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September 22, 2019

Characterization of a novel SXT/R391 Integrative and Conjugative Element carrying cfr, blaCTX-M-65, fosA3 and aac(6′)-Ib-cr in Proteus mirabilis.

A novel 139,487-bp SXT/R391 integrative and conjugative element, ICEPmiChnBCP11, was characterized in Proteus mirabilis of swine origin in China. ICEPmiChnBCP11 harbors 20 different antimicrobial resistance genes, including the clinically important rRNA methyltransferase gene cfr, the extended-spectrum ß-lactamase gene blaCTX-M-65, fosfomycin resistance gene fosA3, and fluoroquinolone resistance gene aac(6′)-Ib-cr An ISPpu12-mediated composite transposon containing various resistance genes and 10 copies of IS26 is inserted in hot spot 4. ICEPmiChnBCP11 was successfully transferred to Escherichia coli. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Long-term colonization dynamics of Enterococcus faecalis in implanted devices in research macaques.

Enterococcus faecalis is a common opportunistic pathogen that colonizes cephalic recording chambers (CRCs) of macaques used in cognitive neuroscience research. We previously characterized 15 E. faecalis strains isolated from macaques at the Massachusetts Institute of Technology (MIT) in 2011. The goal of this study was to examine how a 2014 protocol change prohibiting the use of antimicrobials within CRCs affected colonizing E. faecalis strains. We collected 20 E. faecalis isolates from 10 macaques between 2013 and 2017 for comparison to 4 isolates previously characterized in 2011 with respect to the sequence type (ST) distribution, antimicrobial resistance, biofilm formation, and changes in genes that might confer a survival advantage. ST4 and ST55 were predominant among the isolates characterized in 2011, whereas the less antimicrobial-resistant lineage ST48 emerged to dominance after 2013. Two macaques remained colonized by ST4 and ST55 strains for 5 and 4 years, respectively. While the antimicrobial resistance and virulence factors identified in these ST4 and ST55 strains remained relatively stable, we detected an increase in biofilm formation ability over time in both isolates. We also found that ST48 strains were typically robust biofilm formers, which could explain why this ST increased in prevalence. Finally, we identified mutations in the DNA mismatch repair genes mutS and mutL in separate ST55 and ST4 strains and confirmed that strains bearing these mutations displayed a hypermutator phenotype. The presence of a hypermutator phenotype may complicate future antimicrobial treatment for clinically relevant E. faecalis infections in macaques.IMPORTANCEEnterococcus faecalis is a common cause of health care-associated infections in humans, largely due to its ability to persist in the hospital environment, colonize patients, acquire antimicrobial resistance, and form biofilms. Understanding how enterococci evolve in health care settings provides insight into factors affecting enterococcal survival and persistence. Macaques used in neuroscience research have long-term cranial implants that, despite best practices, often become colonized by E. faecalis This provides a unique opportunity to noninvasively examine the evolution of enterococci on a long-term indwelling device. We collected E. faecalis strains from cephalic implants over a 7-year period and characterized the sequence type, antimicrobial resistance, virulence factors, biofilm production, and hypermutator phenotypes. Improved antimicrobial stewardship allowed a less-antimicrobial-resistant E. faecalis strain to predominate at the implant interface, potentially improving antimicrobial treatment outcomes if future clinical infections occur. Biofilm formation appears to play an important role in the persistence of the E. faecalis strains associated with these implants. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

A model for the evolution of prokaryotic DNA restriction-modification systems based upon the structural malleability of Type I restriction-modification enzymes.

Restriction Modification (RM) systems prevent the invasion of foreign genetic material into bacterial cells by restriction and protect the host’s genetic material by methylation. They are therefore important in maintaining the integrity of the host genome. RM systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Comparing the structures of the different types, similarities can be observed suggesting an evolutionary link between these different types. This work describes the ‘deconstruction’ of a large Type I RM enzyme into forms structurally similar to smaller Type II RM enzymes in an effort to elucidate the pathway taken by Nature to form these different RM enzymes. Based upon the ability to engineer new enzymes from the Type I ‘scaffold’, an evolutionary pathway and the evolutionary pressures required to move along the pathway from Type I RM systems to Type II RM systems are proposed. Experiments to test the evolutionary model are discussed.

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September 22, 2019

Comparative genome analysis of jujube witches’-broom Phytoplasma, an obligate pathogen that causes jujube witches’-broom disease.

JWB phytoplasma is a kind of insect-transmitted and uncultivable bacterial plant pathogen causeing a destructive Jujube disease. To date, no genome information about JWB phytoplasma has been published, which hindered its characterization at genomic level. To understand its pathogenicity and ecology, the genome of a JWB phytoplasma isolate jwb-nky was sequenced and compared with other phytoplasmas enabled us to explore the mechanisms of genomic rearrangement.The complete genome sequence of JWB phytoplasma (jwb-nky) was determined, which consisting of one circular chromosome of 750,803 bp with a GC content of 23.3%. 694 protein-encoding genes, 2 operons for rRNA genes and 31 tRNA genes as well as 4 potential mobile units (PMUs) containing clusters of DNA repeats were identified. Based on PHIbaes analysis, a large number of genes were genome-specific and approximately 13% of JWB phytoplasma genes were predicted to be associated with virulence. Although transporters for maltose, dipeptides/oligopeptides, spermidine/putrescine, cobalt, Mn/Zn and methionine were identified, KEGG pathway analysis revealed the reduced metabolic capabilities of JWB phytoplasma. Comparative genome analyses between JWB phytoplasma and other phytoplasmas shows the occurrence of large-scale gene rearrangements. The low synteny with other phytoplasmas indicated that the expansion of multiple gene families/duplication probably occurred separately after differentiation.In this study, the complete genome sequence of a JWB phytoplasma isolate jwb-nky that causing JWB disease was reported for the first time and a number of species-specific genes were identified in the genome. The study enhanced our understandings about genomic basis and the pathogenicity mechanism of this pathogen, which will aid in the development of improved strategies for efficient management of JWB diseases.

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September 22, 2019

Genes significantly associated with lineage II food isolates of Listeria monocytogenes.

Listeria monocytogenes is a widespread foodborne pathogen that can cause listeriosis, a potentially fatal infection. L. monocytogenes is subdivided into four phylogenetic lineages, with the highest incidence of listeriosis occurring within lineage I followed by lineage II. Strains of L. monocytogenes differ in their phenotypic characteristics, including virulence. However, the genetic bases for these observed differences are not well understood, and current efforts to monitor L. monocytogenes in food consider all strains to be equally virulent. We use a comparative genomics approach to identify genes and single nucleotide polymorphisms (SNPs) in 174 clinical and food isolates of L. monocytogenes that potentially contribute to virulence or the capacity to adapt to food environments.No SNPs are significantly associated with food or clinical isolates. No genes are significantly associated with food or clinical isolates from lineage I, but eight genes consisting of multiple homologues are associated with lineage II food isolates. These include three genes which encode hypothetical proteins, the cadmium resistance genes cadA and cadC, the multi-drug resistance gene ebrB, a quaternary ammonium compound resistance gene qac, and a regulatory gene. All eight genes are plasmid-borne, and most closed L. monocytogenes plasmids carry at least five of the genes (24/27). In addition, plasmids are more frequently associated with lineage II food isolates than with lineage II clinical isolates.We identify eight genes that are significantly associated with food isolates in lineage II. Interestingly, the eight genes are virtually absent in lineage II outbreak isolates, are composed of homologues which show a nonrandom distribution among lineage I serotypes, and the sequences are highly conserved across 27 closed Listeria plasmids. The functions of these genes should be explored further and will contribute to our understanding of how L. monocytogenes adapts to the host and food environments. Moreover, these genes may also be useful as markers for risk assessment models of either pathogenicity or the ability to proliferate in food and the food processing environment.

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September 22, 2019

Novel clade C-I Clostridium difficile strains escape diagnostic tests, differ in pathogenicity potential and carry toxins on extrachromosomal elements.

The population structure of Clostridium difficile currently comprises eight major genomic clades. For the highly divergent C-I clade, only two toxigenic strains have been reported, which lack the tcdA and tcdC genes and carry a complete locus for the binary toxin (CDT) next to an atypical TcdB monotoxin pathogenicity locus (PaLoc). As part of a routine surveillance of C. difficile in stool samples from diarrheic human patients, we discovered three isolates that consistently gave negative results in a PCR-based screening for tcdC. Through phenotypic assays, whole-genome sequencing, experiments in cell cultures, and infection biomodels we show that these three isolates (i) escape common laboratory diagnostic procedures, (ii) represent new ribotypes, PFGE-types, and sequence types within the Clade C-I, (iii) carry chromosomal or plasmidal TcdBs that induce classical or variant cytopathic effects (CPE), and (iv) cause different levels of cytotoxicity and hamster mortality rates. These results show that new strains of C. difficile can be detected by more refined techniques and raise questions on the origin, evolution, and distribution of the toxin loci of C. difficile and the mechanisms by which this emerging pathogen causes disease.

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September 22, 2019

Genome sequence analysis of Zooshikella ganghwensis strain VG4 and its potential for the synthesis of antimicrobial metabolites.

With antimicrobial resistance on the rise, the discovery of new compounds with novel structural scaffolds exhibiting antimicrobial properties has become an important area of research. Such compounds can serve as starting points for the development of new antimicrobials. In this report, we present the draft genome sequence of the Zooshikella ganghwensis strain VG4, isolated from Red Sea sediments, that produces metabolites with antimicrobial properties. A genomic analysis reveals that it carries at least five gene clusters that have the potential to direct biosynthesis of bioactive secondary metabolites such as polyketides and nonribosomal peptides. By using in-silico approaches, we predict the structure of these metabolites.

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September 22, 2019

Closed genome and comparative phylogenetic analysis of the clinical multidrug resistant Shigella sonnei strain 866.

Shigella sonnei is responsible for the majority of shigellosis infections in the US with over 500,000 cases reported annually. Here, we present the complete genome of the clinical multidrug resistant (MDR) strain 866, which is highly susceptible to bacteriophage infections. The strain has a circular chromosome of 4.85?Mb and carries a 113?kb MDR plasmid. This IncB/O/K/Z-type plasmid, termed p866, confers resistance to five different classes of antibiotics including ß-lactamase, sulfonamide, tetracycline, aminoglycoside, and trimethoprim. Comparative analysis of the plasmid architecture and gene inventory revealed that p866 shares its plasmid backbone with previously described IncB/O/K/Z-type Shigella spp. and Escherichia coli plasmids, but is differentiated by the insertion of antibiotic resistance cassettes, which we found associated with mobile genetic elements such as Tn3, Tn7, and Tn10. A whole genome-derived phylogenetic reconstruction showed the evolutionary relationships of S. sonnei strain 866 and the four established Shigella species, highlighting the clonal nature of S. sonnei.

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September 22, 2019

Involvement of PorK, a component of the type IX secretion system, in Prevotella melaninogenica pathogenicity.

Prevotella melaninogenica is a gram-negative anaerobic commensal bacterium that resides in the human oral cavity and is isolated as a pathogen of suppurative diseases both inside and outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica. The periodontal pathogens Porphyromonas gingivalis and Tanerella forsythia secrete virulence factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS) that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the T9SS-encoding gene, porK, was constructed. Hemagglutination and biofilm formation were decreased in the porK mutant. Furthermore, following growth on skim milk-containing medium, the diameters of the halos surrounding the porK mutant were smaller than those of the wild-type strain, suggesting a decrease in secretion of proteases outside the bacterium. To investigate this in detail, culture supernatants of wild-type and porK mutant strains were purified and compared by two-dimensional electrophoresis. In the mutant strain, fewer spots were detected, indicating fewer secreted proteins. In infection experiments, the mortality rate of mice inoculated with the porK mutant strain was significantly lower than in the wild-type strain. These results suggest that P. melaninogenica secretes potent virulence factors via the T9SS that contribute to its pathogenic ability.© 2018 The Societies and John Wiley & Sons Australia, Ltd.

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September 22, 2019

Genome-wide analysis of Borrelia turcica and ‘Candidatus Borrelia tachyglossi’ shows relapsing fever-like genomes with unique genomic links to Lyme disease Borrelia.

Borrelia are tick-borne bacteria that in humans are the aetiological agents of Lyme disease and relapsing fever. Here we present the first genomes of B. turcica and B. tachyglossi, members of a recently described and rapidly expanding Borrelia clade associated with reptile (B. turcica) or echidna (B. tachyglossi) hosts, transmitted by hard ticks, and of unknown pathogenicity. Borrelia tachyglossi and B. turcica genomes are similar to those of relapsing fever Borrelia species, containing a linear ~ 900?kb chromosome, a single long (> 70?kb) linear plasmid, and numerous short (< 40?kb) linear and circular plasmids, as well as a suite of housekeeping and macronutrient biosynthesis genes which are not found in Lyme disease Borrelia. Additionally, both B. tachyglossi and B. turcica contain paralogous vsp and vlp proteins homologous to those used in the multiphasic antigen-switching system used by relapsing fever Borrelia to evade vertebrate immune responses, although their number was greatly reduced compared to human-infectious species. However, B. tachyglossi and B. turcica chromosomes also contain numerous genes orthologous to Lyme disease Borrelia-specific genes, demonstrating a unique evolutionary, and potentially phenotypic link between these groups. Borrelia tachyglossi and B. turcica genomes also have unique genetic features, including degraded and deleted tRNA modification genes, and an expanded range of macronutrient salvage and biosynthesis genes compared to relapsing fever and Lyme disease Borrelia. These genomes and genomic comparisons provide an insight into the biology and evolutionary origin of these Borrelia, and provide a valuable resource for future work. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

Complete genome sequence of a blaKPC-2-positive Klebsiella pneumoniae strain isolated from the effluent of an urban sewage treatment plant in Japan.

Antimicrobial resistance genes (ARGs) and the bacteria that harbor them are widely distributed in the environment, especially in surface water, sewage treatment plant effluent, soil, and animal waste. In this study, we isolated a KPC-2-producing Klebsiella pneumoniae strain (GSU10-3) from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP) and determined its complete genome sequence. Strain GSU10-3 is resistant to most ß-lactam antibiotics and other antimicrobial agents (quinolones and aminoglycosides). This strain is classified as sequence type 11 (ST11), and a core genome phylogenetic analysis indicated that strain GSU10-3 is closely related to KPC-2-positive Chinese clinical isolates from 2011 to 2017 and is clearly distinct from strains isolated from the European Union (EU), United States, and other Asian countries. Strain GSU10-3 harbors four plasmids, including a blaKPC-2-positive plasmid, pGSU10-3-3 (66.2?kb), which is smaller than other blaKPC-2-positive plasmids and notably carries dual replicons (IncFII [pHN7A8] and IncN). Such downsizing and the presence of dual replicons may promote its maintenance and stable replication, contributing to its broad host range with low fitness costs. A second plasmid, pGSU10-3-1 (159.0?kb), an IncA/C2 replicon, carries a class 1 integron (containing intI1, dfrA12, aadA2, qacE?1, and sul1) with a high degree of similarity to a broad-host-range plasmid present in the family Enterobacteriaceae The plasmid pGSU10-3-2 (134.8?kb), an IncFII(K) replicon, carries the IS26-mediated ARGs [aac(6′)Ib-cr,blaOXA-1, catB4 (truncated), and aac(3)-IId], tet(A), and a copper/arsenate resistance locus. GSU10-3 is the first nonclinical KPC-2-producing environmental Enterobacteriaceae isolate from Japan for which the whole genome has been sequenced.IMPORTANCE We isolated and determined the complete genome sequence of a KPC-2-producing K. pneumoniae strain from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP). In Japan, the KPC type has been very rarely detected, while IMP is the most predominant type of carbapenemase in clinical carbapenemase-producing Enterobacteriaceae (CPE) isolates. Although laboratory testing thus far suggested that Japan may be virtually free of KPC-producing Enterobacteriaceae, we have detected it from effluent from a WWTP. Antimicrobial resistance (AMR) monitoring of WWTP effluent may contribute to the early detection of future AMR bacterial dissemination in clinical settings and communities; indeed, it will help illuminate the whole picture in which environmental contamination through WWTP effluent plays a part. Copyright © 2018 Sekizuka et al.

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September 22, 2019

Antagonistic pleiotropy in the bifunctional surface protein FadL (OmpP1) during adaptation of Haemophilus influenzae to chronic lung infection associated with chronic obstructive pulmonary disease.

Tracking bacterial evolution during chronic infection provides insights into how host selection pressures shape bacterial genomes. The human-restricted opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) infects the lower airways of patients suffering chronic obstructive pulmonary disease (COPD) and contributes to disease progression. To identify bacterial genetic variation associated with bacterial adaptation to the COPD lung, we sequenced the genomes of 92 isolates collected from the sputum of 13 COPD patients over 1 to 9?years. Individuals were colonized by distinct clonal types (CTs) over time, but the same CT was often reisolated at a later time or found in different patients. Although genomes from the same CT were nearly identical, intra-CT variation due to mutation and recombination occurred. Recurrent mutations in several genes were likely involved in COPD lung adaptation. Notably, nearly a third of CTs were polymorphic for null alleles of ompP1 (also called fadL), which encodes a bifunctional membrane protein that both binds the human carcinoembryonic antigen-related cell adhesion molecule 1 (hCEACAM1) receptor and imports long-chain fatty acids (LCFAs). Our computational studies provide plausible three-dimensional models for FadL’s interaction with hCEACAM1 and LCFA binding. We show that recurrent fadL mutations are likely a case of antagonistic pleiotropy, since loss of FadL reduces NTHi’s ability to infect epithelia but also increases its resistance to bactericidal LCFAs enriched within the COPD lung. Supporting this interpretation, truncated fadL alleles are common in publicly available NTHi genomes isolated from the lower airway tract but rare in others. These results shed light on molecular mechanisms of bacterial pathoadaptation and guide future research toward developing novel COPD therapeutics.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidate the bacterial pathways undergoing in vivo evolutionary adaptation, we compared bacterial genomes collected over time from 13 COPD patients and identified recurrent genetic changes arising in independent bacterial lineages colonizing different patients. Besides finding changes in phase-variable genes, we found recurrent loss-of-function mutations in the ompP1 (fadL) gene. We show that loss of OmpP1/FadL function reduces this bacterium’s ability to infect cells via the hCEACAM1 epithelial receptor but also increases its resistance to bactericidal fatty acids enriched within the COPD lung, suggesting a case of antagonistic pleiotropy that restricts ?fadL strains’ niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways. Copyright © 2018 Moleres et al.

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September 22, 2019

Genomic characterization and probiotic potency of Bacillus sp. DU-106, a highly effective producer of L-lactic acid isolated from fermented yogurt.

Bacillus sp. DU-106, a newly isolated member of Bacillus cereus group, exhibits the predominant ability to produce L-lactic acid. The probiotic potency of test strain revealed its survivability at acidic pH, bile salts and viability in simulated gastric juice in vitro. The acute oral toxicity test indicated its no toxicity to laboratory mice in vivo. We further determined the complete genome of strain DU-106 to understand genetic basis as a potential probiotic. It has a circular chromosome and three plasmids for a total genome 5,758,208 bp in size with a G + C content of 35.10%. Genes associated with lactate synthesis were found in the DU-106 genome. We also annotated various stress-related, bile salt resistance, and adhesion-related domains in this strain, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival under gastrointestinal tract. Moreover, strain DU-106 genome lacks the virulence genes encodes cereulide synthetase, enterotoxin FM, and cytotoxin K. These phenotypic and genomic probiotic potencies facilitate its potential candidate as probiotic starter in food industry.

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September 22, 2019

Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages.

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial- susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85% of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99% sensitivity and 100% specificity. Approximately 10% showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

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September 22, 2019

Screening and whole-genome sequencing of two Streptomyces species from the rhizosphere soil of peony reveal their characteristics as plant growth-promoting Rhizobacteria.

Two bacteria, Streptomyces albireticuli MDJK11 and S. alboflavus MDJK44, which are potential plant growth-promoting rhizobacteria against pathogenic fungi were isolated from the rhizosphere soil of peony in Shandong, China. Their biological characteristics and complete genome sequences were reported in this study. The total genome size of MDJK11 was only 8.14?Mb with 6,550 protein-coding genes and a high GC content of 72.8?mol%. The MDJK44 genome comprises a 9.62 Mb chromosome with 72.1?mol% GC content, 7,285 protein-coding genes, and two plasmids. Some gene sequences in these two genomes were analyzed to be heterologously obtained by horizontal transfer. Gene or gene cluster candidates responding to secondary metabolites production, antimicrobial activities, and plant growth-promoting capacities were also analyzed in this paper. The genomic information and biological characteristics will facilitate the understanding and application of S. albireticuli and S. alboflavus species as biocontrol agents in future agriculture.

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