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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

Hepacivirus A infection in horses defines distinct envelope hypervariable regions and elucidates potential roles of viral strain and adaptive immune status in determining envelope diversity and infection outcome.

Hepacivirus A (also known as nonprimate hepacivirus and equine hepacivirus) is a hepatotropic virus that can cause both transient and persistent infections in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A, and its roles in immune evasion and persistence are unknown. To address these knowledge gaps, we first evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals), and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than that observed in horses who spontaneously cleared infection (P = 0.0002) or in SCID foals (P < 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P < 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcomes and could be dependent on strain-specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank. This analysis defined three novel hypervariable regions (HVRs) in E2, at residues 391 to 402 (HVR1), 450 to 461 (HVR2), and 550 to 562 (HVR3). For the experimentally infected horses, entropy analysis focusing on the HVRs demonstrated that these regions were under increased selective pressure during persistent infection. Increased diversity in the HVRs was also temporally associated with seroconversion in some horses, suggesting that these regions may be targets of neutralizing antibody and may play a role in immune evasion.IMPORTANCE Hepacivirus C (hepatitis C virus) is estimated to infect 150 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. In contrast, its closest relative, hepacivirus A, causes relatively mild disease in horses and is frequently cleared. The relationship between quasispecies evolution and infection outcome has not been explored for hepacivirus A. To address this knowledge gap, we examined envelope gene diversity in horses with resolving and persistent infections. Interestingly, two strain-specific patterns of quasispecies diversity emerged. Persistence of the WSU strain was associated with increased quasispecies diversity and the accumulation of amino acid changes within three novel hypervariable regions following seroconversion. These findings provided evidence that envelope gene mutation is influenced by adaptive immune pressure and may contribute to hepacivirus persistence. However, the CU strain persisted despite relative evolutionary stasis, suggesting that some hepacivirus strains may use alternative mechanisms to persist in the host. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.

Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis.To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms “Membrane” and “Protein Secretion.”In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis.

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September 22, 2019

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d’Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences. However, compared with the ape parasites, human strains of P. vivax exhibit about 10-fold less diversity and have a relative excess of nonsynonymous nucleotide polymorphisms, with site-frequency spectra suggesting they are subject to greatly relaxed purifying selection. These data suggest that human P. vivax has undergone an extreme bottleneck, followed by rapid population expansion. Investigating potential host-specificity determinants, we found that ape P. vivax parasites encode intact orthologs of three reticulocyte-binding protein genes (rbp2d, rbp2e, and rbp3), which are pseudogenes in all human P. vivax strains. However, binding studies of recombinant RBP2e and RBP3 proteins to human, chimpanzee, and gorilla erythrocytes revealed no evidence of host-specific barriers to red blood cell invasion. These data suggest that, from an ancient stock of P. vivax parasites capable of infecting both humans and apes, a severely bottlenecked lineage emerged out of Africa and underwent rapid population growth as it spread globally. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

The structure of a conserved telomeric region associated with variant antigen loci in the blood parasite Trypanosoma congolense

African trypanosomiasis is a vector-borne disease of humans and livestock caused by African trypanosomes (Trypanosoma spp.). Survival in the vertebrate bloodstream depends on antigenic variation of Variant Surface Glycoproteins (VSGs) coating the parasite surface. In T. brucei, a model for antigenic variation, monoallelic VSG expression originates from dedicated VSG expression sites (VES). Trypanosoma brucei VES have a conserved structure consisting of a telomeric VSG locus downstream of unique, repeat sequences, and an independent promoter. Additional protein-coding sequences, known as “Expression Site Associated Genes (ESAGs)”, are also often present and are implicated in diverse, bloodstream-stage functions. Trypanosoma congolense is a related veterinary pathogen, also displaying VSG-mediated antigenic variation. A T. congolense VES has not been described, making it unclear if regulation of VSG expression is conserved between species. Here, we describe a conserved telomeric region associated with VSG loci from long-read DNA sequencing of two T. congolense strains, which consists of a distal repeat, conserved noncoding elements and other genes besides the VSG; although these are not orthologous to T. brucei ESAGs. Most conserved telomeric regions are associated with accessory minichromosomes, but the same structure may also be associated with megabase chromosomes. We propose that this region represents the T. congolense VES, and through comparison with T. brucei, we discuss the parallel evolution of antigenic switching mechanisms, and unique adaptation of the T. brucei VES for developmental regulation of bloodstream-stage genes. Hence, we provide a basis for understanding antigenic switching in T. congolense and the origins of the African trypanosome VES.

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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

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

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 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

A comparison of genotypic and phenotypic methods for analyzing the susceptibility to sulfamethoxazole and trimethoprim in Edwardsiella piscicida.

In a study of 39 isolates of Edwardsiella piscicida made from Korean aquaculture sites, sul genes were detected in 16 isolates and dfr genes in 19. Ten isolates were shown to contain both sul and dfr genes. MIC and disc diffusion zones assays were performed to measure the phenotypic susceptibilities of the 39 isolates. Normalized resistance interpretation was applied to these data to categorize isolates as either fully susceptible or as manifesting reduced susceptibility. The standard CLSI protocols specify the use of a mixture of sulfamethoxazole/trimethoprim (20:1) in both MIC and disc diffusion tests. Using the CLSI MIC protocol, 100% of the isolates containing dfr genes, but only 75% of the isolates containing sul genes, were categorized as manifesting reduced susceptibility. Using the CLSI disc diffusion protocol, only 58% of the isolates containing dfr genes and 69% of those containing sul genes were categorized as manifesting reduced susceptibility. When the single agent trimethoprim was substituted for the combined mixture in both the MIC and disc diffusion protocols, 100% of the dfr-positive isolates were categorized as NWT. When the single-agent sulfamethoxazole was substituted, the analysis of the MIC characterized 100% and the disc zone data 94% of the sul-positive isolates as manifesting reduced susceptibility. It is argued that the use of trimethoprim and sulfamethoxazole as single agents in phenotypic susceptibility tests would provide more meaningful data than the currently recommended use of these two agents combined.

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September 22, 2019

Identification of the KPC plasmid pCT-KPC334: New insights on the evolutionary pathway of epidemic plasmids harboring fosA3-blaKPC-2 genes.

A novel, non-conjugative plasmid pKP1034 isolated from a fosfomycin-resistant, carbapenemase-producing Klebsiella pneumonia strain KP1034 was recently reported to carry fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12 and rmtB genes, and was hypothesized to evolve from several recombination events of two closely related plasmids, pHN7A8 and pKPC-LK30 [1]. In this study, a plasmid pCT-KPC334 carrying fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, blaTEM-1, and rmtB genes was identified, providing evidence on the evolutionary pathway of plasmids harboring fosA3-blaKPC-2 genes.

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September 22, 2019

Extraordinary genome instability and widespread chromosome rearrangements during vegetative growth

The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on “core” and “accessory” chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro, but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability. Copyright © 2018 by the Genetics Society of America.

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September 22, 2019

4.5 years within-patient evolution of a colistin resistant KPC-producing Klebsiella pneumoniae ST258.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) has emerged globally over the last decade as a major nosocomial pathogen that threatens patient care. These highly resistant bacteria are mostly associated with a single Kp clonal group, CG258, but the reasons for its host and hospital adaptation remain largely unknown.We analyzed the in vivo evolution of a colistin-resistant KPC-Kp CG258 strain that contaminated a patient following an endoscopy and was responsible for a fatal bacteremia 4.5 years later. Whole-genome sequencing was performed on 17 KPC-Kp isolates from this patient; single-nucleotide polymorphisms were analyzed and their implication in antimicrobial resistance and bacterial host adaptation investigated.The patient KPC-Kp strain diversified over 4.5 years at a rate of 7.5 substitutions per genome per year, resulting in broad phenotypic modifications. After 2 years of carriage, all isolates restored susceptibility to colistin. Higher expression of the fimbriae conferred the ability to produce more biofilm, and the isolate responsible for a bacteremia grew in human serum. The convergent mutations occurring in specific pathways, such as the respiratory chain and the cell envelope, revealed a complex long-term adaptation of KPC-Kp.Broad genomic and phenotypic diversification and the parallel selection of pathoadaptive mutations might contribute to long-term carriage and virulence of KPC-Kp CG258 strains and to the dissemination of this clone.

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September 22, 2019

Genome plasticity of agr-defective Staphylococcus aureus during clinical infection.

Therapy for bacteremia caused by Staphylococcus aureus is often ineffective, even when treatment conditions are optimal according to experimental protocols. Adapted subclones, such as those bearing mutations that attenuate agr-mediated virulence activation, are associated with persistent infection and patient mortality. To identify additional alterations in agr-defective mutants, we sequenced and assembled the complete genomes of clone pairs from colonizing and infected sites of several patients in whom S. aureus demonstrated a within-host loss of agr function. We report that events associated with agr inactivation result in agr-defective blood and nares strain pairs that are enriched in mutations compared to pairs from wild-type controls. The random distribution of mutations between colonizing and infecting strains from the same patient, and between strains from different patients, suggests that much of the genetic complexity of agr-defective strains results from prolonged infection or therapy-induced stress. However, in one of the agr-defective infecting strains, multiple genetic changes resulted in increased virulence in a murine model of bloodstream infection, bypassing the mutation of agr and raising the possibility that some changes were selected. Expression profiling correlated the elevated virulence of this agr-defective mutant to restored expression of the agr-regulated ESAT6-like type VII secretion system, a known virulence factor. Thus, additional mutations outside the agr locus can contribute to diversification and adaptation during infection by S. aureus agr mutants associated with poor patient outcomes. Copyright © 2018 Altman et al.

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