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September 22, 2019

Comparative heterochromatin profiling reveals conserved and unique epigenome signatures linked to adaptation and development of malaria parasites.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite’s adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Primordial origin and diversification of plasmids in Lyme disease agent bacteria.

With approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species.We sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii, B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non-B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi. These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types.The primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

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September 22, 2019

Repeat-driven generation of antigenic diversity in a major human pathogen, Trypanosoma cruzi

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan with a complex genome, is the causative agent of American trypanosomiasis (Chagas disease). The parasite uses a highly diverse repertoire of surface molecules, with roles in cell invasion, immune evasion and pathogenesis. Thus far, the genomic regions containing these genes have been impossible to resolve and it has been impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here present an improved genome assembly of a T. cruzi clade I (TcI) strain using high coverage PacBio single molecule sequencing, together with Illumina sequencing of 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene structure reveals an unusual duality in the organisation of the parasite genome, a core genomic region syntenous with related protozoa flanked by unique and highly plastic subtelomeric regions encoding surface antigens. The presence of abundant interspersed retrotransposons in the subtelomeres suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure.

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September 22, 2019

Genetic basis of chromosomally-encoded mcr-1 gene.

Compared with plasmid-borne mcr-1, the occurrence of chromosomally-encoded mcr-1 is rare although it has been reported in several cases. This study aimed to investigate the genetic features of chromosomally-encoded mcr-1 among Escherichia coli strains as well as the potential genetic basis governing mobilisation of mcr-1 in bacterial chromosomes. The genome sequences of 16 E. coli strains containing a chromosomal mcr-1 gene were obtained and analysed. Phylogenetic and whole-genome sequencing (WGS) analysis demonstrated that mcr-1 was associated with four major types of genetic arrangements, namely ISApl1-mcr1-orf, Tn6330, complex Tn6330 and ?Tn6330 in chromosomes of genetically unrelated E. coli strains. The mcr-1-carrying mobile elements were shown to insert into the AT-rich region, which was also the case for ISApl1. Analysis of complete E. coli genome sequences showed that there were multiple copies of ISApl1 present in E. coli chromosomes that also carried mcr-1, whilst all mcr-1-negative chromosomes were absent of any copy of ISApl1, suggesting the strong association of ISApl1 and mcr-1. Insertion of ISApl1 into E. coli chromosomes may be a prerequisite for the insertion of mcr-1-carrying mobile elements. Insertion of mcr-1 into E. coli chromosomes would enable it to become intrinsically resistant, which is expected to become more prevalent. Policy on the prudent use of colistin both in veterinary and clinical settings should be imposed globally to further prevent dissemination of mcr-1 in E. coli and other bacterial pathogens. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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September 22, 2019

Plasmid-mediated quinolone resistance in Shigella flexneriisolated from macaques.

Non-human primates (NHPs) for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants isolation and antibiotic testing of enteric pathogens before treating macaques with quinolones prophylactically or therapeutically.

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September 22, 2019

A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions.

Many bacteria utilize simple DNA sequence repeats as a mechanism to randomly switch genes on and off. This process is called phase variation. Several phase-variable N6-adenine DNA-methyltransferases from Type III restriction-modification systems have been reported in bacterial pathogens. Random switching of DNA methyltransferases changes the global DNA methylation pattern, leading to changes in gene expression. These epigenetic regulatory systems are called phasevarions – phase-variable regulons. The extent of these phase-variable genes in the bacterial kingdom is unknown. Here, we interrogated a database of restriction-modification systems, REBASE, by searching for all simple DNA sequence repeats in mod genes that encode Type III N6-adenine DNA-methyltransferases. We report that 17.4% of Type III mod genes (662/3805) contain simple sequence repeats. Of these, only one-fifth have been previously identified. The newly discovered examples are widely distributed and include many examples in opportunistic pathogens as well as in environmental species. In many cases, multiple phasevarions exist in one genome, with examples of up to 4 independent phasevarions in some species. We found several new types of phase-variable mod genes, including the first example of a phase-variable methyltransferase in pathogenic Escherichia coli. Phasevarions are a common epigenetic regulation contingency strategy used by both pathogenic and non-pathogenic bacteria.

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September 22, 2019

Genomic structural variations affecting virulence during clonal expansion of Pseudomonas syringae pv. actinidiae biovar 3 in Europe.

Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

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September 22, 2019

Complete genomic analysis of a Salmonella enterica Serovar Typhimurium isolate cultured from ready-to-eat pork in China carrying one large plasmid containing mcr-1.

One mcr-1-carrying ST34-type Salmonella Typhimurium WW012 was cultured from 3,200 ready-to-eat (RTE) pork samples in 2014 in China. Broth dilution method was applied to obtain the antimicrobial susceptibility of Salmonella Typhimurium WW012. Broth matting assays were carried out to detect transferability of this phenotype and whole-genome sequencing was performed to analyze its genomic characteristic. Thirty out of 3,200 RTE samples were positive for Salmonella and the three most frequent serotypes were identified as S. Derby (n = 8), S. Typhimurium (n = 6), and S. Enteritidis (n = 6). One S. Typhimurium isolate (S. Typhimurium WW012) cultured from RTE prepared pork was found to contain the mcr-1 gene. S. Typhimurium WW012 expressed a level of high resistance to seven different antimicrobial compounds in addition to colistin (MIC = 8 mg/L). A single plasmid, pWW012 (151,609-bp) was identified and found to be of an IncHI2/HI2A type that encoded a mcr-1 gene along with six additional antimicrobial resistance genes. Plasmid pWW012 contained an IS30-mcr-1-orf-orf-IS30 composite transposon that can be successfully transferred to Escherichia coli J53. When assessed further, the latter demonstrated considerable similarity to three plasmids pHYEC7-mcr-1, pSCC4, and pHNSHP45-2, respectively. Furthermore, plasmid pWW012 also contained a multidrug resistance (MDR) genetic structure IS26-aadA2-cmlA2-aadA1-IS406-sul3-IS26-dfrA12-aadA2-IS26, which showed high similarity to two plasmids, pHNLDF400 and pHNSHP45-2, respectively. Moreover, genes mapping to the chromosome (4,991,167-bp) were found to carry 28 mutations, related to two component regulatory systems (pmrAB, phoPQ) leading to modifications of lipid A component of the lipopolysaccharide structure. Additionally, one mutation (D87N) in the quinolone resistance determining region (QRDR) gene of gyrA was identified in this mcr-1 harboring S. Typhimurium. In addition, various virulence factors and heavy metal resistance-encoding genes were also identified on the genome of S. Typhimurium WW012. This is the first report of the complete nucleotide sequence of mcr-1-carrying MDR S. Typhimurium strain from RTE pork in China.

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September 22, 2019

Evaluation of WGS based approaches for investigating a food-borne outbreak caused by Salmonella enterica serovar Derby in Germany.

In Germany salmonellosis still represents the 2nd most common bacterial foodborne disease. The majority of infections are caused by Salmonella (S.) Typhimurium and S. Enteritidis followed by a variety of other broad host-range serovars. Salmonella Derby is one of the five top-ranked serovars isolated from humans and it represents one of the most prevalent serovars in pigs, thus bearing the potential risk for transmission to humans upon consumption of pig meat and products thereof. From November 2013 to January 2014 S. Derby caused a large outbreak that affected 145 primarily elderly people. Epidemiological investigations identified raw pork sausage as the probable source of infection, which was confirmed by microbiological evidence. During the outbreak isolates from patients, food specimen and asymptomatic carriers were investigated by conventional typing methods. However, the quantity and quality of available microbiological and epidemiological data made this outbreak highly suitable for retrospective investigation by Whole Genome Sequencing (WGS) and subsequent evaluation of different bioinformatics approaches for cluster definition. Overall the WGS-based methods confirmed the results of the conventional typing but were of significant higher discriminatory power. That was particularly beneficial for strains with incomplete epidemiological data. For our data set both, single nucleotide polymorphism (SNP)- and core genome multilocus sequence typing (cgMLST)-based methods proved to be appropriate tools for cluster definition. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 22, 2019

The Egyptian rousette genome reveals unexpected features of bat antiviral immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

The presence of colistin resistance gene mcr-1 and -3 in ESBL producing Escherichia coli isolated from food in Ho Chi Minh City, Vietnam.

Colistin is indicated for the treatment of multidrug-resistant gram-negative bacterial infections. However, the spread of colistin-resistant bacteria harbouring an mcr gene has become a serious concern. This study investigated local foods in Vietnam for contamination with colistin-resistant bacteria. A total of 261 extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Escherichia coli isolates from 330 meat and seafood products were analysed for colistin susceptibility and the presence of mcr genes. Approximately, 24% (62/261) of ESBL- or AmpC-producing E. coli isolates showed colistin resistance; 97% (60/62) of colistin-resistant isolates harboured mcr-1, whereas 3% (2/62) harboured mcr-3. As the result of plasmid analysis of two strains, both plasmids harbouring mcr-3 revealed that plasmid replicon type was IncFII. Sequencing analysis indicated that an insertion sequence was present near mcr-3, suggesting that IncFII plasmids harbouring mcr-3 could be transferred to other bacterial species by horizontal transfer of the plasmid or transfer with some insertion sequence. In conclusion, ESBL-producing E. coli and AmpC-producing E. coli have acquired colistin resistance because 24% of such isolates show colistin resistance and 3% of the colistin-resistant strains harbour mcr-3. We reported the present of the mcr-3-carrying ESBL-producing E. coli isolated from pork in Vietnam.

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September 22, 2019

First report of the occurrence and whole-genome characterization of Edwardsiella tarda in the false killer whale (Pseudorca crassidens).

Although several Edwardsiella tarda infections have been reported, its pathogenic role in marine mammals has not been investigated at the genome level. We investigated the genome of E. tarda strain KC-Pc-HB1, isolated from the false killer whale (Pseudorca crassidens) found bycaught in South Korea. The obtained genome was similar to that of human pathogenic E. tarda strains, but distinct from other Edwardsiella species. Although type III and VI secretion systems, which are essential for the virulence of other Edwardsiella species, were absent, several virulence-related genes involved in the pathogenesis of E. tarda were found in the genome. These results provide important insights into the E. tarda infecting marine mammals and give valuable information on potential virulence factors in this pathogen.

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September 22, 2019

Otitis in a cat associated with Corynebacterium provencense.

The role of corynebacteria in canine and feline otitis has not been investigated in detail; however, members of this genus are increasingly recognized as pathogens of otitis in both human and veterinary medicine.Here we report the first case of feline otitis associated with the recently described species Corynebacterium provencense. A seven-month old cat presented with a head tilt and ataxia was diagnosed with peripheral vestibular syndrome associated with an otitis media/interna. This took place 6 weeks after resection of a polyp, having initially shown a full recovery with topical ofloxacin and glucocorticoid treatment. Bacteriology of an ear swab yielded a pure culture of corynebacteria, which could not be identified at the species level using routine methods. However, the 16S rRNA gene sequence was 100% identical to the recently published novel corynebacterium species, Corynebacterium provencense. Whole genome sequencing of the cat isolate and calculation of average nucleotide identity (99.1%) confirmed this finding. The cat isolate was found to contain additional presumptive iron acquisition genes that are likely to encode virulence factors. Furthermore, the strain tested resistant to clindamycin, penicillin and ciprofloxacin. The cat was subsequently treated with chloramphenicol, which lead to clinical improvement.Corynebacteria from otitis cases are not routinely identified at the species level and not tested for antimicrobial susceptibility in veterinary laboratories, as they are not considered major pathogens. This may lead to underreporting of this genus or animals being treated with inappropriate antimicrobials since corynebacteria are often resistant to multiple drugs.

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September 22, 2019

Characteristics of carbapenem-resistant Enterobacteriaceae in ready-to-eat vegetables in China.

Vegetables harboring bacteria resistant to antibiotics are a growing food safety issue. However, data concerning carbapenem-resistant Enterobacteriaceae (CRE) in ready-to-eat fresh vegetables is still rare. In this study, 411 vegetable samples from 36 supermarkets or farmer’s markets in 18 cities in China, were analyzed for CRE. Carbapenemase-encoding genes and other resistance genes were analyzed among the CRE isolates. Plasmids carrying carbapenemase genes were studied by conjugation, replicon typing, S1-PFGE southern blot, restriction fragment length polymorphism (RFLP), and sequencing. CRE isolates were also analyzed by pulsed-field gel electrophoresis (PFGE). Ten vegetable samples yielded one or more CRE isolates. The highest detection rate of CRE (14.3%, 4/28) was found in curly endive. Twelve CRE isolates were obtained and all showed multidrug resistance: Escherichia coli, 5; Citrobacter freundii, 5; and Klebsiella pneumoniae, 2. All E. coli and C. freundii carried blaNDM, while K. pneumoniae harbored blaKPC-2. Notably, E. coli with blaNDM and ST23 hypervirulent Klebsiella pneumoniae (hvKP) carrying blaKPC-2 were found in the same cucumber sample and clonal spread of E. coli, C. freundii, and K. pneumoniae isolates were all observed between vegetable types and/or cities. IncX3 plasmids carrying blaNDM from E. coli and C. freundii showed identical or highly similar RFLP patterns, and the sequenced IncX3 plasmid from cucumber was also identical or highly similar (99%) to the IncX3 plasmids from clinical patients reported in other countries, while blaKPC-2 in K. pneumoniae was mediated by similar F35:A-:B1 plasmids. Our results suggest that both clonal expansion and horizontal transmission of IncX3- or F35:A-:B1-type plasmids may mediate the spread of CRE in ready-to-eat vegetables in China. The presence of CRE in ready-to-eat vegetables is alarming and constitutes a food safety issue. To our knowledge, this is the first report of either the C. freundii carrying blaNDM, or K. pneumoniae harboring blaKPC-2 in vegetables. This is also the first report of ST23 carbapenem-resistant hvKP strain in vegetables.

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September 22, 2019

Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen Orientia tsutsugamushi.

Orientia tsutsugamushi is a clinically important but neglected obligate intracellular bacterial pathogen of the Rickettsiaceae family that causes the potentially life-threatening human disease scrub typhus. In contrast to the genome reduction seen in many obligate intracellular bacteria, early genetic studies of Orientia have revealed one of the most repetitive bacterial genomes sequenced to date. The dramatic expansion of mobile elements has hampered efforts to generate complete genome sequences using short read sequencing methodologies, and consequently there have been few studies of the comparative genomics of this neglected species.We report new high-quality genomes of O. tsutsugamushi, generated using PacBio single molecule long read sequencing, for six strains: Karp, Kato, Gilliam, TA686, UT76 and UT176. In comparative genomics analyses of these strains together with existing reference genomes from Ikeda and Boryong strains, we identify a relatively small core genome of 657 genes, grouped into core gene islands and separated by repeat regions, and use the core genes to infer the first whole-genome phylogeny of Orientia.Complete assemblies of multiple Orientia genomes verify initial suggestions that these are remarkable organisms. They have larger genomes compared with most other Rickettsiaceae, with widespread amplification of repeat elements and massive chromosomal rearrangements between strains. At the gene level, Orientia has a relatively small set of universally conserved genes, similar to other obligate intracellular bacteria, and the relative expansion in genome size can be accounted for by gene duplication and repeat amplification. Our study demonstrates the utility of long read sequencing to investigate complex bacterial genomes and characterise genomic variation.

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