ICESp1109, a novel hybrid Integrative Conjugative Element of macrolide-resistant Streptococcus pyogenes serotype M77 collected between 2003 and 2017 in Poland.

OBJECTIVES: Genetic characterization of the antibiotic resistance determinants and associated mobile genetic elements (MGEs) among Streptococcus pyogenes [Group A streptococci (GAS)] clinical isolates of an M77 serotype collected in Poland between 2003 and 2017. METHODS: The genomes of 136 M77 GAS isolates were sequenced using Illumina, and selected with long-read approach (Oxford Nanopore). Whole genome sequences were analyzed to determine the presence of macrolide resistance determinants, and their genetic context. RESULTS: The strains used in the study were collected in the two multicenter surveys from in- and outpatients. Sequencing data analysis revealed that all strains carried the tet(O) gene (100%, N=136). They were classified as a single sequence type ST63. For erythromycin resistance, the unique determinant was erm(TR) detected in 76.5% (N=104) isolates. A single appearance of tet(M) and erm(B) on Tn3872 was noticed. The mefA, mefE, and msr(D) genes were detected in neither of the genomes. This correlated with the detected strain phenotypes - 11 exhibited cMLSB, 93 - iMLSB, and no M phenotype.The erm(TR) gene was predominantly (N=74) found within a novel hybrid Integrative Conjugative Element composed of the ICESp1108-like sequence and ICESp2906 variant which was then named ICESp1109. However, in strains isolated before 2008, erm(TR) was located within ICESp2905 (N=27). The erm(TR) gene was detected within stand-alone ICESp1108-like sequences in 3 strains. CONCLUSIONS: Based on phylogenetic analysis results the clonal dissemination of the macrolide-resistant S. pyogenes M77/ST63 strain with hybrid ICESp1109 was observed between 2008 and 2017. ICESp1109 is the novel hybrid ICE in Gram-positive bacteria.

Polar-Region Soils as Novel Reservoir of Lactic Acid Bacteria from the Genus Carnobacterium.

Polar habitats offer excellent sites to isolate unique bacterial strains due to their diverse physical, geochemical, and biological factors. We hypothesize that the unique environmental conditions of polar regions select for distinct strains of lactic acid bacteria (LAB) with novel biochemical properties. In this study, we characterized ten strains of psychrotrophic LAB isolated from hitherto poorly described sources-High Arctic and maritime Antarctic soils and soil-like materials, including ornithogenic soils, cryoconites, elephant seal colonies, and postglacial moraines. We evaluated the physiological and biochemical properties of the isolates. Based on 16S rRNA and housekeeping genes, the four LAB strains were assigned to three Carnobacterium species: C. alterfunditum, C. maltaromaticum, and C. jeotgali. The remaining strains may represent three new species of the Carnobacterium genus. All isolates were neutrophilic and halophilic psychrotrophs capable of fermenting various carbohydrates, organic acids, and alcohols. The identified metabolic properties of the isolated Carnobacterium strains suggest possible syntrophic interactions with other microorganisms in polar habitats. Some showed antimicrobial activity against food pathogens such as Listeria monocytogenes and human pathogens like Staphylococcus spp. Several isolates exhibited unique metabolic traits with potential biotechnological applications that could be more effectively exploited under less stringent technological conditions compared to thermophilic LAB strains, such as lower temperatures and reduced nutrient concentrations. Analysis of extrachromosomal genetic elements revealed 13 plasmids ranging from 4.5 to 79.5 kb in five isolates, featuring unique genetic structures and high levels of previously uncharacterized genes. This work is the first comprehensive study of the biochemical properties of both known and new Carnobacterium species and enhances our understanding of bacterial communities in harsh and highly selective polar soil ecosystems.

Genome Mining of Pseudanabaena galeata CCNP1313 Indicates a New Scope in the Search for Antiproliferative and Antiviral Agents.

Compounds derived from natural sources pave the way for novel drug development. Cyanobacteria is an ubiquitous phylum found in various habitats. The fitness of those microorganisms, within different biotopes, is partially dependent on secondary metabolite production. Their enhanced production under biotic/abiotic stress factors accounts for better survival rates of cells, and thereby cyanobacteria are as an enticing source of bioactive compounds. Previous studies have shown the potent activity of extracts and fractions from Pseudanabaena galeata (Böcher 1949) strain CCNP1313 against cancer cells and viruses. However, active agents remain unknown, as the selected peptides had no effect on the tested cell lines. Here, we present a bottom-up approach, pinpointing key structures involved in secondary metabolite production. Consisting of six replicons, a complete genome sequence of P. galeata strain CCNP1313 was found to carry genes for non-ribosomal peptide/polyketide synthetases embedded within chromosome spans (4.9 Mbp) and for a ribosomally synthesized peptide located on one of the plasmids (0.2 Mbp). Elucidation of metabolite synthesis pathways led to prediction of their structure. While none of the synthesis-predicted products were found in mass spectrometry analysis, unexplored synthetases are characterized by structural similarities to those producing potent bioactive compounds.

Native and Alien Antarctic Grasses as a Habitat for Fungi.

Biological invasions are now seen as one of the main threats to the Antarctic ecosystem. An example of such an invasion is the recent colonization of the H. Arctowski Polish Antarctic Station area by the non-native grass Poa annua. This site was previously occupied only by native plants like the Antarctic hair grass Deschampsia antarctica. To adapt successfully to new conditions, plants interact with soil microorganisms, including fungi. The aim of this study was to determine how the newly introduced grass P. annua established an interaction with fungi compared to resident grass D. antarctica. We found that fungal diversity in D. antarctica roots was significantly higher compared with P. annua roots. D. antarctica managed a biodiverse microbiome because of its ability to recruit fungal biocontrol agents from the soil, thus maintaining a beneficial nature of the endophyte community. P. annua relied on a set of specific fungal taxa, which likely modulated its cold response, increasing its competitiveness in Antarctic conditions. Cultivated endophytic fungi displayed strong chitinolysis, pointing towards their role as phytopathogenic fungi, nematode, and insect antagonists. This is the first study to compare the root mycobiomes of both grass species by direct culture-independent techniques as well as culture-based methods.

The IncC and IncX1 resistance plasmids present in multi-drug resistant Escherichia coli strains isolated from poultry manure in Poland.

The study describes the whole-genome sequencing of two antibiotic-resistant representative Escherichia coli strains, isolated from poultry manure in 2020. The samples were obtained from a commercial chicken meat production facility in Poland. The antibiotic resistance profile was characterized by co-resistance to ß-lactam antibiotics, aminoglycosides, and fluoroquinolones. The three identified resistance plasmids (R-plasmids), pECmdr13.2, pECmdr13.3, and pECmdr14.1, harbored various genes conferring resistance to tetracyclines (tetR[A]) for, aminoglycoside (aph, aac, and aad families), ß-lactam (blaCMY-2, blaTEM-176), sulfonamide (sul1, sul2), fluoroquinolone (qnrS1), and phenicol (floR). These plasmids, which have not been previously reported in Poland, were found to carry IS26 insertion elements, the intI1-integrase gene, and conjugal transfer genes, facilitating horizontal gene transfer. Plasmids pECmdr13.2 and pECmdr14.1 also possessed a mercury resistance gene operon related to transposon Tn6196; this promotes plasmid persistence even without antibiotic selection pressure due to co-selection mechanisms such as co-resistance. The chicken manure-derived plasmids belonged to the IncX1 (narrow host range) and IncC (broad host range) incompatibility groups. Similar plasmids have been identified in various environments, clinical isolates, and farm animals, including cattle, swine, and poultry. This study holds significant importance for the One Health approach, as it highlights the potential for antibiotic-resistant bacteria from livestock and food sources, particularly E. coli, to transfer through the food chain to humans and vice versa.

A newly identified IncY plasmid from multi-drug-resistant Escherichia coli isolated from dairy cattle feces in Poland.

Comprehensive whole-genome sequencing was performed on two multi-drug-resistant Escherichia coli strains isolated from cattle manure from a typical dairy farm in Poland in 2020. The identified strains are resistant to beta-lactams, aminoglycosides, tetracyclines, trimethoprim/sulfamethoxazole, and fluoroquinolones. The complete sequences of the harbored plasmids revealed antibiotic-resistance genes located within many mobile genetic elements (e.g., insertional sequences or transposons) and genes facilitating conjugal transfer or promoting horizontal gene transfer. These plasmids are hitherto undescribed. Similar plasmids have been identified, but not in Poland. The identified plasmids carried resistance genes, including the tetracycline resistance gene tet(A), aph family aminoglycoside resistance genes aph(3″)-lb and aph (6)-ld, beta-lactam resistance genes blaTEM-1 and blaCTX-M-15, sulfonamide resistance gene sul2, fluoroquinolone resistance gene qnrS1, and the trimethoprim resistance gene dfrA14. The characterized resistance plasmids were categorized into the IncY incompatibility group, indicating a high possibility for dissemination among the Enterobacteriaceae. While similar plasmids (99% identity) have been found in environmental and clinical samples, none have been identified in farm animals. These findings are significant within the One Health framework, as they underline the potential for antimicrobial-resistant E. coli from livestock and food sources to be transmitted to humans and vice versa. It highlights the need for careful monitoring and strategies to limit the spread of antibiotic resistance in the One Health approach. IMPORTANCE: This study reveals the identification of new strains of antibiotic-resistant Escherichia coli in cattle manure from a dairy farm in Poland, offering critical insights into the spread of drug resistance. Through whole-genome sequencing, researchers discovered novel plasmids within these bacteria, which carry genes resistant to multiple antibiotics. These findings are particularly alarming, as these plasmids can transfer between different bacterial species, potentially escalating the spread of antibiotic resistance. This research underscores the vital connection between the health of humans, animals, and the environment, emphasizing the concept of One Health. It points to the critical need for global vigilance and strategies to curb the proliferation of antibiotic resistance. By showcasing the presence of these strains and their advanced resistance mechanisms, the study calls for enhanced surveillance and preventive actions in both agricultural practices and healthcare settings to address the imminent challenge of antibiotic-resistant bacteria.

Does Salmonella diarizonae 58:r:z53 Isolated from a Mallard Duck Pose a Threat to Human Health?

Salmonella diarizonae (IIIb) is frequently isolated from reptiles and less frequently from birds and mammals. However, its isolation from invasive human infections has not been widely reported. Migratory mallard ducks are excellent bioindicators of pathogen presence and pathogen antibiotic resistance (AMR). We present the first isolation from a mallard duck in central Europe of the antibiotic-resistant Salmonella enterica subsp. diarizonae with the unique antigenic pattern 58:r:z53 and report its whole-genome sequencing, serosequencing, and genotyping, which enabled the prediction of its pathogenicity and comparison with phenotypic AMR. The isolated strain was highly similar to S. diarizonae isolated from humans and food. Twenty-four AMR genes were detected, including those encoding aminoglycoside, fluoroquinolone, macrolide, carbapenem, tetracycline, cephalosporin, nitroimidazole, peptide antibiotic, and disinfecting agent/antiseptic resistance. Six Salmonella pathogenicity islands were found (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13). An iron transport system was detected in SPI-1 centisome C63PI. Plasmid profile analyses showed three to be present. Sequence mutations in the invA and invF genes were noted, which truncated and elongated the proteins, respectively. The strain also harbored genes encoding type-III secretion-system effector proteins and many virulence factors found in S. diarizonae associated with human infections. This study aims to elucidate the AMR and virulence genes in S. enterica subsp. diarizonae that may most seriously threaten human health.

Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

Comparative genomics, pangenomics, and phenomic studies of Pectobacterium betavasculorum strains isolated from sugar beet, potato, sunflower, and artichoke: insights into pathogenicity, virulence determinants, and adaptation to the host plant.

Introduction: Bacteria of genus Pectobacterium, encompassing economically significant pathogens affecting various plants, includes the species P. betavasculorum, initially associated with beetroot infection. However, its host range is much broader. It causes diseases of sunflower, potato, tomato, carrots, sweet potato, radish, squash, cucumber, and chrysanthemum. To explain this phenomenon, a comprehensive pathogenomic and phenomic characterisation of P. betavasculorum species was performed. Methods: Genomes of P. betavasculorum strains isolated from potato, sunflower, and artichoke were sequenced and compared with those from sugar beet isolates. Metabolic profiling and pathogenomic analyses were conducted to assess virulence determinants and adaptation potential. Pathogenicity assays were performed on potato tubers and chicory leaves to confirm in silico predictions of disease symptoms. Phenotypic assays were also conducted to assess the strains ability to synthesise homoserine lactones and siderophores. Results: The genome size ranged from 4.675 to 4.931 kbp, and GC % was between 51.0% and 51.2%. The pangenome of P. betavasculorum is open and comprises, on average, 4,220 gene families. Of these, 83% of genes are the core genome, and 2% of the entire pangenome are unique genes. Strains isolated from sugar beet have a smaller pangenome size and a higher number of unique genes than those from other plants. Interestingly, genomes of strains from artichoke and sunflower share 391 common CDS that are not present in the genomes of other strains from sugar beet or potato. Those strains have only one unique gene. All strains could use numerous sugars as building materials and energy sources and possessed a high repertoire of virulence determinants in the genomes. P. betavasculorum strains were able to cause disease symptoms on potato tubers and chicory leaves. They were also able to synthesise homoserine lactones and siderophores. Discussion: The findings underscore the adaptability of P. betavasculorum to diverse hosts and environments. Strains adapted to plants with high sugar content in tissues have a different composition of fatty acids in membranes and a different mechanism of replenishing nitrogen in case of deficiency of this compound than strains derived from other plant species. Extensive phenomics and genomic analyses performed in this study have shown that P. betavasculorum species is an agronomically relevant pathogen.

Interaction of bacteriophage P1 with an epiphytic Pantoea agglomerans strain-the role of the interplay between various mobilome elements.

P1 is a model, temperate bacteriophage of the 94 kb genome. It can lysogenize representatives of the Enterobacterales order. In lysogens, it is maintained as a plasmid. We tested P1 interactions with the biocontrol P. agglomerans L15 strain to explore the utility of P1 in P. agglomerans genome engineering. A P1 derivative carrying the Tn9 (cmR) transposon could transfer a plasmid from Escherichia coli to the L15 cells. The L15 cells infected with this derivative formed chloramphenicol-resistant colonies. They could grow in a liquid medium with chloramphenicol after adaptation and did not contain prophage P1 but the chromosomally inserted cmR marker of P1 Tn9 (cat). The insertions were accompanied by various rearrangements upstream of the Tn9 cat gene promoter and the loss of IS1 (IS1L) from the corresponding region. Sequence analysis of the L15 strain genome revealed a chromosome and three plasmids of 0.58, 0.18, and 0.07 Mb. The largest and the smallest plasmid appeared to encode partition and replication incompatibility determinants similar to those of prophage P1, respectively. In the L15 derivatives cured of the largest plasmid, P1 with Tn9 could not replace the smallest plasmid even if selected. However, it could replace the smallest and the largest plasmid of L15 if its Tn9 IS1L sequence driving the Tn9 mobility was inactivated or if it was enriched with an immobile kanamycin resistance marker. Moreover, it could develop lytically in the L15 derivatives cured of both these plasmids. Clearly, under conditions of selection for P1, the mobility of the P1 selective marker determines whether or not the incoming P1 can outcompete the incompatible L15 resident plasmids. Our results demonstrate that P. agglomerans can serve as a host for bacteriophage P1 and can be engineered with the help of this phage. They also provide an example of how antibiotics can modify the outcome of horizontal gene transfer in natural environments. Numerous plasmids of Pantoea strains appear to contain determinants of replication or partition incompatibility with P1. Therefore, P1 with an immobile selective marker may be a tool of choice in curing these strains from the respective plasmids to facilitate their functional analysis.

Polyhydroxyalkanoate involvement in stress-survival of two psychrophilic bacterial strains from the High Arctic.

An ever-growing body of literature evidences the protective role of polyhydroxyalkanoates (PHAs) against a plethora of mostly physical stressors in prokaryotic cells. To date, most of the research done involved bacterial strains isolated from habitats not considered to be life-challenging or extremely impacted by abiotic environmental factors. Polar region microorganisms experience a multitude of damaging factors in combinations rarely seen in other of Earth's environments. Therefore, the main objective of this investigation was to examine the role of PHAs in the adaptation of psychrophilic, Arctic-derived bacteria to stress conditions. Arctic PHA producers: Acidovorax sp. A1169 and Collimonas sp. A2191, were chosen and their genes involved in PHB metabolism were deactivated making them unable to accumulate PHAs (ΔphaC) or to utilize them (Δi-phaZ) as a carbon source. Varying stressors were applied to the wild-type and the prepared mutant strains and their survival rates were assessed based on CFU count. Wild-type strains with a functional PHA metabolism were best suited to survive the freeze-thaw cycle - a common feature of polar region habitats. However, the majority of stresses were best survived by the ΔphaC mutants, suggesting that the biochemical imbalance caused by the lack of PHAs induced a permanent cell-wide stress response thus causing them to better withstand the stressor application. Δi-phaZ mutants were superior in surviving UV irradiation, hinting that PHA granule presence in bacterial cells is beneficial despite it being biologically inaccessible. Obtained data suggests that the ability to metabolize PHA although important for survival, probably is not the most crucial mechanism in the stress-resistance strategies arsenal of cold-loving bacteria. KEY POINTS: • PHA metabolism helps psychrophiles survive freezing • PHA-lacking psychrophile mutants cope better with oxidative and heat stresses • PHA granule presence enhances the UV resistance of psychrophiles.

Comprehensive phenomic and genomic studies of the species, Pectobacterium cacticida and proposal for reclassification as Alcorniella cacticida comb. nov.

Introduction: Pectobacterium cacticida was identified as the causative agent of soft rot disease in cacti. Due to a high potential of spread in the face of global warming, the species poses a significant threat to horticultural and crop industry. The aim of this study was to revise the genomic, physiology and virulence characteristics of P. cacticida and update its phylogenetic position within the Pectobacterium genus. Methods: Whole genome sequences of five P. cacticida strains were obtained and subjected to comprehensive genomic and phylogenomic data analyses. We assessed the presence of virulence determinants and genes associated with host and environmental adaptation. Lipidomic analysis, as well as biochemical and phenotypic assays were performed to correlate genomic findings. Results: Phylogenomic analysis revealed that P. cacticida forms a distinct lineage within the Pectobacterium genus. Genomic evaluation uncovered 516 unique proteins, most of which were involved in cellular metabolism. They included genes of carbohydrate metabolism and transport and ABC transporters. The main differing characteristics from other Pectobacterium species were the lack of a myo-inositol degradation pathway and the presence of the malonate decarboxylase gene. All tested strains were pathogenic towards Opuntia spp., chicory, Chinese cabbage, and potato, but exhibited only mild pathogenicity towards carrot. Discussion: This study sheds light into the genomic characteristics of P. cacticida and highlights the pathogenic potential of the species. Unique genes found in P. cacticida genomes possibly enhance the species' survival and virulence. Based on phylogenomic analyses, we propose the reclassification of P. cacticida to a new genus, Alcorniella comb. nov.

Genomic and transcriptomic analysis of Ligilactobacillus salivarius IBB3154-in search of new promoters for vaccine construction.

IMPORTANCE: The genome of the strain Ligilactobacillus salivarius IBB3154 was sequenced, and transcriptome analysis was carried out at two different temperatures, allowing the determination of gene expression levels in response to environmental changes (temperature). Genes with higher expression at 42°C were identified. The use of a reporter gene (ß- glucuronidase) did not confirm the transcriptomic results; it was found that the promoters of the genes sasA1 and sasA2 were active in the presence of bile salts. This opens up new opportunities for the overexpression of genes of other bacterial species in Ligilactobacillus cells in the intestinal environment.

Genetic engineering of low-temperature polyhydroxyalkanoate production by Acidovorax sp. A1169, a psychrophile isolated from a subglacial outflow.

In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles.

Highly Resistant Serotype 19A Streptococcus pneumoniae of the GPSC1/CC320 Clone from Invasive Infections in Poland Prior to Antipneumococcal Vaccination of Children.

INTRODUCTION: The introduction of pneumococcal conjugate vaccines (PCV) into the national immunization programs (NIPs) has significantly reduced the number of pneumococcal infections. However, infections caused by isolates of non-vaccine serotypes (NVT) started spreading shortly thereafter and strains of NVT 19A have become the main cause of invasive pneumococcal disease burden worldwide. The aim of the study was to characterize serotype 19A invasive pneumococci of GPSC1/CC320 circulating in Poland before the introduction of PCV into the Polish NIP in 2017 and to compare them to isolates from other countries where PCVs were implemented much earlier than in Poland. METHODS: All the GPSC1/CC320 isolates were analyzed by serotyping, susceptibility testing, and whole genome sequencing followed by analyses of resistome, virulome, and core genome multilocus sequence typing (cgMLST), including comparative analysis with isolates with publicly accessible genomic sequences (PubMLST). RESULTS: During continuous surveillance the NRCBM collected 4237 invasive Streptococcus pneumoniae isolates between 1997 and 2016, including 200 isolates (4.7%) of serotype 19A. The most prevalent among 19A pneumococci were highly resistant representatives of Global Pneumococcal Sequence Cluster 1/Clonal Complex 320, GPSC1/CC320 (n = 97, 48.5%). Isolates of GPSC1/CC320 belonged to three sequence types (STs): ST320 (75.2%) ST4768 (23.7%), and ST15047 (1.0%), which all represented the 19A-III cps subtype and had complete loci for both PI-1 and PI-2 pili types. On the basis of the cgMLST analysis the majority of Polish GPSC1/CC320 isolates formed a group clearly distinct from pneumococci of this clone observed in other countries. CONCLUSION: Before introduction of PCV in the Polish NIP we noticed an unexpected increase of serotype 19A in invasive pneumococcal infections, with the most common being representatives of highly drug-resistant GPSC1/CC320 clone, rarely identified in Europe both before and even after PCV introduction.

Escherichia albertii as a Potential Enteropathogen in the Light of Epidemiological and Genomic Studies.

Escherichia albertii is a new enteropathogen of humans and animals. The aim of the study was to assess the prevalence and pathogenicity of E. albertii strains isolated in northeastern Poland using epidemiological and genomic studies. In 2015-2018, a total of 1154 fecal samples from children and adults, 497 bird droppings, 212 food samples, 92 water samples, and 500 lactose-negative E. coli strains were tested. A total of 42 E. albertii strains were isolated. The PCR method was suitable for their rapid identification. In total, 33.3% of E. albertii isolates were resistant to one antibiotic, and 16.7% to two. Isolates were sensitive to cefepime, imipenem, levofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and did not produce ESBL ß-lactamases. High genetic variability of E. albertii has been demonstrated. In the PFGE method, 90.5% of the strains had distinct pulsotypes. In MLST typing, 85.7% of strains were assigned distinct sequence types (STs), of which 64% were novel ST types. Cytolethal distending toxin (CDT) and Paa toxin genes were found in 100% of E. albertii isolates. Genes encoding toxins, IbeA, CdtB type 2, Tsh and Shiga (Stx2f), were found in 26.2%, 9.7%, 1.7%, and 0.4% of E. albertii isolates, respectively. The chromosome size of the tested strains ranged from 4,573,338 to 5,141,010 bp (average 4,784,003 bp), and at least one plasmid was present in all strains. The study contributes to a more accurate assessment of the genetic diversity of E. albertii and the potential threat it poses to public health.

Comparative genomic analysis of two ST320 Streptococcus pneumoniae isolates, representing serotypes 19A and 19F.

BACKGROUND: Streptococcus pneumoniae (pneumococcus) represents an important human pathogen, responsible for respiratory and invasive infections in the community. The efficacy of polysaccharide conjugate vaccines formulated against pneumococci is reduced by the phenomenon of serotype replacement in population of this pathogen. The aim of the current study was to obtain and compare complete genomic sequences of two pneumococcal isolates, both belonging to ST320 but differing by the serotype. RESULTS: Here, we report genomic sequences of two isolates of important human pathogen, S. pneumoniae. Genomic sequencing resulted in complete sequences of chromosomes of both isolates, 2,069,241 bp and 2,103,144 bp in size, and confirmed the presence of cps loci specific for serotypes 19A and 19F. The comparative analysis of these genomes revealed several instances of recombination, which involved not only S. pneumoniae but also presumably other streptococci as donors. CONCLUSIONS: We report the complete genomic sequences of two S. pneumoniae isolates of ST320 and serotypes 19A and 19F. The detailed comparative analysis of these genomes revealed the history of several recombination events, clustered in the region including the cps locus.

Bacterial communities of Antarctic lichens explored by gDNA and cDNA 16S rRNA gene amplicon sequencing.

Recently, lichens came once more into the scientific spotlight due to their unique relations with prokaryotes. Several temperate region lichen species have been thoroughly explored in this regard yet, the information on Antarctic lichens and their associated bacteriobiomes is somewhat lacking. In this paper, we assessed the phylogenetic structure of the whole and active fractions of bacterial communities housed by Antarctic lichens growing in different environmental conditions by targeted 16S rRNA gene amplicon sequencing. Bacterial communities associated with lichens procured from a nitrogen enriched site were very distinct from the communities isolated from lichens of a nitrogen depleted site. The former were characterized by substantial contributions of Bacteroidetes phylum members and the elusive Armatimonadetes. At the nutrient-poor site the lichen-associated bacteriobiome structure was unique for each lichen species, with chlorolichens being occupied largely by Proteobacteria. Lichen species with a pronounced discrepancy in diversity between the whole and active fractions of their bacterial communities had the widest ecological amplitude, hinting that the nonactive part of the community is a reservoir of latent stress coping mechanisms. This is the first investigation to make use of targeted metatranscriptomics to infer the bacterial biodiversity in Antarctic lichens.

Diverse Partners of the Partitioning ParB Protein in Pseudomonas aeruginosa.

In the majority of bacterial species, the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s), assists in the chromosome partitioning. ParB forms large nucleoprotein complexes at parS(s), located in the vicinity of origin of chromosomal replication (oriC), which after replication are subsequently positioned by ParA in cell poles. Remarkably, ParA and ParB participate not only in the chromosome segregation but through interactions with various cellular partners they are also involved in other cell cycle-related processes, in a species-specific manner. In this work, we characterized Pseudomonas aeruginosa ParB interactions with the cognate ParA, showing that the N-terminal motif of ParB is required for these interactions, and demonstrated that ParAB-parS-mediated rapid segregation of newly replicated ori domains prevented structural maintenance of chromosome (SMC)-mediated cohesion of sister chromosomes. Furthermore, using proteome-wide techniques, we have identified other ParB partners in P. aeruginosa, which encompass a number of proteins, including the nucleoid-associated proteins NdpA(PA3849) and NdpA2, MinE (PA3245) of Min system, and transcriptional regulators and various enzymes, e.g., CTP synthetase (PA3637). Among them are also NTPases PA4465, PA5028, PA3481, and FleN (PA1454), three of them displaying polar localization in bacterial cells. Overall, this work presents the spectrum of P. aeruginosa ParB partners and implicates the role of this protein in the cross-talk between chromosome segregation and other cellular processes. IMPORTANCE In Pseudomonas aeruginosa, a Gram-negative pathogen causing life-threatening infections in immunocompromised patients, the ParAB-parS system is involved in the precise separation of newly replicated bacterial chromosomes. In this work, we identified and characterized proteins interacting with partitioning protein ParB. We mapped the domain of interactions with its cognate ParA partner and showed that ParB-ParA interactions are crucial for the chromosome segregation and for proper SMC action on DNA. We also demonstrated ParB interactions with other DNA binding proteins, metabolic enzymes, and NTPases displaying polar localization in the cells. Overall, this study uncovers novel players cooperating with the chromosome partition system in P. aeruginosa, supporting its important regulatory role in the bacterial cell cycle.

Functional Characterization of TetR-like Transcriptional Regulator PA3973 from Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 encodes an uncharacterized TetR family transcriptional regulator. In this study, we applied a transcriptome profiling (RNA-seq), genome-wide identification of binding sites using ChIP-seq, as well as the phenotype analyses to unravel the biological role of PA3973. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome. A 13 bp sequence motif was indicated as the binding site of PA3973. The PA3973 regulon encompasses the PA3972-PA3971 genes encoding a probable acyl-CoA dehydrogenase and a thioesterase. In vitro analysis showed PA3973 binding to PA3973p. Accordingly, the lack of PA3973 triggered increased expression of PA3972 and PA3971. The ∆PA3972-71 PAO1161 strain demonstrated impaired growth in the presence of stress-inducing agents hydroxylamine or hydroxyurea, thus suggesting the role of PA3972-71 in pathogen survival upon stress. Overall our results showed that TetR-type transcriptional regulator PA3973 has multiple binding sites in the P. aeruginosa genome and influences the expression of diverse genes, including PA3972-PA3971, encoding proteins with a proposed role in stress response.