We conducted a global-scale study to identify H. pylori antimicrobial-resistant genes (ARG), address their global distribution, and understand their effect on the antimicrobial resistance (AMR) phenotypes of the clinical isolates. We identified ARG using several well-known tools against extensive bacterial ARG databases, then analyzed their correlation with clinical antibiogram data from dozens of patients across countries. This revealed that combining multiple tools and databases, followed by manual selection of ARG from the annotation results, produces more conclusive results than using a single tool or database alone. After curation, the results showed that H. pylori has 42 ARG against 11 different antibiotic classes (16 genes related to single antibiotic class resistance and 26 genes related to multidrug resistance). Further analysis revealed that H. pylori naturally harbors ARG in the core genome, called the \'Set of ARG commonly found in the Core Genome of H. pylori (ARG-CORE)\', while ARG-ACC-the ARG in the accessory genome-are exclusive to particular strains. In addition, we detected 29 genes of potential efflux pump-related AMR that were mostly categorized as ARG-CORE. The ARG distribution appears to be almost similar either by geographical or H. pylori populations perspective; however, some ARG had a unique distribution since they tend to be found only in a particular region or population. Finally, we demonstrated that the presence of ARG may not directly correlate with the sensitive/resistance phenotype of clinical patient isolates but may influence the minimum inhibitory concentration phenotype.

A genome-wide association study (GWAS) was performed as a preliminary step to identify regions potentially related to ham quality traits. In this research, genomic information was obtained from 238 commercial hybrid pigs utilising the GeneSeek® Genomic Profiler genome-wide porcine genotyping array. Carcasses were tested for hot weight, the thickness of backfat and loin, and lean meat percentage. The corresponding fresh hams were assayed for weight and ultimate pH; the activities of Cathepsin B and Ferrochelatase of Semimembranosus muscle were determined through fluorimetric methods. The lean meat percentage of fresh ham (LMPH), salt absorbed after first (SALT1) and overall salting stages (SALT) were estimated online by the Ham Inspector™ apparatus. Hams were processed in compliance with the procedures established for Protected Designation of Origin Parma ham, and ham weight losses were measured at the main processing stages. Hot carcass weights showed a significant negative correlation with their lean meat percentage and LMPH, while LMPH was correlated positively with carcass lean meat, SALT1, SALT, and weight losses. The GWAS detected genome-wide association for 12 single nucleotide polymorphisms with Ferrochelatase activity. The results obtained in this preliminary study were achieved by combining innovative and non-destructive technologies for screening hams under processing, measures of enzymatic muscle properties relevant to dry-cured ham quality, and genomic information obtained through a GWAS. Additional studies carried out in a larger number of pigs have been planned to investigate the effect of gene variants of Ferrochelatase activity in the dry-cured ham\'s quality with main reference to colour development and to confirm the GWAS results obtained in this study.

Chitin is an abundant natural polysaccharide that is hard to degrade because of its crystalline nature and because it is embedded in robust co-polymeric materials containing other polysaccharides, proteins, and minerals. Thus, it is of interest to study the enzymatic machineries of specialized microbes found in chitin-rich environments. We describe a genomic and proteomic analysis of Andreprevotia ripae, a chitinolytic Gram-negative bacterium isolated from an anthill. The genome of A. ripae encodes four secreted family GH19 chitinases of which two were detected and upregulated during growth on chitin. In addition, the genome encodes as many as 25 secreted GH18 chitinases, of which 17 were detected and 12 were upregulated during growth on chitin. Finally, the single lytic polysaccharide monooxygenase (LPMO) was strongly upregulated during growth on chitin. Whereas 66% of the 29 secreted chitinases contained two carbohydrate-binding modules (CBMs), this fraction was 93% (13 out of 14) for the upregulated chitinases, suggesting an important role for these CBMs. Next to an unprecedented multiplicity of upregulated chitinases, this study reveals several chitin-induced proteins that contain chitin-binding CBMs but lack a known catalytic function. These proteins are interesting targets for discovery of enzymes used by nature to convert chitin-rich biomass. The MS proteomic data have been deposited in the PRIDE database with accession number PXD025087.

The genus Acinetobacter comprises species with ecological significance and opportunistic pathogens and has a complicated taxonomy. Precise species identification is a foundation for understanding bacteria. In this study, we found and characterized two novel Acinetobacter species, namely, Acinetobacter tianfuensis sp. nov. and Acinetobacter rongchengensis sp. nov., based on phenotype examinations and genome analyses of the two strains WCHAc060012T and WCHAc060115T. The two strains had ≤89.69% (mean, 79.28% or 79.72%) average nucleotide identity (ANI) and ≤36.4% (mean, 20.89% or 22.19%) in silico DNA-DNA hybridization (isDDH) values compared with each other and all known Acinetobacter species. Both species can be differentiated from all hitherto known Acinetobacter species by a combination of phenotypic characteristics. We found that Acinetobacter pullorum B301T and Acinetobacter portensis AC 877T are actually the same species with 98.59% ANI and 90.4% isDDH values. We then applied the updated taxonomy to curate 3,956 Acinetobacter genomes in GenBank and found that 6% of Acinetobacter genomes (n = 234) are required to be corrected or updated. We identified 56 novel tentative Acinetobacter species, extending the number of Acinetobacter species to 144, including 68 with species names and 76 unnamed taxa. We also found that ANI and the average amino acid identity (AAI) values among type or reference strains of all Acinetobacter species and taxa are ≥76.97% and ≥66.5%, respectively, which are higher than the proposed cutoffs to define the genus boundary. This study highlights the complex taxonomy of Acinetobacter as a single genus and the paramount importance of precise species identification. The newly identified unnamed taxa warrant further studies. IMPORTANCE Acinetobacter species are widely distributed in nature and are of important ecological significance and clinical relevance. In this study, first, we significantly update the taxonomy of Acinetobacter by reporting two novel Acinetobacter species, namely, Acinetobacter tianfuensis and Acinetobacter rongchengensis, and by identifying Acinetobacter portensis as a synonym of Acinetobacter pullorum. Second, we curated Acinetobacter genome sequences deposited in GenBank (n = 3,956) using the updated taxonomy by correcting species assignations for 6% (n = 234) genomes and by assigning 94 (2.4%) to 56 previously unknown tentative species (taxa). Therefore, after curation, we further update the genus Acinetobacter to comprise 144 species, including 68 with species names and 76 unnamed taxa. Third, we addressed the question of whether such a large number of species should be divided in different genera and found that Acinetobacter is indeed a single genus. Our study significantly advanced the taxonomy of Acinetobacter, an important genus with science and health implications.

Diarrhea is a major cause of both morbidity and mortality worldwide, especially among young children. Cryptosporidiosis is a leading cause of diarrhea in children, particularly in South Asia and sub-Saharan Africa, where it is responsible for over 200,000 deaths per year. Beyond the initial clinical presentation of diarrhea, it is associated with long-term sequelae such as malnutrition and neurocognitive developmental deficits. Risk factors include poverty and overcrowding, and yet not all children with these risk factors and exposure are infected, nor do all infected children develop symptomatic disease. One potential risk factor to explain these differences is their human genome. To identify genetic variants associated with symptomatic cryptosporidiosis, we conducted a genome-wide association study (GWAS) examining 6.5 million single nucleotide polymorphisms (SNPs) in 873 children from three independent cohorts in Dhaka, Bangladesh, namely, the Dhaka Birth Cohort (DBC), the Performance of Rotavirus and Oral Polio Vaccines in Developing Countries (PROVIDE) study, and the Cryptosporidiosis Birth Cohort (CBC). Associations were estimated separately for each cohort under an additive model, adjusting for length-for-age Z-score at 12 months of age, the first two principal components to account for population substructure, and genotyping batch. The strongest meta-analytic association was with rs58296998 (P = 3.73 × 10-8), an intronic SNP and expression quantitative trait locus (eQTL) of protein kinase C alpha (PRKCA). Each additional risk allele conferred 2.4 times the odds of Cryptosporidium-associated diarrhea in the first year of life. This genetic association suggests a role for protein kinase C alpha in pediatric cryptosporidiosis and warrants further investigation.IMPORTANCE Globally, diarrhea remains one of the major causes of pediatric morbidity and mortality. The initial symptoms of diarrhea can often lead to long-term consequences for the health of young children, such as malnutrition and neurocognitive developmental deficits. Despite many children having similar exposures to infectious causes of diarrhea, not all develop symptomatic disease, indicating a possible role for human genetic variation. Here, we conducted a genetic study of susceptibility to symptomatic disease associated with Cryptosporidium infection (a leading cause of diarrhea) in three independent cohorts of infants from Dhaka, Bangladesh. We identified a genetic variant within protein kinase C alpha (PRKCA) associated with higher risk of cryptosporidiosis in the first year of life. These results indicate a role for human genetics in susceptibility to cryptosporidiosis and warrant further research to elucidate the mechanism.

Tumor heterogeneity has been suggested based on clinical and pathological findings. Several clinical findings can be explained by tumor evolution during progression and metastasis. We herein report a case of metastatic breast cancer indicated tumor heterogeneity by clinical findings and a genomic analysis. A 64-year-old woman with advanced breast cancer was treated with primary chemotherapy, to which primary tumor responded. After a 6 month treatment pause, lung, liver, and skin metastases developed and her serum tumor markers were elevated. None of those serum markers had been elevated before the treatment, despite the large tumor burden. Notably, there was discordance in the expression of human epidermal growth factor receptor 2 (HER2) between the primary tumor and metastatic skin lesions, with the former being negative and the latter positive. A genomic analysis was performed by in-house Breast Cancer Panel, which consisted of 53 pre-selected genes. Twenty-three somatic mutations were found in primary breast tumor and 7 in the skin metastasis. None of these 30 genes matched. However, the cell-free (cf) DNA in the plasma taken at the time of skin metastasis contained 10 mutations, 7 from the primary lesion and 3 from the metastasis. These data indicate that the clonal changes or tumor heterogeneity was shown in two solid tumors by clinical and the result of a genomic analysis. Of particular interest was that cell-free DNA could be a powerful tool to look into these dynamic changes.

Mixed fibrolamellar hepatocellular carcinoma (mFL-HCC) is a rare liver tumor defined by the presence of both pure FL-HCC and conventional HCC components, represents up to 25% of cases of FL-HCC, and has been associated with worse prognosis. Recent genomic characterization of pure FL-HCC identified a highly recurrent transcript fusion (DNAJB1:PRKACA) not found in conventional HCC.
We performed exome and transcriptome sequencing of a case of mFL-HCC. A novel BAC-capture approach was developed to identify a 400 kb deletion as the underlying genomic mechanism for a DNAJB1:PRKACA fusion in this case. A sensitive Nanostring Elements assay was used to screen for this transcript fusion in a second case of mFL-HCC, 112 additional HCC samples and 44 adjacent non-tumor liver samples.
We report the first comprehensive genomic analysis of a case of mFL-HCC. No common HCC-associated mutations were identified. The very low mutation rate of this case, large number of mostly single-copy, long-range copy number variants, and high expression of ERBB2 were more consistent with previous reports of pure FL-HCC than conventional HCC. In particular, the DNAJB1:PRKACA fusion transcript specifically associated with pure FL-HCC was detected at very high expression levels. Subsequent analysis revealed the presence of this fusion in all primary and metastatic samples, including those with mixed or conventional HCC pathology. A second case of mFL-HCC confirmed our finding that the fusion was detectable in conventional components. An expanded screen identified a third case of fusion-positive HCC, which upon review, also had both conventional and fibrolamellar features. This screen confirmed the absence of the fusion in all conventional HCC and adjacent non-tumor liver samples.
These results indicate that mFL-HCC is similar to pure FL-HCC at the genomic level and the DNAJB1:PRKACA fusion can be used as a diagnostic tool for both pure and mFL-HCC.

The emergence of colistin-resistant Klebsiella pneumoniae (CRKP) is a major public health concern worldwide. In this study, the prevalence and molecular basis of colistin resistance in CRKP isolated from healthy individuals and patients in Lao PDR, Thailand, Nigeria and France were investigated. Stool samples were screened by culture for the presence of colistin-resistant Klebsiella spp. Whole-genome sequence analysis was used to decipher the molecular mechanism of colistin resistance in a blaNDM-1-positive in vitro-selected CRKP mutant. PCR amplification and sequencing of the mgrB genetic environment was performed for all CRKP isolates as well as control colistin-susceptible K. pneumoniae (CSKP) isolates recovered from the same stools. A total of 869 stool samples were screened for colistin-resistant Klebsiella spp., yielding 32 CRKP and 2 colistin-resistant Klebsiella oxytoca. Comparative whole-genome sequence analysis revealed that an in vitro-selected CRKP mutant had an insertion sequence in its mgrB gene, as well as missense mutations in other selected clones. Of the 34 colistin-resistant Klebsiella spp. isolates, 14 (41.2%; 13 CRKP and 1 K. oxytoca) from the four countries also had various defects in their mgrB genes, but no such defects were found in the CSKP controls (P<10(-4)). Few mutations were observed in pmrAB compared with mgrB among the CRKP isolates. The worldwide emergence of CRKP is a major public health concern. Detection and surveillance of such strains are warranted to prevent an uncontrollable pandemic. Inactivation of the PhoP/PhoQ regulator gene mgrB is associated with ≥40% of colistin resistance among the CRKP isolates observed in this study.