Pawel Parniewski

(CGG)4-Based PCR as a Novel Tool for Discrimination of Uropathogenic Escherichia coli Strains: Comparison with Enterobacterial Repetitive Intergenic Consensus-PCR

ABSTRACT Urinary tract infections are one of the most frequent bacterial diseases in humans, and Escherichia coli is most often the relevant pathogen. A specific pathotype of E. coli, known as uropathogenic E. coli (UPEC), often causes serious and difficult-to-treat infections of the urinary tract. We propose a new single-tube screening tool that uses an (N)6(CGG)4 primer to generate fingerprint profiles that allow rapid discrimination and epidemiology of this group of bacteria. We found 71 different CGG-PCR profiles among 127 E. coli strains, while enterobacterial repetitive intergenic consensus (ERIC)-PCR of the same group yielded only 28 profiles. Additionally, the (CGG)4-based PCR test turned out to be very effective for clustering UPEC strains exhibiting multiple virulence genes and usually belonging to the B2 phylogenetic group, and it separated these strains from E. coli strains lacking most of the UPEC-specific virulence factors. Since the reproducibility of the CGG-PCR screen is higher than that of ERIC-PCR, our test should be a valuable means of increasing the discriminatory power of current UPEC typing schemes.

Characterization of a Trinucleotide Repeat Sequence (CGG)5 and Potential Use in Restriction Fragment Length Polymorphism Typing of Mycobacterium tuberculosis

ABSTRACT The genomes of 28 bacterial strains, including mycobacterial species Mycobacterium tuberculosis and Mycobacterium bovis, were analyzed for the presence of a special class of microsatellite, that of trinucleotide repeat sequences (TRS). Results of a search of all 10 possible TRS motifs (i.e., CCT, CGG, CTG, GAA, GAT, GTA, GTC, GTG, GTT, and TAT) with five or more repeating units showed that (CGG)5 was highly represented within the genomic DNA of M. tuberculosis and M. bovis. Most of the (CGG)5 repeats in the genome were within the open reading frames of two large gene families encoding PE_PGRS and PPE proteins that have the motifs Pro-Glu (PE) and Pro-Pro-Glu (PPE). (CGG)5-probed Southern hybridization showed that some mycobacterial species, such as Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium szulgai, possess many copies of (CGG)5 in their genomes. Analysis of clinical isolates obtained from Tokyo and Warsaw with both IS6110 and (CGG)5 probes showed that there is an association between the fingerprinting patterns and the geographic origin of the isolates and that (CGG)5 fingerprinting patterns were relatively more stable than IS6110 patterns. The (CGG)5 repeat is a unique sequence for some mycobacterial species, and (CGG)5 fingerprinting can be used as an epidemiologic method for these species as well as IS6110 fingerprinting can. If these two fingerprinting methods are used together, the precise analysis of M. tuberculosis isolates will be accomplished. (CGG)5-based fingerprinting is particularly useful for M. tuberculosis isolates with few or no insertion elements and for the identification of other mycobacterial species when informative probes are lacking.

Comparison of antibiotic resistance patterns in collections of Escherichia coli and Proteus mirabilis uropathogenic strains

Escherichia coli and Proteus mirabilis are important urinary tract pathogens. The constant increase in the antibiotic resistance of clinical bacterial strains has become an important clinical problem. The aim of this study was to compare the antibiotic resistance of 141 clinical (Sweden and Poland) and 42 laboratory (Czech Republic) P.mirabilis strains and 129 clinical (Poland) uropathogenic E. coli strains. The proportion of unique versus diverse patterns in Swedish clinical and laboratory P. mirabilis strain collections was comparable. Notably, a similar proportion of unique versus diverse patterns was observed in Polish clinical P. mirabilis and E. coli strain collections. Mathematical models of the antibiotic resistance of E. coli and P. mirabilis strains based on Kohonen networks and association analysis are presented. In contrast to the three clinical strain collections, which revealed complex associations with the antibiotics tested, laboratory P. mirabilis strains provided simple antibiotic association diagrams. The monitoring of antibiotic resistance patterns of clinical E. coli and P. mirabilis strains plays an important role in the treatment procedures for urinary tract infections and is important in the context of the spreading drug resistance in uropathogenic strain populations. The adaptability and flexibility of the genomes of E. coli and P.mirabilis strains are discussed.

Comparison of the (CCG) 4 -based PCR and MIRU-VNTR for molecular typing of Mycobacterium avium strains

Mycobacterium avium, a member of the group of non-tuberculous mycobacteria, is most often responsible for the serious diseases in humans and is frequently isolated from NTM-caused pulmonary events. In this connection the epidemiological aspect is also of great importance. Here we present a useful genetic assay that uses (CCG)4-based PCR for genotyping M. avium. After applying this test to 33 strains of M. avium, we found a discriminatory index of 0.979. The accuracy of this analysis was supported by a reasonable reproducibility of 95.1%. These results were compared with the Mycobacterial Intergenic Repeat Unit-Variable Number Tandem Repeats (MIRU-VNTR) typing scheme which had slightly lower discriminatory index of 0.945 however, the method was able to cluster different strains compared to CCG-PCR. Taking into account high discriminatory index and reproducibility, this test scheme has the potential as a screening tool in the investigation of M. avium infections, especially if combined with MIRU-VNTR.

Trinucleotide repeat sequence-based PCR as a potential approach for genotyping Mycobacterium gordonae strains.

Diseases that are caused by non-tuberculous mycobacteria (NTM) continue to pose difficult clinical problems, and the epidemiological aspect of NTM-caused diseases is of great importance. In the case of Mycobacterium gordonae there is no adequate genotyping scheme. Here we present a potential rapid and reproducible genetic assay that uses trinucleotide repeat sequence-based PCR (TRS-PCR) for genotyping M. gordonae. The proposed method constitutes a useful single-primer PCR screen for genotyping this species. Among 10 TRS-containing primers, after applying (CAC)₄-based PCR to 36 strains of M. gordonae, we found a discriminatory index of 0.975. The accuracy of this analysis was supported by a reasonable reproducibility of 92%. These results were compared with the Enterobacterial Repetitive Intergenic Consensus Sequences (ERIC)-PCR typing scheme which had lower discriminatory index of 0.93 and its reproducibility was only 86.3%.

TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum

Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N6(GTG)4, N6(CAC)4, N6(CGG)4, N6(CCG)4 and N6(CTG)4, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N6(GTG)4 primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.

Predicting pathogenicity behavior in Escherichia coli population through a state dependent model and TRS profiling

The Binary State Speciation and Extinction (BiSSE) model is a branching process based model that allows the diversification rates to be controlled by a binary trait. We develop a general approach, based on the BiSSE model, for predicting pathogenicity in bacterial populations from microsatellites profiling data. A comprehensive approach for predicting pathogenicity in E. coli populations is proposed using the state-dependent branching process model combined with microsatellites TRS-PCR profiling. Additionally, we have evaluated the possibility of using the BiSSE model for estimating parameters from genetic data. We analyzed a real dataset (from 251 E. coli strains) and confirmed previous biological observations demonstrating a prevalence of some virulence traits in specific bacterial sub-groups. The method may be used to predict pathogenicity of other bacterial taxa.