Isolation and characterization of a phage collection against Pseudomonas putida.

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides.

Rhodotorula toruloides is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.

Molecular Characterization of Enterococcus Isolates From Different Sources in Estonia Reveals Potential Transmission of Resistance Genes Among Different Reservoirs.

In this study, we aimed to characterize the population structure, drug resistance mechanisms, and virulence genes of Enterococcus isolates in Estonia. Sixty-one Enterococcus faecalis and 34 Enterococcus faecium isolates were collected between 2012 and 2014 across the country from various sites and sources, including farm animals and poultry (n = 53), humans (n = 12), environment (n = 24), and wild birds (n = 44). Clonal relationships of the strains were determined by whole-genome sequencing and analyzed by multi-locus sequence typing. We determined the presence of acquired antimicrobial resistance genes and 23S rRNA mutations, virulence genes, and also the plasmid or chromosomal origin of the genes using dedicated DNA sequence analysis tools available and/or homology search against an ad hoc compiled database of relevant sequences. Two E. faecalis isolates from human with vanB genes were highly resistant to vancomycin. Closely related E. faecalis strains were isolated from different host species. This indicates interspecies spread of strains and potential transfer of antibiotic resistance. Genomic context analysis of the resistance genes indicated frequent association with plasmids and mobile genetic elements. Resistance genes are often present in the identical genetic context in strains with diverse origins, suggesting the occurrence of transfer events.

Characteristics of Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae and Contact to Animals in Estonia.

We have attempted to define the prevalence and risk factors of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-Enterobacteriaceae) carriage, and to characterize antimicrobial susceptibility, beta-lactamase genes, and major types of isolated strains in volunteers, with a specific focus on humans in contact with animals. Samples were collected from 207 volunteers (veterinarians, pig farmers, dog owners, etc.) and cultured on selective agar. Clonal relationships of the isolated ESBL-Enterobacteriaceae were determined by whole genome sequencing and multi-locus sequence typing. Beta-lactamases were detected using a homology search. Subjects filled in questionnaires analyzed by univariate and multiple logistic regression. Colonization with ESBL-Enterobacteriaceae was found in fecal samples of 14 individuals (6.8%; 95%CI: 3.75-11.09%). In multiple regression analysis, working as a pig farmer was a significant risk factor for ESBL-Enterobacteriaceae carriage (OR 4.8; 95%CI 1.2-19.1). The only species isolated was Escherichia coli that distributed into 11 sequence types. All ESBL-Enterobacteriaceae isolates were of CTX-M genotype, with the blaCTX-M-1 being the most prevalent and more common in pig farmers than in other groups. Despite the generally low prevalence of ESBL-Enterobacteriaceae in Estonia, the pig farmers may still pose a threat to transfer resistant microorganisms. The clinical relevance of predominant blaCTX-M-1 carrying E. coli is still unclear and needs further studies.

Chromosomal toxin-antitoxin systems in Pseudomonas putida are rather selfish than beneficial.

Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. The ability of TA-encoded toxins to affect stress tolerance when overexpressed supports the hypothesis of TA systems being associated with stress adaptation. However, the deletion of TA genes has usually no effects on stress tolerance, supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of chromosomal TA systems to Pseudomonas putida. We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence or biofilm formation. Our results instead show that TA loci are costly and decrease the competitive fitness of P. putida. Still, the cost of multiple TA systems is low and detectable in certain conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or moderately potent proteins. Analysis of P. putida TA systems' homologs among fully sequenced Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems spread among bacteria by horizontal gene transfer.

Muropeptides Stimulate Growth Resumption from Stationary Phase in Escherichia coli.

When nutrients run out, bacteria enter a dormant metabolic state. This low or undetectable metabolic activity helps bacteria to preserve their scant reserves for the future needs, yet it also diminishes their ability to scan the environment for new growth-promoting substrates. However, neighboring microbial growth is a reliable indicator of a favorable environment and can thus serve as a cue for exiting dormancy. Here we report that for Escherichia coli and Pseudomonas aeruginosa this cue is provided by the basic peptidoglycan unit (i.e. muropeptide). We show that several forms of muropeptides from a variety of bacterial species can stimulate growth resumption of dormant cells and the sugar - peptide bond is crucial for this activity. These results, together with previous research that identifies muropeptides as a germination signal for bacterial spores, and their detection by mammalian immune cells, show that muropeptides are a universal cue for bacterial growth.

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.

Multidrug resistant Pseudomonas aeruginosa in Estonian hospitals.

BACKGROUND: We aimed to identify the main spreading clones, describe the resistance mechanisms associated with carbapenem- and/or multidrug-resistant P. aeruginosa and characterize patients at risk of acquiring these strains in Estonian hospitals. METHODS: Ninety-two non-duplicated carbapenem- and/or multidrug-resistant P. aeruginosa strains were collected between 27th March 2012 and 30th April 2013. Clinical data of the patients was obtained retrospectively from the medical charts. Clonal relationships of the strains were determined by whole genome sequencing and analyzed by multi-locus sequence typing. The presence of resistance genes and beta-lactamases and their origin was determined. Combined-disk method and PCR was used to evaluate carbapenemase and metallo-beta-lactamase production. RESULTS: Forty-three strains were carbapenem-resistant, 11 were multidrug-resistant and 38 were both carbapenem- and multidrug-resistant. Most strains (54%) were isolated from respiratory secretions and caused an infection (74%). Over half of the patients (57%) were ≥ 65 years old and 85% had ≥1 co-morbidity; 96% had contacts with healthcare and/or had received antimicrobial treatment in the previous 90 days. Clinically relevant beta-lactamases (OXA-101, OXA-2 and GES-5) were found in 12% of strains, 27% of which were located in plasmids. No Ambler class B beta-lactamases were detected. Aminoglycoside modifying enzymes were found in 15% of the strains. OprD was defective in 13% of the strains (all with CR phenotype); carbapenem resistance triggering mutations (F170 L, W277X, S403P) were present in 29% of the strains. Ciprofloxacin resistance correlated well with mutations in topoisomerase genes gyrA (T83I, D87N) and parC (S87 L). Almost all strains (97%) with these mutations showed ciprofloxacin-resistant phenotype. Multi-locus sequence type analysis indicated high diversity at the strain level - 36 different sequence types being detected. Two sequence types (ST108 (n = 23) and ST260 (n = 18)) predominated. Whereas ST108 was associated with localized spread in one hospital and mostly carbapenem-resistant phenotype, ST260 strains occurred in all hospitals, mostly with multi-resistant phenotype and carried different resistance genotype/machinery. CONCLUSIONS: Diverse spread of local rather than international P. aeruginosa strains harboring multiple chromosomal mutations, but not plasmid-mediated Ambler class B beta-lactamases, were found in Estonian hospitals. TRIAL REGISTRATION: This trial was registered retrospectively in ClinicalTrials.gov ( NCT03343119 ).

A k-mer-based method for the identification of phenotype-associated genomic biomarkers and predicting phenotypes of sequenced bacteria.

We have developed an easy-to-use and memory-efficient method called PhenotypeSeeker that (a) identifies phenotype-specific k-mers, (b) generates a k-mer-based statistical model for predicting a given phenotype and (c) predicts the phenotype from the sequencing data of a given bacterial isolate. The method was validated on 167 Klebsiella pneumoniae isolates (virulence), 200 Pseudomonas aeruginosa isolates (ciprofloxacin resistance) and 459 Clostridium difficile isolates (azithromycin resistance). The phenotype prediction models trained from these datasets obtained the F1-measure of 0.88 on the K. pneumoniae test set, 0.88 on the P. aeruginosa test set and 0.97 on the C. difficile test set. The F1-measures were the same for assembled sequences and raw sequencing data; however, building the model from assembled genomes is significantly faster. On these datasets, the model building on a mid-range Linux server takes approximately 3 to 5 hours per phenotype if assembled genomes are used and 10 hours per phenotype if raw sequencing data are used. The phenotype prediction from assembled genomes takes less than one second per isolate. Thus, PhenotypeSeeker should be well-suited for predicting phenotypes from large sequencing datasets. PhenotypeSeeker is implemented in Python programming language, is open-source software and is available at GitHub (https://github.com/bioinfo-ut/PhenotypeSeeker/).

Plasmid with Colistin Resistance Gene mcr-1 in Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Strains Isolated from Pig Slurry in Estonia.

A plasmid carrying the colistin resistance gene mcr-1 was isolated from a pig slurry sample in Estonia. The gene was present on a 33,311-bp plasmid of the IncX4 group. mcr-1 is the only antibiotic resistance gene on the plasmid, with the other genes mainly coding for proteins involved in conjugative DNA transfer (taxA, taxB, taxC, trbM, and the pilX operon). The plasmid pESTMCR was present in three phylogenetically very different Escherichia coli strains, suggesting that it has high potential for horizontal transfer.

Macrolide antibiotics allosterically predispose the ribosome for translation arrest.

Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.

The mitochondrial genome of the venomous cone snail Conus consors.

Cone snails are venomous predatory marine neogastropods that belong to the species-rich superfamily of the Conoidea. So far, the mitochondrial genomes of two cone snail species (Conus textile and Conus borgesi) have been described, and these feed on snails and worms, respectively. Here, we report the mitochondrial genome sequence of the fish-hunting cone snail Conus consors and describe a novel putative control region (CR) which seems to be absent in the mitochondrial DNA (mtDNA) of other cone snail species. This possible CR spans about 700 base pairs (bp) and is located between the genes encoding the transfer RNA for phenylalanine (tRNA-Phe, trnF) and cytochrome c oxidase subunit III (cox3). The novel putative CR contains several sequence motifs that suggest a role in mitochondrial replication and transcription.

ConoDictor: a tool for prediction of conopeptide superfamilies.

ConoDictor is a tool that enables fast and accurate classification of conopeptides into superfamilies based on their amino acid sequence. ConoDictor combines predictions from two complementary approaches-profile hidden Markov models and generalized profiles. Results appear in a browser as tables that can be downloaded in various formats. This application is particularly valuable in view of the exponentially increasing number of conopeptides that are being identified. ConoDictor was written in Perl using the common gateway interface module with a php submission page. Sequence matching is performed with hmmsearch from HMMER 3 and ps_scan.pl from the pftools 2.3 package. ConoDictor is freely accessible at http://conco.ebc.ee.