|Year : 2016 | Volume
| Issue : 3 | Page : 322-326
|Distribution of plasmid-mediated quinolone resistance in Gram-negative bacteria from a tertiary hospital in Nigeria
David Olusoga Ogbolu1, Armstrong Oyebode Alli2, Michael C Anorue3, Oluwole Adebayo Daini4, Afolabi Oluwadun5
1 Department of Biomedical Sciences, Molecular Biology Unit, Ladoke Akintola University of Technology, Osogbo Campus, Ogbomoso, Nigeria; Antimicrobials Research Group, Institute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom
2 Department of Biomedical Sciences, Molecular Biology Unit, Ladoke Akintola University of Technology, Osogbo Campus, Ogbomoso, Nigeria
3 Department of Medical Microbiology and Parasitology, Federal Medical Centre, Ebonyi; Department of Medical Microbiology and Parasitology, Olabisi Onabanjo University, Remo Campus, Ago Iwoye, Nigeria
4 Department of Biochemistry, Kampala International University, Kampala, Uganda
5 Department of Medical Microbiology and Parasitology, Olabisi Onabanjo University, Remo Campus, Ago Iwoye, Nigeria
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|Date of Web Publication||10-Aug-2016|
| Abstract|| |
Background: Until recently, mechanisms of resistance to quinolones in Gram-negative bacteria were believed to be only chromosome encoded. However, emergence of plasmid-mediated quinolone resistance (PMQR) has been reported worldwide. Aim: This study investigated distribution of PMQR in Gram-negative bacteria from a tertiary hospital in eastern part of Nigeria. Materials and Methods: Seventy-one nonduplicate Gram-negative bacterial isolates of eight species were analyzed for antimicrobial susceptibility, genotypic detection of various PMQRs, typed by random amplified polymorphic DNA (RAPD) and analysis of plasmids present, including replicon typing. Results: The minimum inhibitory concentrations showed MIC90values as high as 256 μg/ml for fluoroquinolones. Carriage of PMQR was found to be 35.2%. Twenty (28.2%) isolates carried various qnr genes, of which seven (9.9%) qnrA1; four (5.6%) qnrB1; eight (11.3%) qnrS1 while one (1.4%) encoded qnrD1. Eighteen (25.4%) isolates were positive for aac(6')-Ib-cr while carriage of multiple genes exists in some strains. Similarly, 13 isolates (18.7%) were found to carry PMQR efflux pump gene, qepA. Conjugation experiments revealed that the plasmids once transferred coded for fluoroquinolone resistance. The transconjugant strains carried a common plasmid estimated to be 65 kb. These plasmids were untypable for replicon/incompatibility. Typing revealed high diversity among all species tested with no identical RAPD pattern seen. Conclusion: This study further confirms high level resistance to many antimicrobials in different species of Gram-negative bacteria including fluoroquinolones and spread of PMQR genes in Southern Nigeria.
Keywords: Gram-negative bacteria, Nigeria, plasmid-mediated quinolone resistance, quinolone, resistance
|How to cite this article:|
Ogbolu DO, Alli AO, Anorue MC, Daini OA, Oluwadun A. Distribution of plasmid-mediated quinolone resistance in Gram-negative bacteria from a tertiary hospital in Nigeria. Indian J Pathol Microbiol 2016;59:322-6
|How to cite this URL:|
Ogbolu DO, Alli AO, Anorue MC, Daini OA, Oluwadun A. Distribution of plasmid-mediated quinolone resistance in Gram-negative bacteria from a tertiary hospital in Nigeria. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Jun 19];59:322-6. Available from: http://www.ijpmonline.org/text.asp?2016/59/3/322/188108
| Introduction|| |
Many of the most important members of Gram-negative bacteria are becoming increasingly resistant to currently available antibiotics including fluoroquinolones with threat of ineffective drugs to combat bacterial diseases. Until recently, the mechanisms of resistance to quinolones in Enterobacteriaceae were believed to be only chromosome encoded, i.e., related to modifications of the molecular targets (DNA gyrase and topoisomerase intravenous), decreased outer membrane permeability (porin defect), and overexpression of naturally occurring efflux pumps. Plasmid-mediated quinolone resistance (PMQR) has been reported with at least three known resistance mechanisms, qnr, aminoglycoside acetyltransferase aac(6')-Ib-cr, and qepA underpinning fluoroquinolone resistance.
In the last few years, several qnr genes, qnrA, qnrB, qnrS including their alleles, have been identified from different parts of the world; two additional qnr have been described; qnrC was described by Wang et al., obtained from a Proteus mirabilis clinical isolate from China, described as a 221-amino acid protein.QnrD was also identified and found to cause reduced susceptibility to fluoroquinolones in isolates of Salmonella More Details enterica serovar bovismorbificans and Kentucky isolated from humans in the Henan Province of China. Although multiple mechanisms of quinolone resistance have been reported from other continents, there are few data from Sub-Saharan Africa on the molecular basis for quinolone resistance. One of such few studies was high level of resistant genes including qnr reported by Ogbolu et al. in Nigeria where all the qnr genes were found including qnrD which was only reported in China. Although these PMQR determinants confer low-level resistance to quinolones or fluoroquinolones, they may provide a favorable background in which the selection of additional chromosomally encoded quinolone resistance mechanisms can occur. In view of the dearth of information in Nigeria, particularly southeastern part, we determined the distribution of PMQR genes in Gram-negative bacteria.
| Materials and Methods|| |
Seventy-one Gram-negative bacterial isolates of eight species were obtained from 142 nonduplicate clinical specimens including ear swab, wound swab, semen, high vaginal or cervical swab, sputum, urine, and blood culture in 2012. Single isolates from each specimen were retained. The isolates were from a tertiary hospital in southeastern part of Nigeria. All isolates were identified using API 20E strips (BioMerieux, France).
The antibiotic susceptibility patterns of all 71 isolates were determined by disk diffusion method in Mueller–Hinton agar. Resistant isolates were selected for minimum inhibitory concentrations (MICs) using fluoroquinolone drugs, ciprofloxacin and levofloxacin. MIC testing was performed using the agar dilution method according to the guidelines of the British Society for Antimicrobial Chemotherapy (http://www.bsac.org.uk/susceptibility_testing/guide_to_antimicrobial_susceptibility_testing.cfm). All experiments included the control organisms, Escherichia More Details coli (NCTC 10418) and Pseudomonas aeruginosa (NCTC 10662). A start and finish plate without antibiotic was also included as a growth control.
Polymerase chain reaction (PCR) was used to amplify PMQR genes; multiplex PCR was used to amplify variants of qnrA, qnrB, and qnrS in one reaction  while specific PCR was performed to detect qnrC, qnrD, qepA, and aac(6')-Ib-cr as previously described.,,, To confirm the identity and specific variant of each gene identified, amplimers resulting from these PCRs were sequenced with capillary sequencer ABI 3730 using the primers for amplification. The nucleotide sequences were analyzed with software available through the internet at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov) and aligned to known reference sequences using ClustalW website (http://www.ebi.ac.uk/Tools/msa/clustalw2/help/index.html).
Conjugation experiments were carried out by broth and filter mating assay using E. coli DH5α, with a chromosomal mutation conferring rifampicin resistance as recipient cells. Suspensions of 200 μl were plated out onto selective plate containing rifampicin (100 μg/ml) and ciprofloxacin (2 μg/ml). For controls, test strains were plated on rifampicin plates, recipient cells on separate plates of rifampicin and ciprofloxacin.
Transformation experiments were performed when conjugation was not successful for some isolates. This was achieved by electroporation (Gene Pulser; Bio-Rad, Hemel Hempstead, UK) of purified plasmid DNA into competent E. coli α-select (Bioline, London, UK, efficiency of >109). The protocol of the manufacturer was followed to determine the properties of plasmids. The mixtures (200 μl) containing transformants were plated onto selective agar (ciprofloxacin, 2 μg/ml), allowed to stand for a few minutes, and incubated at 37°C overnight. To control for antibiotic activity, the wild-type strain was also inoculated onto the selective plates. MICs of the transformants and recipient strain were measured.
Plasmid DNA from the donor, transformants, or transconjugants strains was extracted by QIAGEN Plasmid Midi Kit (Crawley, U.K.) following the manufacturer's instruction and restricted with Eco RV and Bam HI. Plasmid size was estimated as previously described.
Incompatibility/replicon PCR-based typing was used to trace plasmids-conferring drug resistance. In this method, 18 pairs of primers were designed for 5 multiplex and 3 simplex PCRs, recognizing the FIA, FIB, FIC, HI1, HI2, I1-Iy, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA replicons, representing the major plasmid incompatibility groups circulating among the Enterobacteriaceae. Because of the high level of homology between the K and B/O replicons, the same forward primer was used in both these simplex PCRs. Plasmid DNA of transformants and transconjugants was generated as a DNA template for PCR-based replicon typing.
The epidemiological relationships between multiple strains of E. coli and Klebsiella pneumoniae were analyzed by random amplified polymorphic DNA (RAPD). The primers sequence and PCR running conditions used were according to Vogel et al. modified to use 1 μl of 100 μm of primers at a concentration of 0.02 μM. The experiment was repeated to ensure reproducibility. DNA fingerprints were compared by visual inspection to assign similar banding patterns to the same RAPD type.
| Results|| |
The in vitro disk susceptibility pattern of all isolates to eight antibiotics used for this study revealed that high level of resistance to majority of the drugs tested was seen for all species. About 50% of the isolates were resistant to each of the fluoroquinolones; ciprofloxacin, 50.7% and levofloxacin, 52.8%. The most active antibiotic in this study was imipenem with resistance of 1.4%, while gentamicin has the highest bacterial resistance of 66.7% [Table 1]. The MICs were determined for fluoroquinolone drugs, ciprofloxacin and levofloxacin using agar dilution method. Determination of precise MIC values confirmed the number of strains resistant to clinical breakpoint concentration for the fluoroquinolones used. The MIC result also showed that the level of resistance to fluoroquinolone antibiotic was high having MIC90 values as high as 256 μg/ml for the fluoroquinolones.
Presence of plasmid-mediated quinolone resistance genes
PMQR genes detected in this study include qnrA, B, S, D, C, aac(6')-Ib-cr, and qepA which were found to be 35.2% (25/71). Twenty (28.2%) of the isolates carried various qnr genes except qnrC, of which seven (9.9%) qnrA1; four (5.6%) qnrB1; eight (11.3%) qnrS1 while one (1.4%) encoded qnrD1. Eighteen (25.4%) of the isolates were positive for aac(6')-Ib-cr gene. This was included in the screening for PMQR genes because the “cr” variant has been found to confer reduced susceptibility to ciprofloxacin by enzymatic inactivation. Similarly, 13 (18.7%) isolates were found to carry PMQR efflux pump gene qepA. Four (13.3%) qnr-positive isolates carried more than one qnr genes with the following association, 2 qnrA1 + qnrS1; 1 qnrA1 + qnrB1; 1 qnrD1 + qnrS1 while some other qnr genes were co-harbored with aac(6')-Ib-cr, 11 or qepA, 7. The PMQR genes are well distributed among the isolates and the clinical specimens where strains were isolated from [Table 2].
|Table 2: The presence of plasmid-mediated quinolone resistance genes in fluoroquinolone-resistant strains|
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Transfer of resistance genes
Conjugation/transformation experiments that were successful for 9/15 selected isolates (selection based more on isolates with multiple genes) revealed that the plasmids once transferred coded for reduced fluoroquinolone resistance (0.5–2 μg/ml) [Table 3]. MB95, MB101, MB121 used transformation experiment. The transconjugants/transformants carried a common plasmid-encoding PMQR as a single or multiple genes. Restriction digestion of the transferred plasmids estimated to be 65 kb. These plasmids were untypable for replicon/incompatibility.
|Table 3: Conjugation/transformation showing low-level quinolone resistance|
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Typing of isolates
To determine the degree of clonality among fluoroquinolone-resistant strains, RAPD was used. The data revealed high diversity among all species tested with no identical RAPD pattern seen. This suggests that the spread of resistance genes is underpinning the fluoroquinolone resistance rather than expression of dominant clone.
| Discussion|| |
Fluoroquinolones are broad-spectrum antibiotics and have become the most commonly prescribed class of antibiotics including in Nigeria and are frequently prescribed for medical conditions of viral origin that are not responsive to antibiotics. Inappropriate use in the form of over- or under-use of this class of antibiotic has already rendered them useless for treating serious common acute bacterial infections such as urinary tract or wound infections, which are common sources of bacterial infections in Nigeria as obtained in this study. Most strains have MICs >4 μg/ml and some values are very high, 256 μg/ml for either of fluoroquinolone used. This is similar to the trends being reported all over the world for multidrug-resistant strains. This is an indication of high level increase in fluoroquinolone resistance that has progressed rapidly over a decade.
PMQR due to DNA gyrase protection by a protein from the pentapeptide repeat family called qnr has been described in many clinical isolates of several species., The carriage of a variety of transferable quinolone-resistant genes was detected in these strains including qnrA, qnrB, qnrS, and qnrD. This further confirms that these genes are truly global and have spread to Sub-Saharan Africa as described previously. qnr D which has previously only been described in an isolate of Salmonella from China  and recently in Nigeria  in Proteus spp. and Pseudomonas spp. was also identified in this study in strains of E. coli demonstrated that it is well distributed in another continent apart from Asia as well as its presence in three species in addition to Salmonella where it was first described; this may likely be due to global dissemination of this gene or due to identification of separate acquisition events from environmental species. Wang et al. described another qnr gene, qnrC which was found in P. mirabilis; qnrC encodes a 221-amino acid protein with different amino acid identities from qnrD which indicates that the gene is different from qnrD. In this study, no qnrC was detected in any of the isolates. Eight (25.9%) isolates encoded qnrS genes which was not detected in a previous study in southwestern part of the country.
aac(6')-Ib-cr has been found to confer reduced susceptibility to ciprofloxacin by enzymatic inactivation due to the activity of “cr” variant. Eighteen (25.4%) isolates were positive for aac(6')-Ib-cr. In Shangai, China, Robicsek et al. discovered that 51% (40/78) resistant strains of Gram-negative bacteria isolates harbored the “cr” variant aminoglycoside acetyltransferase. Further, a prevalence of 50.5% has been reported in the United States  while in Southwestern Nigeria, a prevalence of 54.5% of qnr positive strains carrying aac(6')-Ib-cr was also reported. Thirteen strains co-harbored the aac(6')-Ib-cr variant in addition to other qnr genes. This shows that aac(6)-lb-cr is highly prevalent and circulates either with or independent of qnr.
Similarly, PMQR efflux pump gene, qepA was also found in this study. The accumulation of antibiotics within cells is determined by the relative rates of influx and efflux across the cell envelope. However, quinolone accumulation is reduced by an active efflux system which is especially prominent in quinolone-resistant strains. No plasmid-mediated fluoroquinolone efflux pump has been seen until recently when a novel qepA, a plasmid-mediated efflux pump was found in an E. coli clinical isolate from Japan, from various Gram-negative bacteria isolated in many countries of Asia  and most recently in Nigeria, Africa.
Although carriage of multiple genes was found in most of the isolates, no particular order of combination was observed except that only 4/18 aac(6')-Ib-cr did not encode any other PMQR gene. aac(6')-Ib-cr is the more frequent PMQR gene present in this study followed by qepA. qnrS1 is also more prevalent than the other qnr genes, 11.3%. qnrS was not detected in the previous study done in the Western part of the country. Several studies have shown that other genes such as qnrS, aac(6')-Ib-cr, and alleles of qnrB are more common despite qnrA1 being the first PMQR gene discovered.
Transfer of genes showed reduced susceptibility or low-level resistance to fluoroquinolones by PMQR determinants. They may however provide a favorable background in which the selection of additional chromosomally encoded quinolone resistance mechanisms can occur that can escalate high-level resistance. The transconjugants/transformants carried a common plasmid-encoding PMQR as a single or multiple genes. Restriction digestion of the transferred plasmids estimated to be 65 kb. These plasmids were untypable for replicon/incompatibility.
| Conclusion|| |
This study further confirmed there is high level of resistance to many antimicrobials in different species of Gram-negative bacteria, most critically to fluoroquinolones antibiotics and are underpinned by spread of PMQR genes. The transferable nature of these genes is particularly worrisome, and treatment options for infections caused by these organisms are very limited.
We thank the technical staff of our department for their unflinching support and Dr. Mark Webber of University of Birmingham, UK, for the kind provision of control strains.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. David Olusoga Ogbolu
Department of Biomedical Sciences, Ladoke Akintola University of Technology, Osogbo Campus, Ogbomoso, Osun, Nigeria
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]
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