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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

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in Borrelia burgdorferi.

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease, Borrelia burgdorferi B31, possesses two RM systems with N6-methyladenosine (m6A) MTase activity, which are encoded by the bbe02 gene located on linear plasmid lp25 and bbq67 on lp56. The specific recognition and/or methylation sequences had not been identified for either of these B. burgdorferi MTases, and it was not previously known whether these RM systems influence transcript levels. In the current study, single-molecule real-time sequencing was utilized to map genome-wide m6A sites and to identify consensus modified motifs in wild-type B. burgdorferi as well as MTase mutants lacking either the bbe02 gene alone or both bbe02 and bbq67 genes. Four novel conserved m6A motifs were identified and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by the RM systems has effects on gene expression. Genes with altered transcription in MTase mutants include those involved in vertebrate host colonization (e.g., rpoS regulon) and acquisition by/transmission from the tick vector (e.g., rrp1 and pdeB). The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria.IMPORTANCE Lyme disease is the most prevalent vector-borne disease in North America and is classified by the Centers for Disease Control and Prevention (CDC) as an emerging infectious disease with an expanding geographical area of occurrence. Previous studies have shown that the causative bacterium, Borrelia burgdorferi, methylates its genome using restriction modification systems that enable the distinction from foreign DNA. Although much research has focused on the regulation of gene expression in B. burgdorferi, the effect of DNA methylation on gene regulation has not been evaluated. The current study characterizes the patterns of DNA methylation by restriction modification systems in B. burgdorferi and evaluates the resulting effects on gene regulation in this important pathogen. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Emergence of an extensively drug-resistant (XDR) Streptococcus pneumoniae serotype 15A by capsular switching.

Recently, we have identified an extensively drug-resistant (XDR) Streptococcus pneumoniae serotype 15A isolate from a patient with bacterial meningitis. It belonged to sequence type 8279 (ST8279), a clone identified as XDR serotype 11A isolated in South Korea. We obtained and compared the genome sequences of an XDR 15A and an XDR 11A isolate. The genomes of two XDR isolates were highly identical, except for the capsular polysaccharide (cps) locus and another small region. Capsular switching from 11A to 15A may have occurred via recombination of the cps locus. The emergence of a new XDR clone via capsular switching would be a great concern for public health and in clinical settings. Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019

Cloning and characterization of short-chain N-acyl homoserine lactone-producing Enterobacter asburiae strain L1 from lettuce leaves.

In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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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

The changing landscape of vancomycin-resistant Enterococcus faecium in Australia: a population-level genomic study.

Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm.A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid.vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission.Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.

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September 22, 2019

Discovery of the actinoplanic acid pathway in Streptomyces rapamycinicus reveals a genetically conserved synergism with rapamycin.

Actinobacteria possess a great wealth of pathways for production of bioactive compounds. Following advances in genome mining, dozens of natural product (NP) gene clusters are routinely found in each actinobacterial genome; however, the modus operandi of this large arsenal is poorly understood. During investigations of the secondary metabolome of Streptomyces rapamycinicus, the producer of rapamycin, we observed accumulation of two compounds never before reported from this organism. Structural elucidation revealed actinoplanic acid A and its demethyl analogue. Actinoplanic acids (APLs) are potent inhibitors of Ras farnesyltransferase and therefore represent bioactive compounds of medicinal interest. Supported with the unique structure of these polyketides and using genome mining, we identified a gene cluster responsible for their biosynthesis in S. rapamycinicus Based on experimental evidence and genetic organization of the cluster, we propose a stepwise biosynthesis of APL, the first bacterial example of a pathway incorporating the rare tricarballylic moiety into an NP. Although phylogenetically distant, the pathway shares some of the biosynthetic principles with the mycotoxins fumonisins. Namely, the core polyketide is acylated with the tricarballylate by an atypical nonribosomal peptide synthetase-catalyzed ester formation. Finally, motivated by the conserved colocalization of the rapamycin and APL pathway clusters in S. rapamycinicus and all other rapamycin-producing actinobacteria, we confirmed a strong synergism of these compounds in antifungal assays. Mining for such evolutionarily conserved coharboring of pathways would likely reveal further examples of NP sets, attacking multiple targets on the same foe. These could then serve as a guide for development of new combination therapies.© 2018 Mrak et al.

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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

Staying alive: growth and survival of Bifidobacterium animalis subsp. animalis under in vitro and in vivo conditions.

Members of the Bifidobacterium genus are widely used as probiotics in fermented milk products. Bifidobacterium animalis subsp. animalis CNCM I-4602 grows and survives poorly in reconstituted skimmed milk (RSM). Availing of genome and transcriptome information, this poor growth and survival phenotype in milk was substantially improved by the addition of certain compounds, such as yeast extract, uric acid, glutathione, cysteine, ferrous sulfate, and a combination of magnesium sulfate and manganese sulfate. Carbohydrate utilization of CNCM I-4602 was also investigated, allowing the identification of several carbohydrate utilization gene clusters, and highlighting this strain’s inability to utilize lactose, unlike the type strain of this subspecies, B. animalis subsp. animalis ATCC25527 and the B. animalis subsp. lactis subspecies. In addition, the ability of B. animalis subsp. animalis CNCM I-4602 to colonize a murine model was investigated, which showed that this strain persists in the murine gut for a period of at least 4 weeks. Associated in vivo transcriptome analysis revealed that, among other genes, a gene cluster encoding a predicted type IVb tight adherence (Tad) pilus was upregulated, indicating that this extracellular structure plays a role in the colonization/adaptation of the murine gastrointestinal tract by this strain.

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September 22, 2019

Report from the Killer-cell Immunoglobulin-like Receptors (KIR) component of the 17th International HLA and Immunogenetics Workshop.

The goals of the KIR component of the 17th International HLA and Immunogenetics Workshop (IHIW) were to encourage and educate researchers to begin analyzing KIR at allelic resolution, and to survey the nature and extent of KIR allelic diversity across human populations. To represent worldwide diversity, we analyzed 1269 individuals from ten populations, focusing on the most polymorphic KIR genes, which express receptors having three immunoglobulin (Ig)-like domains (KIR3DL1/S1, KIR3DL2 and KIR3DL3). We identified 13 novel alleles of KIR3DL1/S1, 13 of KIR3DL2 and 18 of KIR3DL3. Previously identified alleles, corresponding to 33 alleles of KIR3DL1/S1, 38 of KIR3DL2, and 43 of KIR3DL3, represented over 90% of the observed allele frequencies for these genes. In total we observed 37 KIR3DL1/S1 allotypes, 40 for KIR3DL2 and 44 for KIR3DL3. As KIR allotype diversity can affect NK cell function, this demonstrates potential for high functional diversity worldwide. Allelic variation further diversifies KIR haplotypes. We determined KIR3DL3?~?KIR3DL1/S1?~?KIR3DL2 haplotypes from five of the studied populations, and observed multiple population-specific haplotypes in each. This included 234 distinct haplotypes in European Americans, 191 in Ugandans, 35 in Papuans, 95 in Egyptians and 86 in Spanish populations. For another 35 populations, encompassing 642,105 individuals we focused on KIR3DL2 and identified another 375 novel alleles, with approximately half of them observed in more than one individual. The KIR allelic level data gathered from this project represents the most comprehensive summary of global KIR allelic diversity to date, and continued analysis will improve understanding of KIR allelic polymorphism in global populations. Further, the wealth of new data gathered in the course of this workshop component highlights the value of collaborative, community-based efforts in immunogenetics research, exemplified by the IHIW.Copyright © 2018. Published by Elsevier Inc.

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September 22, 2019

Biparental Inheritance of Mitochondrial DNA in Humans.

Although there has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. Here, we identified three unrelated multigeneration families with a high level of mtDNA heteroplasmy (ranging from 24 to 76%) in a total of 17 individuals. Heteroplasmy of mtDNA was independently examined by high-depth whole mtDNA sequencing analysis in our research laboratory and in two Clinical Laboratory Improvement Amendments and College of American Pathologists-accredited laboratories using multiple approaches. A comprehensive exploration of mtDNA segregation in these families shows biparental mtDNA transmission with an autosomal dominantlike inheritance mode. Our results suggest that, although the central dogma of maternal inheritance of mtDNA remains valid, there are some exceptional cases where paternal mtDNA could be passed to the offspring. Elucidating the molecular mechanism for this unusual mode of inheritance will provide new insights into how mtDNA is passed on from parent to offspring and may even lead to the development of new avenues for the therapeutic treatment for pathogenic mtDNA transmission.

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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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