Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV.

The locus nfxD, which contributes to high-level quinolone resistance in Escherichia coli KF111b (gyrAr nfxB nfxD), is only expressed in the presence of a gyrA mutation, and maps to the region of the parC and parE genes, was outcrossed into strain KF130, creating strain DH161 (gyrAr nfxD). DNA sequence analysis of DH161 revealed no changes in the topoisomerase IV parC quinolone resistance-determining region but did identify a single T-to-A mutation in parE at codon 445, leading to a change from Leu to His. Full-length cloned parE+ partially complemented the resistance phenotype in KF111b and DH161, but did not complement the resistance phenotype in strain KF130 (gyrAr). No complementation was seen with cloned, truncated parE+. To confirm these findings, gyrAr was first outcrossed from KF130 into E. coli W3110parE10 [parE temperature sensitive(Ts)] and KL16. The transduced strains KL16 and W3110parE10 were subsequently transformed with plasmids containing cloned parE from DH161 or KL16. Cloned parE from DH161 increased norfloxacin resistance in the parE(Ts) background twofold at 30 degrees C and fourfold at 42 degrees C compared to those for cloned parE from KL16. The same experiment with a non-Ts background revealed a twofold increase in the norfloxacin MIC at both 30 and 42 degrees C. These data identify the nfxD conditional resistance locus as a mutant allele of parE. This report is the first of a quinolone-resistant parE mutant and confirms the role of topoisomerase IV as a secondary target of norfloxacin in E. coli.

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Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.10.2380 ◽

1996 ◽

Vol 40 (10) ◽

pp. 2380-2386 ◽

Cited By ~ 217

Author(s):

M J Everett ◽

Y F Jin ◽

V Ricci ◽

L J Piddock

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Quinolone Resistance ◽

Fluoroquinolone Resistance ◽

Polymorphism Analysis ◽

Single Mutation ◽

Wild Type ◽

Single Strand Conformational Polymorphism ◽

E Coli ◽

High Level

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

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Antibiotic resistance and virulence of Escherichia coli strains isolated from animal rendering plant

Scientific Reports ◽

10.1038/s41598-020-72851-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Gabriela Gregova ◽

Vladimir Kmet

Keyword(s):

Escherichia Coli ◽

Antibacterial Agents ◽

Virulence Genes ◽

Gene Encoding ◽

Antibiotic Resistant ◽

Beta Lactamases ◽

E Coli ◽

Animal Wastes ◽

Extended Spectrum Beta Lactamases ◽

High Level

Abstract Processing of animal carcasses and other animal wastes in rendering plants is a significant source of antibiotic resistant microorganisms. The main goal of this study was to investigate the resistance to 18 antibacterial agents including β-lactams, fluoroquinolones, colistin and virulence factors (iss, tsh, cvaC, iutA, papC, kps and ibeA genes) in 88 Escherichia coli strains isolated from a rendering plant over 1 year period. ESBL (Extended-spectrum beta-lactamases) and plasmid-mediated Amp were screened by interpretative reading of MIC. ESBL phenotype was detected in 20.4% of samples and high level of resistance to fluoroquinolone was found in 27.2% of strains. Cephalosporinase CTX-M1, cephamycinase CMY-2, integrase 1 and transposon 3 genes were detected by PCR. Furthermore, there were found three CMY-2 producing E. coli with O25b-ST131, resistant to the high level of enrofloxacin and containing the gene encoding the ferric aerobactin receptor (iutA). One enrofloxacin resistant E. coli strain possessed iss, ibeA, kps and papC virulence genes also with CMY-2, integrase1 and Tn3. ST131 E. coli with CMY-2 has a zoonotic potential and presents a serious health risk to humans.

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Pandemic fluoroquinolone resistant Escherichia coli clone ST1193 emerged via simultaneous homologous recombinations in 11 gene loci

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903002116 ◽

2019 ◽

Vol 116 (29) ◽

pp. 14740-14748 ◽

Cited By ~ 12

Author(s):

Veronika Tchesnokova ◽

Matthew Radey ◽

Sujay Chattopadhyay ◽

Lydia Larson ◽

Jamie Lee Weaver ◽

...

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Resistance Mutations ◽

Chromosomal Dna ◽

Topoisomerase Iv ◽

E Coli ◽

Naturally Occurring ◽

High Level

Global growth in antibiotic resistance is a major social problem. A high level of resistance to fluoroquinolones requires the concurrent presence of at least 3 mutations in the target proteins—2 in DNA gyrase (GyrA) and 1 in topoisomerase IV (ParC), which occur in a stepwise manner. In the Escherichia coli chromosome, the gyrA and parC loci are positioned about 1 Mb away from each other. Here we show that the 3 fluoroquinolone resistance mutations are tightly associated genetically in naturally occurring strains. In the latest pandemic uropathogenic and multidrug-resistant E. coli clonal group ST1193, the mutant variants of gyrA and parC were acquired not by a typical gradual, stepwise evolution but all at once. This happened as part of 11 simultaneous homologous recombination events involving 2 phylogenetically distant strains of E. coli, from an uropathogenic clonal complex ST14 and fluoroquinolone-resistant ST10. The gene exchanges swapped regions between 0.5 and 139 Kb in length (183 Kb total) spread along 976 Kb of chromosomal DNA around and between gyrA and parC loci. As a result, all 3 fluoroquinolone resistance mutations in GyrA and ParC have simultaneously appeared in ST1193. Based on molecular clock estimates, this potentially happened as recently as <12 y ago. Thus, naturally occurring homologous recombination events between 2 strains can involve numerous chromosomal gene locations simultaneously, resulting in the transfer of distant but tightly associated genetic mutations and emergence of a both highly pathogenic and antibiotic-resistant strain with a rapid global spread capability.

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High Carriage of Plasmid-Mediated Quinolone Resistance (PMQR) Genes by Cefotaxime-Resistant Escherichia Coli Recovered from Surface-Leaking Sanitary Sewers

10.21203/rs.3.rs-699642/v1 ◽

2021 ◽

Author(s):

Abimbola Olumide Adekanmbi ◽

Olabisi C. Akinlabi ◽

Adedolapo V. Olaposi

Keyword(s):

Escherichia Coli ◽

Brain Heart Infusion ◽

Quinolone Resistance ◽

Health Concern ◽

Diffusion Method ◽

Resistant Bacteria ◽

E Coli ◽

Specific Pcr ◽

Sanitary Sewers ◽

High Level

Abstract There is a rapid rise in the incident of quinolone resistant bacteria in Nigeria. Most studies in Nigeria have focused on isolates from the clinical settings, with few focusing on isolates of environmental origin. This study aimed to investigate the antibiogram and carriage of plasmid-mediated quinolone resistance (PMQR) genes by quinolone-resistant isolates obtained from a pool of cefotaxime-resistant Escherichia coli (E. coli) recovered from sewage leaking out of some broken sanitary sewers in a University community in Nigeria. Isolation of E. coli from the sewage samples was done on CHROMagar E. coli after enrichment of the samples was done in Brain Heart Infusion amended with 6µg/mL of cefotaxime. Identification of presumptive E. coli was done using molecular methods (detection of uidA gene), while susceptibility to antibiotics was carried out using disc-diffusion method. Detection of PMQR genes (qnrA, qnrB, qnrS, aac(6')-lb-cr, qepA and oqxAB) was done using primer-specific PCR. A total of 32 non-repetitive cefotaxime-resistant E. coli were obtained from the sewage, with 21 being quinolone-resistant. The quinolone-resistant isolates showed varying level of resistance to the tested antibiotics, with imipenem being the only exception with 0% resistance. The PMQR genes: aac(6')-lb-cr, qnrA, qnrB, qnrS and qepA and oqxAB were detected in 90.5%, 61.9%, 47.6%, 38.1%, 4.8% and 0% respectively of the isolates. The findings of this study showed a high level of resistance to antibiotics and carriage of PMQR genes by quinolone-resistant E. coli obtained from the leaking sanitary sewers, suggesting a potential environmental and public health concern.

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Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.2.491 ◽

1996 ◽

Vol 40 (2) ◽

pp. 491-493 ◽

Cited By ~ 138

Author(s):

J Vila ◽

J Ruiz ◽

P Goñi ◽

M T De Anta

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Quinolone Resistance ◽

Clinical Isolates ◽

Serine Residue ◽

Topoisomerase Iv ◽

E Coli ◽

A Subunit ◽

Detection Of Mutations

The gene parC encodes the A subunit of topoisomerase IV of Escherichia coli. Mutations in the parC region analogous to those in the quinolone resistance-determining region of gyrA were investigated in 27 clinical isolates of E. coli for which ciprofloxacin MICs were 0.0007 to 128 micrograms/ml. Of 15 isolates for which ciprofloxacin MICs were > or = 1 microgram/ml, 8 showed a change in the serine residue at position 80 (Ser-80), 4 showed a change in Glu-84, and 3 showed changes in both amino acids. No mutations were detected in 12 clinical isolates for which ciprofloxacin MICs were < or = 0.25 micrograms/ml. These findings suggest that ParC from E. coli may be another target for quinolones and that mutations at residues Ser-80 and Glu-84 may contribute to decreased fluoroquinolone susceptibility.

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High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli

Biochemistry and Cell Biology ◽

10.1139/o90-069 ◽

1990 ◽

Vol 68 (2) ◽

pp. 492-495 ◽

Cited By ~ 11

Author(s):

Wen Shi ◽

King-Chuen Chow ◽

J. Tze-Fei Wong

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Large Scale ◽

Trna Synthetase ◽

Start Codon ◽

Temperature Sensitive ◽

Gene Encoding ◽

Scale Preparation ◽

High Level

The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid. Upon induction with isopropyl-β-D-thiogalactopyranoside, Escherichia coli JM109[pKSW1] cells synthesized TrpRS to a level corresponding to 45% of total cell proteins. This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by [3H]Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization. Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E. coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be detectible on the basis of complementation testing.Key words: tryptophan-tRNA, aminoacyl-tRNA synthetase, Bacillus subtilis.

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The effect of some factors on expression of gene encoding endoglucanase from dna metagenome of Binh Chau hot spring in Escherichia coli

TAP CHI SINH HOC ◽

10.15625/0866-7160/v41n2.13726 ◽

2019 ◽

Vol 41 (2) ◽

Author(s):

Tran Thanh Thuy ◽

Lai Thi Hong Nhung ◽

Tran Dinh Man ◽

Le Thi Thanh Xuan ◽

Nguyen Kim Thoa

Keyword(s):

Escherichia Coli ◽

Target Genes ◽

Hot Springs ◽

Hot Spring ◽

Gene Encoding ◽

Culture Volume ◽

E Coli ◽

Cell Densities ◽

High Level ◽

Expression Time

Expression of microbial target genes in Escherichia coli is broadly used due to its advantages namely: well established system, easy to manipulate, a huge biomass, high level productivity, safe and inexpensive to grow. Metagenomic technique has been applying in Vietnam recently for effective mining of uncultured gene resources, especially in endemic mini-ecologies such as hot springs where the cell densities are low. DNA metagenome of Binh Chau hot spring was isolated and sequenced by Illumia HiseqTM. Based on analyses of databases of cellulase-encoded genes, denovogenes 18736 gene sequence for thermal endoglucanase was selected for expression in E. coli. In this paper, some factors for expression of endoglucanase have been investigated. The results show that appropriate gene expression conditions are: Expression performed in E. coli C43 (DE3) on TB medium at 30oC with 0.25 mM of IPTG as inducer, the culture volume of 20% compared with the bottle volume and the expression time is 42–48 hours. In this condition, the biomass production and soluble enzyme activity can reached up to 5.54–5.58 g /L and 1.92–1.98 U/mL, respectively. Our results show the prospect of exploiting microbial genes without culture.

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Adenylate Cyclase Mutations Rescue the degP Temperature-Sensitive Phenotype and Induce the Sigma E and Cpx Extracytoplasmic Stress Regulons in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.18.6309-6316.2005 ◽

2005 ◽

Vol 187 (18) ◽

pp. 6309-6316 ◽

Cited By ~ 9

Author(s):

Timothy G. Strozen ◽

Geoffrey R. Langen ◽

S. Peter Howard

Keyword(s):

Escherichia Coli ◽

Adenylate Cyclase ◽

Temperature Sensitive ◽

Null Mutant ◽

Gene Encoding ◽

E Coli ◽

Constitutive Activation ◽

Sigma E ◽

Temperature Sensitive Phenotype

ABSTRACT Inactivation of the gene encoding the periplasmic protease DegP confers a high-temperature-sensitive phenotype in Escherichia coli. We have previously demonstrated that a degP mutant of E. coli strain CBM (W3110 pldA1) is not temperature sensitive and showed that this was most likely due to constitutive activation of the sigma E and Cpx extracytoplasmic stress regulons in the parent strain. In this study, further characterization of this strain revealed a previously unknown cryptic mutation that rescued the degP temperature-sensitive phenotype by inducing the extracytoplasmic stress regulons. We identified the cryptic mutation as an 11-bp deletion of nucleotides 1884 to 1894 of the adenylate cyclase-encoding cyaA gene (cyaAΔ11). The mechanism in which cyaAΔ11 induces the sigma E and Cpx regulons involves decreased activity of the mutant adenylate cyclase. Addition of exogenous cyclic AMP (cAMP) to the growth medium of a cyaAΔ11 mutant strain that contains a Cpx- and sigma E-inducible degP-lacZ reporter fusion decreased β-galactosidase expression to levels observed in a cyaA + strain. We also found that a cyaA null mutant displayed even higher levels of extracytoplasmic stress regulon activation compared to a cyaAΔ11 mutant. Thus, we conclude that the lowered concentration of cAMP in cyaA mutants induces both sigma E and Cpx extracytoplasmic stress regulons and thereby rescues the degP temperature-sensitive phenotype.

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Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.4.879 ◽

1996 ◽

Vol 40 (4) ◽

pp. 879-885 ◽

Cited By ~ 167

Author(s):

P Heisig

Keyword(s):

Escherichia Coli ◽

Fluoroquinolone Resistance ◽

Gyra Gene ◽

Topoisomerase Iv ◽

E Coli ◽

Resistant Strains ◽

Gyra Mutation ◽

High Level ◽

Moderately Resistant

Fifteen strains of Escherichia coli with MICs of ciprofloxacin (CIP) between 0.015 and 256 micrograms/ml were examined for the presence of mutations in the quinolone resistance-determining region of the gyrA gene and in an analogous region of the parC gene. No mutation was found in a susceptible isolate (MIC of CIP, 0.015 microgram/ml). Four moderately resistant strains (MIC of CIP 0.06 to 4 micrograms/ml) carried one gyrA mutation affecting serine 83, but in only one strain was an additional parC mutation (Gly-78 to Asp) detected. All ten highly resistant strains examined (MIC of CIP, > 4 micrograms/ml) carried two gyrA mutations affecting residues serine 83 and aspartate 87, and at least one parC mutation. These parC mutations included alterations of serine 80 to arginine or isoleucine and glutamate 84 to glycine or lysine. The parC+ and two mutant alleles (parCI-80 and parCI-80,G-84) were inserted into the mobilizable vector pBP507. Transfer of a plasmid-coded parC+ allele into parC+ strains did not alter the susceptibilities towards ciprofloxacin or nalidixic acid, while a significant increase in susceptibility was detectable for parC mutants. This increase, however, did not restore wild-type susceptibility, whereas transfer of a plasmid-coded gyrA+ allele alone or in combination with parC+ did. These data are in agreement with the view that topoisomerase IV is a secondary, less sensitive target for quinolone action in Escherichia coli and that the development of high-level fluoroquinolone resistance in E. coli requires at least one parC mutation in addition to the gyrA mutation(s).

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Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2343-2351.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2343-2351 ◽

Cited By ~ 91

Author(s):

Patricia Komp Lindgren ◽

Linda L. Marcusson ◽

Dorthe Sandvang ◽

Niels Frimodt-Møller ◽

Diarmaid Hughes

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Infection Model ◽

Resistance Mutations ◽

Topoisomerase Iv ◽

E Coli ◽

Tract Infections ◽

Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

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