Detection of ESBLs and NDM-1 genes among urinary Escherichia coli and Klebsiella pneumoniae from healthy students in Niger Delta University, Amassoma, Bayelsa State, Nigeria

¹Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria, ²Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Gombe State University, Tudun-Wada, Gombe State, Nigeria, ³Department of Medical Microbiology and Parasitology, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria

^&Corresponding author
Adebola Onanuga, Department of Pharmaceutical Microbiology and Biotechnology Faculty of Pharmacy, Niger Delta University, Wilberforce Island Bayelsa State, Nigeria

Introduction    

Enterobacteriaceae are common commensals of the human gastrointestinal tract rendering few benefits to the host as long as they do not acquire virulent traits from invading pathogens in the environment. However, at different anatomical sites, they can become virulent causing varying serious infections which include urinary tract infections (UTIs), sepsis, meningitis, and soft tissue infections depending on their loads and host´s immune system [1]. Notable Enterobacteriaceae that have been commonly implicated in UTIs among humans of all ages and genders are Escherichia coli and Klebsiella pneumoniae [1,2]. UTIs are one of the most common bacterial infections with high economic burden requiring medical care leading to high antibiotic prescription after a visit to a clinician, and occurring more frequently in women than in men because of their shorter urethra that is of closer proximity to the opening of the intestinal tract [3,4]. The high volume of antibiotic prescriptions witnessed in UTIs´ treatment has led to the increasing emergence of antibiotic resistance in the uropathogens causing increased morbidity, delay hospital discharge and expensive cost of treatment which is a serious public health concern [1,5]. Asymptomatic bacteriuria (ASB), a condition that is characterized by the presence of bacteria in two consecutive clear voided urine specimens with both yielding positive cultures of at least 10⁵ CFU/ml of the same uropathogen in an individual without urinary symptoms, occurs at varying frequencies among humans of varying ages but highly prevalent among pregnant women, diabetics and people with genitourinary abnormalities who are more likely to experience symptomatic UTIs along with complications at their much lowered immune status, and can equally become potential transmitters of these causative pathogens to more vulnerable populations in the society [3,6,7]. The UTIs´ aetiologic agents, among which Escherichia coli and Klebsiella pneumoniae are most prevalent, have been reported to be frequently encountered in individuals with ASB [5].

The increasing rate at which Escherichia coli and Klebsiella pneumoniae develop multiple resistance to most of the common antibiotics has seriously hampered the effective treatment of UTIs in both hospital and community settings [1,8,9]. Strains of these organisms that produce extended-spectrum beta-lactamases (ESBLs) have been reported to be generally resistant to many important classes of antibiotics like aminoglycosides and fluoroquinolones besides the beta-lactam antibiotics because of their rapid acquisition of multiple antimicrobial resistance genes on mobile genetic elements aiding their rapid transmission among different bacterial species thereby making UTIs often difficult and expensive to treat [1,8]. The genes encoding the ESBLs production in these bacteria have been identified to be mainly blaTEM, blaSHV and blaCTX-M with varying proportions across different geographical areas of the world [10-12]. The last resort for most ESBLs infections that defer treatment by common antibiotics has been the carbapenems, which are a class of potent beta-lactams with much resistance to most beta-lactamases including ESBLs [13,14]. However, the increasing emergence and rapid dissemination of genes encoding the production of carbapenem-hydrolyzing beta-lactamases among E. coli and K. pneumoniae implicated in UTIs has become one of the most worrisome global public health problems because of the grave consequences of treatment failure such as increased rates of morbidity and mortality [2,9,14,15]. Hence, frequent screening of asymptomatic individuals for surveillance of multiple antimicrobial-resistant uropathogens is therefore, pivotal to current appropriate therapy and strategies for containment of antibiotic resistance in our environments. There is a paucity of data on the study of ASB and the molecular characteristics of its etiologic agents in healthy individuals, even though it is a common occurrence in most African communities. This study, therefore, investigated the phenotypic and molecular characteristics of antibiotic-resistant Escherichia coli and Klebsiella pneumoniae from students with asymptomatic bacteriuria in a Nigerian University, as a means of enriching the antimicrobial resistance surveillance information on ASB from healthy population in this region and thus provides current strategies for UTIs treatment in our environments.

Methods     

Study design: a cross-sectional study, which aimed at determining the prevalence of ASB and the antibiotic resistance characteristics of common uropathogens among healthy population was carried out among 303 undergraduate students of Niger Delta University, Bayelsa State, as a representative of sexually active individuals in the community. The sample size of this study was determined using the average prevalence of ASB among similar subjects in the previous studies, as described by Charan and Biswas [16].

Study population: the students of various Faculties of Niger Delta University who lived in the hostels within the University environments that had no symptoms or history of UTIs three months before the commencement of the study were randomly drafted into this study from August to November 2019. Niger Delta University is a fast-growing State University situated at Amassoma area of the Wilberforce Island in Southern Ijaw Local Government Area of Bayelsa State on latitude 4°58'13"N longitude 6°06'32" E and 11 meters elevation above the sea level. The students of this University are predominantly from the Southern part of Nigeria.

Inclusion criteria: the students who were not on antibiotics and had no recent history of UTIs were randomly contacted in their various hostels to give their early morning urine samples for research purposes. The interested students gave written informed consent and filled questionnaire to supply their basic demographic data like their Faculty, gender, and age with the exclusion of their names, registration numbers and addresses.

Ethical approval: the permission to conduct this study was obtained from the Ethics and Research Committee of Niger Delta University, Wilberforce Island, Nigeria, before the commencement of the sample collection.

Collection of samples and bacteriology: each volunteer collected a clean mid-stream early morning urine sample into a wide-open sterile universal bottle after being properly instructed on the procedure of collection without touching the skin. The urine samples were labeled appropriately and transported immediately to the laboratory for processing. A 10 µL dispensing loop was used to aseptically transfer each urine sample onto a sterilized nutrient agar plate and properly streaked for discrete colonies before being incubated at 37^oC for 24 hours. The sample that had a total of 10⁵ CFU/ml or more was regarded as having significant bacteriuria and the resulting discrete colonies from the plates were separately streaked on HiCromeTM Mueller Hinton Agar, Cysteine lactose electrolyte deficient agar (CLED) and Eosin methylene blue agar (EMB). The isolates with characteristic features on these agar plates were presumptively identified as either E. coli or K. pneumoniae, before storing in glycerol broth at 4^oC for further investigations.

Preparation of DNA template for PCR Amplification: the DNA of the isolates was extracted by suspending many colonies from the overnight culture of each isolate on Nutrient agar plates into 100 µL 1X Tris-EDTA buffer, vortexed and boiled at 100^oC for 10 minutes as described by Onanuga et al. [17]. The boilate was transferred immediately to the freezer (-20^oC) for 10 minutes, maintained at room temperature, vortexed, and centrifuged at 10, 000 rpm for 10 minutes. The resulting supernatant containing DNA of each isolate was collected, stored at 4^oC and used as a DNA template for PCR analyses.

Molecular Identification of E. coli through PCR amplification of 16S rRNA gene: the isolates that gave characteristic colours on EMB, CLED, and Chrom agars were subjected to PCR analysis for the presence of E. coli 16S rRNA (Eco-1) gene as a confirmation for E. coli. The E. coli 16SrRNA gene was screened for in each of the isolate´s DNA by amplifying the gene as previously described by Mamun et al. [18] using the primers in Table 1. The PCR was carried out in a thermal cycler using a 25µl reaction mixture containing 12.5 µl 2x master mix, 0.5 µl each of the forward and reverse primers, 8.5 µl of nuclease-free water and 3 µl of the DNA template at specified conditions in Table 1. The amplified PCR products (10 µl) were evaluated on a 1.5% (w/v) agarose gel at 100 mV for 60 min using BIO-RAD Power Pac 3000 with a molecular weight marker (100 bp DNA Ladder). The DNA bands were then visualized and photographed under UV light after staining the gel with ethidium bromide.

Molecular Identification of K. pneumoniae through PCR amplification of TyrB gene: the isolates that gave characteristic colours on CLED and Chrom agars were subjected to PCR analysis for the presence of the TyrB gene as a confirmation for K. pneumoniae isolates. The TyrB gene was screened for in each of the isolate´s DNA as previously described by Jeong et al. [19] using the primers in Table 1. The K. pneumoniae specific TyrB gene was amplified in the PCR using the same reaction mixture at specified conditions in Table 1. The amplified PCR products were separated on a 1.5% (w/v) agarose gel in the same pattern as described above.

Screening of E. coli and K. pneumoniae isolates for Haemolysin production: haemolytic property of the isolates was determined by inoculating the isolates on freshly prepared sterile 5%v/v Blood agar (consisting of 5 ml of human blood in 100 ml of Nutrient Agar) plates using straight wire loop and incubated at 37^oC for 24 hours. Thereafter, the plates were observed for green to black colouration of the agar (partial lysis of red blood cells - alpha haemolysis) and clear zones (complete lysis of red blood cells - beta haemolysis) around inoculated organisms indicating the production of haemolysin.

Screening of E. coli and K. pneumoniae isolates for Biofilm production: biofilm production by the isolates was performed using Congo Red Agar medium which was prepared using the combination of brain heart infusion agar 52 g/L, sucrose 50 g/L and Congo red indicator 8 g/L as described by Mathur et al. [20]. Congo red was prepared as concentrated aqueous solution separately from other medium constituents and then sterilized in different containers before adding them together when the agar had cooled to 55^oC before distributing to sterile plates to solidify. The plates were then inoculated with the test organisms and incubated at 37^oC for 24 hours before examining for black colonies with a dry crystalline consistency indicating biofilm production.

Antimicrobial Susceptibility Testing and Phenotypic detection of ESBLs: antimicrobial susceptibility testing of the isolates of E. coli and K. pneumoniae was performed using the modified Kirby-Bauer disc diffusion method on Mueller-Hinton agar using commercial discs (Oxoid, UK) against the following antimicrobials; nitrofurantoin (F, 300 μg), cefotaxime (CTX, 30 μg), imipenem (IMP, 10 μg), ceftazidime (CAZ, 30 μg), co-trimoxazole (SXT, 25 μg), gentamicin (CN, 10 μg), chloramphenicol (C, 30 μg) and levofloxacin (LEV, 5 μg) following the Clinical and Laboratory Standards Institute guidelines [21]. The isolates that were resistant to at least one agent in three or more classes of antimicrobial agents used were defined as having multi-drug resistance (MDR) [22]. Furthermore, the isolates that were resistant to Ceftazidime and Cefotaxime were screened for phenotypic production of ESBLs enzymes using combination discs method with discs containing cefpodoxime and cefpodoxime plus clavulanic acid in accordance to CLSI guidelines [21].

Screening for ESBLs genes in the isolates of E. coli and K. pneumoniae: the confirmed isolates of E. coli and K. pneumoniae were screened for the presence of blaTEM, blaSHV and blaCTX-M ESBLs genes as previously described by Bebe et al. [23] and Gholipour et al. [24] using the primers in Table 1. The multiplex PCR reactions were carried out using the same reaction mixture at the specified conditions in Table 1. The amplified PCR products were separated on a 1.5% (w/v) agarose gel in the same pattern as described above.

Screening for Carbapenemases (NDM, VIM, KPC, OXA-48) genes in the isolates of E. coli and K. pneumoniae: the isolates of E. coli and K. pneumoniae that exhibited resistance to Imipenem were screened for the presence of any carbapenemases genes by amplifying NDM, VIM, KPC, OXA-48 genes as described by Balkan et al. [25] and Gokmen et al. [26] using the primers in Table 1. The multiplex PCR reactions were carried out using the same reaction mixture at the specified conditions in Table 1. The amplified PCR products were separated on a 1.5% (w/v) agarose gel in the same pattern as described above.

Statistical analysis: the groups´ differences were tested using the Chi square test (or Fisher´s exact test when expected frequencies were too low), with the assumed level of statistical significance at a P < 0.05 while the strength and direction association or relationship was measured using the phi coefficient/Cramer´s V tests where applicable. Data was performed with SPSS for Windows, version 15.0 (SPSS Inc., Chicago, IL).

Results     

A total of 303 urine samples were collected from the healthy University students comprising 144 (47.5%) males and 159 (52.5%) females of ages 15 to 34 years with an average age of 22.7 years. Of all the 303 screened samples, 64 (21.1%) yielded positive significant bacteriuria with a significantly higher prevalence among the females (χ² = 16.51; P < 0.0001; Odds ratio = 3.459). However, the observed differences in the prevalence of ASB across the age groups are not statistically significant (P > 0.05) as shown in Table 2. Twenty-one (6.9%) E. coli and 21 (6.9%) K. pneumoniae isolates were recovered from the volunteers´ samples, with their respective specific target genes (Figure 1 and Figure 2). The prevalence of E. coli isolates in all the samples was significantly higher in the females (11.3%) than the males (2.1%) (χ² = 10.0; P = 0.002; Odds ratio = 6). However, the difference observed in the prevalence of K. pneumoniae among the genders was not statistically significant (P = 0.993) as shown in Table 3.

Seven (16.7%) haemolysin producers comprising 6 (28.6%) E. coli and 1 (4.8%) K. pneumoniae were recovered from all the bacterial isolates. Also, 38 (90.5%; 38/42) biofilm producers comprising 17 (81%; 17/21) E. coli and 21 (100%; 21/21) K. pneumoniae were equally recovered from all the bacterial isolates. A total of 4 E. coli and 1 K. pneumoniae isolates produced both haemolysin and biofilm. The antimicrobial susceptibility testing revealed that E. coli and K. pneumoniae isolates generally exhibited higher resistance (57 - 95%) to cefotaxime, ceftazidime, cotrimoxazole, and lower resistance (10 - 33%) to chloramphenicol, levofloxacin, and imipenem. The K. pneumoniae isolates showed higher rates of resistance to most of the tested agents than the E. coli isolates and they were found to exhibit 100% resistance to nitrofurantoin and 71.4% to gentamicin as shown in Table 4. The prevalence of multiple antibiotic resistance (MDR) among the E. coli and K. pneumoniae isolates was 9 (42.9%; 10/21) and 18 (85.7%; 18/21) respectively. The observed difference in the MDR of these organisms is statistically significant (χ² = 8.4; P = 0.004; Odds ratio = 8). The prevalence of phenotypic ESBLs among the screened isolates of E. coli and K. pneumoniae was 60% and 72.7% respectively. They were all MDR and biofilm-producing isolates.

In all, 28 isolates comprising 10 E. coli and 18 K. pneumoniae were screened for the ESBLs genes. A total of 15 (53.6%; 15/28) blaTEM, 15 (53.6%; 15/28) blaSHV and 3 (10.7%; 3/28) blaCTX-M genes were recovered from both organisms, in which E. coli isolates harboured 5 (50%; 5/10) blaTEM and 3 (30%; 3/10) blaCTX-M genes but no blaSHV, whilst K. pneumoniae isolates on the other hand, harboured 10 (55.6%; 10/18) blaTEM and 15 (83.3%; 15/18) blaSHV genes in which 9 (50%; 9/18) of them harboured both blaTEM and blaSHV genes but no blaCTX-M was detected among them (Figure 3). All the organisms´ isolates with the ESBLs genes (24) were resistant to the tested cephalosporins and 22 (91.7%; 22/24) of them were MDR. All the E. coli isolates that expressed phenotypic ESBLs, harboured blaCTX-M genes while 7 (87.5%) of the K. pneumoniae isolates that expressed phenotypic ESBLs, harboured both blaTEM and blaSHV genes while the remaining 1 (12.5%) haboured only blaSHV gene. A total of 3 (60%; 3/5) of the organisms´ isolates that were resistant to imipenem harboured NDM-1 gene (Figure 4). One (50%; ½) was recovered from E. coli isolates while 2 (66.7%; 2/3) were recovered from K. pneumoniae isolates and all exhibited higher levels of MDR harbouring ESBLs genes.

Discussion     

This study´s prevalence of significant bacteriuria among apparently healthy University students was 21.1% with a significantly higher proportion among the females (P < 0.0001) and with no definite association with the age groups of the subjects (P > 0.05). Our prevalence of ASB is similar to the previous findings of Onifade et al. [27] in Ile-Ife, Ngwai et al. [28] in Keffi, Olowe et al. [29] in Oshogbo and Odetoyin et al. [30] in Ile-Ife who reported 20-28.7% rate of ASB among similar Nigerian populations of no overt clinical symptoms. However, the studies of Varli et al. [31] in Turkey and Nsofor et al. [32] in Owerri, Nigeria reported 6.2% and 13.8% lower rate of ASB respectively. This observed difference might be attributed to the sampling techniques, nature, and environments of the populations. Our finding of higher proportion (30.2%) of ASB among the females with a 3.5 times frequent occurrence (greater risk) than the males (P < 0.0001; Odds ratio = 3.5) has been widely reported by previous researchers [23,27-31]. This might be due to the female´s shorter urethra and its closer proximity to the opening of intestinal tract [3,4]. Our result of no definite association of ASB with the age group of the subjects is in agreement with the report of Varli et al. [31] but is in contrast to the findings of Olowe et al. [29] and Odetoyin et al. [30] who reported an increased ASB with increasing age in males. The higher prevalence (30.2%) of ASB among females in this study therefore, indicates the female gender as a major risk factor among healthy populations and this underscores the need for their frequent screening and possible treatment, considering their important roles in the society.

Escherichia coli and K. pneumoniae which were among the leading etiologic agents of UTIs were detected in the same proportion, with E. coli significantly isolated from the females (P = 0.002). The most implicated uropathogen in ASB from healthy populations has been reported by many studies to be E. coli [27-29,31,32] and its higher prevalence among females has equally been reported by Ngwai et al. [28]. However, the recovery of K. pneumoniae in equal proportion with E. coli in this study suggests the increasing rate at which K. pneumoniae is becoming an important agent of ASB which might result into symptomatic UTIs in humans. This is in agreement with the findings of Onanuga and Selekere, [33] in Bayelsa State who reported K. pneumoniae as the most implicated uropathogen in a similar ASB study. This therefore, emphasizes the need for microbial culture identification for better management of UTIs since different organisms behave differently to treatments.

Haemolysin production by certain strains of bacteria has been attributed to the secretion of alpha-haemolytic toxin which is associated with significant disease burden in healthy humans [34,35]. The screening for haemolysin production among this study´s bacteria revealed a total prevalence of 7 (16.7%) comprising 6 (28.6%) E. coli and 1 (4.8%) K. pneumoniae isolates, with a higher proportion of 6 (85.7%) recovered from the females. The observed low level of these toxin-producing isolates suggests that the bacteria emanated from healthy individuals without overt symptoms. However, it also suggests that the females in this study might be highly predisposed to E. coli associated UTIs in the event of a lowered immune status and might constitute a transmission agent to the vulnerable healthy community. A high prevalence of 38 (90.5%) biofilm producers comprising 17 (81%) E. coli and 21 (100%) K. pneumoniae isolates were recovered in this study. This therefore, suggests the possibility of a high-level multidrug resistance tendency among the isolates since biofilm-producing bacteria have been reported to exhibit varying degrees of resistance to antimicrobial agents and host defense strategies, thereby causing a serious public health problem of treatment failures [35-37]. This observation is an indication that these organisms might have emanated from the population areas where antimicrobials have been grossly misused which therefore support the postulation that healthy members of the population are the highest reservoirs of antimicrobial-resistant bacteria [38,39].

This study´s antimicrobial susceptibility test results revealed that the organisms´ isolates generally exhibited high in vitro resistance of 57 - 95% to cefotaxime, ceftazidime, and co-trimoxazole, a finding that has been widely reported in Gram-negative bacilli from ASB [27,33]. This may be attributed to the extensive use of these agents in the treatment of uncomplicated UTIs in both hospital and community settings since extensive use of antimicrobial agents favours the selection of antibiotic-resistant bacteria and hence, a major risk factor for antibiotic resistance in a community [30,40]. This therefore, implies that these agents should no longer be considered for the empiric therapy of UTIs. The E. coli isolates in this study showed low to moderate (28.6 - 33.3%) in vitro resistance to gentamicin, chloramphenicol, and nitrofurantoin but K. pneumoniae exhibited higher in vitro resistance to gentamicin (71%) and nitrofurantoin (100%). Our result also reveals that K. pneumoniae generally exhibited higher resistance to all the tested agents than E. coli which is in agreement with the reports of previous studies in Africa [2,41]. The most effective agents in this study are levofloxacin and imipenem since the organisms exhibited very low resistance to them. This data therefore, highlights the importance of routine antimicrobial susceptibility testing as a guide to improving infection control and the use of antimicrobial agents in the community.

The prevalence of multiple drug resistance in this study was E. coli (42.9%) and K. pneumoniae (85.7%) which show a significantly higher MDR among K. pneumoniae isolates with 8 times frequent occurrence (greater risk) than E. coli (P = 0.004; χ² = 8.4; Odds ratio = 8) which implies that these organism´ isolates might have been exposed to many classes of antibiotics hence possessing many antibiotic resistance genes and this has been ascribed to the widespread use of antimicrobial agents in both agricultural and health care settings [2]. The observed higher MDR in K. pneumoniae may be attributed to its higher biofilm-forming ability over E. coli as revealed in this study and which has been reported to be a major risk factor for multidrug resistance [35-37]. This non-susceptibility of urinary K. pneumoniae to most commonly used antibiotics is a global health problem that is rapidly increasing with varying dimensions across regions which call for regular surveillance programs to monitor its resistance and spread [2]. ESBLs producing strains of bacteria that were previously associated with nosocomial UTIs were now detected in 60% of E. coli and 72.7% of K. pneumoniae screened isolates in this study. These isolates were all observed to be MDR and biofilm producers, implying that the ESBL enzymes which were primarily produced to hydrolyze beta-lactams antibiotics are mediated by plasmids which also harbour other classes of antibiotics resistance determinants thereby making the bacteria to be non-susceptible to most commonly used antibiotics [42-44]. The detection of a high proportion of this type of bacteria among healthy individuals in a community setting may pose a serious health problem of therapeutic difficulties raising major concern.

The screening for ESBLs genes among these study´s isolates, which is the first report of its kind in Nigeria, revealed a total prevalence of 15 (53.6%) blaTEM, 15 (53.6%) blaSHV and 3 (10.7%) blaCTX-M genes among the screened E. coli and K. pneumoniae isolates that largely exhibited MDR and expressed phenotypic ESBLs. This finding is similar to the reports of Ojdana et al. [45] and Hamad and Mustafa, [46] from hospital-based urine samples in Poland and Iraq respectively. These beta-lactamases genes that encode antibiotic resistance to broad-spectrum beta-lactam drugs have been reported to be located on transferable genetic elements such as plasmids or transposons which can facilitate the horizontal transmission of resistance among other bacteria species [44,45,47].This therefore suggests a possibility of rapid spreading of multi-resistance among bacteria within the host´s community since the isolates harbouring them were MDR.

The gene encoding the New Delhi Metallo-beta-lactamase-1 (NDM-1), a carbapenemase beta-lactamase enzyme of the class B Metallo-beta-lactamase, was screened for in 2 E. coli and 3 K. pneumoniae isolates that exhibited in vitro resistance to imipenem in this study. A total of 3 (7.1%; 3/42) isolates comprising E. coli (1) and K. pneumoniae (2), which exhibited a high level of MDR and possessed ESBLs genes, harboured the NDM-1 gene. The bacteria strains that showed resistance to carbapenems, one of the last resort agents that are used in treating multidrug-resistant ESBLs producing bacterial infections have been reported to be difficult to treat, causing increased death in hospital patients that are infected by them [48,49]. This NDM-1 gene whose emergence has been ascribed to the excessive use of antibiotics, is located on the bacterial plasmids and can be spread from one strain of bacteria to another by horizontal transfer thus, accounts for its increasing emergence across the globe even beyond the hospital settings [49,50]. Hence, the detection of this gene among isolates from healthy individuals is therefore a serious threat to public health which calls for concerted efforts by all health stakeholders to contain its spread in the community through education on the rational use of antibiotics and strict personal proper hand hygiene.

Conclusion     

The study reports a higher burden of ASB among healthy University students that is characterized with high proportions of MDR E. coli and K. pneumoniae isolates that harboured ESBLs and NDM-1 genes thereby predicting a likely outbreak of community-associated UTIs that will be difficult to treat. This therefore, calls for regular surveillance of bacterial resistance coupled with education on prudent use of antibiotics and strategies to contain the spread of MDR pathogens in our community.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

Adebola Onanuga participated in designing the study, performed the experiments, analyzed the data and wrote the manuscript. Darlington Deboh Eboh participated in the experiments and review of the manuscript for publication. Babatunde Odetoyin performed the molecular experiments, participated in data analysis and review of the manuscript for publication. Ocholi Jonathan Adamu participated in the review of the manuscript for publication. All authors read and approved the final manuscript.

Acknowledgments     

The authors thank the technologists of the laboratories for their support and assistance during the period of the study.

Tables and figures     

Table 1: primers used for PCR amplification and sequencing

Table 2: the distribution of significant bacteriuria among the subjects´ age groups

Table 3: the prevalence of E. coli and K. pneumoniae among the healthy volunteers

Table 4: antimicrobial resistance patterns of the volunteers´ urinary bacteria isolates

Figure 1: amplification of 16sRNA of E. coli

Figure 2: amplification of TyrB of Klebsiella pneumoniae

Figure 3: amplification of SHV, TEM, CTX-M

Figure 4: amplification of NDM 1

References