Molecular Characterization of β-lactamase Genes in Antibiotic Resistant Bacteria

Beta-lactamases (bls) are implicated seriously in resistance to β - lactam antibiotics. β- lactam resistance related to Sulfa hydral variabl- beta lactamases (SHV- bls) were detected in E. coli and Klebsiellae isolated from human and chickens. In this work, resistance profile of the isolates, extended spectrum β-lactamases (ESBLs) activity and SHV genes sequencing were examined. E. coli was isolated from humans, broiler and day-old chicks samples, meanwhile, K. pneumonia was isolated only from human samples (88.8%). Sixty-four (88.8 %) of human isolates contained beta-lactamases (iodometric positive) , 28% were ESBLs and 35% contained SHV. In case of day-old chick isolates, 50% contained beta-lactamases (iodometric positive), 5.5% were ESBLs. Out of 45 broiler isolates, 37.7% contained beta-lactmases (iodometric positive) , 8.8% were ESBLs and 80% contained SHV. It was found that SHV were of SHV-5 and SHV-12. It could be concluded that, wide spread of ESBLs SHV-5 and SHV-12 in humans and chicken is a major concern in resistance to β -lactam drugs.


Introduction
Escherichia coli considered one of the major causes of nosocomial infections in humans. E. coli is a common inhabitant in the human and animal gut and an indicator of fecal contamination of food [1]. The β-Lactams are groups of antibiotics extensively used within human and veterinary medicine in treating E. coli infections.
In addition, Klebsiella pneumoniae is a member genus of Enterobacteriaceae and considered as one of normal flora of GIT of humans. It is the most prevalent and clinically important, hospital-acquired and occurred primarily in patients with impaired host defenses [2]. Emergence of resistance against β-lactam antibiotics began prior to the first βlactam, penicillin, was developed. The first βlactamase was identified in Escherichia coli before using penicillin in medical practices [3].
Bacterial genome can acquire resistance genes via integrons, transposons and plasmids because they are not fixed and resistance mechanisms are easily exchanged between different species of bacteria through their sophisticated ways sharing mechanisms essential for survival [4]. The β-lactam antibiotics are classified into several groups according to structural characteristics, and the presence of the four-membered β-lactam (azetidin-2-one) rings is the main structural feature of them. They include Penicillins, Cephalosporins, Cephamycins, Carbapenems, Monobactams and β -lactamase inhibitors. This category of antibiotics is subdivided into functional drug groups to facilitate understanding and prescribing practices, the antibiotics of each functional group differ in pharmacokinetics, safety, and duration of the clinical experience with their use [5]. There are three pathways which have an important role in clinical antibacterial resistance which include destruction of the antibiotics, altered antibiotics targets and decreased intracellular uptake of the drug [6]. Bacterial destruction of β-lactams by β -lactamases considered the most common method of resistance. βlactamses open the β-lactam ring and the altered drug prohibits subsequent effective binding to penicillin binding proteins (PBP), consequently, cell wall synthesis is unable to continue [6].
-Lactamases can be classified according to two main schemes: The Ambler molecular classification scheme and the Bush-Jacoby-Medieros functional classification system. The 56 first scheme divides βlactamases into four major groups (A to D). The main principle of this classification scheme depends upon protein homology (amino acid sequences) and not phenotypic characteristics. In the Ambler classification scheme the β-lactamases of classes A, C and D are serine -lactamases. Meanwhile, the class B enzymes are metallo--lactamases [7]. There are other different βlactamases which may be chromosomally encoded β-lactamases such as AmpC. There are also plasmid-borne genes coding for βlactamases, such as TEM-1, TEM-2 and SHV-1. Mutations in the genes causing generation of the extended-spectrum β-lactamases (ESBLs), which can be divided into at least four groups. Those four groups mediate resistance to oxyimino cephalosporins, e.g. cefuroxime, cefotaxime and ceftazidime [8].
The second prevalent type of β -lactamases is SHV-1, which was described primarily in K. pneumoniae and E. coli. The SHV-1 is responsible for plasmid mediated ampicillin resistance nad amino acids substitution causes change of enzyme structures and activities [9]. The aim of the present study was to identify ESBLs phenotypically and genotypically by PCR and sequence analysis of SHV genes.

Collection of samples
A total of 135 samples were collected from chickens (n=63) reared in poultry farms and humans (n=72) at Sharkia Governorate, Egypt. The chicken samples were obtained from livers and spleens of freshly dead and diseased broilers and one-day old chicks suspected to have colisepticemia. Urine samples were collected from humans suffering from urinary tract infections.

Isolation and identification
The samples were streaked directly onto the surface of nutrient agar according to the method of Calnek et al. [10]. All inoculated plates were incubated at 37ºC for 24 h and examined for bacterial growth. The isolated colonies were sub-cultured again onto MacConkey´s agar and examined for lactose fermentation [11]. The lactose fermenter colonies were then streaked onto Eosin Methylene Blue agar media (EMB) (Oxoid, UK). The characteristic greenish metallic sheen E. coli colonies on EMB media were sub-cultured on soft agar for further identification. The biochemical testes used for E. coli identification were indole, methyl red, Voges-Proskauer and Simmon's citrate (Oxoid, UK) [12,13]. The isolates which were Gram's negative bacilli, Indole +, Methyl red +, Voges-Proskauer and Simmon´s citrate negative were identified as E. coli strains and subjected to further tests. K. pneuomonia colonies were lactose fermenter and was confirmed as indole and Methyl red negative, while Voges-Proskauer and Simmon´s citrate positive [12,13].

Antibiogram testing
The identified isolates were purified again on nutrient agar and tested for antimicrobial susceptibility on 11 different antibiotics including β -lactams, aminoglycosides, penicillins and quinolones (Oxoid, UK). Antibiotic susceptibility profiles were determined using the Kirby-Bauer disk diffusion method and interpretation was based on the CLSI guidelines [14]. All isolates were tested for the detection of β -lactamases by iodometric tube method; briefly, 1 mL of crude or pure β-lactamase enzyme was used as a control positive and 8 bacterial colonies of tested bacteria were emulsified in 250 μL of penicillin solution in small test tubes. Penicillin solution alone was used as a negative control. After 30-60 min, 200 µL of 1% starch solution and 1 mL of iodine solution were added changing the color into blue. Rapid de-colorization of the blue color within 5-10 min was an indicator for β-lactamases production [15]. Isolates that were resistant to third generation cephalosporins and iodometric positive were tested for the presence of the extended spectrum β -lactamases using double disc diffusion (DDD) [14]. In brief, discs of cephalosporin (cefotaxime, ceftazidime and cefepime) (30 μg) alone and cephalosporin + clavulanic acid (30 μg/10 μg) were placed with a distance of 25 mm, center to center, on a Muller Hinton agar plate. The inhibition zone around the cephalosporin disc combined with clavulanic acid is compared to the zone around the disc with the cephalosporin alone. The test is positive if the inhibition zone is ≥ 5 mm larger with clavulanic acid than without) [14].
The isolates were also examined by PCR for detection of SHV resistance genes.

Genotypic detection of ESBL
The DNA from the purified colonies of ESBLs producing E. coli and K. pneumonia was extracted using Wizard ® Chromosomal DNA purification kit (Promega company, USA), following the manufacturer's instructions. The SHV genes were detected as described previously [16]. Specific primers for the SHV genes including forward primer (5'-CACTCAAGGATGTATTGTG-3'); Reverse primer (5'-TTAGCGTTGCCAGTGCTCG-3') were used for PCR amplification that produced 885 bp amplicon. A reaction volume of 25 µL included 6 µL of Master Mix kits (Bioflux company). which consisted of (2.5 µL TAE 10X buffer + 2 µL dNTPs + 1.5 µL Mgcl2 + 0.2 µL Taq polymerase), in addition,2 µL of each primer and 2 µL of DNA template were added and then distilled water was added to reach 25 µL total volume. The cycling conditions started with an initial 10 min denaturation at 94°C, followed by 35 cycles of 45 sec at 94°C, annealing at 42°C for 45 sec, extension at 72°C for 45 sec and a final extension for 10 min. The amplified products were visualized by electrophoresis in 2% agarose gel (BioRad) with ethidium bromide (Invitrogen) (0.5 μg/ mL) for 1 hour (6 Volts/cm), and visualized under UV light and results documented.

Sequencing and sequence analysis
The PCR products of were purified from agarose gel using QIAquick® PCR Purification kit (Qiagen) , and then sequenced by Sanger dideoxy sequencing method (Sigma Company). The nucleotide sequences were identified using NCBI-BLAST and submitted to the GenBank (accession numbers KU096850, E. coli EGY20151// KU096851, pneumoniae EGY201511 were provided) The sequences were then aligned with other sequences in the GenBank by ClustalW method using MegAlign of DNAStar software (Lasergene version 7.2; DNASTAR, Madison, WI, USA).

Results and Discussion
A total of 135 samples from different sources were subjected to bacterial isolation. All the samples were positive for Enterobacteriacea. After bacterial culture on MacConkey's and EMB media and biochemical identification using IMVC tests most of the isolates were Lactose fermenters. E. coli and Klebsiella spp. were isolated from 123 and 12 examined samples, which were used for antimicrobial studies.

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Concerning antimicrobial resistance of E. coli isolates to the βlactam antibiotics, the results revealed that 96% of E. coli isolates were resistant to amoxicillin. This was compatible with the findings obtained by Jorgensen and Turnidge [17], who reported that 92.8% of E. coli isolates were resistant to amoxicillin. In addition, 90% of E. coli isolates were resistant to amoxicillin/ clavulanic acid (Table 1). Similar results were obtained by Jorgensen and Turnidge [17].
Concerning the resistance to extendedspectrum cephalosporins, E. coli isolates showed low resistance to third generation cephalosporins including cefotaxime (31%) and ceftriaxone (30%). Similar results were reported by Li et al. [18], who recorded that 44% of E. coli isolates were resistant to cefotaxime. In contrary, Daniel et al. [19] reported that 2.4% of E. coli isolates were resistant to ceftriaxone (Table 1).

Concerning
fourth generation cephalosporins, 26% of E. coli isolates were resistant to cefepime (Table 1). This result was compatible with that reported by Pai et al. [20], who found that the activity of cefepime was not significantly impaired by AmpC and more than 90% susceptibility had been reported in E. coli isolates from animals. The low resistance rate to cefepime could be explained by the co-presence of ESBL isolates [21]. However, the results revealed no resistance to Cefepim/ tazobactam (Table 1).
Large number of β-lactamases (BLs) has been discovered such as ESBL. The detection of ESBL producing microorganisms is a challenge requiring both phenotypic and genotypic tests for all genes associated with βlactamase production. According to the majority of epidemiological studies on ESBL, K. pneumoniae and E. coli are the most common species implicated in this type of resistance. In this study, E. coli and K. pneumonia were found carrying detectable ESBL. The results agreed with a study done in Rio Grande do Sul by Freitas and co-workers who observed that these two species were the most prevalent among ESBL producing microorganisms [15,26,27]. Concerning the crude β-lactamase assay, iodometric method was sensitive for βlactamase detection by measuring the rate of penicillin hydrolysis to penicilloic acid by β-lactamase enzyme [15]. In this method, iodine binds with penicilloic acid rather than penicillin and this led to rapid de-colorization of blue color of starch iodine complex, which indicated the presence of βlactamase producers (positive result). The isolates showed differences in the decolorization time from those with less inhibition diameters in penicillin sensitivity test than the other, were the faster in the reduction of iodine. These results agreed with Lianes et al. [28].
Awareness of ESBL production by K. pneumoniae and E. coli is very important clinically. Reliable laboratory method is now available, double disc diffusion (DDD), by which ESBL production can be detected. This method has also been promoted by the CLSI [15]. Isolates suspected to produce ESBLs should not be reported as susceptible to thirdgeneration cephalosporins or cefepime until follow-up confirmatory tests are performed.
The results in Table 3 show that 20 out of 72 (27%) human isolates, 1 out of 18 (5%) day old chicken isolates and 4 out of 45 (8.8%) broiler isolates were ESBLs according to the results of DDD test. Concerning studied gene in this investigation the SHV gene was recovered from E. coli and Klebsiella isolates ( Table 2). This result agreed with Kola et al. [29] who found that the most predominant βlactamase-encoding genes identified among the Enterobacteriaceae isolates were TEM, followed by CTX-M, and SHV.
Sequencing of amplified SHV genes was performed for ten β-lactamase resistant E. coli isolates and one β-lactamase resistant K. pneumoniae isolate. Alignment of the sequences with wild type of SHV-5, SHV-12 and SHV-1 in E. coli and K. pneumoniae isolates on the GenBank with accession numbers (E. coli NC_004998.1, E. coli NC_024967.1 and K. pneumoniae X98098.1), respectively, was 90-100% ( Figure 1). This matched with Newire et al. [30], who explained that comparison of the corresponding amino acid sequence of the 11 SHV positive isolates demonstrated the predominance of SHV-12, while SHV-5 and SHV-2a was the most closely related sequencing match in the remaining isolates. SHV-12 is thought to have evolved from SHV-2a, and SHV-5 from SHV-2.

Conclusion
There is a high prevalence of β-lactamase genes in our clinical isolates that are responsible for such resistance. Therefore, it must report β-lactamases production along with routine sensitivity reports, which will help the clinician in prescribing proper antibiotics. Also, the sequence analysis of amplified genes showed differences between multiple SNPs in the same gene among different local isolates and with internationally published sequences. In the end, it has been felt that there is a need to formulate strategies to detect and prevent the emergence of βlactamases producing strains for the effective treatment of infections which are caused by them.