Core Single Nucleotide Polymorphisms Research Articles

Comprehensive whole genome analysis of Staphylococcus aureus isolates from dairy cows with subclinical mastitis.

Staphylococcus species are the primary cause of mastitis in dairy cows across the world. Staphylococcus aureus has recently become a pathogen that is zoonotic and multidrug resistant. This study aimed to sequence whole genomes of 38 S. aureus isolates from 55 subclinical mastitis dairy cows of 7 small-scale farmers in the Free State Province, South Africa and document and their antimicrobial and virulence genes. The 38 isolates were grouped by the in silico multi-locus sequencing types (MLST) into seven sequence types (STs), that is (ST 97, 352, 152, 243) and three new STs (ST8495, ST8500, and ST8501). Thirty-three S. aureus isolates were divided into 7 core single-nucleotide polymorphism (SNP) clusters. Among the 9 distinct spa-types that were detected, Spa-types t2883 accounted for the majority of isolates at 12 (31.57%), followed by t416 with 11 (28.94%) and t2844 with 5 (13.15%). The data also revealed the identification of four (4) plasmids, with Rep_N (rep20) accounting for the majority of isolates with 17 (44.73%), followed by Inc18 (repUS5) with 2 (5.26%). These isolates included 11 distinct antimicrobial resistance genes and 23 genes linked to bacterial virulence. Surprisingly, no methicillin resistance associated genes were detected in these isolates. Genome data of the current study will contribute to understanding epidemiology S. aureus genotypes and ultimately aid in developing treatment and control plans to stop the spread of mastitis in the Free State province and South Africa as a whole.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Evaluation of Genetic Diversity and Agronomic Traits of Germplasm Resources of Stropharia rugosoannulata

China possesses abundant wild germplasm resources and a wide range of cultivated strains of Stropharia rugosoannulata. However, issues such as genetic diversity and unclear genetic relationships have had an impact on the classification and preservation of germplasm resources, the breeding of new varieties, and the promotion of superior strains. There is an urgent need for genetic diversity analysis and assessment of germplasm resources. In this study, we conducted whole-genome resequencing of 50 cultivated and wild strains collected from various regions across the country. After applying a series of filtering parameters, we obtained 888,536 high-quality Single Nucleotide Polymorphism (SNP) markers. Using these SNP markers, we performed principal component analysis, population structure analysis, and phylogenetic clusters analysis on the 50 strains. Most cultivated strains exhibited high genetic similarity, while significant genetic diversity was observed among wild strains. Based on factors such as marker distribution throughout the entire genome and marker quality, we selected 358 core SNP markers to construct SNP fingerprints. Two-dimensional barcodes were generated for each strain to enable specific identification. Additionally, the mycelial growth rate of strains was evaluated on Potato Dextrose Agar (PDA) and substrate culture media. We also assessed their lignin degradation capability using guaiacol agar plates assay. It was observed that the mycelial growth rate on PDA and substrate culture medium exhibited a significant correlation with the diameter of the mycelial colony on guaiacol agar medium. Additionally, the correlation between the mycelial growth rate on the substrate medium and that on the guaiacol agar plate was stronger than that on PDA medium. This study provided molecular-level identification and assessment of germplasm resources. It clarified the genetic relationships among strains and the characteristics of mycelium growth-related agronomic traits of each strain. This research contributed to the enrichment and development of utilizable germplasm resources and breeding materials for S. rugosoannulata, offering a scientific basis for further research.

Optimized Pepper Target SNP-Seq Applied in Population Structure and Genetic Diversity Analysis of 496 Pepper (Capsicum spp.) Lines.

Peppers are a major vegetable crop worldwide. With the completion of additional genome assemblies, a multitude of single-nucleotide polymorphisms (SNPs) can be utilized for population structure and genetic diversity analysis. In this study, we used target SNP-sequencing as a new high-throughput sequencing technology, screening out 425 perfect SNPs for analyzing the genetic diversity and population structure among 496 pepper lines from five pepper species in China and abroad. The perfect SNP panel exhibited commendable discriminative ability, as indicated by the average values of polymorphism information content, observed heterozygosity, minor allele frequency, and genetic diversity, which were 0.346, 0.011, 0.371, and 0.449, respectively. Based on phylogenetic, population structure, and principal component analyses, 484 C. annuum lines were divided into four subpopulations according to the shape of fruit: blocky fruit, wide-horn fruit, narrow-horn fruit, and linear fruit. These subpopulations displayed clear clustering with minimal or no overlap. Moreover, F statistic (Fst) analysis revealed considerable distinctions among these subpopulations. Additionally, we established a set of 47 core SNPs that could effectively differentiate among all pepper lines. This core SNP set could precisely classify the C. annuum lines into four distinct fruit-shape groups. The blocky and narrow-horn fruit subpopulations displayed the lowest and highest genetic diversity, respectively. This study highlights the importance of fruit shape as a crucial trait in pepper breeding. Moreover, this work indicates the immense potential of optimized target SNP technology in the addition of foreground markers of important traits to improve molecular breeding efficiency, and demonstrates its broad application prospects in the genetic analysis and variety identification of peppers.

Construction of SNP fingerprints and genetic diversity analysis of radish (Raphanus sativus L.).

Radish (Raphanus sativus L.) is a vegetable crop with economic value and ecological significance in the genus Radish, family Brassicaceae. In recent years, developed countries have attached great importance to the collection and conservation of radish germplasm resources and their research and utilization, but the lack of population genetic information and molecular markers has hindered the development of the genetic breeding of radish. In this study, we integrated the radish genomic data published in databases for the development of single-nucleotide polymorphism (SNP) markers, and obtained a dataset of 308 high-quality SNPs under strict selection criteria. With the support of Kompetitive Allele-Specific PCR (KASP) technology, we screened a set of 32 candidate core SNP marker sets to analyse the genetic diversity of the collected 356 radish varieties. The results showed that the mean values of polymorphism information content (PIC), minor allele frequency (MAF), gene diversity and heterozygosity of the 32 candidate core SNP markers were 0.32, 0.30, 0.40 and 0.25, respectively. Population structural analysis, principal component analysis and genetic evolutionary tree analysis indicated that the 356 radish materials were best classified into two taxa, and that the two taxa of the material were closely genetically exchanged. Finally, on the basis of 32 candidate core SNP markers we calculated 15 core markers using a computer algorithm to construct a fingerprint map of 356 radish varieties. Furthermore, we constructed a core germplasm population consisting of 71 radish materials using 32 candidate core markers. In this study, we developed SNP markers for radish cultivar identification and genetic diversity analysis, and constructed DNA fingerprints, providing a basis for the identification of radish germplasm resources and molecular marker-assisted breeding as well as genetic research.

Whole Genome-Sequencing and Phylogenetic Analysis of Bacillus anthracis from 2019-2023 in Ningxia Hui Autonomous Region, China.

Since 2019, the incidence of anthrax in the Ningxia Hui Autonomous Region has increased significantly compared with previous years, so in this situation the anthrax in the Ningxia region not only had a detrimental impact on public health, but also inflicted significant economic repercussions. Therefore, we conducted a molecular epidemiological study of 20 strains from 2019-2023 isolates. This study investigated the origin of Bacillus anthracis and its genetic diversity. We conducted canonical single-nucleotide polymorphisms (CanSNPs) typing and whole genome sequencing based on the extracted nucleic acid of Bacillus anthracis. Based on the whole genome drafts, we studied the genomic characteristics of 20 isolates. Meanwhile, we performed phylogenetic studies based on genome-wide core single-nucleotide polymorphisms (SNPs) using MEGA's Maximum Likelihood (ML) method and core-genome-based multilocus sequence typing (cgMLST) of the core genomes of these strains using BioNumerics' minimum spanning tree (MST) model. The 20 isolates were categorized into sub-lineages A.Br.001/002, and comparative genomic analyses of these strains with other isolates from other parts of the world showed that the strains from Ningxia were correlated with isolates from Europe, Indonesia, Georgia (USA), and Beijing (China). For the 20 isolates in Ningxia, the genetic relationship of the isolates isolated from the same year or region was relatively close. The A.Br.001/002 subgroup was the dominant endemic strain in Ningxia. The genetic relationship and phylogenesis between isolates from Ningxia and strains from Europe and Indonesia suggest that anthrax spread around the globe through ancient trade routes.

Adaptive cell wall thickening in Enterococcus faecalis is associated with decreased vancomycin susceptibility

ObjectivesEnterococcus faecalis can adopt both a commensal and a nosocomial lifestyle, resisting numerous antibiotics. In this study, we aim to investigate the relationship between the cell wall (CW) thickness and decreased susceptibility to vancomycin (VD) in van-gene negative clinical isolates of E. faecalis (nMIC 8 = 2, nMIC 4 = 3, ST30, ST40, and ST59). MethodsThe CW thickness was assessed in VD strains and compared with vancomycin susceptible isolates of the same sequence type (ST) (Vancomycin susceptible [VS]; nMIC 2 = 5). The VD and VS strains were subjected to serial passage (evolved [ev]) with and without vancomycin selection. Subsequent measurements of CW thickness and vancomycin MICs were performed. ResultsThe VD strains exhibited increased CW thickness when compared with ST-related VS strains (ΔCW thickness VD vs. VS ST30 25 nm, ST59 15 nm, and ST40 1 nm). Serial passages without vancomycin selection led to a decrease in CW thickness and vancomycin MIC in VD strains (ΔCW thickness VD vs. evVD ST30 22 nm, ST59 3 nm, and ST40 2 nm). Serial passages with vancomycin selection caused an increase in CW thickness and vancomycin MIC in ST-related VS strains (ΔCW thickness VS vs. evVS ST30 22 nm, ST59 16 nm, and ST40 1 nm). DiscussionAdaptive changes in CW thickness were observed in response to vancomycin exposure. Increased CW thickness correlated with decreased vancomycin susceptibility, whereas decreased CW thickness correlated with increased vancomycin susceptibility. Core single nucleotide polymorphisms in the evolved mutants were mostly found in genes encoding proteins associated with the cytoplasm or the cytoplasmic membrane. The potential relevance of these adaptive changes is underlined by the observed phenotypes in clinical isolates. Our findings emphasize the importance of monitoring adaptive changes, as vancomycin-resistant enterococci infections are a growing concern.

Core-genome multilocus sequence typing and core-SNP analysis of Clostridium neonatale strains isolated in different spatio-temporal settings.

Clostridium neonatale was described as a new species within the Clostridium genus cluster I sensu stricto. It was recovered from the gut microbiota of infants and has been associated with necrotizing enterocolitis (NEC), a severe gastrointestinal disease affecting preterm neonates. In the absence of molecular typing methods, we developed a core (cg)-genome multilocus sequence typing (MLST) scheme based on 48 newly sequenced and 12 publicly available genomes of C. neonatale. Using the ChewBBACA algorithm, a stable MLST scheme consisting of 2,350 target genes (with genomes having ≥95% cgMLST targets) is proposed. The strains are distributed among five clades, and while some are clonality related within clades, the C. neonatale strain distribution is independent of geographic or temporal clustering. When considering strains isolated from patients with and without NEC, there were no observable differences in clustering. When compared to a core-single nucleotide polymorphism (SNP) analysis approach (31,248 positions), we showed that both cgMLST and cgSNP have comparable genetic discrimination of strains. In this study, we validated the use of cgMLST and cgSNP-based typing methods for the genetic comparison of clinical isolates of C. neonatale, which will allow for future surveillance and epidemiological clinical investigations of this potential opportunistic pathogen.IMPORTANCEClostridium neonatale has been isolated from the fecal samples of asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Taking advantage of a large collection of independent strains isolated from different spatio-temporal settings, we developed and established a cgMLST scheme for the molecular typing of C. neonatale. Both the cgMLST and cgSNP methods demonstrate comparable discrimination power. Results indicate geographic- and temporal- independent clustering of C. neonatale NEC-associated strains. No specific cgMLST clade of C. neonatale was genetically associated with NEC.

Pathogenic characterization of Klebsiella pneumoniae resistant to carbapenems and polymyxin

Objective: Analysis and investigation of pathogenic characteristics of polymyxin-and carbapenem-resistant Klebsiella pneumoniae (PR-CRKP). Methods: A total of 23 PR-CRKP strains isolated from clinical specimens from the General Hospital of Southern Theater Command from March 2019 to July 2021 were retrospectively collected, Whole-genome sequencing was performed on 23 PR-CRKP strains, resistance genes were identified by comparison of the CARD and the ResFinder database, high-resolution typing of PR-CRKP strains was analyzed by core genomic multilocus sequencing (cgMLST) and single nucleotide polymorphism (SNP); polymyxin resistance genes were determined by PCR and sequencing. Results: All PR-CRKP strains were KPC-2 producing ST11 types. cgMLST results showed that the evolutionary distance between the PR-CRKP strains and Klebsiella pneumoniae in mainland China was 66.44 on average, which is more closely related than foreign strains; the 23 PR-CRKP strains were divided into 3 main subclusters based on SNP phylogenetic trees, with some aggregation among Clade 2-1 in the isolation department and date. The two-component negative regulatory gene mgrB has seven mutation types including point mutations, different insertion fragments and different insertion positions. Conclusion: The close affinity of PR-CRKP strains indicate the possibility of nosocomial clonal transmission and the need to strengthen surveillance of PR-CRKP strains to prevent epidemic transmission of PR-CRKP.

Genetic Diversity and Population Structure Analysis of Hollyhock (Alcea rosea Cavan) Using High-Throughput Sequencing

Hollyhock (Alcea rosea (Linn). Cavan) is an herbaceous flowering plant with significant applications in urban greening, soil remediation, and traditional medicine. However, its genetic diversity and molecular characteristics at the population level have not been explored yet. Here, the phenotypic and genetic diversity of 162 hollyhock accessions from China revealed extensive variation among 11 traits and strong correlations between several quantitative traits. Whole-genome re-sequencing of 32 randomly chosen accessions identified 10,468,760 core single-nucleotide polymorphisms (SNPs) distributed evenly across the genome, except for on chromosome 21, and the average nucleotide diversity (π) was calculated to be 0.00397. Principal component analysis showed that these 32 accessions could be divided into four subpopulations, which was in agreement with the population structure analysis, and the subpopulations were strongly correlated with geographic location. A neighbor-joining dendrogram displayed similar clusters, except for accessions HuB25 and HLJ28, which formed two separate clusters. Our findings illuminate the genetic diversity in hollyhock and provide valuable information for hollyhock breeding.

Whole-genome sequencing of carbapenem-resistant Enterobacterales isolates and evaluation of hospital-acquired infections

Background: Multidrug-resistant organisms (MDROs) are increasingly implicated in nosocomial outbreaks worldwide. We evaluated whole-genome sequencing (WGS) as an adjunctive epidemiological tool to identify infection clusters and MDRO transmission in the healthcare setting. Methods: Clinical isolates of carbapenem-resistant Enterobacterales (CRE) from July 1, 2021, to June 30, 2022, underwent Illumina WGS. Assembled genomes were taxonomically classified with GTDB-Tk software and were typed using multilocus sequence typing (MLST). Average nucleotide identity (ANI) was calculated between genomes. Numbers of differences among core single-nucleotide polymorphisms (SNPs) were calculated for pairs within taxonomic groups, and the data were evaluated in the context of patient dates and locations of care obtained from the electronic medical record. Results: In total, 39 CRE isolates underwent WGS (Fig. 1). Klebsiella pneumoniae represented the largest number of isolates (n = 18). Using MLST, 2 distinct groups of K. pneumoniae were identified (ST307 and ST258) with 5 and 4 isolates, respectively (Fig. 2). Within ST307, SNP differences ranged between 8 and 115. 3 isolates (CRE8, CRE10, and CRE12) were collected within 4 weeks of each other and had ≤26 pairwise SNP differences. Notably, CRE8 and CRE10 were located on the same unit at the same time and used the same MRI scanner on the same day. CRE35 had >95 SNP differences and was admitted 8 months prior to others in ST307 but had surgery in the same OR as CRE8. Within ST258, pairwise comparison of samples revealed 139–588 SNP differences. CRE21, CRE31, and CRE33 had SNP differences of ≤150. These patients were in the same hospital room (CRE33 and CRE21) and unit (CRE31 and CRE33), but they did not overlap temporally. CRE37 had >580 SNP differences, with no overlap in hospitalization dates or locations with other patients. Conclusions: Two closely related K. pneumoniae isolate populations were identified using WGS. Strong temporal and spatial commonalities were identified among isolates with few SNP differences. Isolate pairs with intermediate SNP differences shared spatial commonalities, suggesting possible indirect transmission between patients. No common exposures were identified for pairs with large numbers of SNP differences. WGS is an evolving tool to detect outbreak clonal populations of MDRO not identified through traditional epidemiologic techniques. WGS can provide insight into transmission patterns and the role of environmental contamination in propagating these nosocomial infections. More studies are needed to define the role and clinical significance of isolates with intermediate SNP differences in transmission of these pathogens between hosts and the healthcare environment.Disclosures: None

Top-Down Genomic Surveillance Approach To Investigate the Genomic Epidemiology and Antibiotic Resistance Patterns of Enterococcus faecium Detected in Cancer Patients in Arkansas.

Control of hospital-associated Enterococcus faecium infection is a strenuous task due to the difficulty of identifying transmission routes and the persistence of this nosocomial pathogen despite the implementation of infection control measures that have been successful with other important nosocomial pathogens. This study provides a comprehensive analysis of over 100 E. faecium isolates collected from 66 cancer patients at the University of Arkansas for Medical Sciences (UAMS) between June 2018 and May 2019. In the top-down approach used in this study, we employed, in addition to the 106 E. faecium UAMS isolates, a filtered set of 2,167 E. faecium strains from the GenBank database to assess the current population structure of E. faecium species and, consequently, to identify the lineages associated with our clinical isolates. We then evaluated the antibiotic resistance and virulence profiles of hospital-associated strains from the species pool, focusing on antibiotics of last resort, to establish an updated classification of high-risk and multidrug-resistant nosocomial clones. Further investigation of the clinical isolates collected from UAMS patients using whole-genome sequencing analytical methodologies (core genome multilocus sequence typing [cgMLST], core single nucleotide polymorphism [coreSNP] analysis, and phylogenomics), with the addition of patient epidemiological data, revealed a polyclonal outbreak of three sequence types occurring simultaneously in different patient wards. The integration of genomic and epidemiological data collected from the patients increased our understanding of the relationships and transmission dynamics of the E. faecium isolates. Our study provides new insights into genomic surveillance of E. faecium to assist in monitoring and further limiting the spread of multidrug-resistant E. faecium. IMPORTANCE Enterococcus faecium is a member of the gastrointestinal microbiota. Although its virulence is low in healthy, immunocompetent individuals, E. faecium has become the third leading cause of health care-associated infections in the United States. This study provides a comprehensive analysis of over 100 E. faecium isolates collected from cancer patients at the University of Arkansas for Medical Sciences (UAMS). We employed a top-down analytical approach (from population genomics to molecular biology) to classify our clinical isolates into their genetic lineages and thoroughly evaluate their antibiotic resistance and virulence profiles. The addition of patient epidemiological data to the whole-genome sequencing analytical methodologies performed in the study allowed us to increase our understanding of the relationships and transmission dynamics of the E. faecium isolates. This study provides new insights into genomic surveillance of E. faecium to help monitor and further limit the spread of multidrug-resistant E. faecium.

Spontaneous Genomic Variation as a Survival Strategy of Nosocomial Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most important nosocomial human pathogens frequently isolated in bloodstream and medical device-related infections. However, its mechanisms of evolution and adaptation are still poorly explored. To characterize the strategies of genetic and phenotypic diversity in S. haemolyticus, we analyzed an invasive strain for genetic and phenotypic stability after serial passage in vitro in the absence and presence of beta-lactam antibiotics. We performed pulsed-field gel electrophoresis (PFGE) of the culture and analyzed five colonies at seven time points during stability assays for beta-lactam susceptibility, hemolysis, mannitol fermentation, and biofilm production. We compared their whole genomes and performed phylogenetic analysis based on core single-nucleotide polymorphisms (SNPs). We observed a high instability in the PFGE profiles at the different time points in the absence of antibiotic. Analysis of WGS data for individual colonies showed the occurrence of six large-scale genomic deletions within the oriC environ, smaller deletions in non-oriC environ regions, and nonsynonymous mutations in clinically relevant genes. The regions of deletion and point mutations included genes encoding amino acid and metal transporters, resistance to environmental stress and beta-lactams, virulence, mannitol fermentation, metabolic processes, and insertion sequence (IS) elements. Parallel variation was detected in clinically significant phenotypic traits such as mannitol fermentation, hemolysis, and biofilm formation. In the presence of oxacillin, PFGE profiles were overall stable over time and mainly corresponded to a single genomic variant. Our results suggest that S. haemolyticus populations are composed of subpopulations of genetic and phenotypic variants. The maintenance of subpopulations in different physiological states may be a strategy to adapt rapidly to stress situations imposed by the host, particularly in the hospital environment. IMPORTANCE The introduction of medical devices and antibiotics into clinical practice have substantially improved patient quality of life and contributed to extended life expectancy. One of its most cumbersome consequences was the emergence of medical device-associated infections caused by multidrug-resistant and opportunistic bacteria such as Staphylococcus haemolyticus. However, the reason for this bacterium's success is still elusive. We found that in the absence of environmental stresses, S. haemolyticus can spontaneously produce subpopulations of genomic and phenotypic variants with deletions/mutations in clinically relevant genes. However, when exposed to selective pressures, such as the presence of antibiotics, a single genomic variant will be recruited and become dominant. We suggest that the maintenance of these cell subpopulations in different physiological states is an extremely effective strategy to adapt to stresses imposed by the host or the infection environment and might contribute for S. haemolyticus survival and persistence in the hospital.

Core single nucleotide polymorphism analysis reveals transmission of Mycobacterium marinum between animal and environmental sources in two aquaria.

Mycobacterium marinum is a slow-growing, photochromogenic nontuberculous mycobacterium, which can cause mycobacteriosis in various animals, including humans. Several cases of fish mycobacteriosis have been reported to date. Mycobacterium marinum has also been isolated from aquatic environmental sources such as water, sand, biofilms, and plants in the natural environments. Hence, we hypothesized that a wide variety of sources could be involved in the transmission of M.marinum. In this study, we tested this hypothesis by isolating M.marinum from various sources such as fish, invertebrates, seagrass, periphytons, biofilms, sand, and/or water in two aquaria in Japan and conducting a phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) using whole-genome sequences of the isolated strains. The analysis revealed that the strains from animal and environmental sources belonged to the same clusters. This molecular-based study epidemiologically confirmed that various sources, including fish, invertebrates, and environmental sources, could be involved in transmission of M.marinum in a closed-rearing environment. This is the first report where M.marinum was isolated from different sources, and various transmission routes were confirmed in actual cases, which provided essential information to improve the epidemiology of M.marinum.

Molecular epidemiology of Clostridioides difficile in companion animals: Genetic overlap with human strains and public health concerns.

The changing epidemiology of Clostridioides difficile reflects a well-established and intricate community transmission network. With rising numbers of reported community-acquired infections, recent studies tried to identify the role played by non-human reservoirs in the pathogen's transmission chain. This study aimed at describing the C. difficile strains circulating in canine and feline populations, and to evaluate their genetic overlap with human strains to assess the possibility of interspecies transmission. Fecal samples from dogs (n = 335) and cats (n = 140) were collected from two populations (group A and group B) in Portugal. C. difficile isolates were characterized for toxigenic profile and PCR-ribotyping. The presence of genetic determinants of antimicrobial resistance was assessed in all phenotypically resistant isolates. To evaluate the genetic overlap between companion animals and human isolates from Portugal, RT106 (n = 42) and RT014/020 (n = 41) strains from both sources were subjected to whole genome sequencing and integrated with previously sequenced RT106 (n = 43) and RT014/020 (n = 142) genomes from different countries. The genetic overlap was assessed based on core-single nucleotide polymorphism (SNP) using a threshold of 2 SNP. The overall positivity rate for C. difficile was 26% (76/292) in group A and 18.6% (34/183) in group B. Toxigenic strains accounted for 50% (38/76) and 52.9% (18/34) of animal carriage rates, respectively. The most prevalent ribotypes (RT) were the toxigenic RT106 and RT014/020, and the non-toxigenic RT010 and RT009. Antimicrobial resistance was found for clindamycin (27.9%), metronidazole (17.1%) and moxifloxacin (12.4%), associated with the presence of the ermB gene, the pCD-METRO plasmid and point mutations in the gyrA gene, respectively. Both RT106 and RT014/020 genetic analysis revealed several clusters integrating isolates from animal and human sources, supporting the possibility of clonal interspecies transmission or a shared environmental contamination source. This study shows that companion animals may constitute a source of infection of toxigenic and antimicrobial resistant human associated C. difficile isolates. Additionally, it contributes with important data on the genetic proximity between C. difficile isolates from both sources, adding new information to guide future work on the role of animal reservoirs in the establishment of community associated transmission networks and alerting for potential public health risk.

Varicella Vaccine: a Molecular Variant That May Contribute to Attenuation.

Varicella was troublesome when varicella vaccine (vOka) was licensed in the United States. Varicella's yearly death toll was ~100, indirect costs were massive, and varicella threatened immunocompromised children. Since licensure, varicella has almost disappeared; nevertheless, vOka attenuation has lacked a molecular explanation. Sadaoka et al. (T. Sadaoka, D. P. Depledge, L. Rajbhandari, J. Breuer, et al., mBio 13:e0186422, 2022, https://doi.org/10.1128/mbio.01864-22), however, have now identified 6 core single nucleotide polymorphisms (SNPs), which singly or in combination may contribute to VOka attenuation; moreover, they found a predominant variant allele of vOka encoding the viral glycoprotein gB that results in glutamine instead of arginine at amino acid 699. This change impairs fusion activity and the ability of varicella-zoster virus (VZV) to infect human neurons from axon terminals. Molecular virological studies of vOka are reassuring in suggesting that reversion to virulence is unlikely and should also help assuage current fears about VZV vaccination and alleviate unanticipated future problems. The impressive work of Sadaoka et al. thus represents an auspicious advance in knowledge.

Genetic characteristics, antimicrobial resistance, and prevalence of Arcobacter spp. isolated from various sources in Shenzhen, China.

Arcobacter spp. is a globally emerging zoonotic and foodborne pathogen. However, little is known about its prevalence and antimicrobial resistance in China. To investigate the prevalence of Arcobacter spp. isolated from various sources, 396 samples were collected from human feces, chicken cecum, and food specimens including chicken meat, beef, pork, lettuce, and seafood. Arcobacter spp. was isolated by the membrane filtration method. For 92 strains, the agar dilution method and next-generation sequencing were used to investigate their antimicrobial resistance and to obtain whole genome data, respectively. The virulence factor database (VFDB) was queried to identify virulence genes. ResFinder and the Comprehensive Antibiotic Resistance Database (CARD) were used to predict resistance genes. A phylogenetic tree was constructed using the maximum likelihood (ML) method with core single-nucleotide polymorphisms (SNPs). We found that 27.5% of the samples (n = 109) were positive for Arcobacter spp., comprising Arcobacter butzleri (53.0%), Arcobacter cryaerophilus (39.6%), and Arcobacter skirrowii (7.4%). Chicken meat had the highest prevalence (81.2%), followed by seafood (51.9%), pork (43.3%), beef (36.7%), lettuce (35.5%), chicken cecum (8%), and human fecal samples (0%, 0/159). Antimicrobial susceptibility tests revealed that 51 A. butzleri and 40 A. cryaerophilus strains were resistant to streptomycin (98.1, 70%), clindamycin (94.1, 90%), tetracycline (64.7, 52.5%), azithromycin (43.1%, 15%), nalidixic acid (33.4, 35%), and ciprofloxacin (31.3, 35%) but were susceptible to erythromycin, gentamicin, chloramphenicol, telithromycin, and clindamycin (≤10%). A. skirrowii was sensitive to all experimental antibiotics. The virulence factors tlyA, mviN, cj1349, ciaB, and pldA were carried by all Arcobacter spp. strains at 100%, and the following percentages were cadF (95.7%), iroE (23.9%), hecB (2.2%), hecA, and irgA (1.1%). Only one A. butzleri strain (F061-2G) carried a macrolide resistance gene (ereA). One A. butzleri and one A. cryaerophilus harbored resistance island gene clusters, which were isolated from pork and chicken. Phylogenetic tree analysis revealed that A. butzleri, A. cryaerophilus, and A. skirrowii were separated from each other. To our knowledge, this is the first report of the isolation of Arcobacter spp. from vegetables and seafood in China. The resistance island gene cluster found in pork and chicken meat and the presence of virulence factors could be a potential risk to human health.

Species-informative SNP markers for characterising freshwater prawns of genus Macrobrachium in Cameroon.

Single Nucleotide Polymorphisms (SNPs) are now popular for a myriad of applications in animal and plant species including, ancestry assignment, conservation genetics, breeding, and traceability of animal products. The objective of this study was to develop a customized cost-effective SNP panel for genetic characterisation of Macrobrachium species in Cameroon. The SNPs identified in a previous characterization study were screened as viable candidates for the reduced panel. Starting from a full set of 1,814 SNPs, a total of 72 core SNPs were chosen using conventional approaches: allele frequency differentials, minor allele frequency profiles, and Wright’s Fst statistics. The discriminatory power of reduced set of informative SNPs were then tested using the admixture analysis, principal component analysis, and discriminant analysis of principal components. The panel of prioritised SNP markers (i.e., N = 72 SNPs) distinguished Macrobrachium species with 100% accuracy. However, large sample size is needed to identify more informative SNPs for discriminating genetically closely related species, including M. macrobrachion versus M. vollenhovenii and M. sollaudii versus M. dux. Overall, the findings in this study show that we can accurately characterise Macrobrachium using a small set of core SNPs which could be useful for this economically important species in Cameroon. Given the results obtained in this study, a larger independent validation sample set will be needed to confirm the discriminative capacity of this SNP panel for wider commercial and research applications.

Evolutionary origin of pathogenic GJB2 alleles in China.

The frequency of the pathogenic allele of the autosomal recessive deafness gene GJB2 varies among different populations in the world, and accumulates to a sufficiently high frequency in certain population. The purpose of this study is to investigate the origin and evolution of GJB2 pathogenic alleles in Chinese deaf patients. Children with non-syndromic hearing loss, and their parents, from 295 families were recruited. Customized capture probes targeted at 943 single nucleotide polymorphisms (SNPs) related to GJB2 gene were designed for sequencing of genomic DNA in blood samples. Haplotypes carrying pathogenic allele were analyzed through linkage disequilibrium block building, ancestry tracing, and extended haplotype heterozygosity calculation. Two pathogenic GJB2 alleles, c.235delC (18.41%) and c.109G > A (15.57%), were observed in 867 donors. For c.235delC allele, three different core haplotypes with one major haplotype (97.32%) were found, and their core SNPs were 100% conserved. For c.109G > A allele, six different haplotypes with one major haplotype (93.28%) were found and the major c.109G > A allele evolved from a specific ancestral haplotype. Geographical origins of donors carrying GJB2 c.109G > A and c.235delC core haplotypes centered between Qinghai and Neimenggu. GJB2 c.235delC has long-range linkage disequilibrium. No positive selection signature was found for GJB2 c.235delC or c.109G > A in the studied population. In conclusion, we discovered a single origin of GJB2 c.235delC allele and multiple independent origins of GJB2 c.109G > A allele. Alternative to positive selection or multiple independent recurrent mutation event, population bottleneck effect might account for the observed high population frequency of these pathogenic alleles.

Comparison of DUS testing and SNP fingerprinting for variety identification in cucumber

Variety identification plays an important role in protecting the intellectual property of varieties, ensuring seed quality, and encouraging breeding innovation. Currently, morphological evaluation in the field, such as distinctness, uniformity, and stability (DUS) testing, and DNA fingerprinting in the laboratory using molecular markers are two dominant methods used for variety identification. Few studies have compared the results of these approaches, and the relationship between the two methods is obscure. In this study, 134 dominant cucumber varieties were evaluated using 50 DUS testing traits and genotyped by 40 single nucleotide polymorphisms (SNPs). The 40 SNPs were developed in our previous study and are well suited for variety identification. In the DUS testing, significant positive or negative correlations among 50 DUS traits were observed, and 20 core traits, including 15 fruit traits, were further selected to increase field inspection efficiency. This suggested that fruit shape plays an important role in variety identification. The ratio of fruit length/diameter was the most important trait, explaining 9.2% of the phenotypic variation. In the DNA fingerprinting test, the 40 SNPs were highly polymorphic and could distinguish all of the 134 cucumber varieties, and 14 core SNPs were selected to improve the identification rate. Interestingly, the population structure analysis of 134 cucumber varieties by phenotypic data in the DUS test was in accordance with the genotypic data from the DNA fingerprinting, indicating that all varieties could be divided into the same four subgroups: European type, North China type, South China type, and hybrids of the North China and South China types. Moreover, linear correlativity of distinguishment for each pair of varieties was observed between the DUS test and the DNA fingerprinting. These results indicated that these two methods have good application in future research, especially for the scaled-up analysis of hundreds of varieties.