Susceptibility To Enrofloxacin Research Articles

Evaluation of a fluorescence resonance energy transfer quantitative polymerase chain reaction assay for identification of gyrA mutations conferring enrofloxacin resistance in canine urinary Escherichia coli isolates and canine urine specimens.

OBJECTIVE To evaluate a fluorescence resonance energy transfer quantitative PCR (FRET-qPCR) assay for detection of gyrA mutations conferring fluoroquinolone resistance in canine urinary Escherichia coli isolates and canine urine specimens. SAMPLE 264 canine urinary E coli isolates and 283 clinical canine urine specimens. PROCEDURES The E coli isolates were used to validate the FRET-qPCR assay. Urine specimens were evaluated by bacterial culture and identification, isolate enrofloxacin susceptibility testing, and FRET-qPCR assay. Sensitivity and specificity of the FRET-qPCR assay for detection of gyrA mutations in urine specimens and in E coli isolated from urine specimens were computed, with results of enrofloxacin susceptibility testing used as the reference standard. RESULTS The validated FRET-qPCR assay discriminated between enrofloxacin-resistant and enrofloxacin-susceptible E coli isolates with an area under the receiver operating characteristic curve of 0.92. The assay accurately identified 25 of 40 urine specimens as containing enrofloxacin-resistant isolates (sensitivity, 62.5%) and 226 of 243 urine specimens as containing enrofloxacin-susceptible isolates (specificity, 93.0%). When the same assay was performed on E coli isolates recovered from these specimens, sensitivity (77.8%) and specificity (94.8%) increased. Moderate agreement was achieved between results of the FRET-qPCR assay and enrofloxacin susceptibility testing for E coli isolates recovered from urine specimens. CONCLUSIONS AND CLINICAL RELEVANCE The FRET-qPCR assay was able to rapidly distinguish between enrofloxacin-resistant and enrofloxacin-susceptible E coli in canine clinical urine specimens through detection of gyrA mutations. Therefore, the assay may be useful in clinical settings to screen such specimens for enrofloxacin-resistant E coli to avoid inappropriate use of enrofloxacin and contributing to antimicrobial resistance.

Detecção de resistência às fluoroquinolonas em Campylobacter isolados de frangos de criação orgânica

Resumo Estudos têm revelado que a resistência às quinolonas em cepas de Campylobacter está relacionada à presença da mutação Treonina-86 para Isoleucina. Com o objetivo de investigar a presença dessa mutação em cepas de Campylobacter sensíveis e resistentes à ciprofloxacina e enrofloxacina, o conteúdo cecal de 80 frangos de corte de criação orgânica, abatidos sob Serviço de Inspeção Estadual (S.I.E.) do Estado do Rio de Janeiro, foram coletados e investigados para a presença de Campylobacter. A determinação da resistência à ciprofloxacina e enrofloxacina foi feita pela técnica de difusão em disco e de diluição em ágar para determinação da Concentração Inibitória Mínima (CIM). A detecção da mutação na Região Determinante de Resistencia às Quinolonas (RDRQ) no gene gyrA foi realizada através de sequenciamento. Campylobacter foi isolado a partir de 100% das amostras avaliadas, sendo 68,75% correspondente à C. jejuni e 31,25% à C. coli. No teste de difusão em disco, 100% das cepas foram resistentes à ciprofloxacina e 56,25% das cepas foram resistentes à enrofloxacina. No teste de diluição em ágar, todas as cepas foram resistentes à ciprofloxacina apresentando CIM variando de ≥ 16-64μg/mL, e resistência ou resistência intermediaria à enrofloxacina foi detectada em 42,50% (CIM ≥ 4-32μg/mL) e 38,75% (CIM = 2μg/mL) das cepas, respectivamente. A mutação Tre-86-Ile, foi observada em 100% das cepas analisadas. Além dessa mutação, foram observadas outras mutações não silenciosas (Val-73-Glu, Ser-114-Leu, Val-88-Asp, Ala-75-Asp, Ser-119-Gli, Arg-79-Lis) e mutações silenciosas (His-81-His, Ser-119-Ser, Ala-120-Ala, Fen-99-Fen, Ala-122-Ala, Gli-74-Gli, Ile-77-Ile, Ala-91-Ala, Leu-92-Leu, Val-93-Val, Ile-106-Ile, Tre-107-Tre, Gli-113-Gli, Ile-115-Ile, Gli-110-Gli). A observação de que cepas sensíveis à enrofloxacina pelos testes fenotípicos apresentavam a substituição Tre-86 para Ile sugere que outros mecanismos podem contribuir para a resistência à enrofloxacina em Campylobacter.

Molecular characterization of enrofloxacin resistant Actinobacillus pleuropneumoniae isolates

Enrofloxacin (ER) resistant Actinobacillus pleuropneumoniae strains emerged in Taiwan in 2002. The mechanism of ER resistance in A. pleuropneumoniae has not yet been reported. A total of 48 A. pleuropneumoniae isolates were obtained from the lungs of pigs with pleuropneumonia in Taiwan between September 2007 and April 2008. Twenty-nine isolates were found to be resistant to enrofloxacin. To understand the mechanisms of A. pleuropneumoniae's resistance to ER, enrofloxacin susceptibility of the isolates along with the mutations of the quinolone resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV, qnr genes were analyzed. Enrofloxacin resistant isolates were found to carry at least one mutation in the QRDR of gyrA, leading to amino acid changes at codon 83 or 87. Efflux pump inhibitor (Phe-Arg-β-naphthylamide) decreased enrofloxacin minimum inhibitory concentration 2–16-fold, suggesting participation of efflux in ER resistance. Plasmid mediated quinolone resistance genes qnr were not detected in these isolates. In conclusion, enrofloxacin resistance of A. pleuropneumoniae may be linked to multiple target gene mutations and active effluxs.

DETECTION AND IDENTIFICATION OF VIRULENT YERSINIA RUCKERI: THE CAUSATIVE AGENT OF ENTERIC REDMOUTH DISEASE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) CULTURED IN FARS PROVINCE, IRAN

Summary From the winter 2002 to spring 2006, 126 moribund rainbow trout with clinical signs of external body haemorrhages around and within the oral cavity were sampled from 10 rainbow trout farms situated in the northwest and west of Fars province, Iran and examined for the detection and identification of Yersinia ruckeri, the causative agent of enteric redmouth disease. Fish kidneys were cultured aseptically on brain heart infusion (BHI) agar plates and incubated at 25oC for 48 h. Using conventional biochemical tests, Y. ruckeri was detected in 7 fish (5.5% of total fish sampled). This was also confirmed using specific polymerase chain reaction (PCR) assay. The 16S rDNA PCR assays produced amplicons of 409 bp when applied to Y. ruckeri isolates as well as a reference strain. Results of antibiogram tests on Y. ruckeri isolates showed a high susceptibility to enrofloxacin, norfloxacin, ofloxacin, trimethoprim and oxytetracycline. In pathogenicity tests, dilution of 4 × 10 8 colony forming unit/ml of Y. ruckeri by immersion route in challenge experiments showed 70 ± 8.2% mortality during 14 days post-infection. Experimentally infected fish showed typical haemorrhages in mouth, blackening of skin, exophthalmia and a wide haemorrhages on the internal organs.

Comparison of the in Vitro Susceptibility of Rodent Isolates of Pseudomonas Aeruginosa and Pasteurella Pneumotropica to Enrofloxacin

The objectives of this study were to determine and compare the in vitro enrofloxacin susceptibility of 94 Pseudomonas aeruginosa isolates obtained from enrofloxacin-treated and untreated mice and that of 40 Pasteurella pneumotropica strains and also to assess the efficacy and effects of enrofloxacin treatment of laboratory mice. The minimum inhibitory concentrations (MICs) of enrofloxacin against all the Ps. aeruginosa isolates were in the range of 1 to 4 microg/ml, whereas those against all the P. pneumotropica strains were less than 0.5 microg/ml. The mutation frequency in 54% of the Ps. aeruginosa isolates on treatment with enrofloxacin ranged from 10(-6) to 10(-8); however, none of the P. pneumotropica strains could grow on medium containing more than 3 microg/ml enrofloxacin. Comparison of in vitro enrofloxacin susceptibilities suggested that enrofloxacin was effective for eliminating P. pneumotropica but not for eliminating Ps. aeruginosa for which the MIC of enrofloxacin was more than 1 microg/ml. These results indicated that the enrofloxacin susceptibility of P. pneumotropica was higher than that of Ps. aeruginosa, and that the enrofloxacin treatment might not affect the susceptibility of Ps. aeruginosa.

Mutant-prevention concentrations of enrofloxacin for Escherichia coli isolates from chickens

To investigate the development of enrofloxacin resistance among Escherichia coli isolates obtained from chickens by determining mutant-prevention concentrations (MPCs) and sequence the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes in selected isolates. 15 chicken-derived E coli isolates. For all isolates, MPC and minimal inhibition concentration (MIC) of enrofloxacin were determined. The MPCs and maximum serum drug concentrations attained with enrofloxacin doses recommended for treatment of E coli infections in chickens were compared. Mutation frequencies and QRDR sequence changes in gyrA and parC were also determined. In 2 of 15 E coli strains, MPCs were low (0.016 and 0.062 microg/mL), MPC:MIC ratios were 2 and 4, and the GyrA and ParC proteins had no mutations. In 9 susceptible isolates with a GyrA point mutation, MPCs ranged from 2 to 16 microg/mL. For isolates with double mutations in GyrA and a single mutation in ParC, MPCs were > 32 microg/mL (several fold greater than the maximal plasma concentration of enrofloxacin in chickens); mutation frequencies were also much lower, compared with frequencies for single-mutation isolates. For E coli infections of chickens, MPC appears to be useful for determining enrofloxacin-dosing strategies. The high MPC:MIC ratio may result in enrofloxacin-treatment failure in chickens infected with some wild-type gyrA E coli isolates despite the isolates' enrofloxacin susceptibility (MICs 0.125 to 1 microg/mL). For infections involving isolates with high MPCs, especially those containing mutations in gyrA and parC genes, treatment with combinations of antimicrobials should be adopted.

In VivoSelection of Reduced Enrofloxacin Susceptibility in Ornithobacterium rhinotracheale and Its Resistance-Related Mutations in gyrA

This study determines the genetic background of the change in antimicrobial susceptibility to enrofloxacin of Ornithobacterium rhinotracheale (ORT) isolates with increased MIC values, isolated either from the field or from turkeys treated with enrofloxacin under experimental challenge conditions. In the field strains of ORT that were either less susceptible or, occasionally, resistant to enrofloxacin, point mutations had occurred in amino acids at positions 83 (serine) or 87 (aspartic acid) of the GyrA subunit. In the isolates showing reduced susceptibility following experimental enrofloxacin treatment (increase in MIC from < or =0.03 to 0.25 microg/ml), molecular analysis revealed a constantly recurring point mutation (G-->T) at nucleic acid position 646 (E. coli numbering) of gyrA resulting in an amino acid change from aspartic acid to tyrosine at position 87 of the GyrA subunit, which is a known hot spot for fluoroquinolone resistance. This study indicates that a single course of enrofloxacin treatment may contribute to the selection of the first mutant with reduced fluoroquinolone susceptibility in ORT. Acquired fluoroquinolone resistance is commonly encountered in ORT isolates. This is the first time that the causal mechanism of fluoroquinolone resistance in ORT has been investigated.

Incidence of quinolone resistance over the period 1986 to 1998 in veterinary Salmonella isolates from Germany.

A total of 24,591 nonhuman salmonella strains isolated in Germany between 1986 and 1998 were examined for their resistance to nalidixic acid by an agar diffusion method. The rate of resistance (inhibition zone, </=13 mm) ranged from 0.2% in 1986 to a peak of 14. 8% in 1990. Between 1991 and 1998 the MICs for nalidixic acid-resistant strains ranged from more than 256 microg/ml for nalidixic acid to between 0.25 and 128 microg/ml for enrofloxacin. In the early 1990s a particularly high incidence of fluoroquinolone resistance (49.5%) was seen among isolates of Salmonella enterica serotype Typhimurium (Salmonella Typhimurium) definitive phage type 204c that mainly originated from cattle. Among isolates from poultry an increase in the incidence of nalidixic acid resistance to a peak of 14.4% was observed in 1994. This peak was due to the presence of specific resistant serotypes, mainly serotypes Hadar, Saintpaul, Paratyphi B (D-tartrate positive; formerly serotype Java) and Newport. Such strains exhibited a decreased susceptibility to enrofloxacin (MIC, 1 microg/ml). Among isolates from pigs the peak incidence of resistance was reached in 1993, with 7.5% of isolates resistant to nalidixic acid and enrofloxacin. The study demonstrates an increase in the incidence of strains that are resistant to nalidixic acid and that have decreased susceptibility to enrofloxacin after the licensing of enrofloxacin. In addition, the number of other serotypes that exhibited nalidixic acid resistance or reduced enrofloxacin susceptibility increased among the total number of isolates investigated between 1992 and 1998.