Prevalence of extended spectrum [beta]-lactamase, AmpC [beta]-lactamase and metallo-[beta]-lactamase enzymes among clinical isolates recovered from patients with urinary tract infections in Benin City, Nigeria.
Urinary tract infection (UTI) is one of the commonest type of infection in humans, and a major reason that patients residing in the community seek medical care (1). Infection of the urinary tract is almost exclusively due to bacteria and the rise in antibacterial resistance worldwide means that therapeutic options are getting fewer (1, 2). Beta-lactam antibiotics are drugs of choice for the treatment of many bacterial infections (3). Production of beta-lactamase enzyme(s) is a major mechanism of bacterial resistance to this class of antibiotics (1, 2. The steady rise in the prevalence of Gram-negative bacteria producing extended spectrum beta-lactamase (ESBL), AmpC beta-lactamase and carbapenemases means newer antibiotics like extended spectrum cephalosporins (ESCs) (e.g. ceftriaxone, cefotaxime, ceftazidime), cephamycins (e.g. cefoxitin and cefotetan), monobactams (e.g. aztreonam) and carbapenems (e.g. ertapenem, imipenem and meropenem) may not achieve their therapeutic purpose when they are administered to patients (1, 3, 5).
ESBL enzymes, which are largely plasmid mediated, are capable of hydrolyzing ESCs and aztreonam, but are inhibited by clavulanic acid, tazobactam or sulbactam (5). AmpC [beta]-lactamases are either plasmid- or chromosomally mediated and, when hyper-produced, confer resistance to a wide variety ofp-lactam antibiotics including penicillins, ESCs, cephamycins, and monobactams (3). Metallo-beta-lactamase (MBL) enzymes are a type of carbapenemase enzyme which are also either plasmid or chromosomally mediated (6, 7). They are characterised by their ability to hydrolyse carbapenem antibiotics, inhibition by metal ion chelators such as EDTA, and resistance to all currently available [beta]-lactamase inhibitor drugs (6, 7). Mobile genetic elements harboring genes that code for any of these enzymes have a high propensity for transfer within and between bacterial species.
Any bacterium that harbors any of the aforementioned beta-lactamase enzymes would compulsorily have resistance to a number of antibiotics. This could invariably lead to prolonged hospital stay, worsened health conditions or even death (3, 5, 6). Diagnostic laboratories in Nigeria, however, do not routinely screen for these enzymes (3, 4, 8).
A previous study in Libya showed ESBL prevalence among the predominant uropathogens E. coli and Klebsiella spp as 6.7% and 21.7% respectively (1). A similar study in Nepal showed a prevalence of 13.51% and 16.55% for E. coli and K. pneumoniae respectively (9). A North-West Nigerian study showed a prevalence of 17.6% and 50.0% for ESBL and AmpC respectively among Enterobacteriaceae recovered from urine specimens (10). Another study in the same region showed 38.3% prevalence of MBL among bacterial isolates recovered from urine specimens (4). There is however paucity of information on the distribution of these three enzymes among Gram-negative bacilli (GNB) causing UTI in South-South region, Nigeria. This study was therefore conducted to determine the prevalence rate of ESBL, AmpC [beta]-lactamase and MBL enzymes among bacterial isolates recovered from the urine samples of patients with signs and symptoms of UTI in Benin City, Nigeria.
MATERIALS AND METHODS
A total of 554 patients with signs and symptoms of UTI either admitted in wards or attending out-patient clinics in University of Benin Teaching Hospital (UBTH) were recruited for this study. This comprised 249 males and 305 females. The study was conducted between 15th February and 14th May, 2016. Informed consent was obtained from all patients and research protocols were carried out in accordance with the Helsinki declaration.
Specimen collection and processing
Clean-catch urine samples were collected into sterile screw-cap universal containers containing boric acid crystals and transported to the medical microbiology laboratory, University of Benin Teaching Hospital for processing. A loopful (0.001 ml) of well-mixed un-centrifuged urine was streaked on blood agar and cysteine lactose electrolyte deficient medium (Lab M, United Kingdom). The plates were incubated aerobically for 24 hours at 37[degrees]C and counts were expressed in a colony forming unit (CFU) per milliliter (mL). A count of [greater than or equal to] [10.sup.5] CFU/mL was considered significant to indicate UTI. For the cell count, each urine sample (10 mL) was centrifuged at 1000 g for 5 minutes. The supernatant was thereafter discarded and the deposit was examined microscopically at high magnification for red blood cells, pus cells, casts, crystals, and epithelial cells (11). A count of pus cells [greater than or equal to] 5 per high power field (x 40 objective) was considered to indicate infection. UTI was diagnosed if the bacteria count, pus cells, or both were significantly high in an individual.
Identification of bacterial isolates
Identification of bacterial colonies was performed using standard techniques, including Gram staining, and biochemical tests (12). The isolates were thereafter preserved on nutrient agar slants for further analysis.
Bacterial isolates were screened for ESBL enzymes using the double disc diffusion method as previously described with 30 pg ceftazidime and cefotaxime discs (Abtek Biologicals Ltd, Liverpool, UK) used as the indicator cephalosporins (13). The positive control strain K. pneumoniae ATCC 700603 was included.
Test for AmpC [beta]-lactamase
The cefoxitin-cloxacillin inhibition test was carried out as previously described (14). Briefly, the test isolate was seeded on Mueller Hinton agar (MHA) as for ESBL detection and two 30 pg cefoxitin disks (Abtek Biologicals Ltd, Liverpool, UK) were placed on the surface of the seeded MHA plate. One of the cefoxitin discs was supplemented with 200 pg cloxacillin. The plate was thereafter incubated at 37[degrees]C overnight. Production of AmpC [beta]-lactamase was inferred if the zone of the cefoxitin disc supplemented with cloxacillin was [greater than or equal to] 4 mm greater than that of cefoxitin disc alone.
A modification of a previously described method was used for the detection of metallo-[beta]-lactamase (15). Briefly, each bacterial isolate was seeded on the surface of MHA plate as in ESBL and AmpC detection. Imipenem 10 [micro]g and meropenem 10 [micro]g discs were placed on either side of a 1,900 mg EDTA disc, 10 mm apart from the EDTA disc (edge-to-edge) on the seeded plate. The plate was incubated overnight at 37[degrees]C. A synergistic zone of inhibition between the EDTA disc and one or both discs was taken as indicative of metallo-b-lactamase production when compared with the control strain Escherichia coli ATCC 25922 which did not show synergism.
Disc susceptibility test
Antimicrobial susceptibility tests were performed using antibacterial discs namely imipenem (IPM) (10 [micro]g), meropenem (MEM) (10 [micro]g), Amoxicillin-clavulanate (AMC) (30 [micro]g), ceftazidime (CAZ) (30 [micro]g), cefuroxime (CXM) (5 [micro]g), gentamicin (GEN) (10 [micro]g), ofloxacin (OFX) (5 [micro]g), ceftriaxone (CRO) (30 [micro]g), and ciprofloxacin (CIP) (5 [micro]g), using the British Society for Antimicrobial Chemotherapy (BSAC) method (16). Bacterial isolates which showed resistance to [greater than or equal to] 3 classes of antibacterial agents were deemed multi-drug resistant (MDR).
A total of 554 consecutive non-repetitive urine samples (249 males and 305 females) were received during the study period. Of this number, 205 (37.0%) were culture positive and 126 were infected with GNB.
The distribution of GNB and beta-lactamase enzyme type is shown in Table 1.Escherichia coli showed the highest prevalence among GNB causing UTI (43.7%). Klebsiella spp was the most prevalent ESBL producer (83.3%). P. aeruginosa and Klebsiella spp showed the highest rate of AmpC and MBL production with 30.0% and 63.9 respectively.
In relation to the combined effect of beta-lactamase enzymes, among all bacterial isolates screened, 35 (27.8%) of isolates showed production of two out ofthe three [beta]-lactamase enzymes screened for in this study. Klebsiella spp had the highest prevalence for ESBL + MBL (38.9%), but only one isolate showed simultaneous production of AmpC + MBL enzymes (2.8%). One isolate each of E. coli (1.8%), Klebsiella spp (2.8%) and P. aeruginosa (10.0%) showed simultaneous production of all three enzymes (Table 2).
The distribution of MDR clinical isolates is shown in Table 3. Klebsiella spp showed the highest prevalence among MDR beta-lactamase positive bacteria with 44.9%. Compared to other bacteria, the majority of P. aeruginosa were MDR.
The most effective antibacterial drug for the GNB which produced ESBL, ampC or MBL, were the carbapenems (imipenem-52.8%, meropenem-53.9%), while the least effective was amoxicillin-clavulanate (13.5%). Also, the most effective antibacterial drug for [beta]-lactamase negative GNB were the carbapenems (imipenem-78.4%, meropenem-63.9%) while the least effective was amoxicillin-clavulanate (35.1%) (Figure 1).
In this study, the prevalence of UTI was 37.0%. This observation agrees with a previous study in Benin which observed 39.0% prevalence of UTI among symptomatic patients and another in Okada, a rural community in Edo State, which reported 39.7% prevalence (17,18). E. coli was the most frequently isolated uropathogen among GNB causing UTI during this study. This finding aligns with previous studies in Nigeria and around the world (9,19). The occurrence of ESBL production among uropathogens was 51.6%, with Klebsiella spp showing a significantly high rate of ESBL producers, with 83.3% prevalence. This observation differs slightly from a previous study in Benin which showed an overall prevalence of GNB producing ESBL as 41.6% among uropathogens, with Enterobacter spp showing the highest prevalence (20). Though Enterobacter spp was not isolated during the study period, this study shows a rise in ESBL-producing Klebsiella spp in comparison with the same study in which 30.4% was observed for Klebsiella spp. The prevalence rate is also higher than that found in an Indian study which reported 52.8% for Klebsiella pneumoniae recovered from UTI (21).
The overall prevalence of GNB producing AmpC [beta]-lactamase and MBL was 18.3% and 35.7% respectively. Among the Enterobacteriaceae Klebsiella spp showed the highest rate of AmpC [beta]-lactamase (22.2%) and MBL (63.9%) production respectively. Two previous Northern Nigerian studies showed the prevalence of GNB-producing AmpC [beta]-lactamase and MBL as 50.0% and 38.1% among uropathogens respectively (4,10). Our study therefore shows a comparatively lower prevalence rate of AmpC [beta]-lactamase.
The co-existence of different [beta]-lactamase enzyme phenotypes on a single bacterium was noteworthy in this study and implies markedly limited therapeutic options for patients with UTI. Providencia spp were more likely to show simultaneous production of ESBL and AmpC [beta]-lactamase in comparison with other organisms. Uncommon genetic determinants of ESBL in our region (VEB-1 and OXA-10) have been recently demonstrated among Providencia spp recovered from catheter tips and wound specimens (22). This bacterium has also been previously implicated in UTI in previous studies in Benin (19, 20). The finding of more than one [beta]-lactamase enzyme phenotype in a bacterium therefore underscores the need for screening bacterial isolates for these three enzymes routinely when GNB are isolated from UTI cases in Nigeria as the drugs of choice for treatment, the [beta]-lactam antibiotics, are failing. Over-the-counter sale of antibiotics without prescription is rife in Nigeria (8,19). This may have exerted selective pressure overtime on bacterial pathogens, leading to the proliferation of these multidrug resistant GNB.
The majority of bacterial isolates causing UTI in this study were MDR. Our observation that the majority of Klebsiella spp were MDR and ESBL positive is in tandem with a lot of studies worldwide and shows that more attention should be accorded this organism in our region (7, 9). Noteworthy was the co-resistance to various other classes of antimicrobials, including fluoroquinolones and aminoglycosides. The existence of plasmids which bear genes conferring resistance to multiple classes of antibiotics alongside genes coding for ESBL, AmpC or MBL has been demonstrated in previous studies (5, 6). In comparison with other bacteria, P. aeruginosa was more likely to be MDR. This bacterium has been demonstrated to have intrinsic resistance to different classes of antibacterial drugs through mechanisms such as multidrug resistance efflux pumps, decreased permeability and the loss of the OprD2 (outer-membrane porin) protein (23).
In our region, the drugs of last resort are carbapenem antibiotics (imipenem, ertapenem and meropenem). The poor susceptibility of the carbapenems, the high prevalence of MBL as well as the co-existence with other resistance markers and other [beta]-lactamase enzymes in uropathogens is a wake-up call for the need to review our antibiotic policy at both institutional and national levels.
The limitation of this study is its reliance on phenotypic methods for the detection of ESBL, AmpC and MBL. Molecular techniques remain the gold standard as genes implicated can be detected thereby aiding epidemiological surveys, more so phenotypic methods do not detect all enzymes.
E. coli was the most frequently encountered GNB causing UTI in this study. The overall prevalence of ESBL, AmpC [beta]=lactamase and MBL among GNB was 51.6%, 15.1% and 35.7% respectively. The co-existence of more than one [beta]-lactamase enzyme type in a bacterium was demonstrated. We strongly recommend laboratory guidance in administration of antibacterial drugs and advocate prudence in the use of antibiotics both in the hospital and community setting. Continued surveillance of resistance prevalence is essential in order to monitor the spread of resistance in our region.
Ephraim Ehidiamen Ibadin, BMLS MSc AIMLS, Medical Laboratory Scientist 
Richard Omoregie, MPhil FIMLS, Lecturer [1,2]
Idahosa Onaiwu Enabulele, MSc PhD, Professor  and Lecturer 
 Medical Microbiology Unit, Medical Laboratory Services, University of Benin Teaching Hospital, Benin City, Nigeria
 School of Medical Laboratory Sciences, University of Benin Teaching Hospital, Benin City, Edo State, Nigeria
 Department of Microbiology, Faculty of Life Sciences, University of Benin, Benin City, Nigeria
Corresponding author: Ephraim Ibadin.
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Ephraim Ehidiamen Ibadin , Richard Omoregie [1,2] and Idahosa Onaiwu Enabulele [2,3]
 Medical Microbiology Unit, Medical Laboratory Services, University of Benin Teaching Hospital;  School of Medical Laboratory Sciences, University of Benin Teaching Hospital and  Department of Microbiology, Faculty of Life Sciences, University of Benin, Benin City, Nigeria
Caption: Figure 1. Antimicrobial susceptibility pattern of GNB recovered from patients with UTI according to [beta]-lactamase production.
Table 1. Distribution of Gram-negative bacilli according to beta- lactamase type produced. Number tested (% Organism of total) ESBL AmpC MBL Citrobacter spp 1 (0.8) 1 (100) 0 0 Escherichia coli 55 (43.7) 22 (40.0) 7 (12.7) 15 (27.3) Klebsiella species 36 (28.6) 30 (83.3) 8 (22.2) 23 (63.9) Proteus mirabilis 6 (4.8) 2 (33.3) 0 1 (16.7) Proteus vulgaris 5 (4.0) 2 (40.0) 0 1 (20.0) Providencia spp 6 (4.8) 4 (66.7) 1 (16.7) 1 (16.7) Alcaligenes spp 5 (4.0) 3 (60.0) 0 0 Pseudomonas aeruginosa 10 (7.9) 1 (10.0) 3 (30.0) 4 (40.0) Pseudomonas fluorescens 1 (0.8) 0 0 0 Acinetobacter species 1 (0.8) 0 0 0 Total 126 65 (51.6) 19 (15.1) 45 (35.7) Number in bracket: percentage. ESBL: extended spectrum beta lactamase. MBL: metallo-[beta]-lactamase. AmpC: AmpC [beta]-lactamase Table 2. Proportion of GNB in relation to combined effect of beta- lactamase enzymes. ESBL+ Number ESBL + ESBL + AmpC + AmpC+ Organism tested AmpC MBL MBL MBL Citrobacter spp 1 0 0 0 0 E. coli 55 3(5.5) 10 (18.2) 0 1 (1.8) Klebsiella spp 36 2(5.6) 14 (38.9) 1 (2.8) 1 (2.8) Proteus mirabilis 6 0 1 (16.7) 0 0 Proteus vulgaris 5 0 1(20.0) 0 0 Providencia spp 6 1 (16.7) 0 0 0 Alcaligenes spp 5 0 0 0 0 Pseudomonas aeruginosa 10 0 2 (20.0) 0 1 (10.0) Pseudomonas fluorescens 1 0 0 0 0 Acinetobacter spp 1 0 0 0 0 Total 126 6 (4.8) 28 (22.2) 1 (0.8) 3 (2.4) Number in bracket: percentage. ESBL: extended spectrum beta lactamase. MBL: metallo-[beta]-lactamase. AmpC: AmpC [beta]-lactamase. Table 3. Proportion of multidrug resistant (MDR) bacterial isolates recovered from uropathogens according to beta-lactamase production. Organism MDR with ESBL/ampC/ MDR ESBL/ampC/MBL or MBL (n = 78) negative (n = 18) Citrobacter spp 1 (1.3) 0 E. coli 26 (33.3) 5 (27.8) Klebsiella spp 35 (44.9) 1 (5.5) Proteus mirabilis 2 (2.6) 2 (11.1) Proteus vulgaris 2 (2.6) 2 (11.1) Providencia spp 4 (5.1) 0 Alcaligenes spp 4 (5.1) 1 (5.6) Pseudomonas aeruginosa 4 (5.1) 6 (33.6) Pseudomonas fluorescens 0 0 Acinetobacter spp 0 1 (5.5) Number in bracket: value in percentage. MDR: multi-drug resistant.
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Ibadin, Ephraim Ehidiamen; Omoregie, Richard; Enabulele, Idahosa Onaiwu|
|Publication:||New Zealand Journal of Medical Laboratory Science|
|Date:||Apr 1, 2018|
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