Occurrence of Extended Spectrum Beta Lactamase Encoding Genes among Urinary Pathogenic Escherichia coli and Klebsiella pneumoniae Isolates Obtained from a Tertiary Hospital in Gombe Nigeria

This study was conducted to assess the occurrence and nature of extended-spectrum beta lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae isolates from patients who presented with urinary tract infection at Federal Teaching Hospital Gombe. Isolates collected were recovered on MacConkey agar at 35˚C and were identified as members of Enterobacteriaceae, and further screened for antimicrobial susceptibility and resistance by disc diffusion method. Isolates resistant to oxyimino-cephalosporins were confirmed as ESBL producers using Double Disks Synergy Test (DDST). The study shows 66% resistance to ceftriaxone (30 µg) in K. pneumoniae, which was the highest value recorded and a 51% resistance to cefpodoxime (10 µg) in E. coli. The sensitivity of E. coli and K. pneumoniae isolates to cefpodoxime (10 µg) were 49% and 33.9% respectively. ESBLs were detected among 40% (40/100) of E. coli and 54.13% (59/109) of K. pneumoniae isolates. Molecular characterization of ESBL encoding genes among E. coli isolates using multiplex-PCR showed 10% prevalence of SHV gene and 5% prevalence for CTX-M gene while TEM gene was not detected. In K. pneumoniae isolates, 5% prevalence was recorded for each of the three genes screened. The study revealed a co-occurrence of SHV and CTX-M in 75% of the E. coli and 70% of the K. pneumoniae isolates; the occurrence of all the three genes was seen in 10% and 5% of K. pneumoniae and E. coli respective-How


Introduction
Beta-lactam antibiotics are a class of broad-spectrum antibiotics, consisting of all antibiotic agents that contain a beta-lactam ring in their molecular structures, and are frequently prescribed antimicrobial agents all over the world in treatment of infections caused by Gram positive and Gram negative bacteria [1]. These agents are widely used antimicrobials for humans as well as animals [2]. Beta-lactam antibiotics are named after their molecular core structure, the beta-lactam ring, which forms the center and active part of the drug [2]. There are six different groups of beta-lactam antibiotics based on their mode of actions: Penicillins, Cephalosporins (1 st , 2 nd , 3 rd , 4 th and 5 th generations), Carbapenems, Penems, Monobactams and beta lactamase inhibitors like clavulanic acid and sulbactam [3]. The latter group has no antibacterial activity on its own but inhibits the activity of beta lactamase enzymes and is often given in combination with other beta-lactam antibiotics. Beta lactam antibiotics are known to irreversibly inhibit enzymes involved in the final steps of cell wall synthesis [4]. Beta lactamases are bacterial enzymes that provide multi-resistance to beta lactam antibiotics such as Penicillins, Cephalosporins and Carbapenems, although Carbapenems are relatively resistant to beta lactamase. Beta lactamase enzymes break down the antibiotics structure thereby rendering them ineffective against the bacteria.
As bacteria continue to develop resistance against beta lactam antibiotics such as Penicillins and Cephalosporins newer antibiotics were discovered and used in the treatment. However, the bacteria incessantly evolved and changed the existing beta-lactamase enzymes to break down these new antibiotics. This further class of enzymes that can break down these newer antibiotics was referred to as Extended-Spectrum Beta Lactamases (ESBLs) [5]. Extended-spectrum beta lactamases are plasmid-mediated enzymes which have the ability to hydrolyze and inactivate broad spectrum of beta lactam antibiotics such as penicillins, cephalosporins (3 rd generation) and monobactams, but are inhibited by beta lactamase inhibitors like clavulanic acid [6]. The beta lactamase enzymes like TEM-1 were first reported in 1965 [7], SHV-1 in 1972 [8] and CTX-M in 1986 [9]. The spread of beta-lactamases may be chromosomal or plasmid mediated [10].
The occurrence of ESBL among pathogenic bacteria is rising and is associated  [11]. Most ESBL plasmids also carry genes conferring resistance to several non-beta-lactam antibiotics apart from encoding genes conferring resistance to the extended spectrum antibiotics [4] [12]. Presently, more than 400 different ESBLs have been identified, and these are clustered into three major groups: TEM, SHV and CTX-M, with 183, 134 and 103 variants, respectively [13]. Among the mentioned ESBL variants, TEM and SHV were the major types in some countries [14]. Klebsiella pneumoniae and Escherichia coli remain the major ESBL-producing organisms isolated worldwide, but these enzymes have also been identified in several other members of the Enterobacteriaceae family and in certain non-fermentors [15].
Urinary Tract Infection (UTI) is one of the most common infections prevalent among both females and males [16]. It is also a common bacterial infection among infants and young children [17].  [21]. These infections are treated with a variety of antibiotics including beta lactams, beta lactam/beta lactamase inhibitor combinations, fluoroquinolones and carbapenems [22]. Recent studies revealed that, there is an increase in the antibiotic resistance among the urinary tract pathogens worldwide [22] [23] [24]. This increase in antibiotic resistance is associated with increase in Extended Spectrum Beta Lactamase producing isolates, which are a class of organisms that produce beta lactamase enzymes involved in the hydrolysis of extended spectrum beta lactam antibiotics [6]. Identified risk factors for developing ESBL producers include prolonged hospitalization and indiscriminate use/abuse of antibiotics [25]. This work, to the best of our knowledge is the first report of the molecular characterization of urinary tract pathogenic E. coli and K. pneumonia from patients who presented at Federal Teaching Hospital, Gombe, North Eastern Nigeria.
The economic and healthcare burden resulting from infections due to multidrug resistant bacteria are estimated to be at least €1.5 billion every year [31].
WHO [32] reported that about 1.8 million children are being killed every year due to increase in resistance among bacteria causing pneumonia. Mortality due to resistant bacterial infections exceeds 25,000 annually [31] in Europe. Production of beta-lactamase enzymes remains the most important contributing factor

Study Area
Federal Teaching Hospital (FTH) Gombe is a 450 bedded tertiary healthcare institution that was established in 1996 and is located within Gombe, the capital city of Gombe State Nigeria. It has the full complement of almost all medical/surgical specialties including the clinical microbiology laboratory where cultures and antibiotic susceptibility tests are routinely performed.

Ethical Approval
This study was approved by the Research and Ethics Committee of the Federal Teaching Hospital Gombe.

Sample Collection and Identification
Two hundred and nine (209)

Phenotypic Detection of ESBL Production
Two tests were done; initial screen test using the indicator cephalosporins (such as ceftazidime) and phenotypic confirmatory test as described by Elsayed [34].

Screening Test
The screening was done by disc diffusion technique. This involves screening for reduced susceptibility to more than one of the indicator antimicrobials (ceftazidime 30 μg, ceftriaxone 30 μg and cefpodoxime 10 μg

Confirmatory Test Using Double Disk Synergy Test (DDST)
The procedure of Amiri [36]  hours. An enhanced zone of inhibition as shown in Figure 1 towards the centrally placed disk was considered positive for ESBL [37]. K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as quality control strains [35].

Molecular Identification of SHV, TEM and CTX-M Bla Genes Using Multiplex PCR
The genomic DNA of forty (40) representative samples was extracted using the commercially available kit (Bioneer, USA) for molecular characterization. The genomic DNA extracts were thereafter stored at −20˚C until required for PCR.
The primers employed in the multiplex PCR were those described by Kaur and Aggarwal [38] for detection of blaTEM, blaSHV and blaCTX-M genes. The sequences of the forward and reverse primers are given in Table 1.  Multiplex-PCR was performed using PTC-100 thermal cycler (USA) to detect beta lactamase genes (blaTEM, blaCTX-M and blaSHV) using PCR conditions as described by [39]. The M-PCR was carried out using 1.5 µl of extracted genomic DNA in a 20 µl PCR reaction mixture consisting of 2.5 µl 10× PCR buffer, 1.5 µl MgCl 2 (50 mM), 0.5 µl dNTPs (10 mM), 1.5 µl of each primer, 0.5 µl of Taq DNA polymerase, and 9 µl sterile distilled water. M-PCR was performed under the following conditions: Initial denaturation at 94˚C for 1 minute, denaturation at 94˚C for 30 seconds, annealing at 60˚C for 30 seconds, and extension at 72˚C for 1 minute and a final extension at 72˚C for 6 minutes. PCR products were determined by electrophoresis in a 1.5% (g/v) agarose gel.

Statistical Analysis
Data obtained were statistically described in forms of frequencies, relative frequencies, figures and tables where appropriate. Differences between proportions were analyzed using ANOVA, in which the P values of the null hypothesis when less than 0.05 were considered significant. All statistical calculations were done using IBM SPSS statistic 23 software.

Antibiotic Susceptibility Pattern of K. pneumoniae and E. coli Isolates to Common Indicator Cephalosporins
The antibiotic susceptibility pattern of K. pneumoniae and E. coli isolates is presented in Table 2. Results showed that the highest resistance (66%) to Ceftriaxone (30 µg) and 61.5% to Ceftadizime (30 µg) were both observed in K. pneumoniae, while the least resistance of 51% to Cefpodoxime (10 µg) was recorded in E. coli. The highest sensitivity to Cefpodoxime (10 µg) of 49% followed by Ceftadizime (30 µg) of 48% were both recorded in E. coli, while the least sensitivity to Ceftriaxone (30 µg) of 33.9% followed by Ceftadizime (30 µg) of 38.5% occurred both in K. pneumoniae.

Molecular Characterization of ESBL Related Genes
Result of the percentage distribution of genotypes among E. coli and K. pneumoniae isolated from urinary clinical samples is presented in Figure 2. From the 40 randomly chosen representative samples (20 for each isolate) subjected to multiplex-PCR to ascertain the possible gene types (blaCTX-M, blaSHV and blaTEM) responsible for the production of beta-lactamases, test isolates were found to harbour one or more genes. The most prevalent among the detected genes ( Figure 3) is SHV, followed by CTX-M and TEM with 10%, 5% and 0% prevalence in E. coli respectively. TEM gene alone was not detected in E. coli but in association with other genes. While in K. pneumoniae 5% prevalence recorded for all the three genes. SHV + CTX-M was 75% in E. coli and 70% in K. pneumoniae, while the combination of SHV + TEM showed 5% in both of the isolates. Co-occurrence of all three genes recorded 5% prevalence in E. coli and 10% in K. pneumoniae.

Discussion
This study was designed to investigate the occurrence, prevalence and molecular nature of ESBL-mediated drug resistance associated with some urinary tract clinical isolates. The resistance of K. pneumoniae and E. coli isolates to antimicrobial agents tested (ceftriaxone, ceftazidime and cefpodoxime) were observed to be high in this study with the highest resistance recorded in K. pneumoniae to ceftriaxone and ceftazidime 66% and 61.5%, respectively. This is consistent with observations made by [40] [41] who reported that Klebsiella species were more resistance to third generation cephalosporins compared to E. coli. In addition, Chourasia et al. [42] buttressed our finding that Klebsiella species were the most frequent ESBL producers relative to E. coli. In contrast, Farzana et al., [43] reported higher ESBL production in E. coli over K. pneumoniae, Proteus spp. and Pseudomonas spp. K. pneumoniae is known to have some virulence factors like hyperviscosity, polysaccharide capsule, production of endotoxin and carbapenemases and their presence may partly account for the higher occurrence of the resistance seen [44]. The antimicrobial susceptibility pattern observed in this study, in addition to the roles played by the factors mentioned above, may be attributable to the large amount of third generation cephalosporins consumed in our locality as they are relatively cheap and, being an oral antibiotics, easy to administer [18]. Other contributors to this include indiscriminate use/abuse of antibiotics, prolonged hospitalization, incomplete/subtherapeutic antibiotic dose regimens, and the use/misuse of antimicrobials in animal husbandry [25].  [47]. This approach to identification and detection of disease-causing pathogens is generally adjudged to be very sensitive, fast and accurate [48]. Several PCR types exist with emphasis on specific feature(s)/aspects of the technique, one of which, the multiplex PCR, allows for simultaneous amplification of two or more genes by optimizing conditions that favors annealing of primers to the genes in a single amplification protocol [49]. Therefore, multiplex PCR has had a wide useful application in molecular identification of disease-causing pa-  [38]. In this study, attempt was made to identify, singly or in combination, ESBL related genes using Multiplex-PCR. The study has successfully detected co-occurrence of three major genes related to ESBL in both studied organisms. The occurrence of SHV and CTX-M genes with 75% prevalence in E. coli and 70% in K. pneumoniae, appear to be the most predominant molecular manifestation of this drug resistance among K. pneumonia and E. coli isolated from urinary tract-infected individuals. Work by Lal et al. [50], reported 67.3% of the two genes occurring together in India and seems to agree with the observation made in this study.
According to Zongo et al. [51], the coproduction of all the three genes (TEM + SHV + CTX-M) was 10.52% prevalent in the samples they studied in Burkina Faso, and this low figure seems to tally with the 10% and 5% prevalence seen in this work in K. pneumonia and E. coli respectively. Similar result was also reported by [18] in which all the three genes carried 9.09% prevalence. The most common single gene occurrence observed in this study was with respect to SHV (10% prevalence in E. coli and 5% in K. pneumonia). Al-Agamy et al. [52]  These variations that exist between this study and other findings indicated that the prevalence and type of ESBL and their related genes varied from one geographical region to another [56]. Use and or misuse of antibiotics favour the emergence and spread of drug resistant bacteria globally [32]. This ever-increasing health and economic burden of antimicrobial resistance requires urgent action worldwide to control the situation. Identification of these ESBL-producing isolates and the knowledge of their rates of resistance are of prime importance for the selection of an appropriate antibiotics to be used in the treatment of infectious diseases especially empirically. The predominant factor for escalation of antibiotic resistance is the acquisition of plasmid encoding anti-  [53]. Such plasmid can be easily transferred from one organism to another and as such can incorporate genetic material coding for resistance against other antimicrobial classes [18]. Such antibiotic resistance has been reported by Falodun et al. [57], to be the outcome of the acquisition of resistance genes through genetic exchange and mutation as well as physiological mechanisms, such as the possession of specific proteins and efflux pump.

Conclusion
In conclusion, by this study, we have demonstrated, for the first time, the molecular nature of the occurrence of ESBL among E. coli and K. pneumoniae isolates causing urinary tract infection in Gombe North-eastern Nigeria. The study showed the co-occurrence of ESBL-related genes in some of the studied isolates and is probably responsible for the observed high resistance to third generation cephalosporins in the locality. To avoid poor identification of antibiotic resistance and the attendant inappropriate antibiotic prescription which may in turn select for new resistance genes, it is recommended that the use of phenotypic tests for ESBL detection be accompanied by the more efficient PCR technique wherever possible. Such molecular methods of detecting ESBL related genes, though sensitive, fast and accurate, are relatively expensive and require specialized equipment and expertise and this should be bored in mind especially when being adopted by countries having many social issues competing for attention from the available limited resource.