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July 7, 2019

Species- and strain-specific adaptation of the HSP70 super family in pathogenic trypanosomatids.

All eukaryotic genomes encode multiple members of the heat shock protein 70 (HSP70) family, which evolved distinctive structural and functional features in response to specific environmental constraints. Phylogenetic analysis of this protein family thus can inform on genetic and molecular mechanisms that drive species-specific environmental adaptation. Here we use the eukaryotic pathogen Leishmania spp. as a model system to investigate the evolution of the HSP70 protein family in an early-branching eukaryote that is prone to gene amplification and adapts to cytotoxic host environments by stress-induced and chaperone-dependent stage differentiation. Combining phylogenetic and comparative analyses of trypanosomatid genomes, draft genome of Paratrypanosoma and recently published genome sequences of 204 L. donovani field isolates, we gained unique insight into the evolutionary dynamics of the Leishmania HSP70 protein family. We provide evidence for (i) significant evolutionary expansion of this protein family in Leishmania through gene amplification and functional specialization of highly conserved canonical HSP70 members, (ii) evolution of trypanosomatid-specific, non-canonical family members that likely gained ATPase-independent functions, and (iii) loss of one atypical HSP70 member in the Trypanosoma genus. Finally, we reveal considerable copy number variation of canonical cytoplasmic HSP70 in highly related L. donovani field isolates, thus identifying this locus as a potential hot spot of environment-genotype interaction. Our data draw a complex picture of the genetic history of HSP70 in trypanosomatids that is driven by the remarkable plasticity of the Leishmania genome to undergo massive intra-chromosomal gene amplification to compensate for the absence of regulated transcriptional control in these parasites. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Complete genome sequence of Lysinibacillus sphaericus WHO reference strain 2362.

Lysinibacillus sphaericus is a species that contains strains widely used in the biological control of mosquitoes. Here, we present the complete 4.67-Mb genome of the WHO entomopathogenic reference strain L. sphaericus 2362, which is probably one of the most commercialized and studied strains. Genes coding for mosquitocidal toxin proteins were detected. Copyright © 2016 Hernández-Santana et al.

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July 7, 2019

Complete genome sequence of Vibrio vulnificus FORC_017 isolated from a patient with a hemorrhagic rash after consuming raw dotted gizzard shad.

Vibrio vulnificus, a resident in the human gut, is frequently found in seafood, causing food-borne illnesses including gastroenteritis and severe septicemia. While V. vulnificus has been known to be one of the major food-borne pathogens, pathogenicity and virulence factors are not fully understood yet. To extend our understanding of the pathogenesis of V. vulnificus at the genomic level, the genome of V. vulnificus FORC_017 isolated from a female patient experiencing a hemorrhagic rash was completely sequenced and analyzed.Three discontinuous contigs were generated from a hybrid assembly using Illumina MiSeq and PacBio platforms, revealing that the genome of the FORC_017 consists of two circular chromosomes and a plasmid. Chromosome I consists of 3,253,417-bp (GC content 46.49 %) containing 2943 predicted open reading frames (ORFs) and chromosome II of 1,905,745-bp (GC content 46.90 %) containing 1638 ORFs. The plasmid pFORC17 consists of 70,069-bp (GC content 43.77 %) containing 84 ORFs. The average nucleotide identity (ANI) value of the FORC_017 and CMCP6 strains was 98.53, suggesting that they are closely related.Pathogenesis-associated genes including vvhA, rtx gene cluster, and various hemolysin genes were present in FORC_017. In addition, three complete secretion systems (Type I, II and VI) as well as iron uptake-related genes for virulence of the FORC_017 were detected, suggesting that this strain is pathogenic. Further comparative genome analysis revealed that FORC_017 and CMCP6 share major toxin genes including vvhA and rtx for pathogenesis activities. The genome information of the FORC_017 provides novel insights into pathogenicity and virulence factors of V. vulnificus.

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July 7, 2019

Complete genome sequence of Enterococcus hirae R17, a daptomycin-resistant bacterium isolated from retail pork in China.

Daptomycin-resistant Enterococcus hirae R17 was isolated from retail pork sold at a free-trade market in Beijing, China. The complete genome sequence of R17 contains a circular 2,886,481-bp chromosome and a circular 73,574-bp plasmid. Genes involved in cell envelope homeostasis of this bacterium were identified by whole-genome analysis. Copyright © 2016 Peng et al.

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July 7, 2019

Plasmid dynamics in KPC-positive Klebsiella pneumoniae during long-term patient colonization.

Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms.In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care. Copyright © 2016 Conlan et al.

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July 7, 2019

Complete genome sequence of a Klebsiella pneumoniae strain carrying blaNDM-1 on a multidrug resistance plasmid

Here, we report the genome sequence of a blaNDM-1-positive Klebsiella pneumoniae AATZP isolate cultured from a perirectal surveillance swab collected upon admission of a patient to the NIH Clinical Center in 2014. Genome sequencing of this isolate revealed three plasmids, including one carrying the blaNDM-1 gene encoding resistance to carbapenems. Copyright © 2016 Conlan et al.

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July 7, 2019

Lactobacillus rhamnosus GG outcompetes Enterococcus faecium by mucus-binding pili – Evidence for a novel probiotic mechanism on a distance.

Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections. IMPORTANCE Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.

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July 7, 2019

Structural basis for recombinatorial permissiveness in the generation of Anaplasma marginale Msp2 antigenic variants.

Sequential expression of outer membrane protein antigenic variants is an evolutionarily convergent mechanism used by bacterial pathogens to escape host immune clearance and establish persistent infection. Variants must be sufficiently structurally distinct to escape existing immune effectors yet retain core structural elements required for localization and function within the outer membrane. We examined this balance using Anaplasma marginale, which generates antigenic variants in the outer membrane protein Msp2 using gene conversion. The overwhelming majority of Msp2 variants expressed during long-term persistent infection are mosaics, derived by recombination of oligonucleotide segments from multiple alleles to form unique hypervariable regions (HVR). As a result, the mosaics are not under long-term selective pressure to encode a functional protein; consequently, we hypothesized that the Msp2 HVR is structurally permissive for mosaic expression. Using an integrated approach of predictive modeling with determination of native Msp2 protein structure and function, we demonstrate that structured elements, most notably ß-sheets, are significantly concentrated in the highly conserved N- and C-terminal domains. In contrast the HVR is overwhelmingly random coil with the structured a-helices and ß-sheets confined to the genomically defined “structural tethers” that separate the antigenically variable microdomains. This structure is supported by the surface exposure of the HVR microdomains and the slow diffusion type porin function in native Msp2. Importantly, the predominance of random coil provides plasticity for formation of functional HVR mosaics and realization of the full potential of segmental gene conversion to dramatically expand the variant repertoire. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization.

Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide.The 2016 WHO reference strains (n?=?14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n?=?23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing.The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n?=?14) were presented.The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Distinct Salmonella enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.

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July 7, 2019

Chromosomal locations of mcr-1 and bla CTX-M-15 in fluoroquinolone-resistant Escherichia coli ST410.

To the Editor: Recently, Yi-Yun Liu et al. reported on the discovery of mcr-1, a plasmidborne resistance gene mediating resistance to colistin, in isolates obtained from humans and animals (1). Since the original publication, mcr-1 with or without the insertion element ISApl1 has been detected on plasmids of different incompatibility groups, including IncI2, IncHI2, and IncX4, and in many different countries (1–3). Because colistin is a last-resort parenteral antimicrobial drug, the transfer of mcr-1 by conjugation or through mobilizable plasmids raises concern about the emergence of pan-resistant Enterobacteriaceae.

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July 7, 2019

Genome sequence and analysis of Peptoclostridium difficile strain ZJCDC-S82.

Peptoclostridium difficile (Clostridium difficile) is the major pathogen associated with infectious diarrhea in humans. Concomitant with the increased incidence of C. difficile infection worldwide, there is an increasing concern regarding this infection type. This study reports a draft assembly and detailed sequence analysis of C. difficile strain ZJCDC-S82. The de novo assembled genome was 4.19 Mb in size, which includes 4,013 protein-coding genes, 41 rRNA genes, and 84 tRNA genes. Along with the nuclear genome, we also assembled sequencing information for a single plasmid consisting of 11,930 nucleotides. Comparative genomic analysis of C. difficile ZJCDC-S82 and two other previously published strains, such as M120 and CD630, showed extensive similarity. Phylogenetic analysis revealed that genetic diversity among C. difficile strains was not influenced by geographic location. Evolutionary analysis suggested that four genes encoding surface proteins exhibited positive selection in C. difficile ZJCDC-S82. Codon usage analysis indicated that C. difficile ZJCDC-S82 had high codon usage bias toward A/U-ended codons. Furthermore, codon usage patterns in C. difficile ZJCDC-S82 were predominantly affected by mutation pressure. Our results provide detailed information pertaining to the C. difficile genome associated with a strain from mainland China. This analysis will facilitate the understanding of genomic diversity and evolution of C. difficile strains in this region.

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July 7, 2019

Molecular evolution of a Klebsiella pneumoniae ST278 isolate harboring blaNDM-7 and involved in nosocomial transmission.

During 2013, ST278 Klebsiella pneumoniae with blaNDM-7 was isolated from the urine (KpN01) and rectum (KpN02) of a patient in Calgary, Canada. The same strain (KpN04) was subsequently isolated from another patient in the same unit. Interestingly, a carbapenem-susceptible K. pneumoniae ST278 (KpN06) was obtained 1 month later from the blood of the second patient. Next-generation sequencing (NGS) revealed that the loss of carbapenem-resistance in KpN06 was due to a 5-kb deletion on the blaNDM-7-harboring IncX3 plasmid. In addition, an IncFIB plasmid in KpN06 had a 27-kb deletion that removed genes encoding for heavy metal resistance. Phylogenetic analysis showed that the K. pneumoniae ST278 from patient 2 was likely a descendant of KpN02 and that KpN06 was a close progenitor of an environmental ST278. It is unclear whether KpN06 lost the blaNDM-7 gene in vivo. This study detailed the remarkable plasticity and speed of evolutionary changes in multidrug-resistant K. pneumoniae, demonstrating the highly recombinant nature of this species. It also highlights the ability of NGS to clarify molecular microevolutionary events within antibiotic-resistant organisms.© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

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July 7, 2019

Salmonella degrades the host glycocalyx leading to altered infection and glycan remodeling.

Complex glycans cover the gut epithelial surface to protect the cell from the environment. Invasive pathogens must breach the glycan layer before initiating infection. While glycan degradation is crucial for infection, this process is inadequately understood. Salmonella contains 47 glycosyl hydrolases (GHs) that may degrade the glycan. We hypothesized that keystone genes from the entire GH complement of Salmonella are required to degrade glycans to change infection. This study determined that GHs recognize the terminal monosaccharides (N-acetylneuraminic acid (Neu5Ac), galactose, mannose, and fucose) and significantly (p?

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July 7, 2019

Association between progranulin and Gaucher disease.

Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of ß-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD.Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed.Serum PGRN levels were significantly lower in GD patients (96.65±53.45ng/ml) than those in healthy controls of the general population (164.99±43.16ng/ml, p<0.0001) and of Ashkenazi Jews (150.64±33.99ng/ml, p<0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, "aged" and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients.Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCase's lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD. Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.

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