ABSTRACT
The in vitro activity of enrofloxacin against 73 Mycoplasma synoviae field strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 µg/ml. The estimated MIC50 and MIC90 values for enrofloxacin were 2 and 8 µg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) of M. synoviae have MICs of ≥ 2 µg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) in M. synoviae isolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥ 1 µg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison of vlhA molecular profiles revealed the presence of 9 different genotypes in the Israeli M. synoviae field isolates and 10 genotypes in the European isolates; only one vlhA genotype (type 4) was identified in both cohorts. Based on results of vlhA molecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistant M. synoviae isolates are suggested.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Fluoroquinolones/pharmacology , Lectins/genetics , Mycoplasma synoviae/drug effects , Mycoplasma synoviae/genetics , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , Enrofloxacin , Europe , Genotype , Israel , Microbial Sensitivity Tests , Molecular Sequence Data , Molecular Typing , Mycoplasma Infections , Mycoplasma synoviae/isolation & purification , Quinolones/pharmacology , Sequence Analysis, DNAABSTRACT
Molecular characterization of the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV in 93 Mycoplasma gallisepticum field strains isolated in different geographic regions revealed discrepancies between minimal inhibitory concentration values and presence of amino acid substitutions within the QRDRs of GyrA and ParC in 9/93 (10%) strains. This may delimitate applicability of a gene-based assay to detect fluoroquinolone resistance in this avian pathogen.
Subject(s)
Fluoroquinolones/pharmacology , Mutation , Mycoplasma gallisepticum/drug effects , Mycoplasma gallisepticum/genetics , Amino Acid Substitution , Animals , Birds , DNA Gyrase/genetics , DNA Topoisomerase IV/chemistry , DNA Topoisomerase IV/genetics , Enrofloxacin , Microbial Sensitivity Tests , Mycoplasma Infections/microbiology , Mycoplasma gallisepticum/isolation & purification , Poultry Diseases/microbiology , Quinolones/pharmacologyABSTRACT
Monitoring of the susceptibility of Mycoplasma bovis field isolates to antibiotics is important for the appropriate choice of treatment. However, in vitro susceptibility testing of mycoplasmas is technically demanding and time-consuming, especially for clinical isolates, and is rarely performed in mycoplasma diagnostic laboratories. Thus, the development of methods allowing rapid real-time detection of resistant strains of M. bovis in clinical samples is a high priority for successful treatment. In this study, a novel TaqMan single-nucleotide-polymorphism (SNP) real-time PCR assay, which enables the rapid identification of M. bovis strains with different susceptibilities to fluoroquinolones, was developed and evaluated. The TaqMan SNP real-time PCR assay is based on the amplification of a 97-bp fragment of the parC quinolone resistance-determining region (QRDR) and allows the specific detection of four possible genotypes: GAC or GAT (susceptible to fluoroquinolones) and AAC or AAT (resistant to fluoroquinolones). Four TaqMan minor groove binder (MGB) probes identifying 1-base mismatches were designed and applied in a dual-probe assay with two reaction tubes. The TaqMan SNP real-time PCRs developed are highly specific for M. bovis, with a detection limit of 5 fg/microl (about 5 M. bovis genomes). In addition, all four SNP real-time PCR tests have almost the same efficiency (97.7% [GAC], 94% [AAC], 99.99% [GAT], and 98% [AAT]). Taken together, the data suggest that this SNP real-time PCR assay has potential as a routine diagnostic test for the detection of decreased susceptibility of M. bovis to fluoroquinolones.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA, Bacterial/genetics , Fluoroquinolones/pharmacology , Mycoplasma bovis/drug effects , Mycoplasma bovis/genetics , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Animals , Bacterial Proteins/genetics , Cattle , Cattle Diseases/microbiology , DNA Topoisomerase IV/genetics , Genotype , Microbial Sensitivity Tests/methods , Mycoplasma Infections/microbiology , Mycoplasma Infections/veterinary , Mycoplasma bovis/isolation & purification , Oligonucleotide Probes/genetics , Sensitivity and SpecificityABSTRACT
Comparison of the quinolone resistance-determining regions (QRDRs) in 42 Mycoplasma bovis clinical isolates revealed amino acid substitutions at both GyrA (position 83) and ParC (position 84) in 10/11 enrofloxacin-resistant strains. The mutation present in the parC QRDR was discriminative for enrofloxacin resistance by parC PCR-restriction fragment length polymorphism. Comparison of molecular profiles by insertion sequence typing suggests that the currently prevalent enrofloxacin-resistant M. bovis strain evolved by selection under field conditions from one of the susceptible strains.
Subject(s)
Anti-Infective Agents/pharmacology , DNA Topoisomerase IV/genetics , Fluoroquinolones/pharmacology , Mycoplasma bovis/drug effects , Mycoplasma bovis/genetics , Point Mutation , Enrofloxacin , Microbial Sensitivity Tests , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
Emergence of resistance to fluoroquinolones is mainly due to chromosomal mutations in genes encoding the subunits of the drug's target enzymes, DNA gyrase and topoisomerase IV, which are essential for DNA replication. The quinolone resistance-determining regions (QRDRs) of these genes were characterized in 25 Mycoplasma gallisepticum strains isolated from commercial poultry flocks during 1997-2007, which exhibited different levels of susceptibility to fluoroquinolones. All enrofloxacin-resistant isolates harbored amino acid substitutions in the QRDRs of each of three proteins (GyrA, GyrB, and ParC). Molecular typing of those strains by random amplification of polymorphic DNA and gene-targeted sequencing supports ongoing, stepwise selection of resistant strains from the existing reservoir of susceptible M. gallisepticum strains.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Fluoroquinolones/pharmacology , Mycoplasma gallisepticum/drug effects , Mycoplasma gallisepticum/genetics , Amino Acid Substitution/genetics , Animals , Base Sequence , Chickens , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , DNA, Bacterial/chemistry , Enrofloxacin , Genes, Bacterial , Molecular Sequence Data , Mutation , Mycoplasma gallisepticum/classification , Random Amplified Polymorphic DNA Technique/veterinary , TurkeysABSTRACT
This work reports the development of a 16S rRNA-based polymerase chain reaction (PCR) test for the detection of Mycoplasma meleagridis, and the application, together with a previously described multi-species PCR, for the diagnosis of M. meleagridis and M. iowae in dead-in-shell turkey embryos. Forty-six allantoic fluid samples from dead-in-shell turkey embryos from hatcheries in Israel and the United States were tested by PCR and cultured for the detection of M. meleagridis and M. iowae. Three methods of DNA extraction for PCR assay were compared in allantoic fluid samples from turkey embryos experimentally infected with M. iowae. The two PCR assays provided more rapid detection than culture and were more effective in detecting the components of a mixed infection. Furthermore, PCR has the ability to recognize all M. iowae strains irrespective of antigenic variation.
