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Home » Archive » 2015

TDK conference 2015

Examination of fluoroquinolone susceptibility by biological methods in Mycoplasma bovis strains
Nagy Sára Ágnes - year 4
HAS-CAR- Institue for Veterinary Medical Research
Supervisors: Kinga Sulyok, Miklós Gyuranecz DVM

Abstract:

Mycoplasma bovis is a cell wall-less bacterium that causes recognizable economic losses worldwide in dairy and beef cattle as well. At the present time no efficient vaccination is available; therefore antibiotic therapy is the most important tool against M. bovis. Fluoroquinolones, which can be appropriate drugs against the agent, are frequently reported to be less effective because of extensive and irresponsible usage. For successful treatment a time-saving productive diagnostic method is needed to detect drug resistance.

The aim of our study was to develop a proper, rapid and cost-effective molecular diagnostic test that can determine single nucleotide polymorphisms (SNPs) associated with fluoroquinolone resistance in M. bovis strains.

Minimal inhibitory concentration (MIC) of 35 M. bovis strains, acquired from pathological and diagnostic samples originated from different parts of Hungary, were determined by microbroth dilution test to evaluate the efficiency of three different fluoroquinolones (danofloxacin, enrofloxacin and marbofloxacin). For identification of SNPs related to drug resistance we compared whole genome sequences of the 35 Hungarian isolates, the M. bovis reference strain PG45 (NCTC 10131) and nine fluoroquinolone-resistant strains selected in vitro. To detect the identified SNPs we developed real-time PCR based and agarose gel based mismatch amplification mutation assays (MAMA). Finally, the sensitivity and the specificity of these systems were tested by other Mycoplasma species of bovine origin and their efficiency was studied on clinical samples (lung samples and nasal swabs) as well.

Among the 35 strains we found high (>10 µg/ml) MIC values in three cases. In the genes of quinolone resistance determination region (QRDR) we ascertained four SNPs that generate phenotypic manifestation of fluoroquinolone resistance. The sensitivity of MAMA systems based on real-time PCR and agarose gel proved to be 102–104 and 103–105 colour changing unit (CCU) respectively. Neither system gave cross-reaction with genomes of other Mycoplasma species originating from cattle.

Our MAMAs are able to distinguish resistant and susceptible M. bovis strains without time-consuming and expensive isolation and classical drug resistance testing of the bacteria; which can help increase the effectivity of the therapy.



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