Rapid Detection of Genomic Mutations in gyrA and parC Genes of Escherichia coli by Multiplex Allele Specific Polymerase Chain Reaction

Onseedaeng, Sukanlayanee; Ratthawongjirakul, Panan

doi:10.1002/jcla.21961

Cited by 17 publications

(14 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Screening of E. coli isolates for ARG types was conducted using PCR for each isolate depending on the antibiotic resistance phenotype. The ARGs we examined were (1) carbapenemase genes bla NDM , bla IMP bla KPC , bla VIM , bla OXA , bla AIM , bla BIC , bla DIM , bla GIM , bla SIM , and bla SPM including bla NDM and bla CTX-M subtyping (Poirel et al, 2011), (2) β-lactamase genes bla SHV , bla TEM , and bla CTX-M (Casella et al, 2018), (3) plasmidmediated AmpC β-lactamase genes bla MOX , bla CMY , bla LAT , bla DHA , bla ACC , bla MIR , bla ACT , and bla FOX (Perez-Perez and Hanson, 2002), (4) colistin resistance genes mcr-1-8 (Rebelo et al, 2018;Yang et al, 2018;Carroll et al, 2019), and (5) plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB and qnrS including whether the gyrA and parC genes in the quinolone resistance determining region (QRDR) were mutated (Komp et al, 2003;Kraychete et al, 2016;Onseedaeng and Ratthawongjirakul, 2016). PCR primers used for screening are shown in Supplementary Table S1.…”

Section: Arg Detectionmentioning

confidence: 99%

Increasing Prevalence of ESBL-Producing Multidrug Resistance Escherichia coli From Diseased Pets in Beijing, China From 2012 to 2017

et al. 2019

View full text Add to dashboard Cite

We investigated antimicrobial resistance trends and characteristics of ESBL-producing Escherichia coli isolates from pets and whether this correlates with antibiotic usage in the clinic. Clinical samples containing E. coli from diseased cats and dogs were screened for antibiotic sensitivity and associated genotypic features. We identified 127 E. coli isolates from 1886 samples from dogs (n = 1565) and cats (n = 321) with the majority from urinary tract infections (n = 108, 85%). High rates of resistance were observed for β-lactams and fluoroquinolones and resistance to > 3 antibiotic classes (MDR) increased from 67% in 2012 to 75% in 2017 (P < 0.0001). This was especially true for strains resistant to 6-9 antibiotics that increased from 26.67 to 60.71%. Increased rates in β-lactam use for clinical treatment accompanied these increasing resistance rates. Accordingly, the most frequently encountered subtypes were bla CTX-M (n = 44, 34.65%), bla CTX-M-65 (n = 19) and bla CTX-M-15 (n = 18) and qnrB (n = 119, 93.70%). The bla CTX-M-isolates possessed 36 unique pulsed field electrophoretic types (PFGEs) and 28 different sequence types (STs) in ST405 (7, 15.9%), ST131 (3, 6.8%), ST73, ST101, ST372, and ST827 (2, 4.5% each) were the most prevalent. This data demonstrated a high level of diversity for the bla CTX-M-positive E. coli isolates. Additionally, bla NDM-5 was detected in three isolates (n = 3, 2.36%), comprised of two ST101 and one ST405 isolates, and mcr-1 was also observed in three colistin-resistant E. coli with three different STs (ST6316, ST405, and ST46). Our study demonstrates an increasing trend in MDR and ESBL-producing E. coli and this correlated with β-lactam antibiotic usage for treatment of these animals. This data indicates that there is significant risk for the spread of resistant bacteria from pets to humans and antibiotic use for pets should be more strictly regulated.

show abstract

Section: Arg Detectionmentioning

confidence: 99%

Increasing Prevalence of ESBL-Producing Multidrug Resistance Escherichia coli From Diseased Pets in Beijing, China From 2012 to 2017

et al. 2019

View full text Add to dashboard Cite

show abstract

“…It can be assumed that this, as a universally functional mechanism, is likely to affect DNA supercoiling, and the expression of several virulence factors and proteins as described in different species is the same ( Ozeki et al., 1997 ; Aubry et al., 2006 ; Abdelbaqi et al., 2007 ; Hashimi et al., 2008 ; Lau et al., 2011 ; Malik et al., 2012 ; Yamachika et al., 2012 ; Sutera et al., 2017 ). Ciprofloxacin resistance usually occurs due to specific point mutations within the DNA gyrase A ( gyrA) and/or topoisomerase IV parC and parE genes, often in combination ( Casin et al., 2003 ; Johnning et al., 2015 ; Onseedaeng and Ratthawongjirakul, 2016 ). Annotation of mutation in the gyrase A gene is considered not to be sufficient to produce resistance to ciprofloxacin ( Hooper and Jacoby, 2015 ).…”

Section: Discussionmentioning

confidence: 99%

Antimicrobial Susceptibility and Genomic Analysis of Aliarcobacter cibarius and Aliarcobacter thereius, Two Rarely Detected Aliarcobacter Species

Hänel

Müller

González-Santamarina

et al. 2021

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

Aliarcobacter cibarius and Aliarcobacter thereius are two rarely detected Aliarcobacter species. In the study, we analyzed the antimicrobial susceptibility and provide detailed insights into the genotype and phylogeny of both species using whole-genome sequencing. Thermophilic Campylobacter species are the most common bacterial foodborne pathogens causing gastroenteritis in humans worldwide. The genus Aliarcobacter is part of the Campylobacteraceae family and includes the species Aliarcobacter butzleri, Aliarcobacter cryaerophilus, Aliarcobacter skirrowii, and the rarely described Aliarcobacter cibarius, Aliarcobacter faecis, Aliarcobacter lanthieri, Aliarcobacter thereius, and Acrobarter trophiarum. Aliarcobacter are emergent enteropathogens and potential zoonotic agents. Here, we generated, analyzed, and characterized whole-genome sequences of Aliarcobacter cibarius and Aliarcobacter thereius. They were isolated from water poultry farms in Germany, cultured and identified by MALDI-TOF MS. With PCR the identity was verified. Antibiotic susceptibility testing was carried out with erythromycin, ciprofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, ampicillin, and cefotaxime using the gradient strip method (E-test). Whole-genome sequences were generated including those of reference strains. Complete genomes for six selected strains are reported. These provide detailed insights into the genotype. With these, we predicted in silico known AMR genes, virulence-associated genes, and plasmid replicons. Phenotypic analysis of resistance showed differences between the presence of resistance genes and the prediction of phenotypic resistance profiles. In Aliarcobacter butzleri, the nucleotide sequence of the gyrA gene (DQ464331) can show a signature mutation resulting in an amino acid change T85>I. Acrobarter cibarius and Acrobarter thereius showed the same gene as assessed by similarity annotation of the mutations 254C>G. Most of the isolates were found to be sensitive to ciprofloxacin. The ciprofloxacin-resistant Aliarcobacter thereius isolate was associated with the amino acid change T85>I. But this was not predicted with antibiotic resistance databases, before. Ultimately, a phylogenetic analysis was done to facilitate in future outbreak analysis.

show abstract

“…Since AS-PCR was first reported in 1989 (Newton et al, 1989), it has been widely used to screen single nucleotide polymorphisms and mutations. Although AS-PCR is considered as low specificity and sensitivity (Sharma et al, 2016), Onseedaeng & Ratthawongjirakul (2016) identified mutations of the gyrA and parC gene in Escherichia coli with high sensitivity and specificity by using AS-PCR. Due to its simple operation, low cost, and relatively high specificity and sensitivity, we successfully used AS-PCR to screen 16 mutations in four first-line DR-genes.…”

Section: Discussionmentioning

confidence: 99%

Rapid screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR

Yang

Song

Xia

et al. 2019

PeerJ

View full text Add to dashboard Cite

Tuberculosis (TB) is a worldwide health, economic, and social burden, especially in developing countries. Drug-resistant TB is the most serious type of this burden. Thus, it is necessary to screen drug-resistant mutations by using a simple and rapid detection method. A total of 32 pairs of allele-specific PCR (AS-PCR) primers were designed to screen mutation and/or wild-type alleles of 16 variations in four first-line drug-resistant genes (katG, rpoB, rpsL, and embB) of TB strains. A pair of primers was designed to amplify 16S rRNA gene and to verify successful amplification. Subsequently, we tested the specificity and sensitivity of these AS-PCR primers. The optimized condition of these AS-PCR primers was first confirmed. All mutations could be screened in general AS-PCR, but only 13 of 16 variations were intuitively investigated by using real-time quantitative PCR (qPCR) and AS-PCR primers. The results of specificity assay suggested that the AS-PCR primers with mutation and/or wildtype alleles could successfully amplify the corresponding allele under optimized PCR conditions. The sensitivity of nine pairs of primers was 500 copy numbers, and the other seven pairs of primers could successfully amplify correct fragments with a template comprising 103 or 104 copy numbers template. An optimized AS-qPCR was established to screen drug-resistant mutations in TB strains with high specificity and sensitivity.

show abstract

Rapid Detection of Genomic Mutations in gyrA and parC Genes of Escherichia coli by Multiplex Allele Specific Polymerase Chain Reaction

Abstract: MAS-PCR may be used for rapid detection of FR resistance in routine laboratory as well as in epidemiology study.

Cited by 17 publications

References 31 publications

Increasing Prevalence of ESBL-Producing Multidrug Resistance Escherichia coli From Diseased Pets in Beijing, China From 2012 to 2017

Increasing Prevalence of ESBL-Producing Multidrug Resistance Escherichia coli From Diseased Pets in Beijing, China From 2012 to 2017

Antimicrobial Susceptibility and Genomic Analysis of Aliarcobacter cibarius and Aliarcobacter thereius, Two Rarely Detected Aliarcobacter Species

Rapid screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR

Contact Info

Product

Resources

About