Occurrence of Escherichia coli producing extended spectrum ß-lactamases in food-producing animals.

Multidrug resistance due to the production of extended-spectrum beta-lactamases (ESBLs) is a major problem in human as well as in veterinary medicine. These strains appear in animal and human microbiomes and can be the source of infection both in animal and in human healthcare, in accordance with the One Health theorem. In this study we examined the prevalence of ESBL-producing bacteria in food-producing animals. We collected 100 porcine and 114 poultry samples to examine the prevalence of ESBL producers. Isolates were identified using the MALDI-TOF system and their antibiotic susceptibility was tested using the disk diffusion method. ESBL gene families and phylogroups were detected by polymerase chain reactions. The prevalence of ESBL producers was relatively high in both sample groups: 72 (72.0%) porcine and 39 (34.2%) poultry isolates were ESBL producers. Escherichia coli isolates were chosen for further investigations. The most common ESBL gene was CTX-M-1 (79.3%). Most of the isolates belong to the commensal E. coli phylogroups. The porcine isolates could be divided into three phylogroups, while the distribution of the poultry isolates was more varied. In summary, ESBL-producing bacteria are prevalent in the faecal samples of the examined food-producing animals, with a dominance of the CTX-M-1 group enzymes and commensal E. coli phylogroups.

Detection of Mycoplasma anatis, M. anseris, M. cloacale and Mycoplasma sp. 1220 in waterfowl using species-specific PCR assays.

Mycoplasma anatis, M. anseris, M. cloacale and M. sp. 1220 colonise geese and ducks, and could be associated with infections of avian respiratory and nervous systems, cause mild to severe inflammation of cloaca and genital tracts, and embryo lethality. Co-occurrence of these Mycoplasma species in waterfowl is frequently detected and the identification of these mycoplasmas to the species level at a regular microbiology laboratory is difficult due to their similar morphological, cultural and biochemical properties. Moreover, species differentiation is only possible based on the sequence analysis of the product of a genus-specific PCR assay. Therefore, the aim of the current study was to develop an effective and robust method for the identification of these species in avian clinical specimens. Polymerase chain reaction (PCR) assays using species-specific primers, which target housekeeping genes in order to identify these species, were designed in the present study. The developed PCR assays can precisely identify these four mycoplasmas to the species level directly from DNA samples extracted from clinical specimens, and no cross-amplification was observed among these species and with other well-known avian mycoplasmas. The average sensitivity of the assays was 101-102 genomic equivalents per reaction. These conventional PCR assays can be run simultaneously at the same PCR cycling program, and the species can be differentiated directly (without sequence analysis) by gel electrophoresis due to the specific sizes of the amplicons. In conclusion, the presented species-specific assays were found to be suitable for routine use at regular veterinary diagnostic laboratories and promote the rapid, simple and cost-effective differentiation of these waterfowl Mycoplasma species.

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.

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

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.

Antibiotic susceptibility profiles of Mycoplasma sp. 1220 strains isolated from geese in Hungary.

BACKGROUND: Mycoplasma sp. 1220 can induce inflammation primarily in the genital and respiratory tracts of waterfowl, leading to serious economic losses. Adequate housing and appropriate antibiotic treatment are promoted in the control of the disease. The aim of the present study was to determine the in vitro susceptibility to thirteen different antibiotics and an antibiotic combination of thirty-eight M. sp. 1220 strains isolated from geese and a duck in several parts of Hungary, Central Europe between 2011 and 2015. RESULTS: High MIC50 values were observed in the cases of tilmicosin (>64 µg/ml), oxytetracycline (64 µg/ml), norfloxacin (>10 µg/ml) and difloxacin (10 µg/ml). The examined strains yielded the same MIC50 values with spectinomycin, tylosin and florfenicol (8 µg/ml), while enrofloxacin (MIC50 5 µg/ml), doxycycline (MIC50 5 µg/ml), lincomycin (MIC50 4 µg/ml) and lincomycin-spectinomycin (1:2) combination (MIC50 4 µg/ml) inhibited the growth of the bacteria with lower concentrations. Tylvalosin (MIC50 0.5 µg/ml) and two pleuromutilins (tiamulin MIC50 0.625 µg/ml; valnemulin MIC50 ≤ 0.039 µg/ml) were found to be the most effective drugs against M. sp. 1220. However, strains with elevated MIC values were detected for all applied antibiotics. CONCLUSIONS: Valnemulin, tiamulin and tylvalosin were found to be the most effective antibiotics in the study. Increasing resistance was observed in the cases of several antibiotics. The results highlight the importance of testing Mycoplasma species for antibiotic susceptibility before therapy.

Antibiotic susceptibility profiles of Mycoplasma bovis strains isolated from cattle in Hungary, Central Europe.

BACKGROUND: Mycoplasma bovis is a worldwide pathogen, causative agent of pneumonia, mastitis, arthritis, and a variety of other symptoms in cattle. The economic losses due to mycoplasma pneumonia could be reduced by antibiotic treatment. The aim of the present study was to determine the in vitro susceptibility of M. bovis strains isolated from cattle in Hungary to eleven antibiotics. RESULTS: Minimal inhibitory concentration (MIC) values of 35 M. bovis strains collected from different parts of Hungary between 2010 and 2013 were determined by the microbroth dilution method. Strains with high MIC values were found in the case of all applied antibiotics. The most effective antibiotics tested in vitro were fluoroquinolones (MIC90 danofloxacin 0.312 µg/ml, enrofloxacin 0.312 µg/ml, marbofloxacin 0.625 µg/ml). Our results confirm the observations of increasing MIC values to antibiotics commonly used in the therapy of mycoplasma infections, primarily to tetracyclines; tetracycline (MIC90 16 µg/ml) and oxytetracycline (MIC90 ≥ 64 µg/ml) and macrolides; tylosin (MIC90 ≥ 128 µg/ml) and tilmicosin (MIC90 ≥ 128 µg/ml). The growth of many M. bovis strains was not inhibited by gentamicin (MIC90 8 µg/ml), spectinomycin (MIC90 ≥ 256 µg/ml), florfenicol (MIC90 8 µg/ml) or lincomycin (MIC90 ≥ 64 µg/ml). CONCLUSIONS: Our results emphasize the necessity of periodic testing for antibiotic susceptibility in this geographic region. Based on our in vitro examinations, fluoroquinolones could be the most effective drugs for the therapy of M. bovis infections in Hungary. However, current antimicrobial use policies have to be taken into account to avoid further antibiotic resistance development and to reserve fluoroquinolones for the treatment of severe infections which have responded poorly to other classes of antimicrobials.

Phylogeny of Mycoplasma bovis isolates from Hungary based on multi locus sequence typing and multiple-locus variable-number tandem repeat analysis.

BACKGROUND: Mycoplasma bovis is an important pathogen causing pneumonia, mastitis and arthritis in cattle worldwide. As this agent is primarily transmitted by direct contact and spread through animal movements, efficient genotyping systems are essential for the monitoring of the disease and for epidemiological investigations. The aim of this study was to compare and evaluate the multi locus sequence typing (MLST) and the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) through the genetic characterization of M. bovis isolates from Hungary. RESULTS: Thirty one Hungarian M. bovis isolates grouped into two clades by MLST. Two strains had the same sequence type (ST) as reference strain PG45, while the other twenty nine Hungarian isolates formed a novel clade comprising five subclades. Isolates originating from the same herds had the same STs except for one case. The same isolates formed two main clades and several subclades and branches by MLVA. One clade contained the reference strain PG45 and three isolates, while the other main clade comprised the rest of the strains. Within-herd strain divergence was also detected by MLVA. Little congruence was found between the results of the two typing systems. CONCLUSIONS: MLST is generally considered an intermediate scale typing method and it was found to be discriminatory among the Hungarian M. bovis isolates. MLVA proved to be an appropriate fine scale typing tool for M. bovis as this method was able to distinguish closely related strains isolated from the same farm. We recommend the combined use of the two methods for the genotyping of M. bovis isolates. Strains have to be characterized first by MLST followed by the fine scale typing of identical STs with MLVA.