL-producing uropathogens.[39] In the same vein, sensitivity of up to 96% and 95% was recorded for this antibiotic in Nigeria and India, respectively.[15],[26] Therefore, Amikacin may be an antibiotic of choice when treating UTIs from ESBL-producing uropathogens.
Conclusion The present study showed ESBL production in about two-thirds of uropathogens with a high susceptibility to amikacin. We recommend strengthening laboratory capacity to improve the surveillance of resistant pathogens in our health facilities, as well as establishment of an efficient antibiotic stewardship program to regulate antibiotics use in our health-care institutions.
Acknowledgment
We would like to appreciate Aminu Kano teaching hospital for providing an enabling environment during the conduct of this research work. We are also very grateful to the Nigeria field epidemiology and laboratory training network/African field epidemiology network for their support during the research work.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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