Usefulness of Capillary Gel Electrophoresis-Based PCR for Detection of Clostridioides difficile Strains with Hypervirulent Ribotypes.

Clostridioides difficile is a complex of anaerobic bacteria responsible for the epidemics of post-antibiotic diarrhea as one of the examples of CDI (Clostridioides difficile infection). As many as 70% of cases concern hospitalized patients, particularly those in intensive care units. Ribotyping is one of the most common methods for differentiating bacterial strains. The purpose of this work was to show the effectiveness of the gel electrophoresis-based PCR ribotyping method and the Webribo database for typing C. difficile isolates, including the hypervirulent 027 ribotype. DNA samples extracted from 69 C. difficile strains with previously marked genotypes were included in this study. PCR was performed using 16S-23S primers, and capillary gel electrophoresis was performed on the Applied Biosystem 3130xl Genetic Analyzer. The Webribo database was applied for ribotype assignment. Out of 69 samples, 48 belonged to already known ribotypes, 13 represented new ribotypes and 8 was indicated as similar to the existing ones, having some differences. Capillary gel electrophoresis-based PCR is an effective method for the differentiation of C. difficile ribotypes and can be recognized as a very useful tool in epidemiological studies, while the Webribo database is a useful and an accessible database for a quick analysis of C. difficile ribotypes.

Deodorizing bacterial consortium: community analysis of biofilms and leachate water collected from an air biofiltration system in a piggery.

Intensive livestock production is a source of water, soil, and air contamination. The first aspect that negatively affects the quality of life of residents in the vicinity of piggeries is malodorous aerosols, which are not only responsible for discomfort but can be an etiological factor in the development of various diseases during prolonged exposure. One of the proven and efficient ways to counteract odor emissions is the usage of air biofiltration. The purpose of this study was to qualitatively analyze the bacterial community colonizing the biofilm of a biofilter operating at an industrial piggery in Switzerland. The study material consisted of biofilm and leachate water samples. The microbiological analysis consisted of DNA isolation, amplification of the bacterial 16S rRNA gene fragment (V3-V4), preparation of a library for high-throughput sequencing, high-throughput NGS sequencing, filtering of the obtained sequencing reads, and evaluation of the species composition in the studied samples. The investigation revealed the presence of the following bacterial genera: Pseudochelatococcus, Methyloversatilis, Flexilinea, Deviosia, Chryseobacterium, Kribbia, Leadbetterella, Corynebacterium, Flavobacterium, Xantobacter, Tessaracoccus, Staphylococcus, Thiobacillus, Enhydrobacter, Proteiniclasticum, and Giesbergeria. Analysis of the microbial composition of biofilters provides the opportunity to improve the biofiltration process.

Comparison of Virulence-Factor-Encoding Genes and Genotype Distribution amongst Clinical Pseudomonas aeruginosa Strains.

Pseudomonas aeruginosa is an opportunistic pathogen encoding several virulence factors in its genome, which is well-known for its ability to cause severe and life-threatening infections, particularly among cystic fibrosis patients. The organism is also a major cause of nosocomial infections, mainly affecting patients with immune deficiencies and burn wounds, ventilator-assisted patients, and patients affected by other malignancies. The extensively reported emergence of multidrug-resistant (MDR) P. aeruginosa strains poses additional challenges to the management of infections. The aim of this study was to compare the incidence rates of selected virulence-factor-encoding genes and the genotype distribution amongst clinical multidrug-sensitive (MDS) and MDR P. aeruginosa strains. The study involved 74 MDS and 57 MDR P. aeruginosa strains and the following virulence-factor-encoding genes: lasB, plC H, plC N, exoU, nan1, pilA, and pilB. The genotype distribution, with respect to the antimicrobial susceptibility profiles of the strains, was also analyzed. The lasB and plC N genes were present amongst several P. aeruginosa strains, including all the MDR P. aeruginosa, suggesting that their presence might be used as a marker for diagnostic purposes. A wide variety of genotype distributions were observed among the investigated isolates, with the MDS and MDR strains exhibiting, respectively, 18 and 9 distinct profiles. A higher prevalence of genes determining the virulence factors in the MDR strains was observed in this study, but more research is needed on the prevalence and expression levels of these genes in additional MDR strains.

Agarose Gel Electrophoresis-Based RAPD-PCR-An Optimization of the Conditions to Rapidly Detect Similarity of the Alert Pathogens for the Purpose of Epidemiological Studies.

Agarose gel electrophoresis is a well-known tool to detect DNA fragments amplified in polymerase chain reaction (PCR). Its usefulness has also been confirmed for epidemiological studies based on restriction fragments length polymorphism (RFLP), usually performed using pulsed-field gel electrophoresis (PFGE). Little is known on the effectiveness for alert-pathogen epidemiological studies of another less time-consuming and costly technique called randomly amplified polymorphic DNA-PCR (RAPD-PCR). Meanwhile, its usefulness is believed to be comparable to RFLP-PFGE. Therefore, the aim of the study was to establish and optimize the conditions of agarose gel electrophoresis following RAPD-PCR for 19 Enterococcus faecium strains derived from epidemic outbreaks at intensive care units. An application of different PCR primers, primer combinations, and, in particular, agarose gel concentrations and electrophoresis conditions revealed the usefulness of this relatively fast and inexpensive method based on RAPD-PCR for epidemiological studies without a compulsion to use the specialized equipment necessary for RFLP-PFGE.

Legionellosis risk-an overview of Legionella spp. habitats in Europe.

An increase in the number of reports of legionellosis in the European Union and the European Economic Area have been recorded in recent years. The increase in cases is significant: from 6947 reports in 2015 to 11,298 in 2019. This is alarming as genus Legionella, which comprises a large group of bacteria inhabiting various aquatic systems, poses a serious threat to human health and life, since more than 20 species can cause legionellosis, with L. pneumophila being responsible for the majority of cases. The ability to colonize diverse ecosystems makes the eradication of these microorganisms difficult. A detailed understanding of the Legionella habitat may be helpful in the effective control of this pathogen. This paper provides an overview of Legionella environments in Europe: natural (lakes, groundwater, rivers, compost, soil) and anthropogenic (fountains, air humidifiers, water supply systems), and the role of Legionella spp. in nosocomial infections, which are potentially fatal for children, the elderly and immunocompromised patients.