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September 22, 2019

Insights into the microbiota of Asian seabass (Lates calcarifer) with tenacibaculosis symptoms and description of sp. nov. Tenacibaculum singaporense

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the Bacteroidetes genus Tenacibaculum, most notably T. maritimum. The impact of tenacibaculosis on fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissue types of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farm fish. The microbiota of diseased farm fish was dominated by Proteobacteria (relative abundancetextpmstandard deviation, 74.5%textpm22.8%) and Bacteroidetes (18.07%textpm21.7%), the latter mainly comprised by a high abundance of Tenacibaculum species (17.6%textpm20.7%). In healthy seabass Proteobacteria had also highest relative abundance (48.04%textpm0.02%), but Firmicutes (34.2%textpm0.02%) and Fusobacteria (12.0%textpm0.03%) were the next two major constituents. Experimentally infected fish developed lesions characteristic for tenacibaculosis, but the microbiota was primarily dominated by Proteobacteria (90.4%textpm0.2%) and Firmicutes (6.2%textpm0.1%). The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in the commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One strain was isolated and is described here as sp. nov. Tenacibaculum singaporense TLL-A1T (=DSM 106434T, KCTC 62393T). The genome of T. singaporense was sequenced and compared to those of T. maritimum DSM 17995T and the newly sequenced T. mesophilum DSM 13764T.

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September 22, 2019

Genomic Tandem Quadruplication is Associated with Ketoconazole Resistance in Malassezia pachydermatis.

Malassezia pachydermatis is a commensal yeast found on the skin of dogs. However, M. pachydermatis is also considered an opportunistic pathogen and is associated with various canine skin diseases including otitis externa and atopic dermatitis, which usually require treatment using an azole antifungal drug, such as ketoconazole. In this study, we isolated a ketoconazole-resistant strain of M. pachydermatis, designated “KCTC 27587,” from the external ear canal of a dog with otitis externa and analyzed its resistance mechanism. To understand the mechanism underlying ketoconazole resistance of the clinical isolate M. pachydermatis KCTC 27587, the whole genome of the yeast was sequenced using the PacBio platform and was compared with M. pachydermatis type strain CBS 1879. We found that a ~84-kb region in chromosome 4 of M. pachydermatis KCTC 27587 was tandemly quadruplicated. The quadruplicated region contains 52 protein coding genes, including the homologs of ERG4 and ERG11, whose overexpression is known to be associated with azole resistance. Our data suggest that the quadruplication of the ~84-kb region may be the cause of the ketoconazole resistance in M. pachydermatis KCTC 27587.

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September 22, 2019

Emergence of pathogenic and multiple-antibiotic-resistant Macrococcus caseolyticus in commercial broiler chickens.

Macrococcus caseolyticus is generally considered to be a non-pathogenic bacterium that does not cause human or animal diseases. However, recently, a strain of M. caseolyticus (SDLY strain) that causes high mortality rates was isolated from commercial broiler chickens in China. The main pathological changes caused by SDLY included caseous exudation in cranial cavities, inflammatory infiltration, haemorrhages and multifocal necrosis in various organs. The whole genome of the SDLY strain was sequenced and was compared with that of the non-pathogenic JCSC5402 strain of M. caseolyticus. The results showed that the SDLY strain harboured a large quantity of mutations, antibiotic resistance genes and numerous insertions and deletions of virulence genes. In particular, among the inserted genes, there is a cluster of eight connected genes associated with the synthesis of capsular polysaccharide. This cluster encodes a transferase and capsular polysaccharide synthase, promotes the formation of capsules and causes changes in pathogenicity. Electron microscopy revealed a distinct capsule surrounding the SDLY strain. The pathogenicity test showed that the SDLY strain could cause significant clinical symptoms and pathological changes in both SPF chickens and mice. In addition, these clinical symptoms and pathological changes were the same as those observed in field cases. Furthermore, the anti-microbial susceptibility test demonstrated that the SDLY strain exhibits multiple-antibiotic resistance. The emergence of pathogenic M. caseolyticus indicates that more attention should be paid to the effects of this micro-organism on both poultry and public health.© 2018 Blackwell Verlag GmbH.

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September 22, 2019

Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio.

Companion animals are likely relevant in the transmission of antimicrobial-resistant bacteria. Enterobacter xiangfangensis sequence type 171 (ST171), a clone that has been implicated in clusters of infections in humans, was isolated from two dogs with clinical disease in Ohio. The canine isolates contained IncHI2 plasmids encoding blaKPC-4 Whole-genome sequencing was used to put the canine isolates in phylogenetic context with available human ST171 sequences, as well as to characterize their blaKPC-4 plasmids. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing.

Inherent sources of error and bias that affect the quality of sequence data include index hopping and bias towards the reference allele. The impact of these artefacts is likely greater for low-coverage data than for high-coverage data because low-coverage data has scant information and many standard tools for processing sequence data were designed for high-coverage data. With the proliferation of cost-effective low-coverage sequencing, there is a need to understand the impact of these errors and bias on resulting genotype calls from low-coverage sequencing.We used a dataset of 26 pigs sequenced both at 2× with multiplexing and at 30× without multiplexing to show that index hopping and bias towards the reference allele due to alignment had little impact on genotype calls. However, pruning of alternative haplotypes supported by a number of reads below a predefined threshold, which is a default and desired step of some variant callers for removing potential sequencing errors in high-coverage data, introduced an unexpected bias towards the reference allele when applied to low-coverage sequence data. This bias reduced best-guess genotype concordance of low-coverage sequence data by 19.0 absolute percentage points.We propose a simple pipeline to correct the preferential bias towards the reference allele that can occur during variant discovery and we recommend that users of low-coverage sequence data be wary of unexpected biases that may be produced by bioinformatic tools that were designed for high-coverage sequence data.

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September 22, 2019

Quaternary ammonium compounds with multiple cationic moieties (multiQACs) provide antimicrobial activity against Campylobacter jejuni

Recently developed quaternary ammonium compounds (QACs) possessing multiple cationic moieties, referred to as multiQACs, were tested with strains of Campylobacter jejuni to determine their potential as antimicrobial compounds against this important foodborne pathogen. Eight multiQACs were tested against a cocktail of six C. jejuni strains isolated from environmental and clinical sources. The resulting reductions in C. jejuni numbers mediated by the multiQACs were compared to the reductions produced by the application of four commercially available QACs, each of which bears a single cation. Multiple concentrations and exposure times were utilized for all compounds. The compounds which yielded the maximum C. jejuni reductions at the lowest concentrations and applied over the shortest exposure times were judged to be the most successful. Of the eight multiQACs investigated, four demonstrated reductions in C. jejuni numbers superior to the commercial QACs; these four are biscationic, and two of them bear an additional uncharged nitrogen atom. The remaining four multiQACs, which contain three or four cations, did not produce reductions in bacterial numbers comparable to commercial QACs in the timeframes tested. At the intermediary compound concentration (0.05?mM) and exposure time (5?min) the most effective multiQACs (PQ-12,12 and 12(3)0(3)12) on average killed over 99% of the Campylobacter cells present while the best commercial compound at those parameters (cetyl pyridinium chloride, CPC) only killed on average 84.56% of the Campylobacter cells. At the highest compound concentration tested (0.1?mM) and shortest exposure time (1?min), the same two biscationic multiQACs averaged mean percent reductions of Campylobacter cell numbers around 99.5% while CPC at the same concentration/exposure only managed a percent reduction of 91.3%. The biscationic multiQACs demonstrate the potential for providing a new group of antimicrobial compounds superior to current commercially available QACs in their effectiveness against C. jejuni.

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September 22, 2019

Novel linezolid resistance plasmids in Enterococcus from food animals in the USA.

To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme.Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible.Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3′)-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant.To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.

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September 22, 2019

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

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September 22, 2019

Hypervirulent group A Streptococcus emergence in an acaspular background is associated with marked remodeling of the bacterial cell surface

Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated–a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.

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September 22, 2019

Evolutionary conservation of Y Chromosome ampliconic gene families despite extensive structural variation.

Despite claims that the mammalian Y Chromosome is on a path to extinction, comparative sequence analysis of primate Y Chromosomes has shown the decay of the ancestral single-copy genes has all but ceased in this eutherian lineage. The suite of single-copy Y-linked genes is highly conserved among the majority of eutherian Y Chromosomes due to strong purifying selection to retain dosage-sensitive genes. In contrast, the ampliconic regions of the Y Chromosome, which contain testis-specific genes that encode the majority of the transcripts on eutherian Y Chromosomes, are rapidly evolving and are thought to undergo species-specific turnover. However, ampliconic genes are known from only a handful of species, limiting insights into their long-term evolutionary dynamics. We used a clone-based sequencing approach employing both long- and short-read sequencing technologies to assemble ~2.4 Mb of representative ampliconic sequence dispersed across the domestic cat Y Chromosome, and identified the major ampliconic gene families and repeat units. We analyzed fluorescence in situ hybridization, qPCR, and whole-genome sequence data from 20 cat species and revealed that ampliconic gene families are conserved across the cat family Felidae but show high transcript diversity, copy number variation, and structural rearrangement. Our analysis of ampliconic gene evolution unveils a complex pattern of long-term gene content stability despite extensive structural variation on a nonrecombining background.© 2018 Brashear et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Emerging multidrug-resistant hybrid pathotype shiga toxin-producing Escherichia coli O80 and related strains of clonal complex 165, Europe.

Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-?, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum ß-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.

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September 22, 2019

A novel probiotic, Lactobacillus johnsonii 456, resists acid and can persist in the human gut beyond the initial ingestion period.

Probiotics are considered to have multiple beneficial effects on the human gastrointestinal tract, including immunomodulation, pathogen inhibition, and improved host nutrient metabolism. However, extensive characterization of these properties is needed to define suitable clinical applications for probiotic candidates. Lactobacillus johnsonii 456 (LBJ 456) was previously demonstrated to have anti-inflammatory and anti-genotoxic effects in a mouse model. Here, we characterize its resistance to gastric and bile acids as well as its ability to inhibit gut pathogens and adhere to host mucosa. While bile resistance and in vitro host attachment properties of LBJ 456 were comparable to other tested probiotics, LBJ 456 maintained higher viability at lower pH conditions compared to other tested strains. LBJ 456 also altered pathogen adhesion to LS 174T monolayers and demonstrated contact-dependent and independent inhibition of pathogen growth. Genome analyses further revealed possible genetic elements involved in host attachment and pathogen inhibition. Importantly, we show that ingestion of Lactobacillus johnsonii 456 over a one week yogurt course leads to persistent viable bacteria detectable even beyond the period of initial ingestion, unlike many other previously described probiotic species of lactic acid bacteria.

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September 22, 2019

Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains.

The recent discovery of the plasmid-mediated mcr-1 gene conferring resistance to colistin is of clinical concern. The worldwide screening of this resistance mechanism among samples of different origins has highlighted the urgent need to improve the detection of colistin-resistant isolates in clinical microbiology laboratories. Currently, phenotypic methods used to detect colistin resistance are not necessarily suitable as the main characteristic of the mcr genes is the low level of resistance that they confer, close to the clinical breakpoint recommended jointly by the CLSI and EUCAST expert systems (S?=?2?mg/L and R?>?2?mg/L). In this context, susceptibility testing recommendations for polymyxins have evolved and are becoming difficult to implement in routine laboratory work. The large number of mechanisms and genes involved in colistin resistance limits the access to rapid detection by molecular biology. It is therefore necessary to implement well-defined protocols using specific tools to detect all colistin-resistant bacteria. This review aims to summarize the current clinical microbiology diagnosis techniques and their ability to detect all colistin resistance mechanisms and describe new tools specifically developed to assess plasmid-mediated colistin resistance. Phenotyping, susceptibility testing, and genotyping methods are presented, including an update on recent studies related to the development of specific techniques.

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September 22, 2019

Mutators as drivers of adaptation in Streptococcus and a risk factor for host jumps and vaccine escape

Heritable hypermutable strains deficient in DNA repair genes (mutators) facilitate microbial adaptation as they may rapidly generate beneficial mutations. Mutators deficient in mismatch (MMR) and oxidised guanine (OG) repair are abundant in clinical samples and show increased adaptive potential in experimental infection models but their role in pathoadaptation is poorly understood. Here we investigate the role of mutators in epidemiology and evolution of the broad host pathogen, Streptococcus iniae, employing 80 strains isolated globally over 40 years. We determine phylogenetic relationship among S. iniae using 10,267 non-recombinant core genome single nucleotide polymorphisms (SNPs), estimate their mutation rate by fluctuation analysis, and detect variation in major MMR (mutS, mutL, dnaN, recD2, rnhC) and OG (mutY, mutM, mutX) genes. S. iniae mutation rate phenotype and genotype are strongly associated with phylogenetic diversification and variation in major streptococcal virulence determinants (capsular polysaccharide, hemolysin, cell chain length, resistance to oxidation, and biofilm formation). Furthermore, profound changes in virulence determinants observed in mammalian isolates (atypical host) and vaccine-escape isolates found in bone (atypical tissue) of vaccinated barramundi are linked to multiple MMR and OG variants and unique mutation rates. This implies that adaptation to new host taxa, new host tissue, and to immunity of a vaccinated host is promoted by mutator strains. Our findings support the importance of mutation rate dynamics in evolution of pathogenic bacteria, in particular adaptation to a drastically different immunological setting that occurs during host jump and vaccine escape events.Importance Host immune response is a powerful selective pressure that drives diversification of pathogenic microorganisms and, ultimately, evolution of new strains. Major adaptive events in pathogen evolution, such as transmission to a new host species or infection of vaccinated hosts, require adaptation to a drastically different immune landscape. Such adaptation may be favoured by hypermutable strains (or mutators) that are defective in normal DNA repair and consequently capable of generating multiple potentially beneficial and compensatory mutations. This permits rapid adjustment of virulence and antigenicity in a new immunological setting. Here we show that mutators, through mutations in DNA repair genes and corresponding shifts in mutation rate, are associated with major diversification events and virulence evolution in the broad host-range pathogen Streptococcus iniae. We show that mutators underpin infection of vaccinated hosts, transmission to new host species and the evolution of new strains.

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September 21, 2019

Complete genome sequence of the type strain of Macrococcus canis.

The first complete genome sequence of the recently describedMacrococcus canisspecies has been determined for the strain KM45013T(=DSM 101690T= CCOS 969T= CCUG 68920T= CCM 8748T). The strain was isolated from a dog with rhinitis and contains a putative ?-hemolysin and amecB-carrying staphylococcal cassette chromosomemecelement (SCCmecKM45013). Copyright © 2018 Gobeli Brawand et al.

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