Use of a rapid mismatch PCR method to detect gyrA and parC mutations in ciprofloxacin-resistant clinical isolates of Escherichia coli

Qiang, Yan Zhi; Tong, Qin; Wang, Fu; Cheng, Wu Pei; Li, Yang Sheng; Gong, Yaoqin

doi:10.1093/jac/49.3.549

Cited by 45 publications

(38 citation statements)

References 10 publications

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“…Previously described parC mutations in E. coli have been at codons 80 and 84 (10). The codon 57 has not previously been associated with quinolone resistance in E. coli; however, the association observed does not confirm that the mutation is the cause of this resistance.…”

Section: Vol 71 2005 Quinolone Resistance In Serovar Enteritidis 2589mentioning

confidence: 82%

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

Kilmartin

Morris

O’Hare

et al. 2005

Appl Environ Microbiol

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We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n ‫؍‬ 22) and nalidixic acid-susceptible (n ‫؍‬ 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates.

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Section: Vol 71 2005 Quinolone Resistance In Serovar Enteritidis 2589mentioning

confidence: 82%

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

Kilmartin

Morris

O’Hare

et al. 2005

Appl Environ Microbiol

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“…For outcrossing and generation of double mutants, the presence of mutant and wild-type alleles was determined in parallel reactions using a PCR-based point-mutant detection method (Qiang et al 2002). For each reaction, either the mutant-specific primer or the wild-type-specific primer was paired with the "common" primer as indicated.…”

Section: Pcr-based Methods For Identification Of Point-mutantsmentioning

confidence: 99%

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

et al. 2012

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The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-offunction mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-toarginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/ VAChT and synaptobrevin. C OORDINATED muscle contraction in both vertebrates and nematodes requires release of the neurotransmitter acetylcholine (ACh) from motor neurons. ACh is synthesized in motor neurons by the enzyme choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular ACh transporter (VAChT). Following transmitter release, cholinergic signaling is terminated primarily through the action of acetylcholinesterase (AChE), which hydrolyses the released ACh into choline and acetyl-CoA. In the nematode Caenorhabditis elegans, the ChAT and VAChT proteins are encoded by the cha-1 and unc-17 genes, respectively (Alfonso et al. , 1994b. Mutations that functionally eliminate either protein are lethal , while reduction-of-function mutations result in animals that are small, slow growing, uncoordinated, and resistant to AChE inhibitors such as aldicarb .The unc-17 missense mutation e245 replaces a glycine with an arginine (G347R) in the ninth transmembrane domain (TMD9) of the VAChT protein . Genetic screens designed to identify mutations that improve the locomotion of e245 homozygotes have identified several loci that suppress the deleterious effects of the VAChT G347R mutation. Sandoval et al. (2006) found that the sup-8 locus corresponds to the previously characterized snb-1 gene, which encodes the v-SNARE synaptobrevin. The single sup-8 allele is associated with a missense mutation that results in a charge alteration (I97D) in the TMD of synaptobrevin. T...

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“…Recombinants were identified by plating on kanamycin, and colonies were screened using MAMA PCR (Qiang et al 2002) to identify which strand-specific mutations were inherited in each colony. Two replicates were performed, and 48 colonies were screened for each recombination (Table 1; detailed results in Table S3).…”

Section: à4mentioning

confidence: 99%

“…The mismatch amplification mutation assay (MAMA) PCR method was used to analyze the genotypes of mismatched lacZ::kanR dsDNA recombinants (Qiang et al 2002).…”

Section: Analysis Of Mismatched Dsdna Recombinants By Mama Pcrmentioning

confidence: 99%

Lambda Red Recombineering inEscherichia coliOccurs Through a Fully Single-Stranded Intermediate

2010

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The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 59 ends of double-stranded DNA are recessed by l exonuclease, leaving behind 39 overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinasecatalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.

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Use of a rapid mismatch PCR method to detect gyrA and parC mutations in ciprofloxacin-resistant clinical isolates of Escherichia coli

Cited by 45 publications

References 10 publications

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

Lambda Red Recombineering inEscherichia coliOccurs Through a Fully Single-Stranded Intermediate

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