Zsuzsanna Rónai

Mutations Associated with Decreased Susceptibility to Seven Antimicrobial Families in Field and Laboratory-Derived Mycoplasma bovis Strains

ABSTRACT The molecular mechanisms of resistance to fluoroquinolones, tetracyclines, an aminocyclitol, macrolides, a lincosamide, a phenicol, and pleuromutilins were investigated in Mycoplasma bovis. For the identification of mutations responsible for the high MICs of certain antibiotics, whole-genome sequencing of 35 M. bovis field isolates and 36 laboratory-derived antibiotic-resistant mutants was performed. In vitro resistant mutants were selected by serial passages of M. bovis in broth medium containing subinhibitory concentrations of the antibiotics. Mutations associated with high fluoroquinolones MICs were found at positions 244 to 260 and at positions 232 to 250 (according to Escherichia coli numbering) of the quinolone resistance-determining regions of the gyrA and parC genes, respectively. Alterations related to elevated tetracycline MICs were described at positions 962 to 967, 1058, 1195, 1196, and 1199 of genes encoding the 16S rRNA and forming the primary tetracycline binding site. Single transversion at position 1192 of the rrs1 gene resulted in a spectinomycin MIC of 256 μg/ml. Mutations responsible for high macrolide, lincomycin, florfenicol, and pleuromutilin antibiotic MICs were identified in genes encoding 23S rRNA. Understanding antibiotic resistance mechanisms is an important tool for future developments of genetic-based diagnostic assays for the rapid detection of resistant M. bovis strains.

Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies Specific real-time PCR

ABSTRACT Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.

Detection of wide genetic diversity and several novel strains among non-avium nontuberculous mycobacteria isolated from farmed and wild animals in Hungary.

Besides Mycobacterium avium numerous nontuberculous Mycobacterium (NTM) species exist, which pose constant health risk to both humans and animals. The aim of our study was to identify non‐avium NTM isolates from veterinary origin in Hungary, and to detect the occurrence of rifampicin resistance among them.

Development of molecular methods for the rapid detection of antibiotic susceptibility of Mycoplasma bovis

Determining the antibiotic susceptibility profile of Mycoplasma bovis isolates in vitro provides the basis for the appropriate choice of antibiotics in the therapy. Traditionally, the antibiotic susceptibility examination of mycoplasmas is technically demanding, time-consuming and rarely performed in diagnostic laboratories. The aim of the present study was to develop rapid molecular assays to determine mutations responsible for elevated minimal inhibitory concentrations (MICs) to fluoroquinolones, tetracyclines, aminocyclitols, macrolides, lincosamides, phenicols and pleuromutilins in M. bovis. The nine mismatch amplification mutation assays (MAMA) and seven high resolution melt (HRM) tests designed in the present study enable the simultaneous detection of these genetic markers. The sensitivity of the assays varied between 102-105 copy numbers/reaction. Cross-reactions with other mycoplasmas occurring in cattle were detected in assays targeting universal regions (e.g. 16S rRNA). Nevertheless, results of the novel method were in accordance with sequence and MICs data of the M. bovis pure cultures. Also, the tests of clinical samples containing high amount of M. bovis DNA were congruent even in the presence of other Mycoplasma spp. The presented method is highly cost-effective and can provide an antibiogram to 12 antibiotics in approximately 3-4 days when previous isolation of M. bovis is applied. In order to assure the proper identification of the genetic markers at issue, the regions examined by the MAMA and HRM tests are overlapping. In conclusion, the developed assays have potential to be used in routine diagnostics for the detection of antibiotic susceptibility in M. bovis.

Comparative analysis of Pasteurella multocida strains isolated from bovine respiratory infections

Bovine respiratory disease (BRD) is the leading cause of significant economic losses in the intensive beef industry worldwide. Beside numerous risk factors Pasteurella multocida, which is regarded as a secondary pathogen, may play a role in the development of the disease. Previous studies of strains from swine pneumonia revealed that there are a few clones associated with clinical disease, suggesting that some strains may be more virulent than others. This linkage may be true in the BRD, however composition of P. multocida populations in the herds are slightly characterized. Thus, we decided to perform phenotypic and genotypic characterisation of strains isolated from calves with respiratory infection at 31 different herds in Hungary. The results demonstrated the presence of two dominant strain types. At the identical taxonomic background (P. multocida subsp. multocida) with slight phenotypic variability they could be separated by trehalose fermentation capacity, α-glucosidase activity and molecular fingerprint patterns of ERIC- and M13-PCR. Independent prevalence and geographical origin of the strain types may refer to their significance in the illness, but their comparison with strains isolated from healthy individuals is taken into consideration.

Natural IS711 insertion causing omp31 gene inactivation in Brucella ovis

The present report describes an atypical Brucella ovis strain (Bo10) isolated from the epididymis and testis of an infected ram. Macroscopic and microscopic lesions characteristic for the infection, including positive Brucella immunostaining, were observed within lesions in the genital organs. Compared to other isolates, strain Bo10 required an additional day (a total of 96 hr) of incubation to form visible colonies, showed a distinct carbon source utilization profile, agglutinated only weakly with rough (R) serum, but showed a high capacity for autoagglutination. Isolate Bo10 failed to produce the 1,071-bp fragment in the outer membrane protein (omp) 31 gene–based part of the “Bruce-ladder” multiplex polymerase chain reaction system but did produce a 1,915-bp amplicon, thus presenting a profile similar to Brucella abortus. Sequence analysis of the 1,915-bp fragment revealed an 842-bp long insertion sequence (IS)711 transposon element inserted into the promoter region of the omp31 gene, immediately upstream from the ribosome binding site (-10 box/Pribnow box). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of a whole-cell lysate showed the absence in Bo10 of the approximately 31-kDa protein fragment associated with omp31. The results demonstrate a natural inactivation of omp31 and, consequently, the absence of the Omp31 protein in this B. ovis isolate. The novel location of IS711 within the genome of a naturally occurring B. ovis strain supports the hypothesis that IS711 could be an active transposon in this Brucella species.

DETECTION OF UREASE-NEGATIVE BORDETELLA BRONCHISEPTICA FROM THE FIELD

Four urease-negative Bordetella bronchiseptica isolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs™ assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific and ureC PCR, and all strains showed uniform bands in PCR-RFLP studies of flaA genes. The reason for the lack of urease activity, a characteristic considered species specific for B. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification of B. bronchiseptica seems questionable.

Antimicrobial susceptibility of Bacillus anthracis strains from Hungary.

The susceptibility of 29 Bacillus anthracis strains, collected in Hungary between 1933 and 2014, was tested to 10 antibiotics with commercially available minimum inhibitory concentration (MIC) test strips. All strains were susceptible to amoxicillin, ciprofloxacin, clindamycin, doxycycline, gentamicin, penicillin, rifampicin, and vancomycin. Intermediate susceptibility to erythromycin and cefotaxime was detected in 17.2% (5/29) and 58.6% (17/29) of the strains, respectively. Correlations were not observed between the isolation date, location, host species, genotype, and antibiotic susceptibility profile of strains.