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 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 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.

scholarly journals Mutation in 23S rRNA Associated with Macrolide Resistance in Neisseria gonorrhoeae

2002 ◽  
Vol 46 (9) ◽  
pp. 3020-3025 ◽  
Author(s):  
Lai-King Ng ◽  
Irene Martin ◽  
Gary Liu ◽  
Louis Bryden
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Promoter Region ◽  
Efflux Pump ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Cross Resistance ◽  
E Coli ◽  
Single Base Pair ◽  
Resistant Strains ◽  
Domain V

ABSTRACT Fifty-six azithromycin-resistant (MICs, 2.0 to 4.0 μg/ml) Neisseria gonorrhoeae strains with cross-resistance to erythromycin (MICs, 2.0 to 64.0 μg/ml), isolated in Canada between 1997 and 1999, were characterized, and their mechanisms of azithromycin resistance were determined. Most (58.9%) of them belonged to auxotype-serotype class NR/IB-03, with a 2.6-mDa plasmid. Based on resistance to crystal violet (MICs ≥ 1 μg/ml), 96.4% of these macrolide-resistant strains appeared to have increased efflux. Nine of the eleven strains selected for further characterization were found to have a promoter region mtrR mutation, a single-base-pair (A) deletion in the 13-bp inverted repeat, which is believed to cause overexpression of the mtrCDE-encoded efflux pump. The two remaining macrolide-resistant strains (erythromycin MIC, 64.0 μg/ml; azithromycin MIC, 4.0 μg/ml), which did not have the mutation in the mtrR promoter region, were found to have a C2611T mutation (Escherichia coli numbering) in the peptidyltransferase loop in domain V of the 23S rRNA alleles. Although mutations in domain V of 23S rRNA alleles had been reported in other bacteria, including E. coli, Streptococcus pneumoniae, and Helicobacter pylori, this is the first observation of these mutations associated with macrolide resistance in N. gonorrhoeae.

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 DESAIN PRIMER UNTUK AMPLIFIKASI REGIO PROMOTER GEN inhA ISOLAT P016 MULTIDRUG RESISTANCE Mycobacterium tuberculosis DENGAN METODE POLYMERASE CHAIN REACTION

2018 ◽  
pp. 34
Author(s):  
Eka Nata Sari
Keyword(s):  
Polymerase Chain Reaction ◽  
Promoter Region ◽  
Pcr Amplification ◽  
Promoter Sequence ◽  
Cross Resistance ◽  
Chain Reaction ◽  
Pcr Products ◽  
Amplification Process ◽  
Polymerase Chain

Mutations in the inhA promoter region are responsible for isoniazid resistance and cross-resistance to ethionamide. To identify mutations with polymerase chain reaction (PCR), a pair of primers that can amplify the target region. This aim of this study was to obtain the best primer pair to amplify the inhA promoter region using the Clone Manager Suite 6 program. The inhA promoter sequence (Genbank: U66801) obtained from the www.ncbi.nlm.nih.gov was used as a template. The design results obtained the best primer pair tested in vitro using Polymerase Chain Reaction (PCR) method. The PCR amplification process was performed for 40 cycles with the following conditions: predenaturase (95oC for 15 minutes), denaturation (94 oC for 1 minute), annealing (56 oC for 1 minute 30 seconds), elongation (72 oC for 2 minutes), and final elongation (72 oC for 10 minutes). Detection of PCR products was performed in agarose gel electrophoresis 1.3% w / v and visualized by UV transluminator tool. The results obtained were forward primers of 5'-GGTCGAAGTGTGCTGAGTC-3 'and reverse primer 5'-TGCTCTTCTACCGCCGTGA-3' which met the good primary criterion based on Clone Manager Suite 6. The pair of primers has been able to amplify the inhA promoter region by the length of product produced at 373 bp.

Detection of the Glanzmann's Thrombasthenia Mutations in Arab and Iraqi-Jewish Patients by Polymerase Chain Reaction and Restriction Analysis of Blood or Urine Samples

1991 ◽  
Vol 66 (04) ◽  
pp. 500-504 ◽  
Author(s):  
H Peretz ◽  
U Seligsohn ◽  
E Zwang ◽  
B S Coller ◽  
P J Newman
Keyword(s):  
Polymerase Chain Reaction ◽  
Base Pair ◽  
Restriction Analysis ◽  
Urine Samples ◽  
Chain Reaction ◽  
Base Pairs ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Base Pair Deletion ◽  
Polymerase Chain

SummarySevere Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews – an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs – a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

scholarly journals Derivation, characterization, and in vitro cell regeneration of canine white adipose tissue-derived mesenchymal stem cells obtained from a mesenteric region

2021 ◽  
Vol 82 (1) ◽  
Author(s):  
Anirban Mandal ◽  
Ajeet Kumar Jha ◽  
Dew Biswas ◽  
Shyamal Kanti Guha
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Stromal Vascular Fraction ◽  
Cell Regeneration ◽  
Wound Healing Assay ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background The study was conducted to assess the characterization, differentiation, and in vitro cell regeneration potential of canine mesenteric white adipose tissue-derived mesenchymal stem cells (AD-MSCs). The tissue was harvested through surgical incision and digested with collagenase to obtain a stromal vascular fraction. Mesenchymal stem cells isolated from the stromal vascular fraction were characterized through flow cytometry and reverse transcription-polymerase chain reaction. Assessment of cell viability, in vitro cell regeneration, and cell senescence were carried out through MTT assay, wound healing assay, and β-galactosidase assay, respectively. To ascertain the trilineage differentiation potential, MSCs were stained with alizarin red for osteocytes, alcian blue for chondrocytes, and oil o red for adipocytes. In addition, differentiated cells were characterized through a reverse transcription-polymerase chain reaction. Results We observed the elongated, spindle-shaped, and fibroblast-like appearance of cells after 72 h of initial culture. Flow cytometry results showed positive expression for CD44, CD90, and negative expression for CD45 surface markers. Population doubling time was found 18–24 h for up to the fourth passage and 30±0.5 h for the fifth passage. A wound-healing assay was used to determine cell migration rate which was found 136.9 ± 4.7 μm/h. We observed long-term in vitro cell proliferation resulted in MSC senescence. Furthermore, we also found that the isolated cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages. Conclusions Mesenteric white adipose tissue was found to be a potential source for isolation, characterization, and differentiation of MSCs. This study might be helpful for resolving the problems regarding the paucity of information concerning the basic biology of stem cells. The large-scale use of AD-MSCs might be a remedial measure in regenerative medicine.

scholarly journals Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

2015 ◽  
Vol 59 (6) ◽  
pp. 3059-3065 ◽  
Author(s):  
C. Pitart ◽  
F. Marco ◽  
T. A. Keating ◽  
W. W. Nichols ◽  
J. Vila
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistant Mutant ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Content Type ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Impact

ABSTRACTCeftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200Enterobacteriaceaeand 25Pseudomonas aeruginosastrains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistantEnterobacteriaceaestrains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBLEscherichia coli(MIC90of 0.25 mg/liter), ESBLKlebsiella pneumoniae(MIC90of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90of 1 mg/liter), non-ESBLE. coli(MIC90of ≤0.125 mg/liter), non-ESBLK. pneumoniae(MIC90of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistantP. aeruginosastrains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtainedin vitrofrom two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains ofEnterobacteriaceaeandP. aeruginosawere ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affectEnterobacteriaceaeandP. aeruginosasusceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.

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