scholarly journals The prevalence and mechanism of triclosan resistance in Escherichia coli isolates from urine samples in Wenzhou, China

2020 ◽  
Author(s):  
Weiliang Zeng ◽  
Wenya Xu ◽  
Ye Xu ◽  
Wenli Liao ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Cross Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Triclosan Resistance ◽  
Encoding Genes

Abstract Background Widespread use of triclosan has been reported to cause its residue in urine, which provides an environment of long-term exposure to triclosan for intestinal Escherichia coli. We aimed to determine the triclosan and antibiotic resistance characteristics of Escherichia coli strains isolated from urine, and further investigate the resistance mechanism and molecular epidemic characteristics of triclosan resistant Escherichia coli isolates. Methods A total of 200 non-repetitive E. coli strains from urine samples were obtained and identified. The minimum inhibitory concentrations (MICs) of triclosan and antibiotics, fabI mutation, efflux pump activity, expression of 14 efflux pump encoding genes and epidemiological characteristics were detected with agar dilution method, polymerase chain reaction (PCR), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibition test, quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing (MLST) and pulse field gel electrophoresis (PFGE) in all triclosan resistant isolates. Furthermore, we also investigated the effect of triclosan exposure in vitro on resistance in susceptible strains by serial passage experiment. Results Of 200 E. coli isolates, 2.5% (n = 5) were resistant to triclosan, multidrug resistance (MDR) and cross-resistance phenotypes were observed in these resistant strains, but not in susceptible strains. We did not observe any sense mutations within fabI gene in triclosan resistant strains. Moreover, except DC8603, all the others enhanced efflux pumps activity. Compared with ATCC 25922, except fabI, increased expression were also found in efflux pump encoding genes ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD and mdfA in studied strains with different PFGE patterns and STs types. Surprised, 5 susceptible E. coli isolates increased rapidly triclosan resistance only 4 days after exposure to subinhibitory triclosan concentration in vitro. Conclusions Our study is the first to be reported that short-term triclosan exposure in vitro increases triclosan resistance in susceptible E. coli isolates. Once strains have acquired resistance, they usually present MDR or cross-resistance phenotypes. Besides, our findings indicate that triclosan resistance were mainly involved by fabI overexpression in E. coli, and there was a close association between overexpression of efflux pumps with triclosan resistance.

scholarly journals The prevalence and mechanism of triclosan resistance in Escherichia coli isolated from urine samples in Wenzhou, China

2020 ◽  
Author(s):  
Weiliang Zeng ◽  
Wenya Xu ◽  
Ye Xu ◽  
Wenli Liao ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Urine Samples ◽  
Cross Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Triclosan Resistance ◽  
Encoding Genes

Abstract Background: The widespread application of triclosan contributes to its residual deposition in urine, which provides an environment of long-term exposure to triclosan for the intestinal Escherichia coli. We determined the triclosan and antibiotic resistance characteristics of E. coli strains isolated from urine samples and further investigated the resistance mechanism and molecular epidemic characteristics of triclosan-resistant E. coli isolates. Methods: A total of 200 non-repetitive E. coli strains were isolated from urine samples and then identified. The minimum inhibitory concentrations (MICs) of triclosan and antibiotics, fabI mutation, efflux pump activity, the expression of 14 efflux pump encoding genes, and epidemiological characteristics were determined by the agar dilution method, polymerase chain reaction (PCR), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibition test, quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing (MLST), and pulse-field gel electrophoresis (PFGE) for all triclosan-resistant isolates. Furthermore, we also investigated the effect of triclosan exposure in vitro on antibiotic susceptibility and the efflux pump encoding gene expressions of triclosan-susceptible strains via serial passage experiments. Results: Of the 200 E. coli isolates, 2.5% (n = 5) were found to be resistant to triclosan, and multidrug resistance (MDR) and cross-resistance phenotypes were noted for these triclosan-resistant strains. The triclosan-sensitive strains also exhibited MDR phenotypes, probably because of the high resistance rate to AMP, CIP, LVX, and GEN. Gly79Ala and Ala69Thr amino acid changes were observed in the triclosan-resistant strains, but these changes may not mediate resistance of E. coli to triclosan, because mutations of these two amino acids has also been detected in triclosan-susceptible strains. Moreover, except for DC8603, all other strains enhanced the efflux pumps activity. As compared with ATCC 25922, except for fabI, increased expressions were noted for all efflux pump encoding genes such as ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD, and mdfA among the studied strains with varying PFGE patterns and STs types. Unexpectedly, 5 susceptible E. coli isolates showed rapidly increasing triclosan resistance after exposure to triclosan in vitro for only 12 days, while MDR or cross-resistance phenotypes and the overexpression of efflux pump genes were recorded among these triclosan-induced resistant isolates. Conclusions: This is the first study to report that short-term triclosan exposure in vitro increases triclosan resistance in susceptible E. coli isolates. After acquiring resistance, these strains may present MDR or cross-resistance phenotypes. Moreover, triclosan resistance mainly involves the overexpression of fabI and efflux pumps in E. coli isolates.

scholarly journals The prevalence and mechanism of triclosan resistance in Escherichia coli isolated from urine samples in Wenzhou, China

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Weiliang Zeng ◽  
Wenya Xu ◽  
Ye Xu ◽  
Wenli Liao ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Urine Samples ◽  
Cross Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Resistant Strains ◽  
Polymerase Chain ◽  
Triclosan Resistance ◽  
Encoding Genes

Abstract Background The widespread application of triclosan contributes to its residual deposition in urine, which provides an environment of long-term exposure to triclosan for the intestinal Escherichia coli. We determined the triclosan and antibiotic resistance characteristics of E. coli strains isolated from urine samples and further investigated the resistance mechanism and molecular epidemic characteristics of triclosan-resistant E. coli isolates. Methods A total of 200 non-repetitive E. coli strains were isolated from urine samples and then identified. The minimum inhibitory concentrations (MICs) of triclosan and antibiotics, fabI mutation, efflux pump activity, the expression of 14 efflux pump encoding genes, and epidemiological characteristics were determined by the agar dilution method, polymerase chain reaction (PCR), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibition test, quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing (MLST), and pulse-field gel electrophoresis (PFGE) for all triclosan-resistant isolates. Furthermore, we also investigated the effect of triclosan exposure in vitro on antibiotic susceptibility and the efflux pump encoding gene expressions of triclosan-susceptible strains via serial passage experiments. Results Of the 200 E. coli isolates, 2.5% (n = 5) were found to be resistant to triclosan, and multidrug resistance (MDR) and cross-resistance phenotypes were noted for these triclosan-resistant strains. The triclosan-sensitive strains also exhibited MDR phenotypes, probably because of the high resistance rate to AMP, CIP, LVX, and GEN. Gly79Ala and Ala69Thr amino acid changes were observed in the triclosan-resistant strains, but these changes may not mediate resistance of E. coli to triclosan, because mutations of these two amino acids has also been detected in triclosan-susceptible strains. Moreover, except for DC8603, all other strains enhanced the efflux pumps activity. As compared with ATCC 25922, except for fabI, increased expressions were noted for all efflux pump encoding genes such as ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD, and mdfA among the studied strains with varying PFGE patterns and STs types. Unexpectedly, 5 susceptible E. coli isolates showed rapidly increasing triclosan resistance after exposure to triclosan in vitro for only 12 days, while MDR or cross-resistance phenotypes and the overexpression of efflux pump genes were recorded among these triclosan-induced resistant isolates. Conclusions This is the first study to report that short-term triclosan exposure in vitro increases triclosan resistance in susceptible E. coli isolates. After acquiring resistance, these strains may present MDR or cross-resistance phenotypes. Moreover, triclosan resistance mainly involves the overexpression of fabI and efflux pumps in E. coli isolates.

scholarly journals Overexpression of target enzyme gene fabI and efflux pump decrease triclosan susceptibility in Escherichia coli

2020 ◽  
Author(s):  
Weiliang Zeng ◽  
Wenya Xu ◽  
Ye Xu ◽  
Wenli Liao ◽  
Yajie Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Close Association ◽  
Opportunistic Pathogen ◽  
Cross Resistance ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
E Coli ◽  
Resistant Strains ◽  
Triclosan Resistance

Abstract Background: Escherichia coli isolates, the most opportunistic pathogen in the gut, are responsible for the most acquired infections. Triclosan is an effective disinfectant for inhibits microorganisms, but its widespread use causes its residue in urine, resulting in long-term exposure of E. coli in the intestine to triclosan environment and increasing triclosan resistance. We aim to provide the mechanism of action of E. coli isolates against triclosan and the molecular epidemiological analysis of triclosan-resistant strains.Results: Five triclosan-resistant isolates were screened out from 200 E. coli isolates by agar dilution method by to further study, interestingly, multidrug-resistant and cross-resistance phenotypes were observed in triclosan-resistant strains, but not in susceptible strains, and all except one exhibited an inhibition of efflux pump activity by efflux pump inhibition testing. Furthermore, compared with susceptible E. coli strain ATCC 25922, except fabI, increased expression were also found in efflux pump encoding genes ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD and mdfA in studied strains which had different PFGE patterns and STs types.Conclusions: These findings indicated that triclosan resistance in E. coli were mainly involved by overexpression of fabI gene, and there was a close association between overexpression of efflux pump with reducing susceptibility to triclosan. Besides, we described cross-resistance between triclosan and antibiotics may be related to the exposure time of triclosan.

Preparation and Standardization of Escherichia coli Nosodes Sourced from Select E. coli Strains

Homeopathy ◽  
2020 ◽  
Vol 109 (04) ◽  
pp. 207-212
Author(s):  
Renuka Munshi ◽  
Gitanjali Talele ◽  
Rajesh Shah
Keyword(s):  
Escherichia Coli ◽  
Preparation Method ◽  
Cell Extract ◽  
Molecular Testing ◽  
Chain Reaction ◽  
E Coli ◽  
Entire Cell ◽  
Polymerase Chain ◽  
Different Strains

Abstract Background The nosodes are well-known preparations in homeopathy that are sourced from organisms and diseased materials. More than 40 known nosodes have been used in homeopathic practice for over a century. Having identified the need for scientifically developed new nosodes sourced from organisms that are currently prevalent, the preparation of Escherichia coli nosodes from different strains of the bacterium is presented in this article. Materials and Methods Escherichia coli strains (E. coli ATCC 11775E, ATCC 25922, and ATCC 8739) were identified, cultured, and tested for purity, and 20 billion cells were processed following the nosode preparation method given in the Homoeopathic Pharmacopoeia of India, group N1. Serial dilution and potentization for liquid potency were done up to 30c potency. Nosodes were prepared by two methods: from cell-free extract (endotoxin) and from entire-cell extract. Result Six nosodes were developed in total. Three univalent nosodes were prepared using individual endotoxins, one from each of the three E. coli strains; those three univalent nosodes were also combined as “Trivalent nosode-I”. “Trivalent nosode-II” was prepared by mixing entire cells of the three E. coli strains. A mix of both Trivalent nosode-I and Trivalent nosode-II was labeled “EC-Polynosode”. The safety profile of the potentized nosodes was documented by the non-detectability of traces of source material (absence of contamination, live organisms, or DNA material) through a culture test, sterility test, and molecular testing (polymerase chain reaction). Conclusion Different variants of E. coli nosodes were systematically and scientifically prepared and standardized using the cultures. Homeopathic pathogenetic trials, in-vitro efficacy studies, and clinical evaluation of E. coli nosodes (single, trivalent, or polyvalent nosodes) will be required in future.

scholarly journals On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

2005 ◽  
Vol 37 (4) ◽  
pp. 265-269 ◽  
Author(s):  
Xi-Qiang Zhu ◽  
Su-Xia Li ◽  
Hua-Jun He ◽  
Qin-Sheng Yuan
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Mass Ratio ◽  
Chain Reaction ◽  
Expression Plasmid ◽  
Protein Subunits ◽  
E Coli ◽  
Metal Affinity ◽  
Polymerase Chain ◽  
Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

scholarly journals Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

2007 ◽  
Vol 59 (2) ◽  
pp. 508-512 ◽  
Author(s):  
B.R. Paneto ◽  
R.P. Schocken-Iturrino ◽  
C. Macedo ◽  
E. Santo ◽  
J.M. Marin
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Chain Reaction ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Polymerase Chain ◽  
Raw Milk Cheese

The occurrence of toxigenic Escherichia coli in raw milk cheese was surveyed in Middle Western Brazil. Fifty samples of cheese from different supermarkets were analyzed for E.coli. The isolates were serotyped and screened for the presence of verotoxigenic E. coli (VTEC) and enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR). The susceptibility to thirteen antimicrobial agents was evaluated by the disk diffusion method. E.coli were recovered from 48 (96.0%) of the samples. The serogroups identified were O125 (6.0%), O111 (4.0%), O55 (2.0%) and O119 (2.0%). Three (6.0%) and 1(2.0%) of the E.coli isolates were VTEC and ETEC, respectively. Most frequent resistance was observed to the following antimicrobials: cephalothin (60.0%), nalidixic acid (40.0%), doxycyclin (33.0%), tetracycline (31.0%) and ampicillin (29.0%).

scholarly journals Multiplex Polymerase Chain Reaction Assay for Identification of Enterotoxigenic Escherichia Coli Strains

2001 ◽  
Vol 13 (4) ◽  
pp. 308-311 ◽  
Author(s):  
Jacek Osek
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Direct Determination ◽  
Enteric Bacteria ◽  
Enterotoxigenic Escherichia Coli ◽  
Chain Reaction ◽  
Gram Negative ◽  
E Coli ◽  
Polymerase Chain

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein ( uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non- E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

scholarly journals Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

2013 ◽  
Vol 11 (3) ◽  
pp. 382-386 ◽  
Author(s):  
Richard Kibbee ◽  
Natalie Linklater ◽  
Banu Örmeci
Keyword(s):  
Escherichia Coli ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Uida Gene ◽  
Chain Reaction ◽  
Taq Polymerase ◽  
E Coli ◽  
Gene Copies ◽  
Polymerase Chain ◽  
Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

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

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.

scholarly journals Prevalence and molecular detection of shiga toxin producing Escherichia coli from diarrheic cattle

2016 ◽  
Vol 14 (1) ◽  
pp. 63-68 ◽  
Author(s):  
MM Akter ◽  
S Majumder ◽  
KH MNH Nazir ◽  
M Rahman
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Molecular Detection ◽  
Chain Reaction ◽  
Specific Primers ◽  
Fecal Samples ◽  
E Coli ◽  
Uremic Syndrome ◽  
Polymerase Chain ◽  
Positive Isolate

Shiga toxin-producing Escherichia coli (STEC) are zoonotically important pathogen which causes hemorrhagic colitis, diarrhea, and hemolytic uremic syndrome in animals and humans. The present study was designed to isolate and identify the STEC from fecal samples of diarrheic cattle. A total of 35 diarrheic fecal samples were collected from Bangladesh Agricultural University (BAU) Veterinary Teaching Hospital. The samples were primarily examined for the detection of E. coli by cultural, morphological and biochemical characteristics, followed by confirmation of the isolates by Polymerase Chain Reaction (PCR) using gene specific primers. Later, the STEC were identified among the isolated E. coli through detection of Stx-1 and Stx-2 genes using duplex PCR. Out of 35 samples, 25 (71.43%) isolates were confirmed to be associated with E. coli, of which only 7 (28%) isolates were shiga toxin producers, and all of them were positive for Stx-1. However, no Stx-2 positive isolate could be detected. From this study, it may be concluded that cattle can act as a reservoir of STEC which may transmit to human or other animals.J. Bangladesh Agril. Univ. 14(1): 63-68, June 2016

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