Distribution of PCR ribotypes among recent Clostridium difficile isolates collected in two districts of Hungary using capillary gel electrophoresis and review of changes in the circulating ribotypes over time.

Following the first description of a Clostridium difficile case caused by ribotype 027 in Hungary in 2007, the rapid spread of C. difficile infection in different hospitals within the country was observed. The aim of this pilot study was to investigate the distribution of different PCR ribotypes among inpatient and outpatient isolates obtained in two geographically different parts of Hungary. One hundred and ninety-two toxigenic C. difficile isolates collected between 1 October and 1 December 2014 were PCR ribotyped using capillary gel electrophoresis and the database of WEBRIBO (http://webribo.ages.at), which allows the automatic analysis and comparison of capillary-sequencer-based PCR ribotyping data. Altogether, 31 different known ribotypes were found, and 16 isolates showed a novel banding pattern, not included in the current library. Besides the dominance of 027 (33.3 %) among all isolates, there were differences in its presence among isolates obtained from the two regions (45.8 % in the central region and 20.8 % in the south-east region, respectively), whereas the second most prevalent ribotype 036 (19.8 %) was more frequently found among isolates obtained in the south-east region compared with the central region of Hungary (29.1 versus 10.4 %). Similar differences in the spread of different ribotypes, in particular 027, which were found during earlier studies in Hungary may be due to the existing order for admissions of patients to hospitals. We also summarized the changing pattern of PCR ribotypes of Hungarian C. difficile isolates over time, based on earlier published data.

Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis.

BACKGROUND: Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus. METHODS: Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing. RESULTS: For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%. CONCLUSION: The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.

Direct detection and drug-resistance profiling of bacteremias using inertial microfluidics.

Detection of bacteria in bloodstream infections and their antibiotic susceptibility patterns is critical to guide therapeutic decision-making for optimal patient care. Current culture-based assays are too slow (>48 h), leading to excessive up-front use of broad-spectrum antibiotics and/or incorrect antibiotic choices due to resistant bacteria, each with deleterious consequences for patient care and public health. To approach this problem, we describe a method to rapidly isolate bacteria from whole blood using inertial microfluidics and directly determine pathogen identity and antibiotic susceptibility with hybridization-based RNA detection. Using the principle of Dean flow fractionation, bacteria are separated from host blood cells in a label-free separation method with efficient recovery of even low abundance bacteria. Ribosomal RNA detection can then be applied for direct identification of low abundance pathogens (~100 per mL) from blood without culturing or enzymatic amplification. Messenger RNA detection of antibiotic-responsive transcripts after brief drug exposure permits rapid susceptibility determination from bacteria with minimal culturing (~10(5) per mL). This unique coupling of microfluidic cell separation with RNA-based molecular detection techniques represents significant progress towards faster diagnostics (~8 hours) to guide antibiotic therapy.

Ribotyping and automated ribotyping of Listeria monocytogenes.

Ribotyping is a molecular technique that allows identification and typing of bacteria to the strain level. It is based on restriction endonuclease cleavage of total genomic DNA followed by electrophoretic separation, Southern blot transfer, and hybridization of transferred DNA fragments with a radiolabeled ribosomal operon probe. Following autoradiography, only those bands containing a portion of the ribosomal operon are visualized. The number of fragments generated by ribotyping is a reflection of the multiplicity of rRNA operons present in a bacterial species.Automated Ribotyping-AR (RiboPrinter) is a commercially available instrument with a high level of reproducibility and standardization. The RiboPrinter automates all of the steps in the process from cell lysis to data capture and database comparisons. Further, reagent cassettes including the enzymes, enzyme conjugated-hybridization probe, electrophoretic gel, and membrane have been developed to deliver consistent performance. Data capture is accomplished via a CCD camera and the gel patterns obtained stored in a digitized format, making it easier to compare results among laboratories and to exchange data electronically.

Succession in the intestinal microbiota of preadolescent turkeys.

In the present study, automated ribosomal intergenic spacer analysis (ARISA), library sequence analysis, real-time PCR detection of Bacteroides uniformis and Campylobacter coli and dot-blot hybridizations of Clostridiaceae were used to identify trends in microbial colonization of the ceca of male turkeys. Two separate trials were performed with six and five birds, respectively. ARISA community profiles identified a period of community transition at week 12 of age in both trials. A significant increase of Ca. coli was also detected at week 12 in one trial, suggesting a possible correlation between microbiota destabilization and pathogen prevalence. Libraries of ribosomal small subunit 16S genes representing weeks 9, 11, 12 and 14 of both trials were sequenced. Whereas fingerprint and sequence analyses indicated significant differences in the species composition between the two trials, in general sequence library and dot-blot analyses indicated that Clostridia-like species decreased in prevalence over time. While B. uniformis prevalence in the two trials rose from 7% and 0% of the library clones at week 9 to 84% and 79% at week 11, real-time PCR did not support these results, with only approximately twofold and sixfold increases in internal transcribed spacer copy numbers observed.

Combined use of 16S ribosomal DNA and automated ribosomal intergenic spacer analysis to study the bacterial community in catfish ponds.

AIMS: To apply culture-independent techniques to explore the bacterial community composition in catfish pond water. METHODS AND RESULTS: 16S rDNA libraries were constructed and sequenced from 15 pond water samples. Automated ribosomal intergenic spacer analysis (ARISA) was used to fingerprint each bacterial community. A broad diversity in bacterial species composition was found by 16S rDNA analysis. Alphaproteobacteria was the most represented class in all ponds, followed by Gammaproteobacteria and Gram-positive high G + C content bacteria. Uniqueness of bacterial communities from each individual pond was confirmed by ARISA. Catfish pathogens were detected sporadically. CONCLUSIONS: Bacterial communities in a catfish aquaculture setting can vary from pond to pond at one given point. No correlation could be made between bacteria composition and fish strain or between bacterial profile and the presence of catfish pathogens in a particular pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing the composition of bacterial communities in catfish ponds. Fish health specialists and catfish aquaculture managers should be aware of the wide differences in bacterial communities between ponds and include this variable in fish husbandry practices.

[Identification of bacteria through 16S rRNA sequencing: principles, methods and applications in clinical microbiology]. / Identificación bacteriana mediante secuenciación del ARNr 16S: fundamento, metodología y aplicaciones en microbiología clínica.

Phylogenetic relationships among prokaryotes can be inferred from comparisons of their 16S rRNA (or 16S rDNA) sequences. This has had an enormous repercussion on bacterial taxonomy, leading to the currently applied system of classification, and allowing a rapid and precise identification of bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques turns out impossible, difficult, or requires a lot of time. Amplification of the gene to be sequenced uses preferably DNA extracted from a bacterial pure culture, but can be achieved also directly from a clinical sample. The latter has led to the discovery of new pathogens. Bearing in mind its potential, as the technical resources improve and the prize becomes more competitive, the identification based on 16S rDNA sequencing will certainly find a wider application in the clinical microbiology laboratory.

Dereplication of Streptomyces soil isolates and detection of specific biosynthetic genes using an automated ribotyping instrument.

The discrimination of distinct cultures among morphologically similar Streptomyces soil isolates (dereplication) and the detection of specific biosynthetic pathways in these strains are important steps in the selection of microorganisms to include in a natural products library. We have developed methods for analysis of actinomycetes using the RiboPrinter microbial characterization system, an automated instrument that performs ribotyping on bacterial samples. To evaluate our dereplication method, 26 Streptomyces isolates, obtained from soil samples collected in Maui, Hawaii, were ribotyped and compared with each other, using the RiboPrinter. The strains were also compared by 16S rDNA sequence analysis, MIDI fatty acid analysis, and LC-MS profiling of fermentation extracts. The RiboPrinter was able to identify closely related isolates and to discriminate between morphologically similar isolates with unique genetic, fatty acid and fermentation profiles. For the detection of biosynthetic genes, a 1,006-bp probe containing a portion of an adenylation domain of a non-ribosomal peptide synthetase (NRPS) was employed. Using this alternate probe in place of the standard ribosomal probe, the RiboPrinter was able to detect NRPS genes in several strains of Streptomyces. These results demonstrate that the RiboPrinter has multiple applications in a natural products research program.