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Research Article
 

Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli Strains Isolated from Abattoir by Multiplex PCR Technique



Ejikeugwu Chika, Esimone Charles, Iroha Ifeanyichukwu, Igwe David Okeh, Ugwu Malachy, Ezeador Chika, Duru Carissa and Adikwu Michael
 
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ABSTRACT

Background and Objective: Globally, infections caused by antibiotic resistant bacteria still pose a threat to public health. The widespread use of antibiotics in food-animal production allows resistant strains of microbes to evolve. The contamination of the environment with animal wastes (containing resistant bacteria) is a major route via which human populations become infected by these microbes. Metallo-beta-lactamase (MBL) is one of the resistance mechanism at the disposal of Gram-negative bacteria including Escherichia coli that is chiefly responsible for bacteria resistance to the carbapenems. This study evaluated the antibiogram and occurrence of MBL genes from E. coli isolates recovered from abattoir. Materials and Methods: A total of 120 rectal swab samples from cows in a local abattoir were used for this study. Each of the rectal swab samples were bacteriologically analyzed for the presence of MBL-producing E. coli using the inhibition based assay and multiplex PCR technique. Specific primers for blaIMP-1 and blaVIM-1 MBL genes were used for the multiplex PCR analysis. The data were analyzed using SPSS with chi-square test and one-way analysis of variance. Results: A total of 48 (40%) isolates of E. coli was recovered from the 120 rectal swab samples. The E. coli isolates were highly resistant to ceftriaxone (72.9%), cefoxitin (75%), ceftazidime (100%), ertapenem (83.3%), oxacillin (81.3%), ciprofloxacin (70.8%), cefotaxime (93.8%), aztreonam (97.9%) and nitrofurantoin (75%). Of the 48 E. coli isolates from abattoir, 15 (31%) E. coli isolates were phenotypically confirmed to produce MBLs. However, only 8 E. coli isolates were genotypically confirmed to harbour blaIMP-1 gene by the multiplex PCR used in this study. None of the E. coli phenotypes harboured the blaVIM-1 MBL gene. Conclusion: This study reported the first multiplex PCR detection of blaIMP-1 MBL gene in E. coli isolates from rectal swab of cows in Abakaliki, Nigeria. The molecular identification of the genes encoding MBL production in Gram-negative bacteria from community samples is vital for a reliable epidemiological investigation, surveillance and the forestalling of the emergence and spread of these organisms through the food chain and food-producing animals.

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Ejikeugwu Chika, Esimone Charles, Iroha Ifeanyichukwu, Igwe David Okeh, Ugwu Malachy, Ezeador Chika, Duru Carissa and Adikwu Michael, 2017. Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli Strains Isolated from Abattoir by Multiplex PCR Technique. Research Journal of Microbiology, 12: 266-273.

URL: https://scialert.net/abstract/?doi=jm.2017.266.273
 
Received: July 24, 2017; Accepted: September 11, 2017; Published: September 15, 2017



INTRODUCTION

The use of antibiotics in animal husbandry allows resistant microbes to evolve through the process of natural selection or selective pressure. These resistant bacteria can persist in meat, poultry/animal products and animal wastes and serve as route via which the environment becomes contaminated with them. Antibiotic resistant microbes from animals represent the most relevant reservoir of resistance to antibiotics and other antimicrobial agents in the community and this is due in part to their ability to acquire and incorporate into their genome, antibiotic resistance genes from their environment. Metallo-beta-lactamases (MBLs) are beta-lactamase enzymes produced by pathogenic bacteria and which hydrolyzes the carbapenems such as imipenem, meropenem and ertapenem and render them ineffective for treatment1,2. MBLs efficiently hydrolyze all beta-lactam drugs except aztreonam, a monobactam. They confer variable range of high levels of resistance to all beta-lactam antibiotics and some non-beta-lactams such as fluoroquinolones and aminoglycosides. Their presence in clinically important Gram-negative bacteria have put the use of the carbapenems under threat1,3. The MBLs have high affinity for zinc ions (Zn2+). The enzyme is largely inhibited by chelating agents such as EDTA and dipicolinic acid in vitro. This informed the basis upon which the expression of MBLs can be detected phenotypically in pathogenic bacteria1,3. Genes responsible for the expression of MBLs can be either chromosomally-mediated or plasmid-mediated1,2. The carbapenems are often the last line of treatment option for a variety of infectious diseases including those caused by multidrug resistant organisms. The occurrence of MBL-producing bacteria in a hospital or non-hospital setting poses not only a therapeutic problem but also serious concern for infection control management in the health system4,5. This is due to the fact that organisms producing MBLs are multidrug resistant in nature. They limit therapeutic options for treatment. MBLs may be disseminated in hospital environment through genetic transfer elements such as transposons, plasmids and integrons amongst clinically important bacteria and the extensive use of the carbapenems especially irrationally has given impetus to the spread of organisms that produce the enzymes1,4,6. Antimicrobial resistance is a natural biological phenomenon but it can be contained and averted through proper drug prescription, rational use of antibiotics and through timely and appropriate detection of antibiotic resistant bacteria. The purported continual usage of antimicrobial agents as growth promoting agents in the production of food-producing animals selects for antimicrobial resistant bacteria that give rise to other resistant microbes. The detection of MBL-producing Gram-negative bacteria from community samples including samples from rectal swabs of cow is important because of the possible transfer of antibiotic resistant bacteria through the food chain and food-producing animals to humans. More so, determining the genetic factors responsible for these resistance determinants is vital to the provision of sound epidemiological data that will assist in the surveillance and control of the emergence and spread of drug resistant bacteria in the community. This study investigated the antibiogram and occurrence of MBL-producing Escherichia coli isolates from rectal swabs of cow using both phenotypic technique and multiplex PCR technique.

MATERIALS AND METHODS

Sample collection and processing: One hundred and twenty (120) rectal swab samples were collected from the anal region of cows using sterile swab stick(s) soaked in normal saline. The samples were collected from January, 2016-May, 2016. All the samples were transported to the Microbiology Laboratory Unit of Ebonyi State University, Abakaliki, Nigeria for bacteriological analysis according to all relevant national and international guidelines. Samples were aseptically inoculated in nutrient broth (Oxoid, UK) and incubated at 30oC for 18-24 h prior to bacterial isolation7. Following overnight incubation at 30°C, the test tubes were examined for visible bacterial growth as evidenced by turbidity. Tubes showing turbidity were each sub-cultured onto freshly prepared solid culture media plates for the isolation of the test bacterium.

Isolation and identification of Escherichia coli : A loopful of the turbid solution from the overnight nutrient broth culture were aseptically plated onto eosin methylene blue (EMB) agar (Oxoid, UK) and MacConkey agar (Oxoid, UK) plates and incubated at 30°C for 18-24 h. After incubation, suspected colonies of E. coli growing on the culture media plates was aseptically subcultured onto freshly prepared EMB and MacConkey agar (MAC) plate(s) for the isolation of pure cultures of E. coli. The following tests were carried out: Indole test, methyl red (MR) test and Gram stain/morphology examination7.

Antimicrobial susceptibility testing: Antimicrobial susceptibility testing was carried out on all E. coli isolates based on the guideline of the Clinical and Laboratory Standard Institute (CLSI) using the modified Kirby-Bauer disk diffusion technique.

Table 1: Multiplex PCR conditions for PCR amplification of MBL genes
Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique
F: Forward primer, R: Reverse primer

Single antibiotic disks comprising imipenem (IPM, 10 μg), meropenem (MEM, 10 μg), ertapenem (ETP, 10 μg), cefoxitin (FOX, 30 μg), ceftazidime (CAZ, 30 μg), sulphamethoxazole-trimethoprim (SXT, 25 μg), gentamicin (CN, 10 μg), cefotaxime (CTX, 30 μg), ceftriaxone (CRO, 30 μg), ciprofloxacin (CIP, 10 μg), ofloxacin (OFX, 10 μg), oxacillin (OX, 10 μg), ampicillin (AMP, 10 μg), cefepime (FEP, 30 μg), aztreonam (ATM, 30 μg), nitrofurantoin (F, 10 μg) and cloxacillin (OB, 200 μg) was used for susceptibility testing. Antimicrobial susceptibility testing was carried out on Mueller-Hinton (MH) agar plates (Oxoid, UK) as was previously described8-10. The test antibiotic disks were each aseptically placed at a distance of 25 mm apart on MH agar plates already swabbed with the test isolates (adjusted to 0.5 McFarland turbidity standards). All culture plates were incubated at 30°C for 18-24 h. The zones of inhibition was recorded and interpreted as susceptible or resistant based on the standard antibiotic breakpoints of CLSI10.

Screening and phenotypic detection of metallo-β-lactamase (MBL): To phenotypically screen for the production of MBL in the test E. coli isolates, the susceptibility of the isolates to the carbapenems including imipenem and meropenem was evaluated as per the CLSI criteria10. Isolates with inhibition zone diameter <23 mm was phenotypically evaluated for MBL production using the inhibition based assay technique. E. coli isolates (adjusted to 0.5 McFarland turbidity standards) were aseptically swabbed on MH agar plates. Imipenem (10 μg) and meropenem (10 μg) disks impregnated with EDTA (1 μL) was aseptically placed on the MH agar plates. Supplementary imipenem (10 μg) and meropenem (10 μg) disks without EDTA was also placed adjacent to the carbapenem disks encumbered with EDTA. All plates were incubated at 30°C for 18-24 h. A difference of >7 mm between the zones of inhibition of any of the carbapenem disks with and without the chelating agent (EDTA) infers MBL production phenotypically3,11-13.

Multiplex PCR amplification of MBL genes: Multiplex PCR technique was performed to amplify the gene sequence of MBL genes, including blaIMP-1 and blaVIM-1 genes, as described previously (Table 1)14. The genotypes of all 15 E. coli isolates were analyzed for the presence of blaIMP-1 and blaVIM-1 genes in a thermal cycler (Lumex instruments, Canada) with a final volume of 26.5 μL master mix comprising 0.2 μL of taq polymerase enzyme U/μL, 2.5 μL of 10X PCR buffer along with 2.5 μL MgCl2, 1 μL of 10 pM from each of the forward and reverse primers, 2.5 μL of dNTPs MIX (2 mM), 3 μL of DNA template (from the test isolates), 14.8 μL of nuclease-free water. The PCR conditions used for gene amplification were as described by Shibata et al.14. A 100 bp DNA molecular marker was used as the positive control (marker) while the negative control was nuclease-free water. Amplified PCR products were separated according to their individual sizes using 1.5% agarose gel electrophoresis which was run for 2 h at 80 V.

Statistical analysis: The data were analyzed using SPSS version 23.0 (SPSS, Chicago, IL, USA) with chi-square test and one-way analysis of variance. The differences in data were considered statistically significant at p<0.05.

RESULTS

Table 2 shows the rate of isolation and identification of Escherichia coli. E. coli was isolated from 48 swab samples out of 120 swab samples from the anal region of cows. The recovery rate of E. coli from the rectal swab samples analyzed in this study was 40% (Table 2). Based on the data obtained in this preliminary result, E. coli ferments lactose and produces pinkish colonies on MAC agar. On EMB agar, E. coli produces colonies with metallic sheen (Table 2). Table 3 shows the result of antimicrobial susceptibility testing of 48 E. coli isolates. The antimicrobial susceptibility test reveals a high rate of resistance among the 48 E. coli isolates. Resistance was noted in the E. coli isolates especially to carbapenems, penicillins, aminoglycosides, fluoroquinolones and cephalosporins used in this study.

A total of 47 (97.9%) E. coli isolates and 48 (100%) E. coli isolates was resistant to aztreonam and ceftazidime. This marked the highest resistant rate of E. coli in this study. It was also found that 36 (75%) isolates of the E. coli, 39 (81.3%) E. coli isolates, 45 (93.8%) E. coli isolates, 34 (70.8%) E. coli isolates and 40 (83.3%) E. coli isolates were resistant to nitrofurantoin, oxacillin, cefotaxime, ciprofloxacin and ertapenem, respectively (Table 3).

Table 2: Isolation and characterization of Escherichia coli
Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique
Keys: n: Number of isolates, %: Percentage, -ve: Negative, +ve: Positive, EMB: Eosin methylene blue agar, MAC: MacConkey agar

Table 3: Susceptibility test results of Escherichia coli
Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique
Key: S: Susceptible, R: Resistant, IPM: Imipenem, MEM: Meropenem, ETP: Ertapenem, FOX: Cefoxitin, CAZ: Ceftazidime, SXT: Sulphamethoxazole-trimethoprim, CN: Gentamicin, CTX: Cefotaxime, CRO: Ceftriaxone, CIP: Ciprofloxacin, OFX: Ofloxacin, OX: Oxacillin, AMP: Ampicillin, ATM: Aztreonam, F: Nitrofurantoin and OB: Cloxacillin

Table 4:
Frequency of MBL-producing E. coli detected by inhibition-based assay
Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique

Table 5: Occurrence of MBL genes in the Escherichia coli phenotypes
Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique
aAntibiogram, IPM: Imipenem (10 μg), ETP: Ertapenem (10 μg), FOX: Cefoxitin (30 μg), CAZ: Ceftazidime (30 μg), CN: Gentamicin (10 μg) and CTX: Cefotaxime (30 μg)

Table 4 shows the result of the phenotypic detection of MBL-producing E. coli isolates using the inhibition-based assay. The MBL production was phenotypically detected in only 15 (31%) E. coli isolates out of the 48 isolates of E. coli that was phenotypically screened for the production of MBL using the inhibition-based assay (Table 4). The production of MBL is phenotypically inhibited by EDTA in vitro since this carbapenem-hydrolyzing enzyme requires zinc ion (Zn2+) for enzyme activity.

Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique
Fig. 1:
A strain of Escherichia coli tested using the inhibition based assay, and showing a positive result for the production of metallo-β-lactamase (MBL) phenotypically. The difference in zone size between imipenem and meropenem disks used alone and imipenem and meropenem impregnated with EDTA differ by >7 mm, Key: 1: Meropenem (10 μg) without EDTA, 2: Imipenem (μg) with EDTA, 3: Meropenem (μg) with EDTA and 4: Imipenem (10 μg) without EDTA

Figure 1 shows a positive test plate of the inhibition based assay in which the production of MBL in the test E. coli isolate was inhibited by the presence of EDTA (as shown in the lower panel of the plate) while the upper panel of the plate lacked EDTA and thus the test isolated showed reduced susceptibility to the test carbapenem. Figure 2 shows a negative test plate in which the production of MBL was not phenotypically detected by the inhibition based assay.

Table 5 shows the frequency of MBL genes detected in the 15 Escherichia coli phenotypes by multiplex PCR technique. Out of the 15 MBL E. coli phenotypes analyzed by multiplex PCR technique for the detection of specific MBL genes including blaIMP-1 and blaVIM-1 MBL genes, only the blaIMP-1 MBL genes was detected in the E. coli phenotypes (Table 5). The blaVIM-1 MBL genes were not detected by the multiplex PCR technique used in this study.

Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique
Fig. 2:
Escherichia coli isolate susceptible to imipenem and meropenem with and without EDTA. This isolate of E. coli did not produce MBL phenotypically, Key: 1: Meropenem (10 μg) without EDTA, 2: Imipenem (μg) with EDTA, 3: Meropenem (μg) with EDTA and 4: Imipenem (10 μg) without EDTA

The prevalence of blaIMP-1 gene in the MBL E. coli phenotypes was 36.4%. This implies that only 8 isolates out of the 15 MBL E. coli phenotypes investigated by multiplex PCR harboured the blaIMP-1 MBL genes (Table 5). The E. coli phenotypes that harboured the blaIMP-1 MBL genes were resistant to imipenem, ertapenem, cefoxitin, ceftazidime, gentamicin and cefotaxime (Table 5). Electrophoretic analysis of the amplified PCR product of the MBL phenotypes after gene amplification using multiplex PCR technique revealed bands in the electrophoretogram. The electrophoretic analysis was positive for blaIMP-1 MBL genes with a base pair size of 587 bp (Fig. 3).

DISCUSSION

Metallo-β-lactamase (MBL) is a carbapenem-hydrolyzing enzyme produced by Gram-negative bacteria and which gives bacteria the exceptional ability to resist the antimicrobial onslaught of carbapenems such as imipenem. The increasing reports of the development and spread of multidrug resistance genes amongst pathogenic bacteria in both the community and hospital environments is alarming11,15-18. A total of 48 E. coli isolates was bacteriologically recovered from the rectal swab samples analyzed in this study. E. coli has been noted as a causative agent in diarrheal and gastrointestinal infections18-22. The prevalence of E. coli (40%) in this study is similar to a previous report by Iroha et al.21 who reported the prevalence of antibiotic resistant E. coli isolates and other Gram-negative bacteria from abattoir effluent samples. Akinniyi et al.22 also reported in their study that E. coli was among the most prevalent bacteria isolated from environmental samples including samples from poultry farms. In Australia, Leung et al.23 also reported similar prevalence of E. coli from environmental samples. The irrational use of antibiotics in the rearing and production of food-producing animals is very strategic for the development and dissemination of antibiotic resistant bacteria in the community. The occurrence of E. coli in environmental samples have been previously reported as important causative factor in some community acquired infections22,16,17,23. The E. coli isolates showed varying levels of susceptibility and resistance to the antimicrobial activity of the antibiotics used in this study. Most importantly, the isolated E. coli was highly resistant to ceftriaxone (72.9%), cefoxitin (75%), ceftazidime (100%) and cefotaxime (93.8%). Reduced susceptibility to cloxacillin (68.8%), nitrofurantoin (75%), aztreonam (97.9%), ampicillin (62.5%), ciprofloxacin (70.8%), oxacillin (81.3%) and ertapenem (83.3%) was also recorded. There was no statistical difference in the antibiogram data of E. coli to the tested antibiotics (p>0.05). The high levels of resistance of E. coli isolates in this study is similar to previous studies reported in the Netherlands, Nigeria and Uganda24-26. Bergenholtz et al.17 also reported high resistance of E. coli isolates from environmental samples to some conventional antibiotics. In Uganda, it was also reported that 168 E. coli isolates out of 182 E. coli isolates were resistant to several antibiotic classes including penicillins, fluoroquinolones, aminoglycosides and tetracyclines25. The negative impact of antimicrobial usage in animals on humans has been elucidated by Hamer and Gill27. E. coli have been previously noted as producers of MBLs and such phenotypes confers on the bacteria the exceptional ability to be resistant to a wide variety of antibiotics28,29. The MBL production was significantly detected in only 15 (31%) isolates of E. coli out of the 48 isolates of E. coli that was phenotypically screened (p<0.05). Multiplex PCR analysis using blaIMP-1 and blaVIM-1 specific primers revealed that the MBL phenotypes in this study harboured only the blaIMP-1 MBL genes. The blaVIM-1 MBL gene was not detected in the E. coli phenotypes confirmed by multiplex PCR in this study. The prevalence of MBL-producing E. coli that were found to harbour MBL genes in this study is 16.7%. This rate is much higher than the result of Adwan et al.30 who reported 87.4% carriage rate of blaIMP-1 genes in E. coli isolates in their study. Similar rates of blaIMP-1 gene prevalence have also been reported by Chouchani et al.31 in Tunisia. Enwuru et al.32 in Lagos, Nigeria reported that the prevalence of MBL positive bacteria harbouring the blaIMP-1 and blaIMP-2 genes in their study was between 40-50%.

Image for - Molecular Identification of MBL Genes blaIMP-1 and blaVIM-1 in Escherichia coli  Strains Isolated from Abattoir by Multiplex PCR Technique
Fig. 3:
Electrophoretogram of amplification of blaIMP-1 MBL genes amplified from different DNA samples from MBL positive E. coli phenotypes, Key: Lane M: DNA markers/ladder, Lanes 1, 2, 3 and 9: Amplified products of blaIMP-1 MBL genes with a base pair size of 587 bp, Lanes 4, 5, 6, 7 and 8: Lanes without gene amplification, Lane 10: Negative control which contains nuclease free water

Mansouri et al.33 in Iran also reported lesser prevalence of MBL genes in their study. The occurrence of MBL-producing E. coli isolates in this study is similar to the work of Leung et al.23 in Australia who reported the occurrence of MBL-producing E. coli from environmental samples. Chakraborty et al.29 also reported similar prevalence of E. coli isolates positive for MBL production in India. This result is also similar to the work of Bashir et al.28 who recorded higher prevalence of MBL-producing E. coli isolates in their study carried out in Kashmir. To our knowledge this is the first description of blaIMP-1 MBL-producing Escherichia coli from rectal swabs of cow in Abakaliki, Nigeria. Our results draw attention to the importance of MBL genes as a key mediator of carbapenem resistance in E. coli isolates from the non-hospital environment. The most interesting finding in the present study was the first multiplex PCR detection of blaIMP-1 genes from E. coli isolates emanating from abattoir samples. blaIMP-1 Genes represents the most common resistance traits in carbapenemase-producing Enterobacteriaceae and this MBL gene is known to be distributed globally1.

CONCLUSION AND RECOMMENDATION

From this study, it was observed for the first time that E. coli from rectal swabs of cow in Abakaliki, Nigeria, harbour blaIMP-1 MBL genes. This gene is responsible for bacterial resistance to the carbapenems. The occurrence of E. coli isolates that harbour genes for metallo β-lactamase (MBL) production in abattoir samples is implicative for carbapenem resistance in the non-hospital environment.

Authors recommend improved personal and environmental hygiene coupled with better alternative growth promoting agents that excludes the use of antibiotics in animal husbandry. A well-organized detection protocol, antibiotic surveillance system and intervention measure is paramount to contain the emergence and spread of Gram-negative bacteria harbouring genes for MBL production in the community.

SIGNIFICANCE STATEMENTS

This study identified the presence of blaIMP-1 MBL genes that is responsible for the resistance of Gram-negative bacteria to carbapenems such as imipenem and meropenem. Carbapenems are used to treat serious bacterial infections including those caused by organisms that produce extended spectrum β-lactamases (ESBLs). This study provides some epidemiological data necessary to take action towards containing the emergence and spread of MBL-positive bacteria in the non-hospital environment.

REFERENCES

  1. Walsh, T.R., A. Bolmstrom and A. Gales, 2002. Evaluation of a new Etest for detecting metallo-β-lactamases in routine clinical testing. J. Clin. Microbiol., 40: 2755-2759.
    CrossRef  |  Direct Link  |  


  2. Rossolini, G.M., M.A. Condemi, F. Pantanella, J.D. Docquier, G. Amicosante and M.C. Thaller, 2001. Metallo-β-lactamase producers in environmental microbiota: New molecular class B enzyme in Janthinobacterium lividum. Antimicrobial Agents Chemother., 45: 837-844.
    CrossRef  |  Direct Link  |  


  3. Chika, E., U. Malachy, I. Ifeanyichukwu, E. Peter, G. Thaddeus and E. Charles, 2014. Phenotypic detection of metallo-β-Lactamase (MBL) enzyme in Enugu, Southeast Nigeria. Am. J. Biol. Chem. Pharm. Sci., 2: 1-6.
    Direct Link  |  


  4. Toleman, M.A., D. Biedenbach, D.M.C. Bennett, R.N. Jones and T.R. Walsh, 2005. Italian metallo-β-Lactamases: A national problem? Report from the SENTRY antimicrobial surveillance programme. J. Antimicrob. Chemother., 55: 61-70.
    CrossRef  |  PubMed  |  Direct Link  |  


  5. Wadekar, M.D., K. Anuradha and D. Venkatesha, 2013. Phenotypic detection of ESBL and MBL in clinical isolates of Enterobacteriaceae. Int. J. Curr. Res. Acad. Rev., 1: 89-95.


  6. Bush, K. and G.A. Jacoby, 2010. Updated functional classification of β-lactamases. Antimicrobial Agents Chemother., 54: 969-976.
    PubMed  |  


  7. Chessbrough, M., 2000. District Laboratory Practice in Tropical Countries. 2nd Edn., Cambridge University Press, UK., ISBN-0-521-66546-9, pp: 178-187


  8. Javeed, I., R. Hafeez and M.S. Anwar, 2011. Antibiotic susceptibility pattern of bacterial isolates from patients admitted to a tertiary care hospital in Lahore. Biomedica, 27: 19-23.
    Direct Link  |  


  9. Iroha, I.R., A.E. Oji and C.O. Esimone, 2008. Antimicrobial resistance pattern of plasmid-mediated extended-spectrum β-lactamase producing strains of Escherichia coli. Scient. Res. Essays, 3: 215-218.
    Direct Link  |  


  10. CLSI., 2011. Performance standards for antimicrobial susceptibility testing; twenty first informational supplement. CLSI Document M100-S21, Clinical and Laboratory Standards Institute, Wayne, PA., USA.


  11. Aibinu, I., T. Nwanneka and T. Odugbemi, 2007. Occurrence of ESBL and MBL in clinical isolates of Pseudomonas aeruginosa from Lagos, Nigeria. J. Am. Sci., 3: 81-85.
    Direct Link  |  


  12. Owlia, P., H. Saderi, Z. Karimi, S.M.B. Akhavi Rad and M.A. Bahar, 2008. Phenotypic detection of metallo-β-lactamase producing Pseudomonas aeruginosa strains isolated from burned patients. Iran. J. Pathol., 3: 20-24.
    Direct Link  |  


  13. Varaiya, A., N. Kulkarni, M. Kulkarni, P. Bhalekar and J. Dogra, 2008. Incidence of metallo β-lactamase producing Pseudomonas aeruginosa in ICU patients. Indian J. Med. Res., 127: 398-402.
    Direct Link  |  


  14. Shibata, N., Y. Doi, K. Yamane, T. Yagi and H. Kurokawa et al., 2003. PCR typing of genetic determinants for metallo-β-lactamases and integrases carried by Gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J. Clin. Microbiol., 41: 5407-5413.
    CrossRef  |  PubMed  |  Direct Link  |  


  15. Akujobi, C.O., N.N. Odu and S.I. Okorondu, 2012. Detection of Amp C β-lactamases in clinical isolates of Escherichia coli and Klebsiella. Afr. J. Clin. Exp. Microbiol., 13: 51-55.
    Direct Link  |  


  16. Slama, K.B., A. Jouini, R.B. Sallem, S. Somalo and Y. Saenz et al., 2010. Prevalence of broad-spectrum cephalosporin-resistant Escherichia coli isolates in food samples in Tunisia and characterization of integrons and antimicrobial resistance mechanisms implicated. Int. J. Food Microbiol., 137: 281-286.
    CrossRef  |  PubMed  |  Direct Link  |  


  17. Bergenholtz, R.D., M.S. Jorgensen, L.H. Hansen, L.B. Jensen and H. Hasman, 2009. Characterization of genetic determinants of Extended-Spectrum Cephalosporinases (ESCs) in Escherichia coli isolates from Danish and imported poultry meat. J. Antimicrobial. Chemother., 64: 207-209.
    CrossRef  |  Direct Link  |  


  18. Usha, P.T.A., S. Jose and A.R. Nisha, 2010. Antimicrobial drug resistance: A global concern. Vet. World, 3: 138-139.
    Direct Link  |  


  19. Madigan, T.M., M.J. Martinko, V.P. Dunlap and P.D Clark, 2009. Brock Biology of Microorganisms. 12th Edn., Pearson Benjamin Cummings Publishers, USA., pp: 795-796


  20. Prescott, L.M., J.P. Harley and D.A. Klein, 2005. Microbiology. 6th Edn., McGraw Hill Publishers, USA., pp: 296-299


  21. Iroha, I.R., O.B. Eromonsele, I.B. Moses, F.N. Afiukwa, A.E. Nwakaeze and P.C. Ejikeugwu, 2016. In-vitro antibiogram of multi-drug resistant bacteria isolated from Ogbete abattoir effluent in Enugu state, Nigeria. Int. Res. J. Public Environ. Health, 3: 1-6.
    Direct Link  |  


  22. Akinniyi, A.P., E. Oluwaseun, B.O. Motayo and A.F. Adeyokinu, 2012. Emerging multidrug resistant AmpC β-lactamase and Carbapenamase Enteric Isolates in Abeokuta, Nigeria. Nat. Sci., 7: 70-74.
    Direct Link  |  


  23. Leung, G.H., T.J. Gray, E.Y. Cheong, P. Haertsch and T. Gottlieb, 2013. Persistence of related bla-IMP-4 metallo-β-lactamase producing Enterobacteriaceae from clinical and environmental specimens within a burns unit in Australia: A six-year retrospective study. Antimicrobial Resistance Infection Control, Vol. 2.
    CrossRef  |  Direct Link  |  


  24. Van den Bogaard, A.E., N. London, C. Driessen and E.E. Stobberingh, 2001. Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. J. Antimicrob. Chemother., 47: 763-771.
    CrossRef  |  PubMed  |  Direct Link  |  


  25. Majalija, S., O. Francis, W.G. Sarah, Musisi-Lubowa, P. Vudriko and F.M. Nakamya, 2010. Antibiotic susceptibility profiles of fecal Escherichia coli isolates from dip-litter broiler chickens in Northern and Central Uganda. Vet. Res., 3: 75-80.
    Direct Link  |  


  26. Ogunleye, A.O., M.A. Oyekunle and A.O. Sonibare, 2008. Multidrug resistant Escherichia coli isolates of poultry origin in Abeokuta, South Western Nigeria. Vet. Arhiv., 78: 501-509.
    Direct Link  |  


  27. Hamer, D.H. and C.J. Gill, 2002. From the farm to the kitchen table: The negative impact of antimicrobial use in animals on humans. Nutr. Rev., 60: 261-264.
    Direct Link  |  


  28. Bashir, D., M.A. Thokar, B.A. Fomda, G. Bashir, D. Zahoor, S. Ahmad and A.S. Toboli, 2011. Detection of metallo-β-lactamase (MBL) producing Pseudomonas aeruginosa at a tertiary care hospital in Kashmir. Afr. J. Microbiol. Res., 5: 164-172.


  29. Chakraborty, D., S. Basu and S. Das, 2010. A study on infections caused by metallo β-lactamase producing gram negative bacteria in intensive care unit patients. Am. J. Infect. Dis., 6: 34-39.
    CrossRef  |  Direct Link  |  


  30. Adwan, G., H. Bourinee and S. Othman, 2016. Prevalence of metallo-β-lactamase producing Escherichia coli isolated from North of Palestine. J. Microbiol. Antimicrobial Agents, 2: 9-15.


  31. Chouchani, C., R. Marrakchi, L. Ferchichi, A. El Salabi and T.R. Walsh, 2011. VIM and IMP metallo-β-lactamases and other extended-spectrum β-lactamases in Escherichia coli and Klebsiella pneumonia from environmental samples in a Tunisian hospital. APMIS., 119: 725-732.
    CrossRef  |  Direct Link  |  


  32. Enwuru, N.V., C.A. Enwuru, S.O. Ogbonna and A.A. Adepoju-Bello, 2011. Metallo-β-lactamase production by Escherichia coli and Klebsiella species isolated from hospital and community subjects in Lagos, Nigeria. Nat. Sci., 9: 1-5.


  33. Mansouri, S., D.K. Neyestanaka, M. Shokoohi, S. Halimi, R. Beigverdi, F. Rezagholezadeh and A. Hashemi, 2014. Characterization of AmpC, CTX-M and MBLs types of β-Lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli producing Extended Spectrum β-lactamases in Kerman, Iran. Jundishapur J. Microbiol., Vol. 7, No. 2.
    CrossRef  |  Direct Link  |  


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