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The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3' proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.
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Objective To analyze the molecular characteristics of qnrS-positive Escherichia coli ( E. coli) strains resistant to quinolone. Methods A total of 57 qnrS1-positive clinical isolates were collect-ed from Fujian Medical University Union Hospital. Plasmid-mediated quinolone resistance ( PMQR) genes [qnrA, qnrB, qnrC, qnrD, aac(6′)-Ib-cr, qepA and oqxAB] andβ-lactamase genes (blaCTX-M-1, blaCTX-M-2, blaCTX-M-8 , blaCTX-M-9 , blaSHV and blaTEM ) were detected by PCR and then sequenced. Agar dilution method was used to analyze the antimicrobial susceptibility of the qnrS1-positive strains. Phylogenetic analysis was conducted using PCR. Multilocus sequence typing ( MLST) was performed for phenotyping. Enterobacterial repetitive intergenic consensus-polymerase chain reaction ( ERIC-PCR) was used to evaluate the genetic sim-ilarity between those isolates. Transferability of the qnrS1 genes carried by the 57 strains was examined by conjugation test with the sodiumazide-resistant E. coli J53 as the recipient strain. Mutations in the quinolone resistance-determining regions ( QRDR) in those strains were analyzed by PCR. Results All of the qnrS1-positive E. coli strains showed high resistance to quinolones. PMQR genes were harbored by 14 (24. 6％) isolates. Extended spectrum β-lactamases (ESBLs)-producing isolates accounted for 68. 4％. Mutations in the QRDR of gyrA, gyrB, parC and parE genes were found in 56 (98. 2％) strains and the most frequent point mutations were S83L (89. 5％) in gyrA gene, S80I (54. 4％) in parC gene and P415V (28. 1％) in parE gene. The qnrS1 gene was successful transferred from 13 (22. 8％) isolates to E. coli J53 by conjuga-tion. Five plasmid incompatibility groups were detected. Phylogenetic analysis showed that there were 36 (63. 2％), 13 (22. 8％), 1 (1. 8％) and 7 (12. 3％) isolates belonging to groups A, B1, B2 and D, respectively. The 57 qnrS1-positive E. coli strains were assigned to 50 ERIC types and 39 sequence types ( ST) based on the results of ERIC-PCR and MLST. Conclusions Mutations in the QRDR in E. coli strains were associated with qnrS1 gene and might play a critical role in the dissemination of quinolone-resistant bacteria.
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Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.
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Background & objectives: Infection with Salmonella enterica serovar Typhi (hereafter S. Typhi) is an important public health problem in India. There has been an increase in the number of reported clinical failures to ciprofloxacin treatment but the data on possible mechanism of failure are limited. One mechanism that has been widely reported and found associated with ciprofloxacin resistance, is the mutations in target genes in QRDR (quinolone resistance determining region). It is hypothesized that mutations in DNA gyrase or topoisomerase IV result in therapeutic failure under selective pressure of antibiotic while the patient is on treatment. We undertook in vitro sequential selection studies to expose the clinical isolates of S. Typhi to different concentration of ciprofloxacin to study the role of antibiotic selective pressure in the development of mutations in QRDR. Methods: Total 26 clinical isolates were divided in to two parts: part I included six isolates obtained from three patients with relapse of enteric fever and part II included 20 isolates with different ciprofloxacin MIC levels. For in vitro induction of mutation experiment, five S. Typhi isolates were selected which included three NAS (nalidixic acid sensitive) and 2 NAR (nalidixic acid resistant) S. Typhi. These isolates were grown under increasing concentrations of ciprofloxacin and mutations acquired in QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) were investigated by sequencing. Results: For the isolates included in the part I of the study, it was found that the MIC to ciprofloxacin increased in the isolates obtained during the relapse of enteric fever as compare to the first isolate. All isolates had single mutation in gyrA gene at S83 without additional mutation in the second isolate. In the second part of the study, the nine isolates with varying MICs to ciprofloxacin also had single mutation in gyrA gene at S83 and another six had triple mutations, two mutations in gyrA gene (at S83 and D87) and one mutation in parC gene (at S80). In in vitro induction of mutation experiment, all mutated isolates showed triple mutation (two mutation in gyrA and one in parC gene) while no mutations were found in wild isolates. Interpretation & conclusions: Upon exposure to the step-wise increased concentration of ciprofloxacin, isolates become more tolerant to the ciprofloxacin and showed 2-4 fold higher MICs without new mutation after 8 μg/ml. So the accumulation of mutations under continuous ciprofloxacin pressure and tolerance of the mutant isolates led to the clinical failure. These results also suggested that there could be another mechanism responsible for resistance.
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Objective To investigate the quinolone resistance determinants in ciprofloxacin-resistant Acinetobacter bau-mannii (ABA)clinical isolates.Methods One hundred and fourteen ciprofloxacin-resistant ABA strains were collected from six Chinese hospitals .The quinolone resistance determining region ( QRDR) of 4 target genes ( gyrA, gyrB, parC and parE) was amplified , sequenced and compared with the reference genome of ATCC 17978 to identify possible resistance-related mutations.Nine plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac(6′)-Ⅰb-cr, oqxA and oqxB) were also amplified, and the amplicons were then sequenced to determine their character-istics.Results Almost all isolates (113/114, 99.1%) harbored a substitution in codon 83 of gyrA gene, leading to a Ser83Leu mutation.Meanwhile,58.8%(67/114) of the isolates possessed dual mutations of GyrA-Ser83Leu and GyrA-Ser80Leu, which were known determinants for ciprofloxacin resistance .There were also multiple non-synonymous substitu-tions in gyrB, leading to Arg393Ser, Arg393Cys, Thr401Ala, Pro406Ser, Val430Phe, Cys440Ser and Gly480Arg muta-tions with prevalence rates of 95.6%, 0.9%, 96.5%, 96.5%, 100%, 96.5%and 96.5%,respectively.For parE, all the seven mutations were synonymous and found in more than 96%of the tested isolates.For PMQR genes, although 83.3%(95/114) of the isolates were positive for aac(6′)-Ⅰb, nocrmutations were identified.None of the other eight PMDR genes were found in our strain collection .Conclusion Although multiple mutations are identified in gyrB and parE, these mutations might be the characteristic SNP markers for specific clones , unlikely linked to quinolone resistance .No PMQR is found in the tested isolates.Mutations in chromosomal QRDR (GyrA-Ser83Leu and ParC-Ser80Leu) are the main determi-nants of ciprofloxacin resistance in our ABA collection .
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Objective To investigate the resistant mechanism of quinolones on multi-drug resistant Klebsiella caused pneumonia(MDR-KPN).Methods From August 2008 to May 2010,47 strains of MDR-KPN were collected from 6 hospitals in Hangzhou and Huzhou in Zhejiang province in China.Drug target genes to quinolones (gyrA,parC) and quinolone-resistance genes mediated by mobile genetic elements [qnrA,qnrB,qnrS,aac (6')-Ⅰ b-cr,qepA] were analyzed by PCR and verified by DNA sequencing.Results Positive results were found in 47 strains of MDR-KPN,43 strains (91.5％) of gyrA mutation,40 strains(85.1％) ofparC mutation,3 strains (6.4％) of qnrB2,1 strain (2.1％) ofqnrB 4,8 strains (17.0％) ofqnrS 1,5 strains (10.6％) of qnrS 4,2 strains (4.3％)of aac (6')-Ⅰ b-cr respectively.Moreover,5 novel variants of gyrA (GenBank accession number:JN811952,JN811953,JN811954,JN811955,JN811956),5 novel variants of parC (GenBank accession number:JN817432,JN817433,JN817434,JN817435,JN817436)were also identified.In addition,qnrS4 (GenBank accession number:JN836269) appeared to be the novel variants of qnrS.Conclusion Quinolone-resistance-determining region played a key role on the resistance to quinolones in this group of MDR-KPN,and quinolone-resistance genes mediated by mobile genetic elements [qnrB2,qnrB4,qnrS1,qnrS4,aac (6')-Ⅰ b-cr] showed positive in some parts of the strains.This was the first report on emergence of qnrS4 in the world.
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Objective To investigate the distribution and variety of quinolone-resistance genes in a group of multiple resistant Klebsiella.Methods A total of 20 strains of multiple resistant Klebsiella were collected from inpatients in Huai' an First People' s Hospital of Nanjing Medical University during February 2010 and March 2012.Strains were identified by molecular identification,and quinolone target genes (gyrA,ParC) and quinolone-resistance genes mediated by mobile genetic elements (qnrA,qnrB,qnrS,aac (6′)-Ⅰ b-Cr,qepA) were analyzed with polymerase chain reaction (PCR).Results In 20 strains of Klebsiella,19 were Klebsiella pneumonia,and 1 was Klebsiella variicola.gyrA and parC genes were found in all 20 strains,and gyrA mutation was found in 11 strains (55.0％),andparC mutation was also found in 11strains (55.0％).aac(6′)-Ⅰ b-cr was positive in 10 strains (50.0％),qnrA was positive in 1 strain (5.0％),and qnrB was positive in 3 strains (15.0％).gyrA and parC in strain No.6 and No.10 were both novel variants (GenBank registration number:JX123016,JX123017,JX144393,JX144394).Conclusion Quinolone-resistance-determining region plays a key role in resistance to quinolones in this group of Klebsiella,and it is the first report on the emergence of novel variants of gyrA and parC in one Klebsiella pneumonia in China.
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Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.
Subject(s)
Humans , /analysis , /isolation & purification , Nalidixic Acid/isolation & purification , Base Sequence , DNA Gyrase/isolation & purification , DNA Topoisomerases/analysis , DNA Topoisomerases/isolation & purification , Genetic Predisposition to Disease , Mutation , Methods , Patients , MethodsABSTRACT
Infections with Salmonella are an important public health problem worldwide. On a global scale, it has been appraised that Salmonella is responsible for an estimated 3 billion human infections each year. The World Health Organization (WHO) has estimated that annually typhoid fever accounts for 21.7 million illnesses (217,000 deaths) and paratyphoid fever accounts for 5.4 million of these cases. Infants, children, and adolescents in south-central and South-eastern Asia experience the greatest burden of illness. In cases of enteric fever, including infections with S. Typhi and S. Paratyphi A and B, it is often necessary to commence treatment before the results of laboratory sensitivity tests are available. Hence, it is important to be aware of options and possible problems before beginning treatment. Ciprofloxacin has become the first-line drug of choice since the widespread emergence and spread of strains resistant to chloramphenicol, ampicillin, and trimethoprim. There is increase in the occurrence of strains resistant to ciprofloxacin. Reports of typhoidal salmonellae with increasing minimum inhibitory concentration (MIC) and resistance to newer quinolones raise the fear of potential treatment failures and necessitate the need for new, alternative antimicrobials. Extended-spectrum cephalosporins and azithromycin are the options available for the treatment of enteric fever. The emergence of broad spectrum β-lactamases in typhoidal salmonellae constitutes a new challenge. Already there are rare reports of azithromycin resistance in typhoidal salmonellae leading to treatment failure. This review is based on published research from our centre and literature from elsewhere in the world. This brief review tries to summarize the history and recent trends in antimicrobial resistance in typhoidal salmonellae.
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Anti-Bacterial Agents/pharmacology , Asia, Southeastern , Drug Resistance, Bacterial , Humans , Salmonella paratyphi A/drug effects , Salmonella paratyphi B/drug effects , Salmonella typhi/drug effects , Typhoid Fever/microbiologyABSTRACT
Mutations into codons Aspartate-87 (62 percent) and Serine-83 (38 percent) in QRDR of gyrA were identified in 105 Salmonella strains resistant to nalidixic acid (94 epidemic and 11 of poultry origin). The results show a high incidence of mutations associated to quinolone resistance but suggest association with others mechanisms of resistance.
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Animals , Chick Embryo , Anti-Bacterial Agents/analysis , Base Sequence , Codon/genetics , Drug Resistance, Microbial , Fluoroquinolones/analysis , In Vitro Techniques , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Poultry , Quinolones/analysis , Salmonella/isolation & purification , Methods , MethodsABSTRACT
The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser83-->Arg83 or Ser83-->Asn83. We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.
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Animals , Aeromonas salmonicida/classification , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Bacterial , Environment , Fish Diseases/microbiology , Fishes , Gram-Negative Bacterial Infections/microbiology , Microbial Sensitivity Tests , Point Mutation , Polymerase Chain Reaction , Quinolones/pharmacology , Republic of Korea , Sequence Analysis , Tetracycline/pharmacology , Tetracycline ResistanceABSTRACT
The quinolones exert their anti-bacterial activity by binding to DNA gyrase A (GyrA), an essential enzyme in maintenance of DNA topology within bacterial cell. The mutations conferring resistance to quinolones arise within the quinolone-resistance-determining region (QRDR) of GyrA. Therefore, quinolones interaction with wild and mutated GyrA can provide the molecular explanation for resistance. Resistant strains of Salmonella enterica of our hospital have shown mutations in the QRDR of GyrA of serine 83 (to phenylalanine or tyrosine) or aspartic acid 87 (to glycine or tyrosine). In order to understand the association between observed resistance and structural alterations of GyrA with respect to quinolone binding, we have studied the interaction of mutated QRDR of GyrA with nalidixic acid and ciprofloxacin by molecular modeling using GLIDE v4. Analysis of interaction parameters like G-score has revealed reduced interaction between nalidixic acid/ciprofloxacin with QRDR of GyrA in all four mutated cases of resistant strains. The mutation of Ser83 to Phe or Tyr shows least binding for nalidixic acid, while Asp87 to Gly or Tyr exhibits minimal binding for ciprofloxacin. The study also highlights the important role of arginines at 21, 91 and His at 45, which form strong hydrogen bonds (at < 3 Å) with quinolones. The hydrophilic OH group of Serine 83, which is in close proximity to the quinolone binding site is replaced by aromatic moieties of Tyr or Phe in mutated GyrA. This replacement leads to steric hindrance for quinolone binding. Therefore, quinolone resistance developed by Salmonella appears to be due to the decreased selectivity and affinity of nalidixic acid/ciprofloxacin to QRDR of GyrA.
Subject(s)
Amino Acid Sequence , Ciprofloxacin/chemistry , Ciprofloxacin/metabolism , DNA Gyrase/chemistry , DNA Gyrase/genetics , DNA Gyrase/metabolism , Drug Resistance, Bacterial/genetics , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Nalidixic Acid/chemistry , Nalidixic Acid/metabolism , Protein BindingABSTRACT
Objective To investigate the importance of plasmid-mediated quinolone resistance in the development of quinolone resistance in clinical isolates of gram-negative bacteria.Methods A total of 541 consecutive clinical isolates of gram-negative ba- cilli resistant or intermediate to ciprofloxacin were screened for the qnrA gene by PCR.Conjugation experiments were carried out with azide-resistant E.coli J53 as a recipient.The aac(6')-Ib-cr gene was detected.The mutations in the quinolone-resist- ance-determining region (QRDR) of the gyrA and parC genes were identified in qnrA positive strains.Results qnrA was identi- fied in 7 of the 541 strains.Among the qnrA positive strains,5 were Enterobacter cloacae.No qnrA was detected in nonfer- menters.Quinolone resistance was transferred in 4 of 7 qnrA positive strains.Transconjugants had 12-to 125-fold increases in MIC of ciprofloxacin relative to that of the recipient.Seven strains contained qnrA with a nucleotide sequence identical to that originally reported.Two transconjugants with higher ciprofloxacin MICs contained aac(6')-Ib-cr gene.Mutations occurred in the QRDR of the gyrA and parC genes in 5 PCR-positive clinical strains.Conclusions Transferable plasmid-mediated quinolone resistance associated with qnrA is highly prevalent in clinical strains of Enterobacter spp.aac(6')-Ib-cr gene and mutations in the quinolone targets may co-exist with qnrA,which may contribute to the further increase of resistance to quinolones.