Anal swabs of 1-month-old Holstein calves with diarrhea were collected from an intensive cattle farm, and a highly pathogenic Escherichia coli strain was obtained by isolation and purification. To study the virulence and resistance genes of pathogenic E. coli that cause diarrhea in calves, a strain of E. coli E12 isolated from calf diarrhea samples was used as experimental material in this experiment, and the virulence of the E12 strain were identified by the mouse infection test, and the whole genome map of the E12 strain were obtained by whole-genome sequencing and analyzed for genome characterization. The results showed that the lethality of strain E12 was 100%, the total length of E12-encoded genes was 4,294,530 bp, Cluster of Orthologous Groups of proteins (COG) annotated to 4,194 functional genes, and the virulence genes of sequenced strain E12 were compared with the virulence genes of sequenced strain E12 from the Virulence Factors of Pathogenic Bacteria (VFDB), which contained a total of 366 virulence genes in sequenced strain E12. The analysis of virulence genes of E12 revealed a total of 52 virulence genes in the iron transferrin system, 56 virulence genes in the secretory system, 41 virulence genes in bacterial toxins, and a total of 217 virulence genes in the Adhesin and Invasins group. The antibiotic resistance genes of sequenced strain E12 were identified through the Antibiotic Resistance Genes Database (ARDB) and Comprehensive Antibiotic Research Database, and it was found that its chromosome and plasmid included a total of 127 antibiotic resistance genes in four classes, and that E12 carried 71 genes related to the antibiotic efflux pumps, 36 genes related to antibiotic inactivation, and 14 antibiotic target alteration and reduced penetration into antibiotics, and 6 antibiotic resistance genes, and the resistance phenotypes were consistent with the genotypes. The pathogenic E. coli that causes diarrhea in calves on this ranch contains a large number of virulence and resistance genes. The results provide a theoretical basis for the prevention and treatment of diarrhea and other diseases caused by E. coli disease.

Variants in cis-regulatory elements link the noncoding genome to human pathology; however, detailed analytic tools for understanding the association between cell-level brain pathology and noncoding variants are lacking. CWAS-Plus, adapted from a Python package for category-wide association testing (CWAS), enhances noncoding variant analysis by integrating both whole-genome sequencing (WGS) and user-provided functional data. With simplified parameter settings and an efficient multiple testing correction method, CWAS-Plus conducts the CWAS workflow 50 times faster than CWAS, making it more accessible and user-friendly for researchers. Here, we used a single-nuclei assay for transposase-accessible chromatin with sequencing to facilitate CWAS-guided noncoding variant analysis at cell-type-specific enhancers and promoters. Examining autism spectrum disorder WGS data (n = 7280), CWAS-Plus identified noncoding de novo variant associations in transcription factor binding sites within conserved loci. Independently, in Alzheimer\'s disease WGS data (n = 1087), CWAS-Plus detected rare noncoding variant associations in microglia-specific regulatory elements. These findings highlight CWAS-Plus\'s utility in genomic disorders and scalability for processing large-scale WGS data and in multiple-testing corrections. CWAS-Plus and its user manual are available at https://github.com/joonan-lab/cwas/ and https://cwas-plus.readthedocs.io/en/latest/, respectively.

Danzhou chicken (DZ) is a local breed in China noted for its strong adaptability, roughage resistance, strong wildness, and delicious taste, thus containing important genetic resources. In this study, genome re-sequencing data was generated from 200 DZ chickens. Combined with previously generated data from 72 additional chickens across six other exotic and local breeds, these data were used to systematically evaluate the germplasm characteristics of DZ chickens from a genomic perspective. Unlike exotic breeds, both DZ and southern local chicken varieties exhibited high genetic diversity, and the genetic distance between DZ and southern local chickens was smaller than the genetic distance between DZ and exotic chickens. A reconstructed Neighbor-Joining phylogenetic tree indicated that all sampled populations clustered into single independent populations, with DZ chickens showing clear evidence of intra-population differentiation, forming 2 subpopulations. Principal component analysis and ADMIXTURE analysis showed that DZ was significantly different from other breeds. These results indicate that DZ is a unique genetic resource that is different from other southern native and exotic chickens. The results of the study will improve our understanding of the genetic structure and current status of the DZ breed, which is of great significance in promoting the conservation of genetic resources of DZ chickens and fostering breed innovations and genetic improvement.

OBJECTIVE: To characterize the clinical relevance of S. saccharolyticus and to identify criteria to distinguish between infection and contamination.
METHODS: We retrospectively investigated clinical features of patients with S. saccharolyticus detection between June 2009 and July 2021. Based on six criteria, infection was considered likely for patients with a score from 3 to 6 points, infection was considered unlikely for patients with a score from 0 to 2 points. We performed group comparison and logistic regression to identify factors than are associated with likely infection. In addition, whole genome sequencing (WGS) of 22 isolates was performed.
RESULTS: Of 93 patients in total, 44 were assigned to the group \"infection likely\" and 49 to the group \"infection unlikely\". Multiple regression analysis revealed \"maximum body temperature during hospital stay\" to have the strongest predictive effect on likely infection (adjusted odds ratio 4.40, 95% confidence interval 2.07-9.23). WGS revealed two different clades. Compared to isolates from clade A, isolates from clade B were more frequently associated with implanted medical devices (3/10 vs. 9/12, p = 0.046) and a shorter time to positivity (TTP) (4.5 vs. 3, p = 0.016). Both clades did neither differ significantly in terms of causing a likely infection (clade A 7/10 vs. clade B 5/12, p = 0.23) nor in median length of hospital stay (28 vs. 15.5 days, p = 0.083) and length of stay at the ICU (21 vs. 3.5 days, p = 0.14).
CONCLUSIONS: These findings indicate that S. saccharolyticus can cause clinically relevant infections. Differentiation between infection and contamination remains challenging.

BACKGROUND: Breeding polled goats is a welfare-friendly approach for horn removal in comparison to invasive methods. To gain a comprehensive understanding of the genetic basis underlying polledness in goats, we conducted whole-genome sequencing of 106 Xinong Saanen dairy goats, including 33 horned individuals, 70 polled individuals, and 3 polled intersexuality syndrome (PIS) individuals.
METHODS: The present study employed a genome-wide association study (GWAS) and linkage disequilibrium (LD) analysis to precisely map the genetic locus underlying the polled phenotype in goats.
RESULTS: The analysis conducted in our study revealed a total of 320 genome-wide significant single nucleotide polymorphisms (SNPs) associated with the horned/polled phenotype in goats. These SNPs exhibited two distinct peaks on chromosome 1, spanning from 128,817,052 to 133,005,441 bp and from 150,336,143 to 150,808,639 bp. The present study identified three genome-wide significant SNPs, namely Chr1:129789816, Chr1:129791507, and Chr1:129791577, as potential markers of PIS-affected goats. The results of our LD analysis suggested a potential association between MRPS22 and infertile intersex individuals, as well as a potential association between ERG and the polled trait in goats.
CONCLUSIONS: We have successfully identified three marker SNPs closely linked to PIS, as well as several candidate genes associated with the polled trait in goats. These results may contribute to the development of SNP chips for early prediction of PIS in goats, thereby facilitating breeding programs aimed at producing fertile herds with polled traits.

UNASSIGNED: The occurrence and dissemination of hypermucoviscous and hypervirulent Klebsiella pneumoniae (hm-hvKp) isolates in clinical settings are a critical public health problem in the world. However, the data on these isolates in community populations are limited. This study aims to understand the prevalence and molecular characteristics of hm-hvKp isolates in community patients in Shanghai, China.
UNASSIGNED: In 2018, an active surveillance system focused on hm-hvKp in community diarrhoeal cases was implemented in Pudong New Area, Shanghai, China, involving 12 sentinel hospitals. The antimicrobial susceptibility of hm-hvKp isolates from fecal samples was tested, and whole-genome sequencing (WGS) was performed to predict the serotypes and sequence types and to identify antimicrobial resistance determinants, virulence determinants, and phylogenetic clusters.
UNASSIGNED: The overall prevalence of hm K. pneumoniae isolates was 2.48% (31/1252), with the proportions of 1.76% (22/1252) for hm-hvKp and 0.72% (9/1252) for hm not hv K. pneumoniae. The prevalence of hm-hvKp isolates among different age groups and different months was statistically significant. All the 22 hm-hvKp isolates were susceptible to 20 antimicrobial agents and only carried bla SHV gene, and KL1 and KL2 accounted for eight (36.36%) cases and seven (31.82%) cases, respectively. The eight ST23/KL1 isolates belonged to the predominant CG23-I clade, which typically possessed the virulence determinants profile of rmpA/rmpA2-iro-iuc-ybt-irp-clb. The five ST86/KL2 isolates were assigned to the global clusters ST86/KL2-1 (n=2), ST86/KL2-2 (n=2), ST86/KL2-3 (n=1), all lack of the clb gene. Shanghai ST23/KL1 and ST86/KL2 isolates were closely related to the global isolates from liver abscesses, blood, and urine.
UNASSIGNED: Hm-hvKp is carried by the community population of Shanghai, with ST23/KL1 and ST86/KL2 isolates predominant. Hm-hvKp isolates of different continents, different sources, and different virulence levels were closely related. Ongoing surveillance of hm-hvKp isolates in the community population is warranted.

A 30-year-old Korean man with myelodysplastic syndrome admitted hospital due to undifferentiated fever and recurrent skin lesions. He received combination therapy with high doses of meropenem, tigecycline and amikacin, yielding carbapenem resistant Klebsiella pneumoniae (CRKP) harboring K. pneumoniae carbapenemase (KPC)-2 from blood cultures on hospital day (HD) 23. Ceftazidime/avibactam was started at HD 37 and CRKP was eradicated from blood cultures after 5 days. However, ceftazidime/avibactam-resistant CRKP carrying KPC-44 emerged after 26 days of ceftazidime/avibactam treatment and then ceftazidime/avibactam-resistant, carbapenem-susceptible K. pneumoniae carrying KPC-135 was isolated on HD 65. The 3-D homology of KPC protein showed that hot spot changes in the omega loop could be attributed to ceftazidime/avibactam resistance and loss of carbapenem resistance. Whole genome sequencing of serial isolates supported that phenotypic variation was due to clonal evolution than clonal replacement. The treatment regimen was changed from CAZ/AVI to meropenem-based therapy (meropenem 1 g iv q 8 hours and amikacin 600 mg iv per day) starting with HD 72. CAZ/AVI-susceptible CRKP was presented again from blood cultures on HD 84, and the patient expired on HD 85. This is the first Korean report on the acquisition of ceftazidime/avibactam resistance through the emergence of blaKPC variants.

Outbreaks are a risk to public health particularly when pathogenic, hypervirulent, and/or multidrug-resistant organisms (MDROs) are involved. In a hospital setting, vulnerable populations such as the immunosuppressed, intensive care patients, and neonates are most at risk. Rapid and accurate outbreak detection is essential to implement effective interventions in clinical areas to control and stop further transmission. Advances in the field of whole genome sequencing (WGS) have resulted in lowered costs, increased capacity, and improved reproducibility of results. WGS now has the potential to revolutionize the investigation and management of outbreaks replacing conventional genotyping and other discrimination systems. Here, we outline specific procedures and protocols to implement WGS into investigation of outbreaks in healthcare settings.

The use of surrogate organisms can enable researchers to safely conduct research on pathogens and in a broader set of conditions. Being able to differentiate between the surrogates used in the experiments and background contamination as well as between different experiments will further improve research efforts. One effective approach is to introduce unique genetic barcodes into the surrogate genome and track their presence using the quantitative polymerase chain reaction (qPCR). In this report, we utilized the CRISPR-Cas9 methodology, which employs a single plasmid and a transformation step to insert five distinct barcodes into Bacillus thuringiensis, a well-established surrogate for Bacillus anthracis when Risk Group 1 organisms are needed. We subsequently developed qPCR assays for barcode detection and successfully demonstrated the stability of the barcodes within the genome through five cycles of sporulation and germination. Additionally, we conducted whole-genome sequencing on these modified strains and analyzed 187 potential Cas9 off-target sites. We found no correlation between the mutations observed in the engineered strains and the predicted off-target sites, suggesting this genome engineering strategy did not directly result in off-target mutations in the genome. This simple approach has the potential to streamline the creation of barcoded B. thuringiensis strains for use in future studies on surrogate genomes.
OBJECTIVE: The use of Bacillus anthracis as a biothreat agent poses significant challenges for public health and national security. Bacillus anthracis surrogates, like Bacillus thuringiensis, are invaluable tools for safely understanding Bacillus anthracis properties without the safety concerns that would arise from using a virulent strain of Bacillus anthracis. We report a simple method for barcode insertion into Bacillus thuringiensis using the CRISPR-Cas9 methodology and subsequent tracking by quantitative polymerase chain reaction (qPCR). Moreover, whole-genome sequencing data and CRISPR-Cas9 off-target analyses in Bacillus thuringiensis suggest that this gene-editing method did not directly cause unwanted mutations in the genome. This study should assist in the facile development of barcoded Bacillus thuringiensis surrogate strains, among other biotechnological applications in Bacillus species.

UNASSIGNED: The coronavirus disease 2019 (COVID-19) is a viral infection characterized by respiratory and gastrointestinal symptoms. The causative agent of this infection is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic study helps in understanding the pathogenesis, epidemiology, and the development of therapeutic and preventive strategies in the combat against COVID-19.
UNASSIGNED: Nasopharyngeal and oropharyngeal swab samples were collected from asymptomatic and symptomatic patients during the time period of 2021-2022 for the detection of SARS-CoV-2 by employing real-time reverse transcriptase, cDNA synthesis, whole-genome sequencing by next-genome sequencing, analysis of SARS-CoV-2 sequence data and lineage and variant of concern assignment along with phylogenetic analysis.
UNASSIGNED: Lineages BA.2.10 and BA.4.1.1 clustered with genomes from Senegal suggested the spread of infections. Similarly, high clustering among delta samples during the second wave showed possible importation and subsequent spread via local transmission.
UNASSIGNED: Studies like these are important to understand the characteristics and origins of locally circulating SARS-CoV-2 diversity in order to prevent further spread.