We describe the development of a multilocus sequence typing (MLST) scheme for Corynebacterium diphtheriae, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize C. diphtheriae, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of C. diphtheriae examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.

Significance and Impact of the Study: The number of opportunistic infections caused by Corynebacteria is increasing due to increase in number of immunocompromised patients. New Corynebacterium species and new human infections, caused by this group of bacteria, has been described recently. However, identification of Corynebacteria is still a challenge despite application of sophisticated laboratory methods. In the study we present possibilities and limitations of various commercial systems for identification of Corynebacteria. AbstractThe Corynebacterium is a genus of bacteria of growing clinical importance. Progress in medicine results in growing population of immunocompromised patients and growing number of infections caused by opportunistic pathogens. A new infections caused by new Corynebacterium species and species previously regarded as commensal micro-organisms have been described. Parallel with changes in Corynebacteria infections, the microbiological laboratory diagnostic possibilities are changing. But identification of this group of bacteria to the species level remains difficult. In the paper, we present various manual, semiautomated and automated assays used in clinical laboratories for Corynebacterium identification, such as API Coryne, RapID CB Plus, BBL Crystal Gram Positive ID System, MICRONAUT-RPO, VITEK 2, BD Phoenix System, Sherlock Microbial ID System, MicroSeq Microbial Identification System, Biolog Microbial Identification Systems, MALDI-TOF MS systems, polymerase chain reaction (PCR)-based and sequencing-based assays. The presented assays are based on various properties, like biochemical tests, specific DNA sequences, composition of cellular fatty acids, protein profiles and have specific limitations.

Most point-of-care tests (POCT) use swabs for sampling and/or for applying a sample on the test. A variety of swabs differing in tip materials is commercially available. Different tip materials have different chemical and physical characteristics which might influence the specimen collection and release. We investigated properties of various types of swabs used in clinical diagnostics with focusing on two kinds of analytes, DNA and proteins, which are most often used targets in POCT. As the model samples we used diphtheria toxoid NIBSC 69/017 for investigating recovery of protein analytes such as antigens and bacterial strains of Escherichia coli ATCC 25922, diphtheria toxin-producing Corynebacterium diphtheriae NCTC 10648, and the clinical isolate nontoxigenic C. diphtheriae 5820/15 for investigating the recovery of nucleic acids. We investigated four types of swabs most commonly used in clinical diagnostics in terms of absorption capacity and efficiency of release of nucleic acids and proteins. Volume uptake was measured in milligrams. For DNA release various washing out buffers were used and the amount of released DNA was measured spectrophotometrically. The amount of protein released from the swabs were examined using the Lowry assay. We observed statistically significant differences (p < 0.05) in the mean weights of absorbed liquid, in the DNA recovery and protein recovery by the four variety of swab examined. However, the efficiency of DNA and protein release was not correlated to the absorbed volume of a sample, but rather to the properties of swabs. The swab composition and structure can have a significant impact on the collection and release efficiency of a sample. Therefore, validation of POCT in relation to the used swabs for sampling is really important. The use of inappropriate swabs could lead to false negative or misleading analysis results.

The multiple locus VNTRs analysis (MLVA) revealed the presence of five genotypes in a group of 10 Bacillus anthracis isolates from epidemiologically unrelated cases of bovine-anthrax in eastern Poland. Eight tested isolates possessed the pagA and capB genes indicating the presence of both virulence plasmids, while two isolates revealed only pagA and lacked pXO2. The MLVA and DNA sequence analysis indicated that seven tested isolates represent four novel genotypes. Five tested strains revealed a unique 144 bp vrrB2 variant as well as 220 bp variant of vrrB1, implying the relatedness to the lineage B2. Consequently, we propose establishing of novel B2 strains sub-lineage. Multiple anthrax outbreaks, which took place in Poland several decades ago were proposed as a cause of intriguing diversity of B. anthracis observed in this study.

Twenty-five cases of nontoxigenic Corynebacterium diphtheriae infection were recorded in Poland during 2004–2012, of which 18 were invasive. Alcoholism, homelessness, hepatic cirrhosis, and dental caries were predisposing factors for infection. However, for 17% of cases, no concomitant diseases or predisposing factors were found.

Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

The genetic similarity of non-toxigenic C. diphtheriae strains causing infection suggests that the strains represent a single clone. They may possess additional virulence genes in a chromosome, related with higher pathogenicity and invasiveness. The genetic changes have not been followed by resistance to antibiotics.