Comparative Whole-genome Sequencing Research Articles

Genomic and biogeographic characterisation of the novel prasinovirus Mantoniella tinhauana virus 1.

Mamiellophyceae are a ubiquitous class of unicellular green algae in the global ocean. Their ecological importance is highlighted in studies focused on the prominent genera Micromonas, Ostreococcus, and Bathycoccus. Mamiellophyceae are susceptible to prasinoviruses, double-stranded DNA viruses belonging to the nucleocytoplasmic large DNA virus group. Our study represents the first isolation of a prasinovirus in the South China Sea and the only one to infect the globally distributed genus Mantoniella. We conducted a comparative analysis with previously identified viral relatives, encompassing morphological characteristics, host specificity, marker-based phylogenetic placement, and whole-genome sequence comparisons. Although it shares morphological and genetic similarities with established prasinoviruses, this novel virus showed distinct genetic traits, confining its infection to the species Mantoniella tinhauana. We also explored the global biogeography of this prasinovirus and its host by mapping metagenomic data and analysing their relationship with various environmental parameters. Our results demonstrate a pronounced link between the virus and its host, both found predominantly in higher latitudes in the surface ocean. By gaining an increased understanding of the relationships between viruses, hosts, and environments, researchers can better make predictions and potentially implement measures to mitigate the consequences of climate change on oceanic processes.

Comparative Whole-Genome Sequencing Analysis of In-situ and Invasive Acral Lentiginous Melanoma: Markedly Increased Copy Number Gains of GAB2 , PAK1 , UCP2 , and CCND1 are Associated with Melanoma Invasion.

Acral lentiginous melanoma (ALM) is the most common subtype of acral melanoma. Even though recent genetic studies are reported in acral melanomas, the genetic differences between in-situ and invasive ALM remain unclear. We aimed to analyze specific genetic changes in ALM and compare genetic differences between in-situ and invasive lesions to identify genetic changes associated with the pathogenesis and progression of ALM. We performed whole genome sequencing of 71 tissue samples from 29 patients with ALM. Comparative analyses were performed, pairing in-situ ALMs with normal tissues and, furthermore, invasive ALMs with normal and in-situ tissues. Among 21 patients with in-situ ALMs, 3 patients (14.3%) had SMIM14 , SLC9B1 , FRG1 , FAM205A , ESRRA , and ESPN mutations, and copy number (CN) gains were identified in only 2 patients (9.5%). Comparing 13 invasive ALMs with in-situ tissues, CN gains were identified in GAB2 in 8 patients (61.5%), PAK1 in 6 patients (46.2%), and UCP2 and CCND1 in 5 patients (38.5%). Structural variants were frequent in in-situ and invasive ALM lesions. Both in-situ and invasive ALMs had very low frequencies of common driver mutations. Structural variants were common in both in-situ and invasive ALMs. Invasive ALMs had markedly increased CN gains, such as GAB2 , PAK1 , UCP2 , and CCND1 , compared with in-situ lesions. These results suggest that they are associated with melanoma invasion.

Psychrosphaera algicola sp. nov. and Paraglaciecola algarum sp. nov., and reclassification of Pseudoalteromonas elyakovii, Pseudoalteromonas flavipulchra, and Pseudoalteromonas profundi as later heterotypic synonyms of P. distincta, P. maricaloris, and P. gelatinilytica.

Two Gram-negative, obligately aerobic, rod-shaped bacteria, strains G1-22T and G1-23T, were isolated from the phycosphere of a marine brown alga. Both strains exhibited catalase- and oxidase-positive activities. Strain G1-22T displayed optimal growth at 25 °C, pH 8.0, and 2.0-3.0% (w/v) NaCl, while strain G1-23T exhibited optimal growth at 25 °C, pH 8.0, and 4.0% NaCl. Ubiquinone-8 was identified as the sole isoprenoid quinone in both strains. As major fatty acids (> 5%), strain G1-22T contained C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C12 : 1 3-OH, and C10 : 0 3-OH, while strain G1-23T contained C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), and C14 : 0. Phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were major polar lipids in both strains. Strains G1-22T and G1-23T had DNA G+C contents of 40.2 and 38.9 mol%, respectively. Phylogenetic analyses based on 16S rRNA and genome sequences revealed that strains G1-22T and G1-23T formed distinct phylogenetic lineages within the genera Psychrosphaera and Paraglaciecola, respectively. Strain G1-22T showed closest relatedness to Psychrosphaera ytuae MTZ26T with 97.8% 16S rRNA gene sequence similarity, 70.2% average nucleotide identity (ANI), and a 21.5% digital DNA-DNA hybridization (dDDH) value, while strain G1-23T was most closely related to Paraglaciecola aquimarina KCTC 32108T with 95.6% 16S rRNA gene sequence similarity, 74.6% ANI, and a 20.1% dDDH value. Based on phenotypic and molecular characteristics, strains G1-22T and G1-23T are proposed to represent two novel species, namely Psychrosphaera algicola sp. nov. (type strain G1-22T=KACC 22486T=JCM 34971T) and Paraglaciecola algarum sp. nov. (type strain G1-23T=KACC 22490T=JCM 34972T), respectively. Additionally, based on the comparison of whole genome sequences, it is proposed that Pseudoalteromonas elyakovii, Pseudoalteromonas flavipulchra, and Pseudoalteromonas profundi are reclassified as later heterotypic synonyms of Pseudoalteromonas distincta, Pseudoalteromonas maricaloris, and Pseudoalteromonas gelatinilytica, respectively.

Physiological and genomic insights into a psychrotrophic drought-tolerant bacterial consortium for crop improvement in cold, semiarid regions

The agricultural land in the Indian Himalayan region (IHR) is susceptible to various spells of snowfall, which can cause nutrient leaching, low temperatures, and drought conditions. The current study, therefore, sought an indigenous psychrotrophic plant growth-promoting (PGP) bacterial inoculant with the potential to alleviate crop productivity under cold and drought stress. Psychrotrophic bacteria preisolated from the night-soil compost of the Lahaul Valley of northwestern Himalaya were screened for phosphate (P) and potash (K) solubilization, nitrogen fixation, indole acetic acid (IAA) production, siderophore and HCN production) in addition to their tolerance to drought conditions for consortia development. Furthermore, the effects of the selected consortium on the growth and development of wheat (Triticum aestivum L.) and maize (Zea mays L.) were assessed in pot experiments under cold semiarid conditions (50 % field capacity). Among 57 bacteria with P and K solubilization, nitrogen fixation, IAA production, siderophore and HCN production, Pseudomonas protegens LPH60, Pseudomonas atacamensis LSH24, Psychrobacter faecalis LUR13, Serratia proteamaculans LUR44, Pseudomonas mucidolens LUR70, and Glutamicibacter bergerei LUR77 exhibited tolerance to drought stress (-0.73 MPa). The colonization of wheat and maize seeds with these drought-tolerant PGP strains resulted in a germination index >150, indicating no phytotoxicity under drought stress. Remarkably, a particular strain, Pseudomonas sp. LPH60 demonstrated antagonistic activity against three phytopathogens Ustilago maydis, Fusarium oxysporum, and Fusarium graminearum. Treatment with the consortium significantly increased the foliage (100 % and 160 %) and root (200 % and 133 %) biomasses of the wheat and maize plants, respectively. Furthermore, whole-genome sequence comparisons of LPH60 and LUR13 with closely related strains revealed genes associated with plant nutrient uptake, phytohormone synthesis, siderophore production, hydrogen cyanide (HCN) synthesis, volatile organic compound production, trehalose and glycine betaine transport, cold shock response, superoxide dismutase activity, and gene clusters for nonribosomal peptide synthases and polyketide synthetases. With their PGP qualities, biocontrol activity, and ability to withstand environmental challenges, the developed consortium represents a promising cold- and drought-active PGP bioinoculant for cereal crops grown in cold semiarid regions.

Characterization of Blood Group Variants in an Omani Population by Comparison of Whole Genome Sequencing and Serology.

Although blood group variation was first described over a century ago, our understanding of the genetic variation affecting antigenic expression on the red blood cell surface in many populations is lacking. This deficit limits the ability to accurately type patients, especially as serological testing is not available for all described blood groups, and targeted genotyping panels may lack rare or population-specific variants. Here, we perform serological assays across 24 antigens and whole genome sequencing on 100 Omanis, a population underrepresented in genomic databases. We inferred blood group phenotypes using the most commonly typed genetic variants. The comparison of serological to inferred phenotypes resulted in an average concordance of 96.9%. Among the 22 discordances, we identify seven known variants in four blood groups that, to our knowledge, have not been previously reported in Omanis. Incorporating these variants for phenotype inference, concordance increases to 98.8%. Additionally, we describe five candidate variants in the Lewis, Lutheran, MNS, and P1 blood groups that may affect antigenic expression, although further functional confirmation is required. Notably, we identify several blood group alleles most common in African populations, likely introduced to Oman by gene flow over the last thousand years. These findings highlight the need to evaluate individual populations and their population history when considering variants to include in genotype panels for blood group typing. This research will inform future work in blood banks and transfusion services.

Development of Attenuated Viruses for Effective Protection against Pepper Veinal Mottle Virus in Tomato Crops.

Tomato (Solanum lycopersicum) is the most important vegetable and fruit crop in the family Solanaceae worldwide. Numerous pests and pathogens, especially viruses, severely affect tomato production, causing immeasurable market losses. In Taiwan, the cultivation of tomato crops is mainly threatened by insect-borne viruses, among which pepper veinal mottle virus (PVMV) is one of the most prevalent. PVMV is a member of the genus Potyvirus of the family Potyviridae and is non-persistently transmitted by aphids. Its infection significantly reduces tomato fruit yield and quality. So far, no PVMV-resistant tomato lines are available. In this study, we performed nitrite-induced mutagenesis of the PVMV tomato isolate Tn to generate attenuated PVMV mutants. PVMV Tn causes necrotic lesions in Chenopodium quinoa leaves and severe mosaic and wilting in Nicotiana benthamiana plants. After nitrite treatment, three attenuated PVMV mutants, m4-8, m10-1, and m10-11, were selected while inducing milder responses to C. quinoa and N. benthamiana with lower accumulation in tomato plants. In greenhouse tests, the three mutants showed different degrees of cross-protection against wild-type PVMV Tn. m4-8 showed the highest protective efficacy against PVMV Tn in N. benthamiana and tomato plants, 100% and 97.9%, respectively. A whole-genome sequence comparison of PVMV Tn and m4-8 revealed that 20 nucleotide substitutions occurred in the m4-8 genome, resulting in 18 amino acid changes. Our results suggest that m4-8 has excellent potential to protect tomato crops from PVMV. The application of m4-8 in protecting other Solanaceae crops, such as peppers, will be studied in the future.

Genomic and phenotypic comparison of two variants of multidrug-resistant Salmonella enterica serovar Heidelberg isolated during the 2015-2017 multi-state outbreak in cattle.

Salmonella enterica subspecies enterica serovar Heidelberg (Salmonella Heidelberg) has caused several multistate foodborne outbreaks in the United States, largely associated with the consumption of poultry. However, a 2015-2017 multidrug-resistant (MDR) Salmonella Heidelberg outbreak was linked to contact with dairy beef calves. Traceback investigations revealed calves infected with outbreak strains of Salmonella Heidelberg exhibited symptoms of disease frequently followed by death from septicemia. To investigate virulence characteristics of Salmonella Heidelberg as a pathogen in bovine, two variants with distinct pulse-field gel electrophoresis (PFGE) patterns that differed in morbidity and mortality during the multistate outbreak were genotypically and phenotypically characterized and compared. Strain SX 245 with PFGE pattern JF6X01.0523 was identified as a dominant and highly pathogenic variant causing high morbidity and mortality in affected calves, whereas strain SX 244 with PFGE pattern JF6X01.0590 was classified as a low pathogenic variant causing less morbidity and mortality. Comparison of whole-genome sequences determined that SX 245 lacked ~200 genes present in SX 244, including genes associated with the IncI1 plasmid and phages; SX 244 lacked eight genes present in SX 245 including a second YdiV Anti-FlhC(2)FlhD(4) factor, a lysin motif domain containing protein, and a pentapeptide repeat protein. RNA-sequencing revealed fimbriae-related, flagella-related, and chemotaxis genes had increased expression in SX 245 compared to SX 244. Furthermore, SX 245 displayed higher invasion of human and bovine epithelial cells than SX 244. These data suggest that the presence and up-regulation of genes involved in type 1 fimbriae production, flagellar regulation and biogenesis, and chemotaxis may play a role in the increased pathogenicity and host range expansion of the Salmonella Heidelberg isolates involved in the bovine-related outbreak.

Highly pathogenic avian influenza A(H5N1) virus infection in foxes with PB2-M535I identified as a novel mammalian adaptation, Northern Ireland, July 2023.

We report cases of mammalian infection with highly pathogenic avian influenza (HPAI) virus A(H5N1) clade 2.3.4.4b in Northern Ireland. Two common gulls (Larus canus) and two red fox kits (Vulpes vulpes), were found dead in close vicinity. Comparison of viral whole genome sequences obtained from the animals identified a novel mammalian adaptation, PB2-M535I. Analysis of genetic sequences from other recent mammalian infections shows that this mutation has arisen on at least five occasions in three European countries since April 2023.

Genomics Reveals Complex Population History and Unexpected Diversity of Eurasian Otters (Lutra lutra) in Britain Relative to Genetic Methods.

Conservation genetic analyses of many endangered species have been based on genotyping of microsatellite loci and sequencing of short fragments of mtDNA. The increase in power and resolution afforded by whole genome approaches may challenge conclusions made on limited numbers of loci and maternally inherited haploid markers. Here, we provide a matched comparison of whole genome sequencing versus microsatellite and control region (CR) genotyping for Eurasian otters (Lutra lutra). Previous work identified four genetically differentiated "stronghold" populations of otter in Britain, derived from regional populations that survived the population crash of the 1950s-1980s. Using whole genome resequencing data from 45 samples from across the British stronghold populations, we confirmed some aspects of population structure derived from previous marker-driven studies. Importantly, we showed that genomic signals of the population crash bottlenecks matched evidence from otter population surveys. Unexpectedly, two strongly divergent mitochondrial lineages were identified that were undetectable using CR fragments, and otters in the east of England were genetically distinct and surprisingly variable. We hypothesize that this previously unsuspected variability may derive from past releases of Eurasian otters from other, non-British source populations in England around the time of the population bottleneck. Our work highlights that even reasonably well-studied species may harbor genetic surprises, if studied using modern high-throughput sequencing methods.

High-Frequency Changes in Pilin Glycosylation Patterns during Neisseria meningitidis Serogroup a Meningitis Outbreaks in the African Meningitis Belt.

In the meningitis belt of sub-Saharan Africa, there are cyclic meningococcal epidemics that coincide with clonal waves of Neisseria meningitidis carriage and invasive disease. In the framework of longitudinal colonization and disease studies in Ghana and Burkina Faso, meningococcal isolates belonging to the closely related hypervirulent A:ST-5, A:ST-7, and A:ST-2859 clones have been collected from 1998 to 2011 during meningococcal outbreaks. A comparative whole-genome sequencing study with 100 of these isolates identified the pilin glycosylation (pgl) locus as one hot spot of recombination. Frequent exchange of pgl genes in N. meningitidis by lateral gene transfer results in differences in the glycosylation patterns of pilin and other cell surface glycoproteins. In this study, we looked at both recombination and phase variation of the pgl genes of these clinical isolates and analyzed the glycan structures resulting from different pgl alleles and their variable expression. Our results indicate that the basal O-linked sugar of the glycans expressed by these isolates is masked by various additional mono- or disaccharide structures whose expression is highly variable due to the phase-variable expression of pgl genes. We also observed a distinct glycoform in two isolates with pgl loci that were modified by recombination. These data suggest that variation in N. meningitidis protein glycosylation could be crucial for bacterial adaptation to evade herd immunity in semi-immune populations. Investigating pilin glycosylation in N. meningitidis can shed light on the mechanisms by which this pathogen evades the host immune response, and may help identify potential targets for novel therapies and vaccines.

Comparative whole-genome sequence analysis of Mycobacterium tuberculosis isolated from pulmonary tuberculosis and tuberculous lymphadenitis patients in Northwest Ethiopia.

Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTBC), is a chronic infectious disease with both pulmonary and extrapulmonary forms. This study set out to investigate and compare the genomic diversity and transmission dynamics of Mycobacterium tuberculosis (Mtb) isolates obtained from tuberculous lymphadenitis (TBLN) and pulmonary TB (PTB) cases in Northwest Ethiopia. A facility-based cross-sectional study was conducted using two groups of samples collected between February 2021 and June 2022 (Group 1) and between June 2020 and June 2022 (Group 2) in Northwest Ethiopia. Deoxyribonucleic acid (DNA) was extracted from 200 heat-inactivated Mtb isolates. Whole-genome sequencing (WGS) was performed from 161 isolates having ≥1 ng DNA/μl using Illumina NovaSeq 6000 technology. From the total 161 isolates sequenced, 146 Mtb isolates were successfully genotyped into three lineages (L) and 18 sub-lineages. The Euro-American (EA, L4) lineage was the prevailing (n = 100; 68.5%) followed by Central Asian (CAS, L3, n = 43; 25.3%) and then L7 (n = 3; 2.05%). The L4.2.2.ETH sub-lineage accounted for 19.9%, while Haarlem estimated at 13.7%. The phylogenetic tree revealed distinct Mtb clusters between PTB and TBLN isolates even though there was no difference at lineages and sub-lineages levels. The clustering rate (CR) and recent transmission index (RTI) for PTB were 30 and 15%, respectively. Similarly, the CR and RTI for TBLN were 31.1 and 18 %, respectively. PTB and TBLN isolates showed no Mtb lineages and sub-lineages difference. However, at the threshold of five allelic distances, Mtb isolates obtained from PTB and TBLN form distinct complexes in the phylogenetic tree, which indicates the presence of Mtb genomic variation among the two clinical forms. The high rate of clustering and RTI among TBLN implied that TBLN was likely the result of recent transmission and/or reactivation from short latency. Hence, the high incidence rate of TBLN in the Amhara region could be the result of Mtb genomic diversity and rapid clinical progression from primary infection and/or short latency. To validate this conclusion, a similar community-based study with a large sample size and better sampling technique is highly desirable. Additionally, analysis of genomic variants other than phylogenetic informative regions could give insightful information. Combined analysis of the host and the pathogen genome (GXG) together with environmental (GxGxE) factors could give comprehensive co-evolutionary information.

Vibrio intestinalis sp. nov., isolated from intestine of seahorse.

A novel Gram-stain-negative, catalase- and oxidase-positive, facultatively anaerobic, and rod-shaped motile bacterial strain, designated as YLB-11T, was isolated from seahorse intestine. The 16S rRNA gene sequencing analysis showed that YLB-11T was most closely related Vibrio mytili LMG 19157T (98.9 % nucleotide sequence identity). Phylogenetic analysis placed strain YLB-11T within the genus Vibrio. The major cellular fatty acids were summed feature 3 (C16: 1 ω6c/C16 : 1 ω7c, 36.4 %), C16 : 0 (19.1 %) and summed feature 8 (C18:1 ω6c/C18:1 ω7c, 12.3 %). The DNA G+C content of YLB-11T was 44.7 mol %. The in silico DNA-DNA hybridization and average nucleotide identity values for whole-genome sequence comparisons between YLB-11T and related species were clearly below the thresholds used for the delineation of a novel species. Therefore, YLB-11T is considered to represent novel species of the genus Vibrio, for which the name Vibrio intestinalis sp. nov. is proposed. The type strain is YLB-11T (=MCCC 1A17441T=KCTC 72604T).

A systematic comparison of whole genome sequencing and targeted panel sequencing for precision oncology.

8546 Background: The analysis of the cancer genome provides clinically meaningful information such as actionable cancer driver mutations. Whole genome sequencing (WGS) provides a complete mutational landscape at an affordable cost thereby having increase clinical utility compared to the usual approach of targeted panel sequencing (TPS). To understand the capabilities of WGS in oncology, we conducted a study in lung adenocarcinomas. Methods: We produced WGS (surgically resected tumor tissues and matched blood; mean coverage 42x) and TPS (tumor only; SNUH panel encompassing 75 cancer genes; mean coverage 904x) through the Illumina sequencing platform and conducted comprehensive bioinformatic analyses. Oncogenic driver events, including single base substitution (SBS), insertion or deletion (INDEL), complex deletion-insertion, and fusion oncogene, were investigated in both datasets. Lastly, we compared the pattern-based mutational features, such as tumor mutational burden (TMB), mutational signatures, and copy number changes which is associated with genomic instability. Results: In 60 lung adenocarcinomas, the TPS method identified 120 driver mutations from the 75 targeted genes. Of these, 114 mutations were also detected by the WGS method, showing 95.0% sensitivity of WGS in detection of core driver mutations. In uncovered genomic regions by TPS, WGS captured 9 additional driver mutations (such as loss-of-function mutations in PIK3R2, ARID2, and APC) and 37 copy number variations (such as amplification of LMO and SMO, deletion of PIK3R1 and APC) in the cancer genes. TMB calculation is straightforward in WGS however, sophisticated considerations were necessary in TPS for TMB, to remove false positives and germline mutations. TPS showed a good capability for identifying hypermutator tumor samples, but a quantitative estimation was not always feasible due to the substantial background fluctuation, particularly for INDELs. Mutational signatures are unique combinations of mutation types in a given sample, which can give important clues to the mechanism underlying mutations. TPS showed adequate mutational signatures (cosine similarity > 0.7), but was not constantly satisfactory in tumors with high TMB ( > 20,000 genome wide SBS and INDELs). In TPS, approximately 30% of high TMB tumors represented marginal cosine similarity (0.5-0.7), and tobacco smoking related signature (SBS 4) found in WGS was captured only in 50% of the associated cases (9 out of 18). For DNA copy number changes and loss-of-heterozygosity analyses, WGS showed a much higher resolution. Conclusions: Our study demonstrates that WGS has comparable capabilities for the detection of driver mutations in core oncogenes and tumor suppressor genes. WGS was superior for pattern-based features, such as TMB, mutational signatures, and copy number changes. WGS is a medically necessary more comprehensive approach to precision oncology.

Seedless fruit in Annona squamosa L. is monogenic and conferred by INO locus deletion in multiple accessions

Key messageInheritance of the presence/absence of seeds in Annona squamosa is mediated by a single fully recessive gene and is caused by a deletion of the INNER NO OUTER (INO) locus.For some fruits, seedless varieties are desirable for consumption and processing. In the sugar apple tree (Annona squamosa L.), the seedless trait in the Thai seedless (Ts) and Brazilian seedless (Bs) accessions was associated with defective ovules and an apparent deletion of the INNER NO OUTER (INO) ovule development gene locus. Segregation analysis of F2 and backcross descendants of crosses of Bs to fertile wild-type varieties in this species with a multi-year generation time showed that seedlessness was recessive and controlled by a single locus. Comparison of whole genome sequence of a wild-type plant and a third accession, Hawaiian seedless (Hs), identified a 16 kilobase deletion including INO in this line. Ts and Bs lines were shown to have an identical deletion, indicating a common origin from a single deletion event. Analysis of microsatellite markers could not preclude the possibility that all three seedless accessions are vegetatively propagated clones. The sequence of the deletion site enabled a codominant assay for the wild-type and mutant genes allowing observation of complete cosegregation of the seedless/defective ovule phenotype with the INO deletion, showing maximal separation of less than 3.5 cM. The observed deletion is the only significant difference between the wild-type and Hs line over 587 kilobases, likely encompassing much more than 3.5 cM, showing that the deletion is the cause of seedless trait. The codominant markers and obtained progenies will be useful for introgression of the seedless trait into elite sugar apple lines and into other Annonas through interspecific crossings.

Salmonella Enteritidis clades in South Africa: why we should be paying more attention

Salmonella Enteritidis is the most reported non-typhoidal Salmonella serovar and is implicated in both gastroenteritis and invasive non-typhoidal disease. Whole-genome sequence comparison of Salmonella Enteritidis isolates from across the world identified four definitive clades: Outlier, Global Epidemic, East African and West African. Three of these clades were implicated in epidemics: the Global Epidemic clade was linked to poultry-associated gastroenteritis, while the two African clades were related to invasive disease. Despite the recognition of different Salmonella Enteritidis clades, the distribution and epidemiology of these clades across Africa is poorly understood. In our proof-of-concept study, we classified 618 Salmonella Enteritidis isolates originating from four South African provinces over a period of two years (2012 – 2013) into clades using a clade-typing real-time PCR assay. Associations between clades and province of collection, antimicrobial resistance and HIV status were explored using statistical analysis. Majority of the South African isolates were classified within the Outlier clade (61.00%), with fewer classified within the Global Epidemic clade (38.83%) and only one isolate classified within the West African Clade (0.16%). Of note, were the isolates within the Global clade, which were strongly associated with ciprofloxacin resistance (15.42%, OR: 7.45, CI: 3.526 – 15.751) and invasive disease (58.33%, OR: 1.57, CI: 1.13 – 2.17) in humans. The increase in poultry consumption and importation in South Africa has placed the country at risk of a Salmonella Enteritidis epidemic. Thus, there is a necessity for routine monitoring of S. Enteritidis along the farm-to-fork continuum to implement preventative measures.

High Concordance between the Whole Genome Sequencing data Analysis Methods for Vibrio Cholerae Surveillance in Finland and Norway

Vibrio cholerae infections, both vibriosis and cholera, are rare in Northern Europe. However, the coastal areas suitable for transmission have increased during recent years. Accessible and validated molecular diagnostic methods are needed to monitor such infections. Here, we describe the comparison and validation of Whole Genome Sequencing (WGS) data analysis methods for V. cholerae characterization in the public health institutes of Finland and Norway. The results showed a concordance of 96.7% between the methods in the two countries.

Sulfurospirillum diekertiae sp. nov., a tetrachloroethene-respiring bacterium isolated from contaminated soil.

Two anaerobic, tetrachloroethene- (PCE-) respiring bacterial isolates, designated strain ACSDCE T and strain ACSTCE, were characterized using a polyphasic approach. Cells were Gram-stain-negative, motile, non-spore-forming and shared a vibrioid- to spirillum-shaped morphology. Optimum growth occurred at 30 °C and 0.1–0.4 % salinity. The pH range for growth was pH 5.5–7.5, with an optimum at pH 7.2. Hydrogen, formate, pyruvate and lactate as electron donors supported respiratory reductive dechlorination of PCE to cis-1,2-dichloroethene (cDCE) in strain ACSDCE T and of PCE to trichloroethene (TCE) in strain ACSTCE. Both strains were able to grow with pyruvate under microaerobic conditions. Nitrate, elemental sulphur, and thiosulphate were alternative electron acceptors. Autotrophic growth was not observed and acetate served as carbon source for both strains. The major cellular fatty acids were C16 : 1 ω7c, C16 : 0, C14 : 0 and C18 : 1 ω7c. Both genomes feature a circular plasmid. Strains ACSDCE T and ACSTCE were previously assigned to the candidate species 'Sulfurospirillum acididehalogenans'. Here, based on key genomic features and pairwise comparisons of whole-genome sequences, including average nucleotide identity, digital DNA–DNA hybridization and average amino acid identity, strains ACSDCE T and ACSTCE, 'Ca. Sulfurospirillum diekertiae' strains SL2-1 and SL2-2, and the unclassified Sulfurospirillum sp. strain SPD-1 are grouped into one distinct species separate from previously described Sulfurospirillum species. Compared to Sulfurospirillum multivorans and Sulfurospirillum halorespirans , which dechlorinate PCE to cDCE without substantial TCE accumulation, these five strains produce TCE or cDCE as the end product. In addition, some cellular fatty acids (e.g., C16 : 0 3OH, C17 : 0 iso 3OH, C17 : 0 2OH) were detected in strains ACSDCE T and ACSTCE but not in other Sulfurospirillum species. On the basis of phylogenetic, physiological and phenotypic characteristics, 'Ca. Sulfurospirillum acididehalogenans' and 'Ca. Sulfurospirillum diekertiae' are proposed to be merged into one novel species within the genus Sulfurospirillum , for which the name Sulfurospirillum diekertiae sp. nov. is proposed. The type strain is ACSDCE T (=JCM 33349T= KCTC 15819T=CGMCC 1.5292T).

Identification of staphylococcal cassette chromosome mec in Staphylococcus aureus and non-aureus staphylococci from dairy cattle in Belgium: Comparison of multiplex PCR and whole genome sequencing

The present study compared multiplex PCR (mPCR) and Whole Genome Sequencing (WGS) using the SCCmecFinder database to identify the Staphylococcal Cassette Chromosome (SCC) mec in five Staphylococcus aureus (SA) and nine non-aureus staphylococci (NAS) isolated from dairy cattle. mPCR identified an SCCmecIV in four SA and one NAS, but could not differentiate between SCCmecII and IV in the fifth SA, that all harbored the mecA gene and were phenotypically resistant to cefoxitin. SCCmecFinder confirmed the presence of an SCCmecIVc(2B) in four SA and of the SCCmecIVa(2B) in the fifth SA and the one NAS. Both methods also detected one untypeable SCCmec in another cefoxitin-resistant NAS harboring the mecA gene and a pseudo SCCmec in one cefoxitin-sensitive NAS harboring one mecC-related gene. No SCCmec elements were identified either in one cefoxitin-sensitive NAS harboring the mecA2 gene, or in five NAS (one resistant and four sensitive to cefoxitin) harboring the mecA1 gene. SCCmecFinder could even not identify the presence of any mecA1 gene in these five NAS, whose presence was nevertheless confirmed by ResFinder. The conclusions of this study are: (i) mPCR and WGS sequencing using SCCmecFinder are complementary methodologies to identify SCCmec; (ii) SCCmecFinder and ResFinder to a lesser extent cannot identify all mec gene allotypes; (iii) a specific classification of the SCCmec in NAS would be epidemiologically helpful; (iv) presence of a mecA gene and a complete SCCmec is linked to cefoxitin resistance, whereas presence of other mec genes and of pseudo or no SCCmec is not.

Pol32, an accessory subunit of DNA polymerase delta, plays an essential role in genome stability and pathogenesis of Candida albicans

ABSTRACT Candida albicans is a pathobiont that inflicts serious bloodstream fungal infections in individuals with compromised immunity and gut dysbiosis. Genomic diversity in the form of copy number alteration, ploidy variation, and loss of heterozygosity as an adaptive mechanism to adverse environments is frequently observed in C. albicans. Such genomic variations also confer a varied degree of fungal virulence and drug resistance, yet the factors propelling these are not completely understood. DNA polymerase delta (Polδ) is an essential replicative DNA polymerase in the eukaryotic cell and is yet to be characterized in C. albicans. Therefore, this study was designed to gain insights into the role of Polδ, especially its non-essential subunit Pol32, in the genome plasticity and life cycle of C. albicans. PCNA, the DNA clamp, recruits Polδ to the replication fork for processive DNA replication. Unlike in Saccharomyces cerevisiae, the PCNA interaction protein (PIP) motif of CaPol32 is critical for Polδ’s activity during DNA replication. Our comparative genetic analyses and whole-genome sequencing of POL32 proficient and deficient C. albicans cells revealed a critical role of Pol32 in DNA replication, cell cycle progression, and genome stability as SNPs, indels, and repeat variations were largely accumulated in pol32 null strain. The loss of pol32 in C. albicans conferred cell wall deformity; Hsp90 mediated azoles resistance, biofilm development, and a complete attenuation of virulence in an animal model of systemic candidiasis. Thus, although Pol32 is dispensable for cell survival, its function is essential for C. albicans pathogenesis; and we discuss its translational implications in antifungal drugs and whole-cell vaccine development.

CRISPR-Based Detection, Identification and Typing of Mycobacterium tuberculosis Complex Lineages.

The polymerase chain reaction (PCR)-based detection of Mycobacterium tuberculosis (M. tuberculosis) complex (MTC) in clinical samples is a first-line approach by which to diagnose tuberculosis in clinical microbiology laboratories. In this study, the genome-wide profiling of 3,156 mycobacterial genomes using Roary determined the CRISPR-csm4 gene as specific for MTB. Real time (RT)-PCR and the PCR-sequencing of CRISPR-csm4, tested on a collection of 20 MTC and 5 nontuberculous mycobacteria, confirmed the 20 MTC isolates, whereas the 5 nontuberculous isolates were not detected. Further, 65 of the leftover clinical samples, including 25 GeneXpert-positive and 40 GeneXpert-negative samples, that were used to evaluate the CRISPR-csm4-MTB assay in the clinical microbiology laboratory setting yielded expected results in every case, further allowing for the identification of the M. tuberculosis Beijing lineage. RT-PCR and the PCR-sequencing of CRISPR-csm4 could be implanted in the clinical microbiology laboratory to complement the currently used assays, with the potential of increasing the specification of the MTC pathogens responsible for tuberculosis. IMPORTANCE The whole-genome sequence comparison of the Mycobacterium tuberculosis complex (MTC) genomic sequences that are available in the NCBI database identified a unique, specific gene to be used directly on clinical diagnostic samples to detect MTC against all species of mycobacteria and to differentiate between MTC species, lineages, and sublineages.