Genomic Analysis of Staphylococcus aureus Strains Originating from Hungarian Rabbit Farms Reinforce the Clonal Origin of Various Virulence Types.

Staphylococcosis is one of the most important infectious diseases in rabbit medicine, especially in commercial farming. Previous studies revealed the existence of virulent variants adapted to rabbits. Typical and atypical, highly virulent as well as low virulent variants have been isolated and reported from industrial units in all major rabbit-meat-producing countries. Preceding the research focused on detecting defined nucleotide sequences, the genome of these organisms as a whole was rarely subjected to scientific investigations. The authors sequenced 51 Staphylococcus strains originating from industrial rabbit farms in Hungary. Another 12 draft genomes of rabbit isolates were constructed from read sequences available in digital repositories, and were compared based on whole-genome multilocus sequence typing. The clonal origin of highly virulent variants is confirmed, the strains from Hungary were closely related with the strains isolated in the UK, Italy, and Spain. Atypical highly virulent strains are the most prevalent in Hungary, they form a separate clonal cluster. The low virulent strains were genetically similar, but more heterogeneous than the highly virulent (HV) and aHV strains even by the traditional MLST typing scheme. Other "non-aureus" Staphylococcus species were also identified.

Outbreaks of a Methicillin-Resistant Staphylococcus aureus Clone ST398-t011 in a Hungarian Equine Clinic: Emergence of Rifampicin and Chloramphenicol Resistance After Treatment with These Antibiotics.

Between July 2011 and May 2016, a total of 40 Staphylococcus aureus strains originating from 36 horses were confirmed as methicillin resistant (methicillin-resistant Staphylococcus aureus [MRSA]) in a university equine clinic. An additional 10 MRSA strains from 36 samples of clinic workers were obtained in October 2017. The first equine isolate represented the sequence type ST398, spa-type t011, and SCCmec IV. This isolate was resistant to a wide spectrum of antimicrobial agents. MRSA strains with the same genotype and with very similar resistance profiles were isolated on 21 more occasions from September 2013 to September 2014. A second outbreak occurred from May 2015 until May 2016. The first isolate in this second outbreak shared the same genotype, but was additionally resistant to chloramphenicol. The second isolate from August 2015 also showed resistance to rifampicin. The clone was isolated 18 times. Most of the human isolates shared the same genotype as the isolates from horses and their resistance patterns showed only slight differences. We can conclude that the MRSA-related cases at the Department and Clinic of Equine Medicine were all nosocomial infections caused by the same clonal lineage belonging to the clonal complex 398. The clonal complex 398 of equine origin is reported for the first time in Hungary. In addition, our observation of the emergence of new resistance to antimicrobial agents within the clonal lineage after treatment with antibiotics is of concern. Strict hygiene regulations have been introduced to lower the incidence of MRSA isolation and the related clinical disease.

Draft Genome Sequence of an Atypical Highly Virulent Rabbit Staphylococcus aureus Strain.

Rabbit staphylococcosis is one of the most important diseases in industrial rabbit production. We report here the draft genome sequence of Staphylococcus aureus strain 380/11, an atypical highly virulent (aHV) rabbit Staphylococcus aureus strain.

Re-emergence of bovine haemorrhagic septicaemia in Hungary.

This paper reports an outbreak of haemorrhagic septicaemia caused by Pasteurella multocida B:2 in beef calves, a disease that has not been described in the Hungarian literature since 1943, and has not been reported to the World Organisation For Animal Health (OIE) since 1970. Acute haemorrhagic septicaemia was confirmed in beef calves on one small farm, and was suspected on two further nearby holdings with concomitant unexplained losses. The source of the infection could not be determined. Apart from a short duration of depression and loss of appetite, the affected calves developed characteristic distal limb oedema. Gross findings in two calves submitted for laboratory examinations included subcutaneous oedema and haemorrhages on serous membranes, and in one case severe pharyngeal lymph node enlargement was observed. Histological examinations revealed lesions characteristic of septicaemia. Moderate to large amounts of Pasteurella antigens were detected in all organs tested by immunohistochemistry. Two isolates of P. multocida (Pm240, Pm241) were cultured from these cases and examined in detail. These were identified as P. multocida ssp. multocida biovar 3. Both were toxA negative and belonged to serotype B:2. Multilocus sequence typing was used to assign these to a new sequence type (ST64) that is closely related to other haemorrhagic septicaemia causing strains of P. multocida regardless of the host.

Virulence type and tissue tropism of Staphylococcus strains originating from Hungarian rabbit farms.

Staphylococcosis has a major economic impact on rabbit farming worldwide. Previous studies described a highly virulent variant, which is disseminated across Europe. Such strains are reported to be capable of inducing uncontrollable outbreaks. The authors describe a survey conducted on 374 Staphylococcus strains isolated from rabbit farms, mostly from Hungary, between 2009 and 2014, from a variety of pathological processes. The virulence type of the strains was determined using a multiplex PCR system. 84.2% of the strains belonged to a previously rarely isolated atypical highly virulent type. Only 6.1% belonged to the typical highly virulent genotype. Even low virulent strains were present at a higher percentage (6.4%). For a small group of strains (3.2%) the detection of the femA gene failed, indicating that these strains probably do not belong to the Staphylococcus aureus species. The results reveal the possibility of the asymptomatic presence of highly virulent strains on rabbit farms. "Non-aureus" Staphylococcus sp. can also have a notable role in the etiology of rabbit staphylococcosis. An association with the lesions and the virulence type was demonstrated. Statistical analysis of data on organotropism showed a significant correlation between septicaemia and the highly virulent genotype.

Draft Genome Sequence of a Highly Virulent Rabbit Staphylococcus aureus Strain.

We report the draft genome sequence of Staphylococcus aureus Sp17, a typical highly virulent (HV) rabbit strain. As current medicine apparently fails to effectively reduce disease and economical losses caused by this organism, it is essential to gain better insight on its genomic arrangement.

Sarcocystis-infection of cattle in Hungary.

BACKGROUND: Reports on Sarcocystis-infection of cattle are outdated or lacking in many European countries, including those in the Central-Eastern part of the continent. Therefore, to assess the prevalence of Sarcocystis spp. among bovids in Hungary, a countrywide survey was initiated. In addition, fulminant deaths of four cattle, that showed clinical signs and post mortem lesions resembling acute sarcocystiosis ("Dalmeny disease"), were investigated. METHODS: During the countrywide survey individual heart and oesophagus samples were collected at slaughterhouses from 151 beef cattle and from 15 buffalo, kept in 31 places of Hungary. Analysis for Sarcocystis spp. was carried out with conventional PCRs for the 18S rDNA gene and gel electrophoresis, followed by sequencing of 36 strongly positive samples. Mortality cases were evaluated by histological, molecular, bacteriological and virological analyses of samples from various organs. RESULTS: Among slaughtered cattle the rate of Sarcocystis-infection was 66%. S. cruzi was identified as the most prevalent species in aurochs-like breed, and the zoonotic S. hominis in Hungarian grey cattle. Concerning the sudden deaths of cattle, Sarcocystis-infection could not be demonstrated in organs showing haemorrhages, but S. cruzi cysts were present in the muscles. In one case "S. sinensis" was molecularly identified in the blood (indicating sarcocystaemia). Results of analyses for bacterial/viral pathogens were negative. CONCLUSIONS: S. cruzi appears to be the most prevalent Sarcocystis sp. in cattle in Hungary, followed by the zoonotic S. hominis. However, the rate of infection with both species was shown to differ between cattle breeds. The suspected role of Sarcocystis spp. as causative agents of the fatal cases could not be confirmed.

Rietveld refinement in the routine quantitative analysis of famotidine polymorphs.

Accurate, precise and reliable X-ray powder diffraction method was developed for the quantitative determination of famotidine polymorphic forms in their binary mixtures, which slightly outperforms the previously established Raman method. The study highlights the advantage of focused beam transmission geometry in diminishing the effect of preferred orientation in general, and the straightforward transmission foil sample preparation technique in facilitating high-throughput measurements in particular. This combination can provide good quality data for Rietveld refinement which assures more reliable quantitative results than utilizing intensity ratios of selected single reflections. After careful adjustment of profile parameters, simple routine application of the method was achieved.

Quantitative determination of famotidine polymorphs: X-ray powder diffractometric and Raman spectrometric study.

X-ray powder diffractometric and Raman spectrometric methods were developed for quantitative measurement of the polymorphic forms of famotidine in their mixtures. This study aims to deduce some useful conclusions regarding quantitative polymorph analysis, which could also be utilized in industrial practice. Both form A and form B of famotidine possess specific X-ray diffraction reflections as well as characteristic Raman vibrational bands, which permits simple determination of the phases in their mixtures. Keeping in mind that multivariate data processing by chemometric approach is thought of nowadays as superior over univariate one, the results of the two evaluation methods were compared by precision, accuracy as well as robustness. It was found that both approaches provide similar results provided analytically useful data regions are properly selected. Overcoming the common problems of quantitative X-ray powder diffractometry and solid state Raman spectrometry both permit accurate quantification of famotidine polymorphs; the latter, however, seems to be more favourable in regular laboratory practice.

Quantifying low levels of polymorphic impurity in clopidogrel bisulphate by vibrational spectroscopy and chemometrics.

Vibrational spectroscopic methods were developed for quantitative analysis of Form II of clopidogrel bisulphate in Form I and Form II polymorphic mixtures. Results show that both IR and Raman spectroscopy combined with chemometrics are suitable to quantify low levels of Form II in Form I, down to 2 and 3%, respectively, with less than 1% limit of detection. Different preprocessing and multivariate methods were applied for spectral processing and were compared to find the best chemometric model. Common problems of quantitative vibrational spectroscopy in the solid phase are discussed; and procedures appropriate to eliminate them are proposed.

Polymorph transitions of bicalutamide: a remarkable example of mechanical activation.

Bicalutamide, an active pharmaceutical ingredient possessing antiandrogenic activity, is known to exhibit polymorphism. The higher melting Form I relates monotropically to the lower melting Form II. The amorphous form can be easily produced by quench cooling the melt, but it is known to crystallize spontaneously to Form II at room temperature within days. Our results show that crystallization of amorphous bicalutamide is greatly influenced by experimental conditions and sample treatment. The effect of mechanical activation on the polymorph transitions is investigated in detail. Seeds of Form I can be formed in the amorphous phase even due to gentle mechanical treatment, which results in crystallization to the more stable structure at elevated temperature. The crystalline Form II may as well be transformed to the stable modification through mechanical activation at elevated temperature.