New Multilocus Sequence Typing Scheme for Enterococcus faecium Based on Whole Genome Sequencing Data.

The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. IMPORTANCE Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.

Rapid Identification of Pseudomonas aeruginosa International High-Risk Clones Based on High-Resolution Melting Analysis.

The Pseudomonas aeruginosa population has a nonclonal epidemic structure. It is generally composed of a limited number of widespread clones selected from a background of many rare and unrelated genotypes recombining at high frequency. Due to the increasing prevalence of nosocomial infections caused by multidrug-resistant/extensively drug-resistant (MDR/XDR) strains, it is advisable to implement infection control measures. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) are considered the gold standard methods in bacterial typing, despite being limited by cost, staff, and instrumental demands. Here, we present a novel mini-MLST scheme for P. aeruginosa rapid genotyping based on high-resolution melting analysis. Using the proposed mini-MLST scheme, 3,955 existing sequence types (STs) were converted into 701 melting types (MelTs), resulting in a discriminatory power of D = 0.993 (95% confidence interval [CI], 0.992 to 0.994). Whole-genome sequencing of 18 clinical isolates was performed to support the newly designed mini-MLST scheme. The clonal analysis of STs belonging to MelTs associated with international high-risk clones (HRCs) performed by goeBURST software revealed that a high proportion of the included STs are highly related to HRCs and have also been witnessed as responsible for serious infections. Therefore, mini-MLST provides a clear warning for the potential spread of P. aeruginosa clones recognized as MDR/XDR strains with possible serious outcomes. IMPORTANCE In this study, we designed a novel mini-MLST typing scheme for Pseudomonas aeruginosa. Its great discriminatory power, together with ease of performance and short processing time, makes this approach attractive for prospective typing of large isolate sets. Integrating the novel P. aeruginosa molecular typing scheme enables the development and spread of MDR/XDR high-risk clones to be investigated.

Sequencing Independent Molecular Typing of Staphylococcus aureus Isolates: Approach for Infection Control and Clonal Characterization.

Staphylococcus aureus is a major bacterial human pathogen that causes a wide variety of clinical manifestations. The main aim of the presented study was to determine and optimize a novel sequencing independent approach that enables molecular typing of S. aureus isolates and elucidates the transmission of emergent clones between patients. In total, 987 S. aureus isolates including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates were used to evaluate the novel typing approach combining high-resolution melting (HRM) analysis of multilocus sequence typing (MLST) genes (mini-MLST) and spa gene (spa-HRM). The novel approach's discriminatory ability was evaluated by whole-genome sequencing (WGS). The clonal relatedness of tested isolates was set by the BURP and BURST approach using spa and MLST data, respectively. Mini-MLST classified the S. aureus isolates into 38 clusters, followed by spa-HRM classifying the isolates into 101 clusters. The WGS proved HRM-based methods to effectively differentiate between related S. aureus isolates. Visualizing evolutionary relationships among different spa-types provided by the BURP algorithm showed comparable results to MLST/mini-MLST clonal clusters. We proved that the combination of mini-MLST and spa-HRM is rapid, reproducible, and cost-efficient. In addition to high discriminatory ability, the correlation between spa evolutionary relationships and mini-MLST clustering allows the variability in population structure to be monitored. IMPORTANCE Rapid and cost-effective molecular typing tools for Staphylococcus aureus epidemiological applications such as transmission tracking, source attribution and outbreak investigations are highly desirable. High-resolution melting based methods are effective alternative to those based on sequencing. Their good reproducibility and easy performance allow prospective typing of large set of isolates while reaching great discriminatory power. In this study, we established a new epidemiological approach to S. aureus typing. This scheme has the potential to greatly improve epidemiological investigations of S. aureus.

[Application of second- and third-generation sequencing for bacterial typing].

Whole-genome sequencing (WGS) is a modern method that allows deep understanding of studied organisms and is currently gaining importance in molecular microbiology. Data obtained by whole-genome sequencing can be used for a number of different analyses, specifically in bacterial epidemiology. The authors provide an overview of the methods that are used for bacterial typing, description of their principles with subsequent possibilities for evaluation of the obtained data and applications in hospital research.

Pyocin-mediated antagonistic interactions in Pseudomonas spp. isolated in James Ross Island, Antarctica.

Interactions within bacterial communities are frequently mediated by the production of antimicrobial agents. Despite the increasing interest in research of new antimicrobials, studies describing antagonistic interactions among cold-adapted microorganisms are still rare. Our study assessed the antimicrobial interactions of 36 Antarctic Pseudomonas spp. and described the genetic background of these interactions in selected strains. The overall bacteriocinogeny was greater compared to mesophilic Pseudomonas non-aeruginosa species. R-type tailocins were detected on transmission electron micrographs in 16 strains (44.4%); phylogenetic analysis of the corresponding gene clusters revealed that the P. prosekii CCM 8878 tailocin was related to the Rp3 group, whereas the tailocin in Pseudomonas sp. CCM 8880 to the Rp4 group. Soluble antimicrobials were produced by eight strains (22.-2%); gene mining found pyocin L homologues in the genomes of P. prosekii CCM 8881 and CCM 8879 and pyocin S9-like homologues in P. prosekii CCM 8881 and Pseudomonas sp. CCM 8880. Analysis of secretomes confirmed the production of all S- and L-type pyocin genes. Our results suggest that bacteriocin-based inhibition plays an important role in interactions among Antarctic soil bacteria, and these native, cold-adapted microorganisms could be a promising source of new antimicrobials.

Prevalence of bacteriocins and their co-association with virulence factors within Pseudomonas aeruginosa catheter isolates.

Urinary tract infections represent common nosocomial infectious diseases. Bacteriocin production has been recently described as a putative virulence factor in these infections but studies focusing particularly on Pseudomonas aeruginosa are not available. Therefore, we assessed the prevalence of the bacteriocin genes, their co-occurrence and their co-association with previously detected virulence factors in a set of 135 P. aeruginosa strains from catheter-associated urinary tract infections (CAUTIs). The overall bacteriocinogeny reached 96.3 % with an average of 3.6 genes per strain. The most frequently detected determinants were the encoded pyocins S4 (76.3 %), R (69.6 %), and S2 (67.4 %). A statistically significant co-occurrence and a negative relationship were observed between several pyocin types. Particular pyocins exhibited associations with biofilm formation, production of pyochelin, pyocyanin, antibiotic-degrading enzymes, overall strain susceptibility and resistance, and motility of the strain. Co-occurrence of the pyocins S2 and S4 (p<<0.0001; Z = 13.15), both utilizating the ferripyoverdine receptor FpvAI, was found but no relation to pyoverdine production was detected. A negative association (p = 0.0047; Z=-2.83) was observed between pyochelin and pyocin S5 utilising the ferripyochelin receptor FptA. Pairwise assays resulted in 52.1 % inhibition which was equally distributed between soluble and particle types of antimicrobials. In conclusion, pyocin determinants appear to be important characteristics of CAUTI-related P. aeruginosa isolates and could contribute to their urovirulence.

Genome sequences of two Antarctic strains of Pseudomonas prosekii: insights into adaptation to extreme conditions.

Pseudomonas prosekii is a recently described species isolated exclusively from James Ross Island close to the Antarctic Peninsula at 64° south latitude. Here, we present two P. prosekii genome sequences and their analyses with respect to phylogeny, low temperature adaptation, and potential biotechnological applications. The genome of P. prosekii P2406 comprised 5,896,482 bp and 5324 genes (GC content of 59.71%); the genome of P. prosekii P2673 consisted of 6,087,670 bp and 5511 genes (GC content of 59.50%). Whole genome sequence comparisons confirmed a close relationship between both investigated strains and strain P. prosekii LMG 26867T. Gene mining revealed the presence of genes involved in stress response, genes encoding cold shock proteins, oxidative stress proteins, osmoregulation proteins, genes for the synthesis of protection molecules, and siderophores. Comparative genome analysis of P. prosekii and P. aeruginosa PAO1 highlighted differences in genome content between extremophile species and a mesophilic opportunistic pathogen.