Molecular Characterization of Extraintestinal Escherichia coli Isolates in Japan: Relationship between Sequence Types and Mutation Patterns of Quinolone Resistance-Determining Regions Analyzed by Pyrosequencing
01 Jun 2013-Journal of Clinical Microbiology (American Society for Microbiology)-Vol. 51, Iss: 6, pp 1692-1698
TL;DR: A pyrosequencing-based high-throughput method for analyzing the nucleotide sequence of the quinolone resistance-determining regions (QRDRs) of gyrA and parC successfully determined the QRDR sequences of 139 out of 140 clinical Escherichia coli isolates, 28% of which were nonsusceptible to ciprofloxacin.
Abstract: Infection from fluoroquinolone-resistant Enterobacteriaceae is an increasing health problem worldwide. In the present study, we developed a pyrosequencing-based high-throughput method for analyzing the nucleotide sequence of the quinolone resistance-determining regions (QRDRs) of gyrA and parC. By using this method, we successfully determined the QRDR sequences of 139 out of 140 clinical Escherichia coli isolates, 28% of which were nonsusceptible to ciprofloxacin. Sequence results obtained by the pyrosequencing method were in complete agreement with those obtained by the Sanger method. All fluoroquinolone-resistant isolates (n = 35; 25%) contained mutations leading to three or four amino acid substitutions in the QRDRs. In contrast, all isolates lacking a mutation in the QRDR (n = 81; 57%) were susceptible to ciprofloxacin, levofloxacin, and nalidixic acid. The qnr determinants, namely, the qnrA, qnrB, and qnrS genes, were not detected in the isolates, and the aac(6')-Ib-cr gene was detected in 2 (1.4%) of the isolates. Multilocus sequence typing of 34 randomly selected isolates revealed that sequence type 131 (ST131) (n = 7; 20%) is the most prevalent lineage and is significantly resistant to quinolones (P < 0.01). The genetic background of quinolone-susceptible isolates seemed more diverse, and interestingly, neighboring STs of ST131 in the phylogenetic tree were all susceptible to ciprofloxacin. In conclusion, our investigation reveals the relationship between fluoroquinolone resistance caused by mutations of QRDRs and the population structure of clinical extraintestinal E. coli isolates. This high-throughput method for analyzing QRDR mutations by pyrosequencing is a powerful tool for epidemiological studies of fluoroquinolone resistance in bacteria.
Citations
More filters
TL;DR: Systematic, unbiased tracking of predominant ExPEC lineages will direct research toward better treatment and prevention strategies for extraintestinal infections.
Abstract: SUMMARY Extraintestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a majority of human extraintestinal infections globally, resulting in enormous direct medical and social costs. ExPEC strains are comprised of many lineages, but only a subset is responsible for the vast majority of infections. Few systematic surveillance systems exist for ExPEC. To address this gap, we systematically reviewed and meta-analyzed 217 studies (1995 to 2018) that performed multilocus sequence typing or whole-genome sequencing to genotype E. coli recovered from extraintestinal infections or the gut. Twenty major ExPEC sequence types (STs) accounted for 85% of E. coli isolates from the included studies. ST131 was the most common ST from 2000 onwards, covering all geographic regions. Antimicrobial resistance-based isolate study inclusion criteria likely led to an overestimation and underestimation of some lineages. European and North American studies showed similar distributions of ExPEC STs, but Asian and African studies diverged. Epidemiology and population dynamics of ExPEC are complex; summary proportion for some STs varied over time (e.g., ST95), while other STs were constant (e.g., ST10). Persistence, adaptation, and predominance in the intestinal reservoir may drive ExPEC success. Systematic, unbiased tracking of predominant ExPEC lineages will direct research toward better treatment and prevention strategies for extraintestinal infections.
274 citations
University College London1, University of East Anglia2, Imperial College Healthcare3, Telford4, Cambridge University Hospitals NHS Foundation Trust5, St Thomas' Hospital6, University Hospitals Birmingham NHS Foundation Trust7, Southern General Hospital8, Public Health England9, University of Leicester10, Leicester Royal Infirmary11, Health Protection Scotland12
TL;DR: Recommendations are made in the following areas: screening, diagnosis and infection control precautions including hand hygiene, single-room accommodation, and environmental screening and cleaning, and where there are species differences.
118 citations
Cites background from "Molecular Characterization of Extra..."
...coli are often resistant to ciprofloxacin, the proportion varies widely by country.(26,177,178) In a multi-centre German study of screening of patients with haematological malignancies, colonization rates with ESBL Enterobacteriaceae varied between 5....
[...]
TL;DR: The results indicate contamination of surface waters by E. coli strains belonging to clinically important clonal groups, demonstrating the clonal distribution of environmental ExPEC strains.
Abstract: Contamination of surface waters by antimicrobial-resistant bacteria and pathogenic bacteria is a great concern. In this study, 531 Escherichia coli isolates obtained from the Yamato River (Japan) were evaluated phenotypically for resistance to 25 antimicrobials. Seventy-six (14.3%) were multidrug resistant (MDR), 66 (12.4%) were non-susceptible to one or two classes of agents, and 389 (73.3%) were susceptible. We performed whole-genome sequencing using Illumina technology on selected strains. In total, genome sequences of 155 strains were analyzed for their antibiotic resistance determinants and phylogenetic characteristics. More than 50 different resistance determinants, including acquired resistance genes and chromosomal resistance mutations, were detected. Among the sequenced MDR strains (n=66), sequence type (ST) 155 complex (n=9), ST10 complex (n=9), and ST69 complex (n=7) were prevalent. Among extraintestinal pathogenic E. coli (ExPEC) strains (n=58), clinically important clonal groups, namely ST95 complex (n=18), ST127 complex (n=8), ST12 complex (n=6), ST14 complex (n=6), and ST131 complex (n=6), were prevalent, demonstrating the clonal distribution of environmental ExPEC strains. fimH (type 1 fimbrial adhesin gene) typing revealed that ST131 complex strains carried fimH 22 or fimH 41, and no strains belonging to the fimH 30 subgroup were detected. Fine-scale phylogenetic analysis and virulence gene content analysis of strains belonging to ST95 complex (one of the major clonal ExPEC groups causing community-onset infection) revealed no significant difference between environmental and clinical strains. The results indicate contamination of surface waters by E. coli strains belonging to clinically important clonal groups. IMPORTANCE Prevalence of antimicrobial-resistant and pathogenic E. coli strains in surface waters is a concern because surface waters are used for sources of drinking water, irrigation, and recreational purposes. In this study, MDR and ExPEC strains in river water were characterized by genomic sequencing and analysis. We detected more than 50 resistance determinants and identified clonal groups specific to MDR and ExPEC strains. This study showed contamination of surface waters by E. coli belonging to clinically important clonal groups. Overall, this study advances our understanding of environmental MDR and ExPEC strains.
57 citations
Cites background or methods from "Molecular Characterization of Extra..."
...The QRDRs of gyrA and parC and chromosomal ampC promoter/attenuator mutations were analyzed as described previously (27, 60)....
[...]
...Fluoroquinolone resistance is conferred mainly by mutations in the gyrA and parC genes (27)....
[...]
TL;DR: The paper shows that favorable fitness in the major STs/lineages of these pathogens was principally attained by the capacity of evolving mutations in the fluoroquinolone-binding serine residues of both the DNA gyrase and topoisomerase IV enzymes.
Abstract: The major international sequence types/lineages of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and ESBL-producing E. coli were demonstrated to have been advanced by favorable fitness balance associated with high-level resistance to fluoroquinolones. The paper shows that favorable fitness in the major STs/lineages of these pathogens was principally attained by the capacity of evolving mutations in the fluoroquinolone-binding serine residues of both the DNA gyrase and topoisomerase IV enzymes. The available information on fitness balance incurred by individual and various combinations of mutations in the enzymes is reviewed in multiple species. Moreover, strong circumstantial evidence is presented that major STs/lineages of other multi-drug resistant bacteria, primarily vancomycin-resistant Enterococcus faecium (VRE), emerged by a similar mechanism. The reason(s) why the major ST/lineage strains of various pathogens proved more adept at evolving favorable mutations than most isolates of the same species remains to be elucidated.
44 citations
Cites background from "Molecular Characterization of Extra..."
...They are, interestingly, sometimes absent from the ST131 major clone strains of ESBLproducing E. coli (Aoike et al., 2013; Paltansing et al., 2013; Reyna-Flores et al., 2013; Guillard et al., 2014)....
[...]
TL;DR: It is indicated that ESBLEC strains belonging to clinically important lineages, including the C1-M27 clade, may disseminate into the environment through wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.
Abstract: Contamination of environmental waters by extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) is of great concern. Wastewater treatment plants (WWTPs) and hospitals release large amounts of ESBLEC into the environment. In the present study, we isolated ESBLEC strains from wastewater collected from a WWTP and a hospital in Japan and performed whole-genome sequencing to characterize these strains. Genomic analysis of 54 strains (32 from the WWTP and 22 from hospital wastewater) revealed the occurrence of clinically important clonal groups with extraintestinal pathogenic E. coli status in the WWTP and hospital wastewater. Fine-scale phylogenetic analysis was performed to further characterize 15 sequence type 131 (ST131) complex strains (11 from the WWTP and 4 from hospital wastewater). These ST131 complex strains were comprised of the following different subgroups: clade A ( n = 2), C1-M27 ( n = 8), and C1 (non-C1-M27) ( n = 1) for strains from the WWTP and clade A ( n = 2), C1-M27 ( n = 1), and C1 (non-C1-M27) ( n = 1) for strains from hospital wastewater. The results indicate that ESBLEC strains belonging to clinically important lineages, including the C1-M27 clade, may disseminate into the environment through wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.
41 citations
References
More filters
TL;DR: The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models, inferring ancestral states and sequences, and estimating evolutionary rates site-by-site.
Abstract: Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
39,110 citations
"Molecular Characterization of Extra..." refers methods in this paper
...Phylogenetic analysis was performed using the maximum-likelihood method by MEGA5 (23)....
[...]
01 Jan 2001
TL;DR: The supplemental information presented in this document is intended for use with the antimicrobial susceptibility testing procedures published in the following Clinical and Laboratory Standards Institute (CLSI)–approved standards.
Abstract: The supplemental information presented in this document is intended for use with the antimicrobial susceptibility testing procedures published in the following Clinical and Laboratory Standards Institute (CLSI)–approved standards: M02-A12—Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Twelfth Edition; M07-A10—Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Tenth Edition; and M11-A8—Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard— Eighth Edition. The standards contain information about both disk (M02) and dilution (M07 and M11) test procedures for aerobic and anaerobic bacteria. Clinicians depend heavily on information from the microbiology laboratory for treatment of their seriously ill patients. The clinical importance of antimicrobial susceptibility test results demands that these tests be performed under optimal conditions and that laboratories have the capability to provide results for the newest antimicrobial agents. The tabular information presented here represents the most current information for drug selection, interpretation, and QC using the procedures standardized in the most current editions of M02, M07, and M11. Users should replace the tables published earlier with these new tables. (Changes in the tables since the previous edition appear in boldface type.) Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 26th ed. CLSI supplement M100S (ISBN 1-56238-923-8 [Print]; ISBN 1-56238924-6 [Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2016. The data in the interpretive tables in this supplement are valid only if the methodologies in M02-A12—Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Twelfth Edition; M07-A10—Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Tenth Edition; and M11-A8—Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard— Eighth Edition are followed.
17,824 citations
01 Jan 2000
16,612 citations
TL;DR: A new implementation of eBURST is presented, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment.
Abstract: The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain(23F)-1 clonal complex of Streptococcus pneumoniae, "population snapshots" of the entire S. pneumoniae and Staphylococcus aureus MLST databases, and the more complicated clonal complexes observed for Campylobacter jejuni and Neisseria meningitidis.
1,800 citations
"Molecular Characterization of Extra..." refers methods in this paper
...Clustering of each sequence type (ST) was determined using the program eBURST version 3, and single-locus variants were used to define the clonal complexes (CCs) (22)....
[...]