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September 22, 2019

Genomic comparison between members of the Salinibacteraceae family, and description of a new species of Salinibacter (Salinibacter altiplanensis sp. nov.) isolated from high altitude hypersaline environments of the Argentinian Altiplano.

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15T=CECT 9105T=IBRC-M 11031T). Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019

Genetic relationships among multidrug-resistant Salmonella enterica serovar Typhimurium strains from humans and animals.

We identified 20 to 22 resistance genes, carried in four incompatibility groups of plasmids, in each of five genetically closely related Salmonella enterica serovar Typhimurium strains recovered from humans, pigs, and chickens. The genes conferred resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim, tetracycline, fluoroquinolones, extended-spectrum cephalosporins and cefoxitin, and azithromycin. This study demonstrates the transmission of multidrug-resistant Salmonella strains among humans and food animals and may be the first identification of mphA in azithromycin-resistant Salmonella strains in Taiwan. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

An improved medium for colistin susceptibility testing.

The plasmid-located colistin resistance gene mcr-1 confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination of mcr-1-containing Enterobacteriaceae Colistin susceptibility testing was performed for 50 mcr-1-containing Escherichia coli and Klebsiella pneumoniae isolates, 7 intrinsically polymyxin-resistant species, K. pneumoniae and E. coli isolates with acquired resistance to polymyxins due to mgrB and pmrB mutations, respectively, and 32 mcr-1-negative, colistin-susceptible isolates of Acinetobacter baumannii, Citrobacter freundii, Enterobacter cloacae, E. coli, K. pneumoniae, and Salmonella enterica serovar Typhimurium. A comparison of the colistin MICs determined in CA-MH medium and those obtained in CE-MH medium was performed using both the BMD and strip-based susceptibility test formats. We validated the data using an isogenic IncX4 plasmid lacking mcr-1 Use of the CE-MH broth provides clear separation between resistant and susceptible isolates in both BMD and gradient diffusion assays; this is true for both mcr-1-containing Enterobacteriaceae isolates and those exhibiting either intrinsic or acquired colistin resistance. CE-MH medium is simple to prepare and overcomes current problems associated with BMD and strip-based colistin susceptibility testing, and use of the medium is easy to implement in routine diagnostic laboratories, even in resource-poor settings. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

New genomic data and analyses challenge the traditional vision of animal epithelium evolution.

The emergence of epithelia was the foundation of metazoan expansion. Epithelial tissues are a hallmark of metazoans deeply rooted in the evolution of their complex developmental morphogenesis processes. However, studies on the epithelial features of non-bilaterians are still sparse and it remains unclear whether the last common metazoan ancestor possessed a fully functional epithelial toolkit or if it was acquired later during metazoan evolution.To investigate the early evolution of animal epithelia, we sequenced the genome and transcriptomes of two new sponge species to characterize epithelial markers such as the E-cadherin complex and the polarity complexes for all classes (Calcarea, Demospongiae, Hexactinellida, Homoscleromorpha) of sponges (phylum Porifera) and compare them with their homologues in Placozoa and in Ctenophora. We found that Placozoa and most sponges possess orthologues of all essential genes encoding proteins characteristic of bilaterian epithelial cells, as well as their conserved interaction domains. In stark contrast, we found that ctenophores lack several major polarity complex components such as the Crumbs complex and Scribble. Furthermore, the E-cadherin ctenophore orthologue exhibits a divergent cytoplasmic domain making it unlikely to interact with its canonical cytoplasmic partners.These unexpected findings challenge the current evolutionary paradigm on the emergence of epithelia. Altogether, our results raise doubt on the homology of protein complexes and structures involved in cell polarity and adhesive-type junctions between Ctenophora and Bilateria epithelia.

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September 22, 2019

Genome-wide analysis of Mycoplasma bovirhinis GS01 reveals potential virulence factors and phylogenetic relationships.

Mycoplasma bovirhinis is a significant etiology in bovine pneumonia and mastitis, but our knowledge about the genetic and pathogenic mechanisms of M. bovirhinis is very limited. In this study, we sequenced the complete genome of M. bovirhinis strain GS01 isolated from the nasal swab of pneumonic calves in Gansu, China, and we found that its genome forms a 847,985 bp single circular chromosome with a GC content of 27.57% and with 707 protein-coding genes. The putative virulence determinants of M. bovirhinis were then analyzed. Results showed that three genomic islands and 16 putative virulence genes, including one adhesion gene enolase, seven surface lipoproteins, proteins involved in glycerol metabolism, and cation transporters, might be potential virulence factors. Glycerol and pyruvate metabolic pathways were defective. Comparative analysis revealed remarkable genome variations between GS01 and a recently reported HAZ141_2 strain, and extremely low homology with others mycoplasma species. Phylogenetic analysis demonstrated that M. bovirhinis was most genetically close to M. canis, distant from other bovine Mycoplasma species. Genomic dissection may provide useful information on the pathogenic mechanisms and genetics of M. bovirhinis. Copyright © 2018 Chen et al.

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September 22, 2019

The FBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia.

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages FC31 and FBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the FBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on FBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a FBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that FBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

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September 22, 2019

A whole genome assembly of the horn fly, Haematobia irritans, and prediction of genes with roles in metabolism and sex determination.

Haematobia irritans, commonly known as the horn fly, is a globally distributed blood-feeding pest of cattle that is responsible for significant economic losses to cattle producers. Chemical insecticides are the primary means for controlling this pest but problems with insecticide resistance have become common in the horn fly. To provide a foundation for identification of genomic loci for insecticide resistance and for discovery of new control technology, we report the sequencing, assembly, and annotation of the horn fly genome. The assembled genome is 1.14 Gb, comprising 76,616 scaffolds with N50 scaffold length of 23 Kb. Using RNA-Seq data, we have predicted 34,413 gene models of which 19,185 have been assigned functional annotations. Comparative genomics analysis with the Dipteran flies Musca domestica L., Drosophila melanogaster, and Lucilia cuprina, show that the horn fly is most closely related to M. domestica, sharing 8,748 orthologous clusters followed by D. melanogaster and L. cuprina, sharing 7,582 and 7,490 orthologous clusters respectively. We also identified a gene locus for the sodium channel protein in which mutations have been previously reported that confers target site resistance to the most common class of pesticides used in fly control. Additionally, we identified 276 genomic loci encoding members of metabolic enzyme gene families such as cytochrome P450s, esterases and glutathione S-transferases, and several genes orthologous to sex determination pathway genes in other Dipteran species. Copyright © 2018 Konganti et al.

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September 22, 2019

Convergent loss of ABC transporter genes from Clostridioides difficile genomes is associated with impaired tyrosine uptake and p-cresol production.

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile, with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile, we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291.

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September 22, 2019

Isolation, functional characterization and transmissibility of p3PS10, a multidrug resistance plasmid of the fish pathogen Piscirickettsia salmonis.

Antibiotic resistance is a major public health concern due to its association with the loss of efficacy of antimicrobial therapies. Horizontal transfer events may play a significant role in the dissemination of resistant bacterial phenotypes, being mobilizable plasmids a well-known mechanism. In this study, we aimed to gain insights into the genetics underlying the development of antibiotic resistance by Piscirickettsia salmonis isolates, a bacterial fish pathogen and causative agent of salmonid piscirickettsiosis, and the main target of antibiotics used in Chilean salmon farming. We provide experimental evidence that the plasmid p3PS10, which harbors multidrug resistance genes for chloramphenicol (cat2), tetracyclines [tet(31)], aminoglycosides (sat1 and aadA1), and sulfonamides (sul2), is carried by a group of P. salmonis isolates exhibiting a markedly reduced susceptibility to oxytetracycline in vitro (128-256 µg/mL of minimal inhibitory concentration, MIC). Antibiotic susceptibility analysis extended to those antibiotics showed that MIC of chloramphenicol, streptomycin, and sulfamethoxazole/trimethoprim were high, but the MIC of florfenicol remained at the wild-type level. By means of molecular cloning, we demonstrate that those genes encoding putative resistance markers are indeed functional. Interestingly, mating assays clearly show that p3PS10 is able to be transferred into and replicate in different hosts, thereby conferring phenotypes similar to those found in the original host. According to epidemiological data, this strain is distributed across aquaculture settings in southern Chile and is likely to be responsible for oxytetracycline treatment failures. This work demonstrates that P. salmonis is more versatile than it was thought, capable of horizontally transferring DNA, and probably playing a role as a vector of resistance traits among the seawater bacterial population. However, the low transmission frequency of p3PS10 suggests a negligible chance of resistance markers being spread to human pathogens.

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September 22, 2019

Nucleotide-binding resistance gene signatures in sugar beet, insights from a new reference genome.

Nucleotide-binding (NB-ARC), leucine-rich-repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long-read sequencing, are improving resolution of repeat-rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB-ARC domain as a model, 231 tentative NB-ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB-ARC signatures as well as full-length sequences. The NB-ARC-associated proteins contained NLR resistance gene domains, including TIR, CC, and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB-ARC domains to validated R genes from monocots and eudicots suggested extensive B. vulgaris-specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR-like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic-acid-based method for NLR prediction in non-model plant species. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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September 22, 2019

Multiple large inversions and breakpoint rewiring of gene expression in the evolution of the fire ant social supergene.

Supergenes consist of co-adapted loci that segregate together and are associated with adaptive traits. In the fire ant Solenopsis invicta, two ‘social’ supergene variants regulate differences in colony queen number and other traits. Suppressed recombination in this system is maintained, in part, by a greater than 9 Mb inversion, but the supergene is larger. Has the supergene in S. invicta undergone multiple large inversions? The initial gene content of the inverted allele of a supergene would be the same as that of the wild-type allele. So, how did the inversion increase in frequency? To address these questions, we cloned one extreme breakpoint in the fire ant supergene. In doing so, we found a second large (greater than 800 Kb) rearrangement. Furthermore, we determined the temporal order of the two big inversions based on the translocation pattern of a third small fragment. Because the S. invicta supergene lacks evolutionary strata, our finding of multiple inversions may support an introgression model of the supergene. Finally, we showed that one of the inversions swapped the promoter of a breakpoint-adjacent gene, which might have conferred a selective advantage relative to the non-inverted allele. Our findings provide a rare example of gene alterations arising directly from an inversion event.© 2018 The Author(s).

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September 22, 2019

Co-occurrence of mcr-1 in the chromosome and on an IncHI2 plasmid: persistence of colistin resistance in Escherichia coli.

Two colistin-resistant Escherichia coli strains (FS13Z2S and FS3Z6C) possessing chromosomally encoded mcr-1 isolated from swine were characterised. Whole-genome sequencing revealed that in strain FS13Z2S mcr-1 occurred in triplicate in the chromosome with another copy encoded on a pHNSHP45-2-like IncHI2 plasmid, whereas in strain FS3Z6C only one copy mcr-1 was inserted in the chromosome. It seems likely that the triplication of chromosomal copies of mcr-1 in FS13Z2S is due to intramolecular transposition events via a composite transposon containing an mcr-1 cassette bracketed by two copies of insertion sequence ISApl1, and the pap2 gene at the insertion site was truncated by an IS1294-like element. In plasmid pFS13Z2S and the chromosome of strain FS3Z6C, only a single copy of ISApl1 was present upstream of the mcr-1 cassette. The two strains exhibited similar colistin minimum inhibitory concentrations (MICs) and featured phosphoethanolamine addition to lipid A, without regard to the copy number of mcr-1. The mcr-1-harbouring plasmid was unstable in wild-type strain FS13Z2S and was quickly lost after 7 days of passage on colistin-free Luria-Bertani broth containing 0.5% SDS, but the mcr-1 copies on the chromosome persisted. These results reveal that the single copy of mcr-1 could result in modification of lipopolysaccharide (LPS) and cause colistin resistance in E. coli. Acquisition of multiple copies of mcr-1, especially on the chromosome, would facilitate stable persistence of colistin resistance in the host strain. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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September 22, 2019

Sequence analysis of IncA/C and IncI1 plasmids isolated from multidrug-resistant Salmonella Newport using Single-Molecule Real-Time Sequencing.

Multidrug-resistant (MDR) plasmids play an important role in disseminating antimicrobial resistance genes. To elucidate the antimicrobial resistance gene compositions in A/C incompatibility complex (IncA/C) plasmids carried by animal-derived MDR Salmonella Newport, and to investigate the spread mechanism of IncA/C plasmids, this study characterizes the complete nucleotide sequences of IncA/C plasmids by comparative analysis. Complete nucleotide sequencing of plasmids and chromosomes of six MDR Salmonella Newport strains was performed using PacBio RSII. Open reading frames were assigned using prokaryotic genome annotation pipeline (PGAP). To understand genomic diversity and evolutionary relationships among Salmonella Newport IncA/C plasmids, we included three complete IncA/C plasmid sequences with similar backbones from Salmonella Newport and Escherichia coli: pSN254, pAM04528, and peH4H, and additional 200 draft chromosomes. With the exception of canine isolate CVM22462, which contained an additional IncI1 plasmid, each of the six MDR Salmonella Newport strains contained only the IncA/C plasmid. These IncA/C plasmids (including references) ranged in size from 80.1 (pCVM21538) to 176.5?kb (pSN254) and carried various resistance genes. Resistance genes floR, tetA, tetR, strA, strB, sul, and mer were identified in all IncA/C plasmids. Additionally, blaCMY-2 and sugE were present in all IncA/C plasmids, excepting pCVM21538. Plasmid pCVM22462 was capable of being transferred by conjugation. The IncI1 plasmid pCVM22462b in CVM22462 carried blaCMY-2 and sugE. Our data showed that MDR Salmonella Newport strains carrying similar IncA/C plasmids clustered together in the phylogenetic tree using chromosome sequences and the IncA/C plasmids from animal-derived Salmonella Newport contained diverse resistance genes. In the current study, we analyzed genomic diversities and phylogenetic relationships among MDR Salmonella Newport using complete plasmids and chromosome sequences and provided possible spread mechanism of IncA/C plasmids in Salmonella Newport Lineage II.

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September 22, 2019

Genetic diversity of Cryptosporidium hominis in a Bangladeshi community as revealed by whole genome sequencing.

We studied the genetic diversity of Cryptosporidium hominis infections in slum-dwelling infants from Dhaka over a 2-year period. Cryptosporidium hominis infections were common during the monsoon, and were genetically diverse as measured by gp60 genotyping and whole-genome resequencing. Recombination in the parasite was evidenced by the decay of linkage disequilibrium in the genome over <300 bp. Regions of the genome with high levels of polymorphism were also identified. Yet to be determined is if genomic diversity is responsible in part for the high rate of reinfection, seasonality, and varied clinical presentations of cryptosporidiosis in this population.

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September 22, 2019

Mycobacterial biomaterials and resources for researchers.

There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.

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