Ljubljana, Slovenia
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Janezic S.,National Laboratory for Health | Indra A.,Austrian Agency for Health and Food Safety AGES | Rattei T.,University of Vienna | Weinmaier T.,University of Vienna | And 2 more authors.
PLoS ONE | Year: 2014

PCR-ribotyping, a typing method based on size variation in 16S-23S rRNA intergenic spacer region (ISR), has been used widely for molecular epidemiological investigations of C. difficile infections. In the present study, we describe the sequence diversity of ISRs from 43 C. difficile strains, representing different PCR-ribotypes and suggest homologous recombination as a possible mechanism driving the evolution of 16S-23S rRNA ISRs. ISRs of 45 different lengths (ranging from 185 bp to 564 bp) were found among 458 ISRs. All ISRs could be described with one of the 22 different structural groups defined by the presence or absence of different sequence modules; tRNAAla genes and different combinations of spacers of different lengths (33 bp, 53 bp or 20 bp) and 9 bp direct repeats separating the spacers. The ISR structural group, in most cases, coincided with the sequence length. ISRs that were of the same lengths had also very similar nucleotide sequence, suggesting that ISRs were not suitable for discriminating between different strains based only on the ISR sequence. Despite large variations in the length, the alignment of ISR sequences, based on the primary sequence and secondary structure information, revealed many conserved regions which were mainly involved in maturation of pre-rRNA. Phylogenetic analysis of the ISR alignment yielded strong evidence for intra- and inter-homologous recombination which could be one of the mechanisms driving the evolution of C. difficile 16S-23S ISRs. The modular structure of the ISR, the high sequence similarities of ISRs of the same sizes and the presence of homologous recombination also suggest that different copies of C. difficile 16S-23S rRNA ISR are evolving in concert. © 2014 Janezic et al.


Janezic S.,National Laboratory for Health | Rupnik M.,National Laboratory for Health | Rupnik M.,University of Maribor
Research in Microbiology | Year: 2015

Approaches to exploring Clostridium difficile genomic diversity have ranged from molecular typing methods to use of comparative genome microarrays and whole genome sequence comparisons. The C. difficile population structure is clonal and distributed into six clades, which correlate well with MLST STs (multilocus sequence types) and PCR ribotypes. However, toxigenic strains and strains with increased virulence are distributed throughout several clades. Here we summarize studies on C. difficile genomic diversity, with emphasis on phylogenetic aspects, epidemiological aspect and variability of some virulence factors. © 2015 Institut Pasteur.


Janezic S.,National Laboratory for Health | Marin M.,Hospital General Universitario Gregorio Maranon | Marin M.,Institute Investigacion Sanitaria Gregorio Maranon | Martin A.,Hospital General Universitario Gregorio Maranon | And 2 more authors.
Journal of Clinical Microbiology | Year: 2015

Toxins A and B are the main virulence factors of Clostridium difficile and are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficile transferase)-negative (A- B+ CDT-) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) without tcdA and with atypical organization of the PaLoc integration site. © 2015, American Society for Microbiology. All Rights Reserved.


Kirincic S.,National Institute of Public Health | Skrjanc B.,National Laboratory for Health | Kos N.,National Laboratory for Health | Kozolc B.,National Laboratory for Health | And 2 more authors.
Food Control | Year: 2015

In this study, 290 different cereals and cereal products, sampled on Slovenian market under official control in the years 2008-2012, were investigated on the presence of mycotoxins: aflatoxins (AF), ochratoxin A (OTA), fumonisins B1 and B2 (FB), deoxynivalenol (DON), zearalenone (ZON) and T-2/HT-2 toxins. Methods used for mycotoxins determination, high performance liquid chromatography (HPLC), liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC-MS), were all in accordance with European Union requirements, therefore accredited or at least validated, performed in the Slovenian accredited official laboratory. Altogether 40% of cereal samples contained one or more mycotoxins and 2.4% of them exceeded European Union maximum levels with one or more mycotoxins. The comparison of results regarding the share of non-compliant results do not show any extreme positive or negative deviations from other EU results. Among all cereal foods, wheat products could contribute most to the exposure of Slovenian inhabitants to mycotoxins, because of their relative high contamination rate (71%), high share of samples exceeding EU maximum levels (6%), predominantly with DON, and their by far the highest consumption, compared to other cereals. Maize products, as second most consumed cereals, could also contribute to exposure a lot, due to their relatively high mean mycotoxin concentrations. The most frequently co-occurred mycotoxin combinations were DON-ZON in wheat, DON-T-2/HT-2 in oat and FB-DON and FB-DON-ZON in maize products. From the point of view of method of farming/processing, where a small number of data were available, cereal samples from conventional farming had the lowest mean mycotoxin concentrations and were the least contaminated/non-compliant (32%/1%), compared to organic (46%/2.2%) and integrated ones (87%/10%); the integrated farming method is based on national legislation. The present study shows the necessity for continuation of official control on mycotoxins in cereals and cereal products and it could be the base for improving control plans and analytics in the future. © 2014 Elsevier Ltd.


Rupnik M.,National Laboratory for Health | Rupnik M.,University of Maribor | Janezic S.,National Laboratory for Health | Janezic S.,University of Maribor
Journal of Clinical Microbiology | Year: 2016

Toxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation of Clostridium difficile strains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis for C. difficile phylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature. © 2015, American Society for Microbiology. All Rights Reserved.


According to the existing literature, a heterogeneous sequence type (ST) or clones of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) circulate in Europe. In Europe, the European clone that belongs to sequence type ST80 is predominant.The aim of the study was to investigate the phenotypic and genotypic characteristics and epidemiological data of CA-MRSA ST80 and its occurrence in Slovenia. We retrospectively analyzed those CA-MRSA isolates that were isolated during microbiological procedures in microbiological laboratories between 2006 and 2013. Only CA-MRSA isolates from the national collection of CA-MRSA strains that belonged to ST80 (European clone) were analyzed. We determined the Pantone-Valentine leukocidin (PVL), mec A genes, exfoliative toxin genes and type of staphylococcal cassette chromosome (SCCmec) by polymerase chain reaction (PCR). We determined also spa type and sequence type.ST80 was confirmed in only 2 (0.5%) out of 385 CA-MRSA isolates, collected in a national collection of CAMRSA. Both isolates were positive for the PVL genes, mec A gene, exfoliative toxin type D gene and SCCmec IV. One CA-MRSA isolate was confirmed in a wound swab taken from a 47-year-old male, and the second was isolated from blood cultures of a 69-year-old female. No epidemiological connections between them were found.In Slovenia CA-MRSA infections caused by ST80 are rare. In the future, it is necessary that a surveillance study of CA-MRSA at the national level continues and CA-MRSA be considered as a public health threat.


PubMed | Vodnikova cesta 243, National Laboratory for Health, University of Ljubljana and Vinharje 6
Type: | Journal: Veterinary research | Year: 2016

Farm animals have been suggested to play an important role in the epidemiology of Clostridium difficile infection (CDI) in the community. The purpose of this study was to evaluate risk factors associated with C. difficile dissemination in family dairy farms, which are the most common farming model in the European Union. Environmental samples and fecal samples from cows and calves were collected repeatedly over a 1 year period on 20 mid-size family dairy farms. Clostridium difficile was detected in cattle feces on all farms using qPCR. The average prevalence between farms was 10% (0-44.4%) and 35.7% (3.7-66.7%) in cows and calves, respectively. Bacterial culture yielded 103 C. difficile isolates from cattle and 61 from the environment. Most C. difficile isolates were PCR-ribotype 033. A univariate mixed effect model analysis of risk factors associated dietary changes with increasing C. difficile prevalence in cows (P = 0.0004); and dietary changes (P = 0.004), breeding Simmental cattle (P = 0.001), mastitis (P = 0.003) and antibiotic treatment (P = 0.003) in calves. Multivariate analysis of risk factors found that dietary changes in cows (P = 0.0001) and calves (P = 0.002) increase C. difficile prevalence; mastitis was identified as a risk factor in calves (P = 0.001). This study shows that C. difficile is common on dairy farms and that shedding is more influenced by farm management than environmental factors. Based on molecular typing of C. difficile isolates, it could also be concluded that family dairy farms are currently not contributing to increased CDI incidence.


PubMed | National Laboratory for Health and University of Maribor
Type: | Journal: International journal of food microbiology | Year: 2016

Unfiltered vinegar samples collected from three oxidation cycles of the submerged industrial production of each, red wine and organic apple cider vinegars, were sampled in a Slovene vinegar producing company. The samples were systematically collected from the beginning to the end of an oxidation cycle and used for culture-independent microbial analyses carried out by denaturing high pressure liquid chromatography (DHPLC) and Illumina MiSeq sequencing of 16S rRNA gene variable regions. Both approaches showed a very homogeneous bacterial structure during wine vinegar production but more heterogeneous during organic apple cider vinegar production. In all wine vinegar samples Komagataeibacter oboediens (formerly Gluconacetobacter oboediens) was a predominating species. In apple cider vinegar the acetic acid and lactic acid bacteria were two major groups of bacteria. The acetic acid bacterial consortium was composed of Acetobacter and Komagataeibacter with the Komagataeibacter genus outcompeting the Acetobacter in all apple cider vinegar samples at the end of oxidation cycle. Among the lactic acid bacterial consortium two dominating genera were identified, Lactobacillus and Oenococcus, with Oenococcus prevailing with increasing concentration of acetic acid in vinegars. Unexpectedly, a minor genus of the acetic acid bacterial consortium in organic apple cider vinegar was Gluconobacter, suggesting a possible development of the Gluconobacter population with a tolerance against ethanol and acetic acid. Among the accompanying bacteria of the wine vinegar, the genus Rhodococcus was detected, but it decreased substantially by the end of oxidation cycles.


PubMed | National Laboratory for Health
Type: Journal Article | Journal: PloS one | Year: 2016

Clostridium difficile is one of the most important human and animal pathogens. However, the bacterium is ubiquitous and can be isolated from various sources. Here we report the prevalence and characterization of C. difficile in less studied environmental samples, puddle water (n = 104) and soil (n = 79). C. difficile was detected in 14.4% of puddle water and in 36.7% of soil samples. Environmental strains displayed antimicrobial resistance patterns comparable to already published data of human and animal isolates. A total of 480 isolates were grouped into 34 different PCR ribotypes. More than half of these (52.9%; 18 of 34) were already described in humans or animals. However, 14 PCR ribotypes were new in our PCR ribotype library and all but one were non-toxigenic. The multilocus sequence analysis of these new PCR ribotypes revealed that non-toxigenic environmental isolates are phylogenetically distinct and belong to three highly divergent clades, two of which have not been described before. Our data suggest that environment is a potential reservoir of genetically diverse population of C. difficile.


PubMed | National Laboratory for Health
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2016

PCR-ribotyping, a method based on heterogeneity of ribosomal intergenic spacer region, is the preferred method for genotyping of Clostridium difficile. Standardly used procedure for PCR-ribotyping is culturing of C. difficile from fecal samples and subsequent typing. In this chapter, we describe a modified PCR-ribotyping method for direct detection of PCR-ribotypes directly in total stool DNA extract, without prior need to isolate C. difficile.

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