Genome-based Analysis Research Articles

Halocatena salina sp. nov., a filamentous halophilic archaeon isolated from Aiding Salt Lake.

A filamentous cell-shaped halophilic archaeon (strain AD-1T) was isolated from Aiding Salt Lake, PR China. Its colonies on HCM7 agar plates were pinkish white, 1-4 mm (diameter), elevated and round. The optimum conditions for growth were observed at 42 °C, 4.3 M NaCl, 0.01 M MgCl2 and pH 7. Strain AD-1T could hydrolyse Tween 60, Tween 80, starch and gelatin. Phylogenetic analysis based on 16S rRNA gene, rpoB' and the concatenated 484 single-copy orthologous proteins revealed that strain AD-1T formed a clade with Halocatena pleomorpha SPP-AMP-1T. The average nucleotide identity and in silico DNA-DNA hybridization values between strain AD-1T and Halocatena pleomorpha SPP-AMP-1T were both below the species delineation thresholds (95~96 and 70 %, respectively). The major phospholipids of strain AD-1T were phosphatidic acid, phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, while the major glycolipids were sulphated galactosyl mannosyl glucosyl diether, galactosyl mannosyl glucosyl diether and glucosyl mannosyl glucosyl diether. The phenotypic, phylogenetic and genome-based analyses suggested that strain AD-1T (=CGMCC 1.13724T=JCM 32960T) represents a novel species, for which the name Halocatena salina sp. nov. is proposed.

Comparative Genomics of Lentilactobacillus parabuchneri isolated from dairy, KEM complex, Makgeolli, and Saliva Microbiomes

BackgroundLentilactobacillus parabuchneri is of particular concern in fermented food bioprocessing due to causing unwanted gas formation, cracks, and off-flavor in fermented dairy foods. This species is also a known culprit of histamine poisonings because of decarboxylating histidine to histamine in ripening cheese. Twenty-eight genomes in NCBI GenBank were evaluated via comparative analysis to determine genomic diversity within this species and identify potential avenues for reducing health associated risks and economic losses in the food industry caused by these organisms.ResultCore genome-based phylogenetic analysis revealed four distinct major clades. Eight dairy isolates, two strains from an unknown source, and a saliva isolate formed the first clade. Three out of five strains clustered on clade 2 belonged to dairy, and the remaining two strains were isolated from the makgeolli and Korean effective microorganisms (KEM) complex. The third and fourth clade members were isolated from Tete de Moine and dairy-associated niches, respectively. Whole genome analysis on twenty-eight genomes showed ~ 40% of all CDS were conserved across entire strains proposing a considerable diversity among L. parabuchneri strains analyzed. After assigning CDS to their corresponding function, ~ 79% of all strains were predicted to carry putative intact prophages, and ~ 43% of the strains harbored at least one plasmid; however, all the strains were predicted to encode genomic island, insertion sequence, and CRISPR-Cas system. A type I-E CRISPR-Cas subgroup was identified in all the strains, with the exception of DSM15352, which carried a type II-A CRISPR-Cas system. Twenty strains were predicted to encode histidine decarboxylase gene cluster that belongs to not only dairy but also saliva, KEM complex, and unknown source. No bacteriocin-encoding gene(s) or antibiotic resistome was found in any of the L. parabuchneri strains screened.ConclusionThe findings of the present work provide in-depth knowledge of the genomics of L. parabuchneri by comparing twenty-eight genomes available to date. For example, the hdc gene cluster was generally reported in cheese isolates; however, our findings in the current work indicated that it could also be encoded in those strains isolated from saliva, KEM complex, and unknown source. We think prophages are critical mobile elements of L. parabuchneri genomes that could pave the way for developing novel tools to reduce the occurrence of this unwanted species in the food industry.

Detection of SARS-CoV-2 infection clusters: The useful combination of spatiotemporal clustering and genomic analyses.

The need for effective public health surveillance systems to track virus spread for targeted interventions was highlighted during the COVID-19 pandemic. It spurred an interest in the use of spatiotemporal clustering and genomic analyses to identify high-risk areas and track the spread of the SARS-CoV-2 virus. However, these two approaches are rarely combined in surveillance systems to complement each one's limitations; spatiotemporal clustering approaches usually consider only one source of virus transmission (i.e., the residential setting) to detect case clusters, while genomic studies require significant resources and processing time that can delay decision-making. Here, we clarify the differences and possible synergies of these two approaches in the context of infectious disease surveillance systems by investigating to what extent geographically-defined clusters are confirmed as transmission clusters based on genome sequences, and how genomic-based analyses can improve the epidemiological investigations associated with spatiotemporal cluster detection. For this purpose, we sequenced the SARS-CoV-2 genomes of 172 cases that were part of a collection of spatiotemporal clusters found in a Swiss state (Vaud) during the first epidemic wave. We subsequently examined intra-cluster genetic similarities and spatiotemporal distributions across virus genotypes. Our results suggest that the congruence between the two approaches might depend on geographic features of the area (rural/urban) and epidemic context (e.g., lockdown). We also identified two potential superspreading events that started from cases in the main urban area of the state, leading to smaller spreading events in neighboring regions, as well as a large spreading in a geographically-isolated area. These superspreading events were characterized by specific mutations assumed to originate from Mulhouse and Milan, respectively. Our analyses propose synergistic benefits of using two complementary approaches in public health surveillance, saving resources and improving surveillance efficiency.

Genomics assisted functional characterization of Bacillus velezensis E as a biocontrol and growth promoting bacterium for lily.

Lily (Lilium spp.) is one of the most famous ornamental flowers globally. Lily basal rot (also known as root rot or stem rot) and lily gray mold have seriously affected the yield and quality of lily, resulting in huge economic losses. In this study, bacterial strain E was isolated from a continuous lily cropping field. Strain E displayed high control efficiency against lily basal rot and gray mold, caused by Fusarium oxysporum and Botrytis cinerea respectively, and promoted the occurrence of scale bulblets. Strain E displayed strong inhibitory effects against several other plant pathogenic fungi and two pathogenic bacteria in dual culture and disc diffusion assays, respectively. Whole genome sequencing revealed that strain E contained a 3,929,247 bp circular chromosome with 4,056 protein-coding genes and an average GC content of 47.32%. Strain E was classified as Bacillus velezensis using genome-based phylogenetic analysis and average nucleotide identity and digital DNA-DNA hybridization analyses. A total of 86 genes and 13 secondary metabolite biosynthetic gene clusters involved in antifungal and antibacterial activity, plant growth promotion, colonization, nutrient uptake and availability were identified in the genome of strain E. In vitro biochemical assays showed that strain E produced siderophores, proteases, cellulases, biofilms, antifungal and antibacterial substances, and exhibited organic phosphate solubilization and swimming and swarming motility, which were consistent with the results of the genome analysis. Colonization analysis showed that strain E could colonize the root of the lily, but not the leaf. Overall, these results demonstrate that B. velezensis strain E can be used as a potential biofertilizer and biocontrol agent for lily production.

Genomic insight into Chryseobacterium turcicum sp. nov. and Chryseobacterium muglaense sp. nov. isolated from farmed rainbow trout in Turkey

Four strains, designated as C-2, C-17T, C-39T and Ch-15, were isolated from farmed rainbow trout samples showing clinical signs during an investigation for a fish-health screening study. The pairwise 16S rRNA gene sequence analysis showed that strain C-17T shared the highest identity level of 98.1 % with the type strain of Chryseobacterium piscium LMG 23089T while strains C-2, C-39T and Ch-15 were closely related to Chryseobacterium balustinum DSM 16775T with an identity level of 99.3 %. A polyphasic approach involving phenotypic, chemotaxonomic and genome-based analyses was employed to determine the taxonomic provenance of the strains. The overall genome relatedness indices including dDDH and ANI analyses confirmed that strains C-2, C-17T, C-39T and Ch-15 formed two novel species within the genus Chryseobacterium. Chemotaxonomic analyses showed that strains C-17T and C-39T have typical characteristics of the genus Chryseobacterium by having phosphatidylethanolamine in their polar lipid profile, MK-6 as only isoprenoid quinone and the presence of iso-C15:0 as major fatty acid. The genome size and G + C content of the strains ranged between 4.4 and 5.0 Mb and 33.5 – 33.6 %, respectively. Comprehensive genome analyses revealed that the strains have antimicrobial resistance genes, prophages and horizontally acquired genes in addition to secondary metabolite-coding gene clusters. In conclusion, based on the polyphasic analyses conducted on the present study, strains C-17T and C-39T are representatives of two novel species within the genus Chryseobacterium, for which the names Chryseobacterium turcicum sp. nov. and Chryseobacterium muglaense sp. nov. with the type strains C-17T (=JCM 34190T = KCTC 82250T) and C-39T (=JCM 34191T = KCTC 822251T), respectively, are proposed.

Functional genomics of the lactic acid bacterium Limosilactobacillus fermentum LAB-1: metabolic, probiotic and biotechnological perspectives

A genome-based systematic analysis was conducted to characterize the metabolic, probiotic, fitness, and safety properties of Limosilactobacillus fermentum LAB-1, a lactic acid bacterium demonstrating strong antimicrobial effects against clinical pathogens. Gene functional characterization revealed a large number of genes for carbohydrate metabolism and a heterofermentative system for carbon dissimilation. Genes for intact pyruvate oxidation, pentose phosphate, and PRPP biosynthetic pathways were identified. Substantial carbohydrate-active enzymes and transporters were also predicted. Metabolic reconstruction revealed complete sets of enzymes for arginine, lysine, methionine, threonine, proline, and ornithine biosynthesis. The bacterium harbors a diverse range of peptidases, and a large variety of peptide and amino acid uptake systems. It encodes restriction-modification and CRISPR-Cas systems for protection against phage infections and carries a wide spectrum of stress proteins for adaptation in the gut and industrial conditions. Genes related to the biosynthesis of B-group and K vitamins were identified allowing its application for novel bio-enriched food production. Other beneficial traits of probiotic and industrial importance such as production of flavor compounds, exopolysaccharide, acetoin, and butanediol were identified. Three antimicrobial peptides were predicted which showed >98% sequence-identity to experimentally validated bacteriocins. Negative traits such as transmissible antibiotic resistance, pathogenicity or virulence appeared to be absent suggesting the strain to be considered safe. The genome analysis will allow precisely targeted laboratory research and full exploitation of the probiotic potentials towards functional-food, biotechnology and health-related applications.

Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China.

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes bla NDM-5 (1.10%, 2/181), mcr-1 (1.10%, 2/181), tet(X4) (1.10%, 2/181), and cfr (6.08%, 2/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified astA (37.02%, 67/181), hlyA (1.66%, 3/181), hlyB (1.66%, 3/181) and hlyD (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.

Genomic and Phylogenetic Characterization of Rhodopseudomonas infernalis sp. nov., Isolated from the Hell Creek Watershed (Nebraska).

The genus Rhodopseudomonas is known for its versatile metabolic capabilities and has been proposed to be used in a wide variety of innovative applications, ranging from biohydrogen and electricity production, bioremediation and as biostimulant in agriculture. Here, we report the isolation, characterization and genome sequence analysis of a novel Rhodopseudomonas species, strain HC1, isolated from the Hell Creek urban native restoration area. Whole genome-based analysis, average nucleotide identity (ANI) comparison, and growth characteristics identified this isolate as a new species of the Rhodopseudomonas genus, for which we propose the name Rhodopseudomonas infernalis sp. nov. Besides containing several nitrogenases for nitrogen fixation and hydrogen production, the HC1 genome encodes a unique gene cluster, not found in any other Rhodopseudomonas species, which encodes genes for the degradation of yet-unidentified aromatic PCB-type chemicals with potentially interesting biotechnological applications. The genomic features of Rps. infernalis HC1 indicate that it plays a positive role in the degradation of anthropogenic substances and aids the restoration of the Hell Creek watershed by contributing to N2 and carbon fixation and plant growth; however, the genome also contains several antibiotic resistance genes, indicating a broad range of antibiotic resistance in this environmental isolate.

P024 Genetic determinants of antifungal drug resistance in Fusarium solani species complex

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMDue to its challenging diagnosis and treatment, fungal keratitis is one of the most serious kinds of corneal infection. The Fusarium solani species complex is responsible for nearly half of all fungal keratitis cases. Fusarium infection is difficult to treat because of the increased antifungal resistance of Fusarium species.ObjectiveTo check the antifungal susceptibility in keratitis-causing isolates of F. solani species complex.To find the genetic determinants of resistance using whole genome sequencing.MethodologyPrior to AFST and according to CLSI, clinical isolates of Fusarium species (n = 5) were cultured on potato dextrose agar for 7 days at room temperature. Fungal spores were harvested using 0.8% NaCl and antifungal drug susceptibility testing of 6 different antifungal drugs were tested using CLSI standards broth microdilution method and minimum inhibitory concentrations were obtained. After 24 and 48 h, broth microdilution plates were manually examined. The wells for growth control were also examined. The minimum inhibitory concentration of anti-fungal drugs was the lowest dose that inhibits growth completely (compared to the growth control well). Minimal effective concentration (MEC) was also determined against antifungal drugs. Whole genome sequencing was done using Illumina Hiseqx10. Bioinformatics analysis was done using different bioinformatics tools and software (FASTQC, SPAdes, OmicsBox, AUGUSTUS, etc.).ResultsIn this study the antifungal drug susceptibility from average MIC value were found as fluconazole (512 μg/ml) > itraconazole (32 μg/ml) > amphotericin B (8 μg/ml) > natamycin (8 μg/ml) > posaconazole (2 μg/ml) > voriconazole (1 μg/ml). Fluconazole had a higher MIC value (512 g/ml) in all isolates, however, voriconazole was shown to be more sensitive, with a lower MIC range (0.25-4 g/ml). Glyoxalase/Bleomycin resistance protein, mfs—multidrug resistance transporter, fusaric acid resistance protein, efflux pump antibiotic resistance protein, and copper resistance protein were found by genome-based analysis.ConclusionAs a conclusion of this study, we observed antifungal drug resistance in Fusarium spp., which is often used to treat keratitis in patients, employing fluconazole, itraconazole, and amphotericin B. Resistance to first-line azoles was as a gene variant which contributed to antifungal drug resistance. This study using the phenotypic and genotypic characterization of drug resistance patterns will help to combat antifungal drug resistance.

Streptococcus canis sequence type 43 may be associated with treatment failure in dogs with corneal ulceration.

To profile Streptococcus canis isolates obtained from corneal ulcers in dogs. 10 dogs. Medical records were searched to identify dogs diagnosed with ulcerative keratitis by a veterinary ophthalmologist and having a positive corneal culture for S canis during the year 2020. For each case, clinical findings and outcome were determined, antimicrobial resistance and sensitivity panels were summarized, whole genome sequencing was performed, and isolates were typed using multi-locus sequence typing and genome-based proteome phylogenetic analysis. 10 S canis isolates were included from dogs diagnosed with ulcerative keratitis. Dogs were either treated surgically via keratectomy and conjunctival grafting (n = 6) or treated medically (4). Three of 10 corneas failed to heal and required enucleation (2/6 conjunctival grafts and 1/4 medically managed corneal ulcers). All three corneal ulcers that failed to heal were associated with S canis sequence type (ST) 43. Sequence types identified from successfully treated cases included ST8 (n = 1), ST50 (1), ST2 (2), ST27 (1), and ST15 (1). One ST43 isolate was obtained from a dog that healed following a conjunctival graft, however this was the only dog that received an oral antibiotic in addition to topical antibiotics. Based on this small dataset, S canis ST43 may be associated with increased virulence and contribute to conjunctival graft failure and progressive corneal collagenolysis. The postoperative administration of an oral antimicrobial may protect against conjunctival graft rejection in dogs specifically due to S canis ST43.

Genome-based analyses reveal heterotypic synonyms of Streptomyces species and associated subspecies.

In the genus Streptomyces, several validly described species have been reduced to synonyms of earlier described species though additional synonyms remain to be detected given the previous dependence on traditional phenotypic methods. In this study, genome-based procedures, including DNA-DNA hybridization analyses, overall genome-related indices, such as ANI, dDDH and AAI, revealed that certain strains recorded genomic indices above the threshold values used to define species boundaries. The results of phylogenetic and phylogenomic trees based on concatenated and phylogenomic analyses showed that 33 out of 364 tested species could be assigned to 15 species groups and that 18 Streptomyces species names be reclassified as later heterotypic synonyms of earlier validly published species. Consequently, it is proposed that S. albaduncus is a later heterotypic synonym of S. griseoloalbus; S. bellus is a synonym of S. coeruleorubidus; S. gancidicus and S. rubiginosus are synonyms of S. pseudogriseolus; S. niveoruber is a synonym of S. griseoviridis; S. griseomycini is a synonym of S. griseostramineus; S. jietaisiensis is a synonym of S. griseoaurantiacus; S. pluricolorescens is a synonym of S. rubiginosohelvolus; S. nashvillensis is a synonym of S. tanashiensis; S. yerevanensis is a synonym of S. flaveus; S. durhamensis is a synonym of S. filipinensis; S. recifensis is a synonym of S. griseoluteus; S. canaries and S. olivaceoviridis are synonyms of S. corchorusii; S. melanosporofaciens is a synonym of S. antimycoticus; S. albulus is a synonym of S. noursei; and S. janthinus and S. violarus are synonyms of S. violaceus. Additionally, seven of these 18 Streptomyces species have been designated subspecies.

Genome-based taxonomic classification of the closest-to-Comamonadaceae group supports a new family Sphaerotilaceae fam. nov. and taxonomic revisions

Many genera closest to the family Comamonadaceae have not been classified into any family; moreover, some of them are not monophyletic groups beyond the genus level. To resolve the taxonomic uncertainty of the closest-to-Comamonadaceae (CTC) group, we performed 16S rRNA gene- and genome-based phylogenetic analyses combined with genome relatedness indices and phenotypic traits comparison. Phylogenies based on the 16S rRNA gene and genome sequences demonstrated that the CTC group formed a coherent and robust monophyletic lineage and was sister to the family Comamonadaceae, thereby proposing the CTC group as a novel family, Sphaerotilaceae fam. nov. The resolved genus- and species-level taxonomic relationships of this new family were then validated by the phylogenomic reconstruction and comparisons of genome relatedness indices including digital DNA-DNA hybridization and average nucleotide identity (ANI) as well as comprehensive phenotypic analysis for type strains. Finally, we reclassified all misidentified genera and species, resulting in 19 new combinations, and proposed Sphaerotilaceae-specific thresholds of ANI and average amino acid identity for genus delineation. Collectively, this study has established a sound taxonomic framework of the novel family Sphaerotilaceae and will help guide future taxonomic efforts and prevent the propagation of taxonomic errors.

Genome-based analysis of infrequent Salmonella serotypes through the Thai pork production chain.

Salmonella is a prevalent zoonotic foodborne pathogen. Swine and pork are implicated as important sources of salmonellosis in humans. In Chiang Mai and Lamphun Provinces in northern Thailand, there has been a high prevalence of Salmonella persistence for over a decade. Infection is usually with dominant S. enterica serotypes, including serotypes Rissen and 1,4,[5],12:i:-. However, other serotypes also contribute to disease but are less well characterized. The whole genome sequencing data of 43 S. enterica serotypes isolated from pork production chain through 2011–2014, were used to evaluate genetic diversity and ascertain the possible source of Salmonella contamination based on Core Genome Multilocus Sequence Typing (cgMLST) approach. The Salmonella serotypes recovered from farms and slaughterhouses were re-circulating by swine environmental contamination. Conversely, the Salmonella contamination in the retail market represents cross-contamination from multiple sources, including contaminated foodstuffs. Salmonella contamination in the pork production chain has the competency for host cell adhesion, host cell invasion, and intracellular survival, which is enough for the pathogenicity of salmonellosis. In addition, all of these isolates were multi-drug resistant Salmonella, which contained at least 10 antimicrobial resistance genes. This result indicated that these S. enterica serotypes also pose a significant public health risk. Our findings support the need for appropriate surveillance of food-animal products going to market to reduce public exposure to highly pathogenic, multi-drug resistant Salmonella. Acquiring information would motivate all stakeholders to reinforce sanitation standards throughout the pork production chain in order to eradicate Salmonella contamination and reduce the risk of salmonellosis in humans.

Open pangenome of Lactococcus lactis generated by a combination of metagenome-assembled genomes and isolate genomes.

Lactococcus lactis (L. lactis) is a well isolated and cultured lactic acid bacterium, but if utilizing the isolate genomes alone, the genome-based analysis of this taxon would be incomplete, because there are still uncultured strains in some ecological niches. In this study, we recovered 93 high-quality metagenome-assembled genomes (MAGs) of L. lactis from food and human gut metagenomes with a culture-independent method. We then constructed a unified genome catalog of L. lactis by integrating these MAGs with 70 publicly available isolated genomes. Having this comprehensive resource, we assessed the genomic diversity and phylogenetic relationships to further explore the genetic and functional properties of L. lactis. An open pangenome of L. lactis was generated using our genome catalog, consisting of 13,066 genes in total, from which 5,448 genes were not identified in the isolate genomes. The core genome-based phylogenetic analysis showed that L. lactis strains we collected were separated into two main subclades corresponding to two subspecies, with some uncultured phylogenetic lineages discovered. The species disparity was also indicated in PCA analysis based on accessory genes of our pangenome. These various analyzes shed further light on unexpectedly high diversity within the taxon at both genome and gene levels and gave clues about its population structure and evolution. Lactococcus lactis has a long history of safe use in food fermentations and is considered as one of the important probiotic microorganisms. Obtaining the complete genetic information of L. lactis is important to the food and health industry. However, it can naturally inhabit many environments other than dairy products, including drain water and human gut samples. Here we presented an open pan-genome of L. lactis constructed from 163 high-quality genomes obtained from various environments, including MAGs recovered from environmental metagenomes and isolate genomes. This study expanded the genetic information of L. lactis about one third, including more than 5,000 novel genes found in uncultured strains. This more complete gene repertoire of L. lactis is crucial to further understanding the genetic and functional properties. These properties may be harnessed to impart additional value to dairy fermentation or other industries.

Parvicella tangerina gen. nov., sp. nov. (Parvicellaceae fam. nov., Flavobacteriales), first cultured representative of the marine clade UBA10066, and Lysobacter luteus sp. nov., from activated sludge of a seawater-processing wastewater treatment plant.

Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88-89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T).

Genome analysis of the candidate phylum MBNT15 bacterium from a boreal peatland predicted its respiratory versatility and dissimilatory iron metabolism.

Uncultured bacteria of the candidate phylum MBNT15, distantly related to Desulfobacterota, have been identified in a broad range of mostly organic-rich aquatic environments. We assembled a near-complete genome of a member of MBNT15 from a boreal peatland metagenome and used genomic data to analyze the metabolic pathways of this bacterium and its ecological role. This bacterium, designated SHF-111, was predicted to be rod shaped, it lacks flagellar machinery but twitching motility is encoded. Genome-based phylogenetic analysis supported the phylum-level classification of the MBNT15 lineage. Genome annotation and metabolic reconstruction revealed the presence of the Embden–Meyerhof, Entner–Doudoroff and pentose phosphate pathways, as well as the complete tricarboxylic acid (TCA) cycle, and suggested a facultatively anaerobic chemoheterotrophic lifestyle with the ability to ferment peptides, amino acids, fatty acids and simple sugars, and completely oxidize these substrates through aerobic and anaerobic respiration. The SHF-111 genome encodes multiple multiheme c-type cytochromes that probably enable dissimilatory iron reduction. Consistently, the relative abundance of MBNT15 in peatlands positively correlated with iron concentration. Apparently, in the wetland ecosystem, MBNT15 representatives play the role of scavengers, carrying out the complete mineralization of low molecular weight organic substances formed as a result of microbial degradation of complex polymeric substrates. Comparative genome analysis of the MBNT15 phylum revealed that vast majority of its members are capable of aerobic respiration and dissimilatory iron reduction and some species also can reduce sulfur and nitrogen compounds, but not sulfate. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Deferrimicrobium borealis, within a candidate phylum Deferrimicrobiota.

Abundance of Poleroviruses within Tasmanian Pea Crops and Surrounding Weeds, and the Genetic Diversity of TuYV Isolates Found.

The genus Polerovirus contains positive-sense, single-stranded RNA plant viruses that cause significant disease in many agricultural crops, including vegetable legumes. This study aimed to identify and determine the abundance of Polerovirus species present within Tasmanian pea crops and surrounding weeds that may act as virus reservoirs. We further sought to examine the genetic diversity of TuYV, the most commonly occurring polerovirus identified. Pea and weed samples were collected during 2019–2020 between October and January from thirty-four sites across three different regions (far northwest, north, and midlands) of Tasmania and tested by RT-PCR assay, with selected samples subject to next-generation sequencing. Results revealed that the presence of polerovirus infection and the prevalence of TuYV in both weeds and pea crops varied across the three Tasmanian cropping regions, with TuYV infection levels in pea crops ranging between 0 and 27.5% of tested plants. Overall, two species members from each genus, Polerovirus and Potyvirus, one member from each of Luteovirus, Potexvirus, and Carlavirus, and an unclassified virus from the family Partitiviridae were also found as a result of NGS data analysis. Analysis of gene sequences of the P0 and P3 genes of Tasmanian TuYV isolates revealed substantial genetic diversity within the collection, with a few isolates appearing more closely aligned with BrYV isolates. Questions remain around the differentiation of TuYV and BrYV species. Phylogenetic inconsistency in the P0 and P3 ORFs supports the concept that recombination may have played a role in TuYV evolution in Tasmania. Results of the evolutionary analysis showed that the selection pressure was higher in the P0 gene than in the P3 gene, and the majority of the codons for each gene are evolving under purifying selection. Future full genome-based analyses of the genetic variations will expand our understanding of the evolutionary patterns existing among TuYV populations in Tasmania.

Microaerobic enrichment of benzene-degrading bacteria and description of Ideonella benzenivorans sp. nov., capable of degrading benzene, toluene and ethylbenzene under microaerobic conditions

In the present study, the bacterial community structure of enrichment cultures degrading benzene under microaerobic conditions was investigated through culturing and 16S rRNA gene Illumina amplicon sequencing. Enrichments were dominated by members of the genus Rhodoferax followed by Pseudomonas and Acidovorax. Additionally, a pale amber-coloured, motile, Gram-stain-negative bacterium, designated B7T was isolated from the microaerobic benzene-degrading enrichment cultures and characterized using a polyphasic approach to determine its taxonomic position. The 16S rRNA gene and whole genome-based phylogenetic analyses revealed that strain B7T formed a lineage within the family Comamonadaceae, clustered as a member of the genus Ideonella and most closely related to Ideonella dechloratans CCUG 30977T. The sole respiratory quinone is ubiquinone-8. The major fatty acids are C16:0 and summed feature 3 (C16:1ω7c/iso-C15:0 2-OH). The DNA G + C content of the type strain is 68.8 mol%. The orthologous average nucleotide identity (OrthoANI) and in silico DNA–DNA hybridization (dDDH) relatedness values between strain B7T and closest relatives were below the threshold values for species demarcation. The genome of strain B7T, which is approximately 4.5 Mb, contains a phenol degradation gene cluster, encoding a multicomponent phenol hydroxylase (mPH) together with a complete meta-cleavage pathway including a I.2.C-type catechol 2,3-dioxygenase (C23O) gene. As predicted by the genome, the type strain is involved in aromatic hydrocarbon-degradation: benzene, toluene and ethylbenzene are degraded aerobically and also microaerobically as sole source of carbon and energy. Based on phenotypic characteristics and phylogenetic analysis, strain B7T is a member of the genus Ideonella and represents a novel species for which the name Ideonella benzenivorans sp. nov. is proposed. The type strain of the species is strain B7T (= LMG 32,345T = NCAIM B.02664T).

Whole Genome Multi-Locus Sequence Typing and Genomic Single Nucleotide Polymorphism Analysis for Epidemiological Typing of Pseudomonas aeruginosa From Indonesian Intensive Care Units

We have previously studied carbapenem non-susceptible Pseudomonas aeruginosa (CNPA) strains from intensive care units (ICUs) in a referral hospital in Jakarta, Indonesia (Pelegrin et al., 2019). We documented that CNPA transmissions and acquisitions among patients were variable over time and that these were not significantly reduced by a set of infection control measures. Three high risk international CNPA clones (sequence type (ST)235, ST823, ST357) dominated, and carbapenem resistance was due to carbapenemase-encoding genes and mutations in the porin OprD. Pelegrin et al. (2019) reported core genome analysis of these strains. We present a more refined and detailed whole genome-based analysis of major clones represented in the same dataset. As per our knowledge, this is the first study reporting Single Nucleotide Polymorphisms (wgSNP) analysis of Pseudomonas strains. With whole genome-based Multi Locus Sequence Typing (wgMLST) of the 3 CNPA clones (ST235, ST357 and ST823), three to eleven subgroups with up to 200 allelic variants were observed for each of the CNPA clones. Furthermore, we analyzed these CNPA clone clusters for the presence of wgSNP to redefine CNPA transmission events during hospitalization. A maximum number 35350 SNPs (including non-informative wgSNPs) and 398 SNPs (ST-specific_informative-wgSNPs) were found in ST235, 34,570 SNPs (including non-informative wgSNPs) and 111 SNPs (ST-specific_informative-wgSNPs) in ST357 and 26,443 SNPs (including non-informative SNPs) and 61 SNPs (ST-specific_informative-wgSNPs) in ST823. ST-specific_Informative-wgSNPs were commonly noticed in sensor-response regulator genes. However, the majority of non-informative wgSNPs was found in conserved hypothetical proteins or in uncharacterized proteins. Of note, antibiotic resistance and virulence genes segregated according to the wgSNP analyses. A total of 8 transmission chains for ST235 strains followed by 9 and 4 possible transmission chains for ST357 and ST823 were traceable on the basis of pairwise distances of informative-wgSNPs (0 to 4 SNPs) among the strains. The present study demonstrates the value of detailed whole genome sequence analysis for highly refined epidemiological analysis of P. aeruginosa.

Genetic diversity and spatial distribution of Burkholderia mallei by core genome-based multilocus sequence typing analysis.

Burkholderia mallei is the etiological agent of glanders, a highly contagious and often fatal disease in equids. Due to the high genetic clonality of B. mallei, high-resolution typing assays are necessary to differentiate between individual strains. Here we report on the development and validation of a robust and reproducible core genome-based Multi Locus Sequence Typing Assay (cgMLST) for B. mallei, which is based on 3328 gene targets and enables high-resolution typing at the strain level. The assay was validated using a set of 120 B. mallei genomes from public databases and 23 newly sequenced outbreak strains from in-house strain collections. In this cgMLST analysis, strains from different geographic regions were clearly distinguished by at least 70 allele differences, allowing spatial clustering while closely related and epidemiologically related strains were separated by only zero to three alleles. Neither the different sequencing technologies nor the assembly strategies had an influence on the cgMLST results. The developed cgMLST is highly robust, reproducible and can be used for outbreak investigations, source tracking and molecular characterization of new B. mallei isolates.