Phenotypic and Molecular Epidemiology of ESBL-, AmpC-, and Carbapenemase-Producing Escherichia coli in Northern and Eastern Europe.

Extended-spectrum beta-lactamases (ESBL) and AmpC producing-Escherichia coli have spread worldwide, but data about ESBL-producing-E. coli in the Northern and Eastern regions of Europe is scant. The aim of this study has been to describe the phenotypical and molecular epidemiology of different ESBL/AmpC/Carbapenemases genes in E. coli strains isolated from the Baltic States (Estonia, Latvia, and Lithuania), Norway and St. Petersburg (Russia), and to determine the predominant multilocus sequence type and single nucleotide polymorphisms diversity of E. coli isolates deduced by whole genome sequencing (WGS). A total of 10,780 clinical E. coli strains were screened for reduced sensitivity to third-generation cephalosporins. They were collected from 21 hospitals located in Estonia, Latvia, Lithuania, Norway and St. Petersburg during a 5 month period in 2012. The overall prevalence of ESBL/AmpC strains was 4.7% by phenotypical test and 3.9% by sequencing. We found more strains with the ESBL/AmpC phenotype and genotype in St. Petersburg and Latvia than other countries. Of phenotypic E. coli strains, 85% contained confirmed ESBL genes (including bla CTX-M, bla TEM- 29, bla TEM- 71), AmpC genes (bla CMY- 59, bla ACT- 12 / - 15 / - 20, bla ESC- 6, bla FEC- 1, bla DHA- 1), or carbapenemase genes (bla NDM- 1). bla CTX-M- 1, bla CTX-M - 14 and bla CTX-M- 15 were found in all countries, but bla CTX-M- 15 prevalence was higher in Latvia than in St. Petersburg (Russia), Estonia, Norway and Lithuania. The dominating AmpC genes were bla CMY- 59 in the Baltic States and Norway, and bla DHA- 1 in St. Petersburg. E. coli strains belonged to 83 different sequence types, of which the most prevalent was ST131 (40%). In conclusion, we generally found low ESBL/AmpC/Carbapenemase prevalence in E. coli strains isolated in Northern/Eastern Europe. However, several inter-country differences in distribution of particular genes and multilocus sequence types were found.

Application of Molecular Methods for Carbapenemase Detection.

This study has evaluated the correlation between different carbapenemases detection methods on carbapenem non-susceptible Klebsiella pneumoniae strains from Northern and Eastern Europe; 31 institutions in 9 countries participated in the research project, namely Finland, Estonia, Latvia, Lithuania, Russia, St. Petersburg, Poland, Belarus, Ukraine, and Georgia. During the research program, a total of 5,001 clinical K. pneumoniae isolates were screened for any carbapenem non-susceptibility by the disk diffusion method, Vitek 2 or Phoenix system following the EUCAST guideline on detection of resistance mechanisms, version 1.0. Strains isolated from outpatients and hospitalized patients from April 2015 to June 2015 were included. All types of samples (blood, pus, urine, etc.) excluding fecal screening or fecal colonization samples have been represented. In total, 171 carbapenemase screening-positive K. pneumoniae isolates (3.42%) were found and characterized. Several methods were used for detection of carbapenemases production, including Luminex assay (PCR and hybridization), whole genome sequencing, MALDI-TOF based Imipenem degradation assay, and immunochromatography testing. Minimal inhibitory concentration determination for Meropenem by agar-based gradient method was also used. Finally, 83 K. pneumoniae strains were carbapenemase negative by all confirmation methods (49.4% of all screening-positive ones), 74 - positive by three methods (44.0%), 8 - positive by two methods (4.8%) and 3 - positive by only one method (1.8%). The sensitivity of the tests was 96.3% for Whole genome sequencing and MALDI-TOF assay (both three undetected cases), and 95.1% for Luminex-Carba (4 undetected cases). The most commonly detected carbapenemases were NDM (n = 54) and OXA-48 (n = 26), followed by KPC-2, VIM-5, and OXA-72 (one case of each). Our results showed that different types of carbapenemases can be detected in the countries involved in the project. The sensitivity of our methods for carbapenemase detection (including screening as a first step and further confirmation tests) was >95%, but we would recommend using different methods to increase the sensitivity of detection and make it more precise.

Primer3_masker: integrating masking of template sequence with primer design software.

Summary: Designing PCR primers for amplifying regions of eukaryotic genomes is a complicated task because the genomes contain a large number of repeat sequences and other regions unsuitable for amplification by PCR. We have developed a novel k-mer based masking method that uses a statistical model to detect and mask failure-prone regions on the DNA template prior to primer design. We implemented the software as a standalone software primer3_masker and integrated it into the primer design program Primer3. Availability and implementation: The standalone version of primer3_masker is implemented in C. The source code is freely available at https://github.com/bioinfo-ut/primer3_masker/ (standalone version for Linux and macOS) and at https://github.com/primer3-org/primer3/ (integrated version). Primer3 web application that allows masking sequences of 196 animal and plant genomes is available at http://primer3.ut.ee/. Contact: maido.remm@ut.ee. Supplementary information: Supplementary data are available at Bioinformatics online.

StrainSeeker: fast identification of bacterial strains from raw sequencing reads using user-provided guide trees.

BACKGROUND: Fast, accurate and high-throughput identification of bacterial isolates is in great demand. The present work was conducted to investigate the possibility of identifying isolates from unassembled next-generation sequencing reads using custom-made guide trees. RESULTS: A tool named StrainSeeker was developed that constructs a list of specific k-mers for each node of any given Newick-format tree and enables the identification of bacterial isolates in 1-2 min. It uses a novel algorithm, which analyses the observed and expected fractions of node-specific k-mers to test the presence of each node in the sample. This allows StrainSeeker to determine where the isolate branches off the guide tree and assign it to a clade whereas other tools assign each read to a reference genome. Using a dataset of 100 Escherichia coli isolates, we demonstrate that StrainSeeker can predict the clades of E. coli with 92% accuracy and correct tree branch assignment with 98% accuracy. Twenty-five thousand Illumina HiSeq reads are sufficient for identification of the strain. CONCLUSION: StrainSeeker is a software program that identifies bacterial isolates by assigning them to nodes or leaves of a custom-made guide tree. StrainSeeker's web interface and pre-computed guide trees are available at http://bioinfo.ut.ee/strainseeker. Source code is stored at GitHub: https://github.com/bioinfo-ut/StrainSeeker.

Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis.

The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants.

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes.

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH(+) phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees.

Primer3--new capabilities and interfaces.

Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3's functionality. In this article, we describe Primer3's current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement-a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.

Characterization of species-specific repeats in 613 prokaryotic species.

Prokaryotes are in general believed to possess small, compactly organized genomes, with repetitive sequences forming only a small part of them. Nonetheless, many prokaryotic genomes in fact contain species-specific repeats (>85 bp long genomic sequences with less than 60% identity to other species) as we have previously demonstrated. However, it is not known at present how frequent such species-specific repeats are and what their functional roles in bacterial genomes may be. Therefore, we have conducted a comprehensive survey of prokaryotic species-specific repeats and characterized them to examine as to whether there are functional classes among different repeats or not and how they are mutually related to each other. Of the 613 distinct prokaryotic species analyzed, 97% were found to contain at least one species-specific repeats. It seems interesting to note that the species-specific repeats thus identified appear to be functionally variable in different genomes: in some genomes, they are mostly associated with duplicated protein-coding genes, whereas in some other genomes with rRNA and tRNA genes. Contrary to what may be expected, only one-fourth of the species-specific repeats were found to be associated with mobile genetic elements.

Automatic identification of species-specific repetitive DNA sequences and their utilization for detecting microbial organisms.

MOTIVATION: The concentration of pathogen DNA in biological samples is often very low. Therefore, the sensitivity of diagnostic tests is always a critical factor. RESULTS: We have developed a novel computational method that identifies species-specific repeats from microbial organisms and automatically designs species-specific PCR primers for these repeats. We tested the methodology on 30 randomly chosen microbial species and we demonstrate that species-specific repeats longer than 300 bp exist in all these genomes. We also used our methodology to design species-specific PCR primers for 86 repeats from five medically relevant microbial species. These PCR primers were tested experimentally. We demonstrate that using species-specific repeats as a PCR template region can increase the sensitivity of PCR in diagnostic tests. AVAILABILITY AND IMPLEMENTATION: A web version of the method called MultiMPrimer3 was implemented and is freely available at (http://bioinfo.ut.ee/multimprimer3/).

Enhancements and modifications of primer design program Primer3.

UNLABELLED: The determination of annealing temperature is a critical step in PCR design. This parameter is typically derived from the melting temperature of the PCR primers, so for successful PCR work it is important to determine the melting temperature of primer accurately. We introduced several enhancements in the widely used primer design program Primer3. The improvements include a formula for calculating melting temperature and a salt correction formula. Also, the new version can take into account the effects of divalent cations, which are included in most PCR buffers. Another modification enables using lowercase masked template sequences for primer design. AVAILABILITY: Features described in this article have been implemented into the development code of Primer3 and will be available in future versions (version 1.1 and newer) of Primer3. Also, a modified version is compiled under the name of mPrimer3 which is distributed independently. The web-based version of mPrimer3 is available at http://bioinfo.ebc.ee/mprimer3/ and the binary code is freely downloadable from the URL http://bioinfo.ebc.ee/download/.