Transcriptomic analysis of gill and kidney from Asian seabass (Lates calcarifer) acclimated to different salinities reveals pathways involved with euryhalinity

Asian seabass (or commonly known as barramundi), Lates calcarifer, is a bony euryhaline teleost from the Family Latidae, inhabiting nearshore, estuarine, and marine connected freshwaters throughout the tropical Indo-West Pacific region. The species is catadromous, whereby adults spawn in salinities between 28 and 34 ppt at the mouth of estuaries, with resultant juveniles usually moving into brackish and freshwater systems to mature, before returning to the sea to spawn again as adults. The species lives in both marine and freshwater habitats and can move quickly between the two; thus, the species’ ability to tolerate changes in salinity makes it a good candidate for studying the salinity acclimation response in teleosts. In this study, the transcriptome of two major osmoregulatory organs (gills and kidneys) of young juvenile Asian seabass reared in freshwater and seawater were compared. The euryhaline nature of Asian seabass was found to be highly pliable and the moldability of the trait was further confirmed by histological analyses of gills and kidneys. Differences in major expression pathways were observed, with differentially expressed genes including those related to osmoregulation, tissue/organ morphogenesis, and cell volume regulation as central to the osmo-adaptive response. Additionally, genes coding for mucins were upregulated specifically under saline conditions, whereas several genes important for growth and development, as well as circadian entrainment were specifically enriched in fish reared in freshwater. Routing of the circadian rhythm mediated by salinity changes could be the initial step in salinity acclimation and possibly migration in euryhaline fish species such as the Asian seabas

BAC-pool sequencing and analysis confirms growth-associated QTLs in the Asian seabass genome

The Asian seabass is an important marine food fish that has been cultured for several decades in Asia Pacific. However, the lack of a high quality reference genome has hampered efforts to improve its selective breeding. A 3D BAC pool set generated in this study was screened using 22 SSR markers located on linkage group 2 which contains a growth-related QTL region. Seventy-two clones corresponding to 22 FPC contigs were sequenced by Illumina MiSeq technology. We co-assembled the MiSeq-derived scaffolds from each FPC contig with error-corrected PacBio reads, resulting in 187 sequences covering 9.7 Mb. Eleven genes annotated within this region were found to be potentially associated with growth and their tissue-specific expression was investigated. Correlation analysis demonstrated that SNPs in ctsb, skp1 and ppp2ca can be potentially used as markers for selecting fast-growing fingerlings. Conserved syntenies between seabass LG2 and five other teleosts were identified. This study i) provided a 10 Mb targeted genome assembly; ii) demonstrated NGS of BAC pools as a potential approach for mining candidates underlying QTLs of this species; iii) detected eleven genes potentially responsible for growth in the QTL region; and iv) identified useful SNP markers for selective breeding programs of Asian seabass

Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

Transcriptomic analysis of gill and kidney from Asian seabass (Lates calcarifer) acclimated to different salinities reveals pathways involved with euryhalinity

Asian seabass (or commonly known as barramundi), Lates calcarifer, is a bony euryhaline teleost from the Family Latidae, inhabiting nearshore, estuarine, and marine connected freshwaters throughout the tropical Indo-West Pacific region. The species is catadromous, whereby adults spawn in salinities between 28 and 34 ppt at the mouth of estuaries, with resultant juveniles usually moving into brackish and freshwater systems to mature, before returning to the sea to spawn again as adults. The species lives in both marine and freshwater habitats and can move quickly between the two; thus, the species’ ability to tolerate changes in salinity makes it a good candidate for studying the salinity acclimation response in teleosts. In this study, the transcriptome of two major osmoregulatory organs (gills and kidneys) of young juvenile Asian seabass reared in freshwater and seawater were compared. The euryhaline nature of Asian seabass was found to be highly pliable and the moldability of the trait was further confirmed by histological analyses of gills and kidneys. Differences in major expression pathways were observed, with differentially expressed genes including those related to osmoregulation, tissue/organ morphogenesis, and cell volume regulation as central to the osmo-adaptive response. Additionally, genes coding for mucins were upregulated specifically under saline conditions, whereas several genes important for growth and development, as well as circadian entrainment were specifically enriched in fish reared in freshwater. Routing of the circadian rhythm mediated by salinity changes could be the initial step in salinity acclimation and possibly migration in euryhaline fish species such as the Asian seabass

Mapping of a major QTL for increased robustness and detection of genome assembly errors in Asian seabass (Lates calcarifer)

Abstract Background For Asian seabass (Lates calcarifer, Bloch 1790) cultured at sea cages various aquatic pathogens, complex environmental and stress factors are considered as leading causes of disease, causing tens of millions of dollars of annual economic losses. Over the years, we conducted farm-based challenges by exposing Asian seabass juveniles to complex natural environmental conditions. In one of these challenges, we collected a total of 1,250 fish classified as either ‘sensitive’ or ‘robust’ individuals during the 28-day observation period. Results We constructed a high-resolution linkage map with 3,089 SNPs for Asian seabass using the double digest Restriction-site Associated DNA (ddRAD) technology and a performed a search for Quantitative Trait Loci (QTL) associated with robustness. The search detected a major genome-wide significant QTL for increased robustness in pathogen-infected marine environment on linkage group 11 (ASB_LG11; 88.9 cM to 93.6 cM) with phenotypic variation explained of 81.0%. The QTL was positioned within a > 800 kb genomic region located at the tip of chromosome ASB_LG11 with two Single Nucleotide Polymorphism markers, R1-38468 and R1-61252, located near to the two ends of the QTL. When the R1-61252 marker was validated experimentally in a different mass cross population, it showed a statistically significant association with increased robustness. The majority of thirty-six potential candidate genes located within the QTL have known functions related to innate immunity, stress response or disease. By utilizing this ddRAD-based map, we detected five mis-assemblies corresponding to four chromosomes, namely ASB_LG8, ASB_LG9, ASB_LG15 and ASB_LG20, in the current Asian seabass reference genome assembly. Conclusion According to our knowledge, the QTL associated with increased robustness is the first such finding from a tropical fish species. Depending on further validation in other stocks and populations, it might be potentially useful for selecting robust Asian seabass lines in selection programs

B Chromosomes of the Asian Seabass (Lates calcarifer) Contribute to Genome Variations at the Level of Individuals and Populations

The Asian seabass (Lates calcarifer) is a bony fish from the Latidae family, which is widely distributed in the tropical Indo-West Pacific region. The karyotype of the Asian seabass contains 24 pairs of A chromosomes and a variable number of AT- and GC-rich B chromosomes (Bchrs or Bs). Dot-like shaped and nucleolus-associated AT-rich Bs were microdissected and sequenced earlier. Here we analyzed DNA fragments from Bs to determine their repeat and gene contents using the Asian seabass genome as a reference. Fragments of 75 genes, including an 18S rRNA gene, were found in the Bs; repeats represented 2% of the Bchr assembly. The 18S rDNA of the standard genome and Bs were similar and enriched with fragments of transposable elements. A higher nuclei DNA content in the male gonad and somatic tissue, compared to the female gonad, was demonstrated by flow cytometry. This variation in DNA content could be associated with the intra-individual variation in the number of Bs. A comparison between the copy number variation among the B-related fragments from whole genome resequencing data of Asian seabass individuals identified similar profiles between those from the South-East Asian/Philippines and Indian region but not the Australian ones. Our results suggest that Bs might cause variations in the genome among the individuals and populations of Asian seabass. A personalized copy number approach for segmental duplication detection offers a suitable tool for population-level analysis across specimens with low coverage genome sequencing

Dissemination of Pseudomonas aeruginosa blaNDM-1-positive ST308 clone in Singapore

Pseudomonas aeruginosa ST308 clone has been reported to carry carbapenemase genes such as blaIMP and blaVIM but has been rarely associated with blaNDM-1. A total of 199 P. aeruginosa ST308 clinical and environmental isolates obtained between April 2019 and November 2020 from a tertiary-care hospital in Singapore were characterized using whole-genome sequencing. In addition, 71 blaNDM-1-positive ST308 whole-genome sequences from two other local tertiary-care hospitals in Singapore and 83 global blaNDM-1-negative ST308 whole-genome sequences in public databases were included to assess phylogenetic relationships and perform genome analyses. Phylogenetic analysis and divergent time estimation revealed that blaNDM-1-positive P. aeruginosa ST308 was introduced into Singapore in 2005 (95 % highest posterior density: 2001 to 2008). Core genome, resistome, and analyses of all local blaNDM-1-positive ST308 isolates showed chromosomal integration of multiple antibiotic resistance genes (ARGs) [aac(3)-Id, aac(6')-Il, aadA6, aadA11, dfrB5, msr(E), floR, sul2, and qnrVC1], which was absent in global blaNDM-1-negative ST308 sequences. Most ARGs and virulence genes were conserved across isolates originating from the three different local hospitals. Close genetic relatedness of the blaNDM-1-positive ST308 clinical and environmental isolates suggests cocirculation between the hospital environment and human hosts with the hospital environment as a potential reservoir. Core genome single nucleotide polymorphism analyses revealed possible clonal transmission of blaNDM-1-positive ST308 isolates between the three hospitals over 7 years. Bloodstream isolates accounted for six of 95 (6.3%) clinical isolates. This study reports the introduction of a pathogenic blaNDM-1-positive P. aeruginosa ST308 more than a decade ago in Singapore and warrants surveillance for wider dissemination.National Medical Research Council (NMRC)Published versionThis research is supported by the Singapore Ministry of Health's (MOH) National Medical Research Council (NMRC) under its NMRC Collaborative Grant: Collaborative Solutions Targeting Antimicrobial Resistance Threats in Health Systems (CoSTAR-HS) (NMRC CG21APR2005), NMRC Clinician Scientist Award (MOH-000276), and NMRC Clinician Scientist Individual Research Grant (MOH-CIRG18Nov-0034). Additional support was provided by the German Federal Ministry of Health (BMG) COVID-19 Research and Development Funding to the World Health Organization (WHO; award 70826)

Acquisition of plasmid with carbapenem-resistance gene blaKPC2 in hypervirulent Klebsiella pneumoniae, Singapore

The convergence of carbapenem-resistance and hypervirulence genes in Klebsiella pneumoniae has led to the emergence of highly drug-resistant superbugs capable of causing invasive disease. We analyzed 556 carbapenem-resistant K. pneumoniae isolates from patients in Singapore hospitals during 2010-2015 and discovered 18 isolates from 7 patients also harbored hypervirulence features. All isolates contained a closely related plasmid (pKPC2) harboring blaKPC-2, a K. pneumoniae carbapenemase gene, and had a hypervirulent background of capsular serotypes K1, K2, and K20. In total, 5 of 7 first patient isolates were hypermucoviscous, and 6 were virulent in mice. The pKPC2 was highly transmissible and remarkably stable, maintained in bacteria within a patient with few changes for months in the absence of antimicrobial drug selection pressure. Intrapatient isolates were also able to acquire additional antimicrobial drug resistance genes when inside human bodies. Our results highlight the potential spread of carbapenem-resistant hypervirulent K. pneumoniae in Singapore.National Medical Research Council (NMRC)Published versionThis work was supported by the National University of Singapore, Yong Loo Lin School of Medicine Aspiration Fund (NUHSRO/2014/068/AF New Idea/03), and National Medical Research Council (NMRC) collaborative grant Collaborative Solutions Targeting Antimicrobial Resistance Threats in Health Systems (CoSTAR-HS, HS/ARGSeedGrant/2017/03) to Y.-H.G. Additional grant support was provided by CoSTAR-HS (NMRC CGAug16C005), NMRC Clinician Scientist Award (NMRC/CSA-INV/0007/2016), and NMRC Clinician Scientist Award (MOH-000276)

Evaluation of NG-Test CARBA 5 version 2, Cepheid Xpert Carba-R, and carbapenem inactivation methods in comparison to whole-genome sequencing for the identification of carbapenemases in non-fermenting Gram-negative bacilli

NG-Test CARBA 5 (NG-Biotech) is a rapid in vitro multiplex immunoassay for the phenotypic detection and differentiation of the "big five" carbapenemase families (KPC, OXA-48-like, VIM, IMP, and NDM). Version 2 of this assay was evaluated alongside the Xpert Carba-R assay (Cepheid, Inc.), the modified carbapenem inactivation method (mCIM), and the CIMTris assay, with a collection of carbapenem-resistant non-fermenting Gram-negative bacilli comprising 138 Pseudomonas aeruginosa and 97 Acinetobacter baumannii isolates. Whole-genome sequencing (WGS) was used as the reference standard. For P. aeruginosa, NG-Test CARBA 5 produced an overall percentage agreement (OPA) with WGS of 97.1%, compared with 92.8% forXpert Carba-R and 90.6% for mCIM. For A. baumannii, as OXA-type carbapenemases (non-OXA-48) are not included, both the NG-Test CARBA 5 and Xpert Carba-R only had an OPA of 6.2%, while the CIMTris performed well with an OPA of 99.0%. The majority of A. baumannii isolates (95.9%) tested falsely positive for IMP on NG-Test CARBA 5; no IMP genes were found on WGS. No clear cause was found for this phenomenon; a cross-reacting protein antigen unique to A. baumannii is a possible culprit. NG-Test CARBA 5 performed well for carbapenemase detection in P. aeruginosa. However, results from A. baumannii isolates should be interpreted with caution.Ministry of Health (MOH)National Medical Research Council (NMRC)Published versionThis work was supported by the Ministry of Health, Singapore, under the FY16 Health Service Development Programme (HSDP) Project 19N01: "Reducing the spread of carbapenemase producing Gram-negative bacteria via rapid and direct detection from surveillance and clinical samples" (S.V.), as well as the National Medical Research Council (NMRC) Centre Grant: “Collaborative Solutions Targeting Antimicrobial Resistance Threats in Health Systems (CoSTAR-HS)" (CG21APR2005), NMRC Clinician Scientist Award (MOH-000276), NMRC Open Fund—Large Collaborative Grant: AntiMicrobial resistance Research & Intervention Alliance Singapore (AMRITAS; MOH-001326-01) (O.T.N.), and NMRC Clinician Scientist Individual Research Grant (CIRG18Nov-0034) (K.M.)

Whole genome sequencing reveals hidden transmission of carbapenemase-producing Enterobacterales

10.1038/s41467-022-30637-5NATURE COMMUNICATIONS131complete