Characterization of plasmid-mediated quinolone resistance by the qnrS gene in Escherichia coli isolated from healthy chickens and pigs

The prevalence of qnr and qepA genes in 660 Escherichia coli isolates was investigated in healthy animals from 30 pig farms and 30 chicken farms in Taiwan from January 2005 to February 2006 by the polymerase chain reaction. The qnrS gene, but not qnrA, qnrB, and qepA were detected in 12/360 pig isolates (3.33%) and in 6/300 chicken isolates (2%). Southern blot hybridization analysis indicated that qnrS was located on plasmids ranging in size from 50–165 kb. Eleven of the 18 qnrS positive isolates which showed a high ciprofloxacin resistance phenotype (minimum inhibitory concentration ≥ 8 mg/l) also had amino acid sequence variations in chromosomal quinolone resistance-determining regions of gyrA and parC. Only two qnrS-positive isolates carried the aac(6’)-Ib-crvariant that mediates FQ acetylation. For the high percentage resistance of cephalosporins, the blaCTX-M gene was also examined in qnrS-positive isolates. The blaCTX-M gene was detected in fifteen isolates (15/18, 83.3%) of which 12 isolates were blaCTX-M-1 and three isolates were blCTX-M-15. This study demonstrated a close linkage between the qnrS gene and blaCTX-M-1, suggesting CTX-M and Qnr-based mechanisms might be co-emerging in E. coli strains isolated from healthy chickens and pigs under selective pressure of quinolone and cephalosporine administration.

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Phenotypic and genotypic quinolone resistance in Escherichia coli underlining GyrA83/87 mutations as a target to detect ciprofloxacin resistance

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa189 ◽

2020 ◽

Vol 75 (9) ◽

pp. 2466-2470

Author(s):

Anaëlle Muggeo ◽

Emmanuelle Cambau ◽

Marlène Amara ◽

Maïté Micaëlo ◽

Béatrice Pangon ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Bloodstream Infections ◽

Blood Cultures ◽

Quinolone Resistance ◽

E Coli ◽

Molecular Determinants ◽

Ciprofloxacin Resistance ◽

First Time

Abstract Background Quinolone resistance (QR) is one component of the MDR emerging in Escherichia coli and is of particular concern given the widespread use of fluoroquinolones. Objectives To characterize the QR phenotypes and genotypes in E. coli responsible for bloodstream infections and to propose molecular determinants that could be targeted to predict ciprofloxacin resistance. Methods E. coli isolates from blood cultures in three French hospitals were studied for quinolone MICs and characterization of genotypic QR determinants (QRg). Results Among 507 isolates tested for MICs, 148 (29.2%) were resistant to quinolones based on EUCAST breakpoints and 143 (28.2%) harboured at least one QRg. QRg were mainly mutations in the QRDR (138 isolates, 27.2%), with 55.8% of these isolates carrying at least three QRDR mutations. gyrA mutations predominated (92.8%) followed by parC (61.6%), parE (32.6%) and gyrB (1.4%) mutations. Only 4.7% of the isolates harboured a plasmid-mediated quinolone resistance (PMQR) gene: aac(6′)-Ib-cr (60.0%) or qnr (qnrS, qnrB) (32.0%). For the first time in France, we reported the qepA4 allele of the plasmid-encoded efflux pump QepA. Only five isolates carried PMQR without a QRDR mutation. The positive predictive value (PPV) for ciprofloxacin resistance was 100% for any QRg and 99.2% for gyrA mutations specifically. Conclusions QR observed in E. coli isolates involved in bloodstream infections is still mainly due to QRDR mutations, especially at codons GyrA83/87, which could be used as a molecular target to rapidly detect resistance.

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IDENTIFICATION OF AMPC Β-LACTAMASE-PRODUCING CLINICAL ISOLATES OF ESCHERICHIA COLI

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2017.v10i12.21648 ◽

2017 ◽

Vol 10 (12) ◽

pp. 357

Author(s):

Tanushree Barua Gupta ◽

Malini Shariff ◽

Thukral Ss ◽

S.s Thukral

Keyword(s):

Escherichia Coli ◽

Detection Method ◽

Clinical Isolates ◽

Confirmatory Test ◽

Chain Reaction ◽

E Coli ◽

Resistance To Penicillin ◽

Polymerase Chain ◽

Third Generation Cephalosporins

Objective: Indiscriminate use of β-lactam antibiotics has resulted in the emergence of β-lactamase enzymes. AmpC β-lactamases, in particular, confer resistance to penicillin, first-, second-, and third-generation cephalosporins as well as monobactams and are responsible for antibiotic resistance in nosocomial pathogens. Therefore, this study was undertaken to screen nosocomial Escherichia coli isolates for the presence and characterization of AmpC β-lactamases. The study also envisaged on the detection of inducible AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) in AmpC β-lactamase-producing E. coli.Methods: A total of 102 clinical isolates of E. coli, were subjected to cefoxitin screening, and screen-positive isolates were further subjected to inhibitor-based detection method, phenotypic confirmatory test, disc antagonism test, polymerase chain reaction (PCR), and isoelectric focusing (IEF).Results: In this study, 33% of E. coli were resistant to cefoxitin, of which 35% were found to be positive for AmpC β-lactamase by inhibitor-based phenotypic test. Of the AmpC-positive isolates, 83% were positive for ESBLs, whereas 25% were producing inducible AmpC β-lactamases. PCR and IEF showed CIT and EBC types of AmpC β-lactamases present in the tested isolates.Conclusion: Our study showed the presence of inducible AmpC enzymes and ESBLs in E. coli isolates and PCR identified more isolates to be AmpC producers.

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Molecular Characterization of Plasmid-Mediated Non-O157 Verotoxigenic Escherichia coli Isolated from Infants and Children with Diarrhea

Baghdad Science Journal ◽

10.21123/bsj.2020.17.3.0710 ◽

2020 ◽

Vol 17 (3) ◽

pp. 0710

Author(s):

Md Fazlul Karim Khan ◽

Shah Samiur Rashid

Keyword(s):

Escherichia Coli ◽

Virulence Gene ◽

Multidrug Resistant ◽

Plasmid Curing ◽

Health Issues ◽

E Coli ◽

Disk Diffusion Assay ◽

Microbiological Techniques ◽

Polymerase Chain

A significant increase in the incidence of non-O157 verotoxigenic Escherichia coli (VTEC) infections have become a serious health issues, and this situation is worsening due to the dissemination of plasmid mediated multidrug-resistant microorganisms worldwide. This study aims to investigate the presence of plasmid-mediated verotoxin gene in non-O157 E. coli. Standard microbiological techniques identified a total of 137 E. coli isolates. The plasmid was detected by Perfectprep Plasmid Mini preparation kit. These isolates were subjected to disk diffusion assay, and plasmid curing with ethidium bromide treatment. The plasmid containing isolates were subjected to a polymerase chain reaction (PCR) for investigating the presence of plasmid mediated verotoxin gene (VT1 and VT2) in non-O157 E. coli. Among the 137 E. coli isolates, 49 isolates were non-O157 E. coli while 29 (59.1%) isolates were verotoxin producing non-O157 serotypes and 26 non-O157 VTEC isolates possessed plasmids. Certain isolates harboured single sized plasmid while others had multiple plasmids with different size varied from 1.8kb to 7.6kb. A plasmid containing all (100%) the isolates was multidrug-resistant. Eight isolates changed their susceptibility patterns while three isolates were found to lose plasmid after post plasmid curing treatment and the rest of the isolates (15) remained constant. Different PCR sets characterized 3 plasmid-mediated verotoxins producing non-O157 E. coli. This current study demonstrated the occurrence of plasmid mediated verotoxin gene in non-O157 E. coli. To the best of our knowledge, this is the first report in the global literature on plasmid-mediated verotoxin gene in non-O157 E. coli. Timely diagnosis and surveillance of VTEC infections should prioritize to stop or slow down the virulence gene for dissemination by plasmid-mediated gene transfer amongst the same bacteria or other species.

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Detection of plasmid-mediated qnr genes among the quinolone-resistant Escherichia coli isolates in Iran

The Journal of Infection in Developing Countries ◽

10.3855/jidc.3746 ◽

2014 ◽

Vol 8 (07) ◽

pp. 818-822 ◽

Cited By ~ 12

Author(s):

Farzaneh Firoozeh ◽

Mohammad Zibaei ◽

Younes Soleimani-Asl

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Quinolone Resistance ◽

Diffusion Method ◽

Chain Reaction ◽

Disk Diffusion Method ◽

E Coli ◽

Polymerase Chain ◽

Tract Infections

Introduction: Plasmid-mediated quinolone resistance, which complicates treatment, has been increasingly identified in Escherichia coli isolates worldwide. The purpose of this study was to identify the plasmid-mediated qnrA and qnrB genes among the quinolone-resistant Escherichia coli isolated from urinary tract infections in Iran. Methodology: A total of 140 Escherichia coli isolates were collected between March and October 2012 from urinary tract infections in Khorram Abad, Iran. All isolates were tested for quinoloe resistance using the disk diffusion method. Also, all quinolone-resistant isolates were screened for the presence of the qnrA and qnrB genes by polymerase chain reaction. Minimum inhibitory concentrations (MICs) of ciprofloxacin for the qnr-positive isolates were determined. Results: One hundred sixteen (82.8%) of 140 Escherichia coli isolates were nalidixic acid-resistant; among them, 14 (12.1%) and 9 (7.8%) were qnrA and qnrB-positive, respectively. Two quinolone-resistant isolates harbored both qnrA and qnrB. Among 63 ciprofloxacin-resistant isolates, 14 (22.2%) and 9 (14.3%) were found to carry qnrA and qnrB genes, respectively. The ciprofloxacin MIC range was 0.25–512 μg/mL for 23 qnr-positive Escherichia coli isolates, 18 of which had MICs values of 4–512 μg/mL. Conclusion: Our study shows that the frequency of plasmid-mediated quinolone resistance genes among E. coli isolates in Iran is high.

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Characterization of the pTFI91 -family replicon of Thiobacillus ferrooxidans plasmids

Canadian Journal of Microbiology ◽

10.1139/m95-048 ◽

1995 ◽

Vol 41 (4-5) ◽

pp. 354-365 ◽

Cited By ~ 6

Author(s):

Leena Chakravarty ◽

Thomas J. Zupancic ◽

Beth Baker ◽

Joseph D. Kittle ◽

Ilona J. Fry ◽

...

Keyword(s):

Dna Sequences ◽

Thiobacillus Ferrooxidans ◽

Blot Hybridization ◽

Marker Gene ◽

Southern Blot Hybridization ◽

Structural Features ◽

Broad Host Range ◽

Origin Of Replication ◽

E Coli ◽

Southern Blot Hybridization Analysis

Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those of T. ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5α cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I in E. coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation of T. ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either in E. coli or in P. putida. Since pTFI91 contains the same origin of replication as other plasmids in several other T. ferrooxidans strains, this replicon may be commonly distributed in T. ferrooxidans.Key words: nucleotide sequence, origin of replication, plasmid DNA, replicon, Thiobacillus ferrooxidans.

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Molecular Detection of Plasmid-Mediated Quinolone Resistance in Ciprofloxacin-Resistant Escherichia coli from Urine of Patients attending Garki Hospital, Abuja, Nigeria

European Journal of Biology and Biotechnology ◽

10.24018/ejbio.2020.1.4.60 ◽

2020 ◽

Vol 1 (4) ◽

Author(s):

Moses Oghenaigah Eghieye ◽

Istifanus Haruna Nkene ◽

Rejoice Helma Abimiku ◽

Yakubu Boyi Ngwai ◽

Ibrahim Yahaya ◽

...

Keyword(s):

Escherichia Coli ◽

Polymerase Chain Reaction ◽

Urinary Tract ◽

Molecular Detection ◽

Quinolone Resistance ◽

Chain Reaction ◽

E Coli ◽

Pcr Method ◽

Polymerase Chain ◽

Tract Infections

Urinary tract infections (UTIs) caused by Escherichia coli (E. coli) is common worldwide; and its successful treatment using antibiotics is limited by acquisition of resistance by the bacteria. This study investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) genes in ciprofloxacin-resistant E. coli from urine of patients with suspected cases of UTIs attending Garki Hospital Abuja (GHA), Nigeria. A total of 8 confirmed ciprofloxacin-resistant E. coli was screened for carriage of PMQR genes using polymerase chain reaction (PCR) method. The occurrences of the PMQR genes detected were in the order: aac-(6′)-Ib-cr (87.5%) > qnrB (50.0%) > qnrS (37.5%) > oqxAB (12.5%) > qnrA(0.0%). qnrB and qnrS did not exist alone, but in combination with other genes; aac-(6′)-Ib-crexisted both alone and in combination with others; the most prevalent patterns of existence were aac-(6′)-Ib-cr alone and aac-(6′)-Ib-cr + qnrB + qnrS at 25.0% each. This study has shown that the ciprofloxacin-resistant E. coli harbored aac-(6′)-Ib-cr, qnrB, qnrS and oqxAB PMQR genes, with aac-(6′)-Ib-cr being the most prevalent. The genes were present either alone or in combination with one another. This has implication for the clinical application of fluoroquinolones to treat UTI in the study location and environs.

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Prevalence and characteristics ofEscherichia coilserotype O157:H7 and other verotoxin-producingE. coliin healthy cattle

Epidemiology and Infection ◽

10.1017/s0950268800001424 ◽

1996 ◽

Vol 117 (2) ◽

pp. 251-257 ◽

Cited By ~ 60

Author(s):

M. Blanco ◽

J. E. Blanco ◽

J. Blanco ◽

E. A. Gonzalez ◽

A. Mora ◽

...

Keyword(s):

Escherichia Coli ◽

Polymerase Chain Reaction ◽

Gene Sequence ◽

Heterogeneous Population ◽

Chain Reaction ◽

E Coli ◽

Healthy Cattle ◽

Faecal Samples ◽

Polymerase Chain

SummaryFrom February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producingEscherichia coli(VTEC) found on 84% of the farms. The proportion of animals infected varied from 0–63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8. O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraernic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagicE. coli(EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacingE. coli (eae)gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes. 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence ofeaegenes in most bovine VTEC strains suggests that they may be less virulent for humans thaneae-positive EHEC.

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Dissemination of Blandm-5 in Escherichia Coli via the Incx3 Plasmid From Different Regions in China

10.21203/rs.3.rs-254667/v1 ◽

2021 ◽

Author(s):

Xiaofeng Hu ◽

Lang Yang ◽

Nian Dong ◽

Yanfeng Lin ◽

Ling Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Blot Hybridization ◽

Carbapenem Resistance ◽

Southern Blot Hybridization ◽

Control Measures ◽

Rrna Gene ◽

Limited Information ◽

E Coli ◽

Carbapenem Resistant ◽

Antimicrobial Resistance Genes

Abstract Background: Recently, the spread of NDM-5-producing Escherichia coli has become a severe challenge in clinical therapy, which necessitates reliable detection and surveillance methods. However, limited information is available regarding the prevalence and dissemination of the blaNDM-5 gene in Escherichia coli in China. Therefore, we investigated the dissemination of the blaNDM-5 gene in carbapenem-resistant Escherichia coli isolates from different regions in China.Methods: A total of 1,180 carbapenem-resistant enterobacteriaceae strains were obtained from patients admitted to the 20 sentinel hospitals in eight cities. Strains positive for blaNDM-5 were detected using the Vitek 2 compact system, 16S rRNA gene sequencing, PCR, the S1-pulsed-field gel electrophoresis assay, and Southern blot hybridization. The horizontal-transfer capability of the blaNDM gene was assessed by filter mating with a standard E. coli J53 azide-resistant strain as the recipient. Genotyping, susceptibility testing, and whole genome sequencing were performed. Results: Seven strains of blaNDM-5-positive E.coli was detected in 1180 clinical strains from different regions in China. The blaNDM-5-carrying strains showed resistance to multiple tested antibiotics and belonged to two widespread sequence types, ST167 and ST405. Antimicrobial resistance genes including blaCTX-M, blaOXA, blaCMY, and two novel blaTEM variants (blaTEM-230 and blaTEM-231) were also identified. Southern blotting located the blaNDM-5 gene on 46-kb IncX3 plasmids in all isolates, which showed only two single nucleotide differences between EJN003 and the other strains. Conclusions: This study further confirms the increasing occurrence of blaNDM-5-carrying IncX3 plasmids and the dissemination of carbapenem resistance in E. coli isolates via the plasmid from different parts in China, which warrants stringent surveillance and control measures.

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Prevalence and Characterization of Plasmid-mediated Quinolone Resistance Genes among Escherichia coli Strains Isolated from Different Water Sources in Alborz Province, Iran

The Indonesian Biomedical Journal ◽

10.18585/inabj.v11i1.484 ◽

2019 ◽

Vol 11 (1) ◽

pp. 36-41

Author(s):

Reza Ranjbar ◽

Shahrzad Tavanania ◽

Azar Sabokbar ◽

Faham Khamesipour

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Surface Water ◽

Nalidixic Acid ◽

Bacterial Species ◽

Water Sources ◽

Quinolone Resistance ◽

E Coli ◽

Different Water Sources

BACKGROUND: This study was conducted to investigate the prevalence of quinolone resistance associated (qnr) antibiotic resistance among Escherichia coli strains isolated from different water sources in Alborz province, Iran.METHODS: E. coli strains were isolated and identified by standard microbiological and biochemical tests from surface water sources in Alborz province, Iran in 2013. Fluoroquinolone-resistant isolates were determined using the antimicrobial susceptibility test determined by the Kirby–Bauer assay. Total genomic and plasmid DNA were extracted by boiling method. The presence of qnr genes in all nalidixic-acid and ciprofloxacin-resistant E. coli strains was determined by Polymerase Chain Reaction (PCR). The PCR amplicons were visualized after electrophoresis stained with ethidium bromide.RESULTS: One hundred E. coli strains were isolated from the water sources examined in this study. As much as 22.7% and 7.3% of the isolates were resistant to nalidixic acid and ciprofloxacin respectively. While qnrS, qnrB and qnrA genes were detected in 28%, 9% and 1% of fluoroquinolone-resistant isolates respectively. All fluoroquinolone-susceptible isolates however did not contain any of the qnr genes.CONCLUSION: This study reflects an increasing prevalence of fluoroquinolone-resistant E. coli strains in surface water sources. Underlining the importance of surface water sources as reservoirs for dissemination of potentially pathogenic E. coli and horizontal gene transfer between other waterborne bacterial species. Other possible mechanisms of resistance should also be investigated for better characterization of quinolone-resistant E. coli isolates. Therefore, immediate measures are needed to control and treat water sources more effectively.KEYWORDS: antibiotic resistance, E. coli, qnr genes, water sources

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Differences in Virulence and Genomic Features of Strains of ‘Candidatus Phytoplasma mali’, the Apple Proliferation Agent

Phytopathology ◽

10.1094/phyto-97-8-0964 ◽

2007 ◽

Vol 97 (8) ◽

pp. 964-970 ◽

Cited By ~ 46

Author(s):

Erich Seemüller ◽

Bernd Schneider

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Blot Hybridization ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Southern Blot Hybridization ◽

Apple Proliferation ◽

Candidatus Phytoplasma Mali ◽

Polymerase Chain

Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with ‘Candidatus Phytoplasma mali’ were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52°C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in ‘Ca. P. mali’. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.

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