Occurrence and characterisation of methicillin-resistant Staphylococcus aureus isolated from bovine milk in Hungary.

The last surveys on methicillin-resistant Staphylococcus aureus (MRSA) isolated from bovine milk in Hungary took place in the 2000s. To elucidate the genetic variability and to estimate the burden of the pathogen, MRSA from our strain collection and prospectively collected Staphylococcus aureus (SA) isolates originating from two milk hygiene laboratories were investigated. Between 2003 and 2018, 27 MRSA strains originating from 10 dairy farms were deposited and characterised. Most strains (n = 20) belonged to ST1-t127-SCCmecIV and were recovered from three unrelated farms. From other farms, variable genotypes were identified sporadically: ST22-t032-SCCmecIV from three farms; a newly described double locus variant of ST97, ST5982-t458-SCCmecIV from two farms; and ST398-t011-SCCmecIV and ST398-t011-SCCmecV from two respective farms. The prospective screening of 626 individual SA isolates originating from 42 dairy farms resulted in four (0.48 %) MRSA strains from three (7.14 %) farms. All MRSA isolates belonged to the clonal complex 398 and a novel spa-type t19251 was also identified. Most isolates were resistant to three or more antimicrobial classes. The occurrence and significance of MRSA of dairy origin seems to be unchanged in the past decade in Hungary. However, the low host specificity and multiresistance of the identified genotypes calls for periodic revision on the role and distribution of the pathogen in the Hungarian dairy sector.

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.

Metagenomic Analysis of Acquired Antibiotic Resistance Determinants in the Gut Microbiota of Wild Boars (Sus Scrofa) - Preliminary Results.

INTRODUCTION: Land application of manure that contains antibiotics and resistant bacteria may facilitate the establishment of an environmental reservoir of antibiotic-resistant microbes, promoting their dissemination into agricultural and natural habitats. The main objective of this study was to search for acquired antibiotic resistance determinants in the gut microbiota of wild boar populations living in natural habitats. MATERIAL AND METHODS: Gastrointestinal samples of free-living wild boars were collected in the Zemplén Mountains in Hungary and were characterised by culture-based, metagenomic, and molecular microbiological methods. Bioinformatic analysis of the faecal microbiome of a hunted wild boar from Japan was used for comparative studies. Also, shotgun metagenomic sequencing data of two untreated sewage wastewater samples from North Pest (Hungary) from 2016 were analysed by bioinformatic methods. Minimum spanning tree diagrams for seven-gene MLST profiles of 104 E. coli strains isolated in Europe from wild boars and domestic pigs were generated in Enterobase. RESULTS: In the ileum of a diarrhoeic boar, a dominant E. coli O112ab:H2 strain with intermediate resistance to gentamicin, tobramycin, and amikacin was identified, displaying sequence type ST388 and harbouring the EAST1 toxin astA gene. Metagenomic analyses of the colon and rectum digesta revealed the presence of the tetQ, tetW, tetO, and mefA antibiotic resistance genes that were also detected in the gut microbiome of four other wild boars from the mountains. Furthermore, the tetQ and cfxA genes were identified in the faecal microbiome of a hunted wild boar from Japan. CONCLUSION: The gastrointestinal microbiota of the free-living wild boars examined in this study carried acquired antibiotic resistance determinants that are highly prevalent among domestic livestock populations.

The relationship between reproductive and biochemical ageing at the time of the menopausal transition.

The biochemical ageing status of women in the menopausal transition was studied using quantitative analysis of age- and autophagy-related gene activities (CDC42 and MAP1LC3 genes were selected as target genes). Free estradiol and progesterone levels in saliva were estimated. General linear models were used to determine the relationship between lifestyle, health status, socioeconomic factors and CDC42 and MAP1LC3 gene expression levels. Gene expression analysis revealed (1) an increasing expression of CDC42 gene after 45years in women, (2) expression level of CDC42 gene associated with menopausal status, (3) while endocrine status was found to associate with the expression of both of the studied age-related genes, (4) the "never used hormonal contraceptives" and "obese nutritional status" were the strongest factors for increased level of age-related gene expressions, and (5) changes in gene expression levels by ageing should be studied by considering not only chronological, but also biological ages. Gene expression profile of ageing has mostly been studied in model systems or human blood samples, but rarely in human saliva samples. The concordance of results between the present and former gene expression analyses, and the simplicity of saliva sample collection emphasizes the importance of saliva tissue samples in gene expression analyses especially in epidemiological surveys.

Ottowia pentelensis sp. nov., a floc-forming betaproteobacterium isolated from an activated sludge system treating coke plant effluent.

A Gram-negative-staining, short-rod-shaped, floc-forming bacterium, designated strain RB3-7(T), was isolated from a laboratory-scale activated sludge system treating coke plant effluent. Comparative analysis of the 16S rRNA gene sequence demonstrated that the novel isolate was distantly related (≤ 95.8 % similarity) to Ottowia thiooxydans K11(T) within the family Comamonadaceae. Strain RB3-7(T) was catalase- and oxidase-positive and non-motile. The predominant fatty acids were C16:0, cyclo C17:0, C18:1ω7c and C16:1ω7c, and the major respiratory quinone was Q-8. The G+C content of the genomic DNA of strain RB3-7(T) was 68.5 mol%. On the basis of phenotypic, chemotaxonomic and molecular data, strain RB3-7(T) is considered to represent a novel species of the genus Ottowia, for which the name Ottowia pentelensis sp. nov. is proposed. The type strain is RB3-7(T) ( = DSM 21699(T) = NCAIM B 02336(T)).

Addressing PCR biases in environmental microbiology studies.

Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition of the sample in the PCR product. Strategies of primer selection and PCR thermal profile design are discussed in detail.

DGGE and T-RFLP analysis of bacterial succession during mushroom compost production and sequence-aided T-RFLP profile of mature compost.

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.

Metabolic activity and phylogenetic diversity of reed (Phragmites australis) periphyton bacterial communities in a hungarian shallow soda lake.

In the present study, the species composition and potential metabolic activities of bacterial communities of reed Phragmites australis (Cav.) (Trin. ex Steudel) periphyton from Lake Velencei were studied by cultivation-based and metabolic fingerprinting methods. Serially diluted spring biofilm samples were used to test the community-level physiological profiling (CLPP) using BIOLOG microplates, and for plating onto different media. On the basis of their morphological, biochemical, and physiological test results, 173 strains were clustered by numerical analysis. Representatives of amplified ribosomal DNA restriction analysis (ARDRA) groups were identified by their 16S rDNA sequence comparison. Based on the results of the CLPP investigations, regional differences were detected among the utilized substrate numbers and types, parallel with the increase in incubation time. The phenotypic test results of the strains showed considerable variability with respect to the sampling sites and the media used for cultivation. The most frequently isolated strains were identified as members of genera Agrobacterium, Pseudomonas (P. anguilliseptica, P. marginalis, P. alcaligenes, P. fragi) with aerobic or facultative anaerobic respiratory metabolism, and the species Aeromonas sobria and A. veronii with strong facultative fermentative metabolism. Other strains were identified as Gram-positive Arthrobacter, Bacillus, and Kocuria species. The rarely isolated strains were members of beta-Proteobacteria (Acidovorax, Delftia, Hydrogenophaga, and Rhodoferax), gamma-Proteobacteria (Psychrobacter and Shewanella), low G + C Gram-positives (Brevibacillus, Paenibacillus, and Exiguobacterium) and high G + C Gram-positives (Aureobacterium and Microbacterium).

Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis.

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.

Observation of bias associated with re-amplification of DNA isolated from denaturing gradient gels.

DNA from environmental PCR products separated by denaturing gradient gel electrophoresis (DGGE) was isolated from the background smear rather than from discrete bands of the DGGE gel. The "interband" region was considered as a potential source of less dominant members of natural microbial communities. Surprisingly, instead of detecting new bands from the re-amplified PCR products, patterns very similar to the original ones were obtained regardless of the position of the "interband" region. The results suggest that the separation of amplicons by DGGE may not be perfect and band re-amplification based sequence analyses need careful interpretation.