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Asset Tag: HGAP

September 22, 2019

Evolution of sequence type 4821 clonal complex meningococcal strains in China from prequinolone to quinolone era, 1972-2013.

The expansion of hypervirulent sequence type 4821 clonal complex (CC4821) lineage Neisseria meningitidis bacteria has led to a shift in meningococcal disease epidemiology in China, from serogroup A (MenA) to MenC. Knowledge of the evolution and genetic origin of the emergent MenC strains is limited. In this study, we subjected 76 CC4821 isolates collected across China during 1972-1977 and 2005-2013 to phylogenetic analysis, traditional genotyping, or both. We show that successive recombination events within genes encoding surface antigens and acquisition of quinolone resistance mutations possibly played a role in the emergence of CC4821 as an epidemic clone in China. MenC and MenB CC4821 strains have spread across China and have been detected in several countries in different continents. Capsular switches involving serogroups B and C occurred among epidemic strains, raising concerns regarding possible increases in MenB disease, given that vaccines in use in China do not protect against MenB.

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September 22, 2019

Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease.

Nontypeable Haemophilus influenzae (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

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September 22, 2019

Distinct evolutionary patterns of Neisseria meningitidis serogroup B disease outbreaks at two universities in the USA.

Neisseria meningitidis serogroup B (MnB) was responsible for two independent meningococcal disease outbreaks at universities in the USA during 2013. The first at University A in New Jersey included nine confirmed cases reported between March 2013 and March 2014. The second outbreak occurred at University B in California, with four confirmed cases during November 2013. The public health response to these outbreaks included the approval and deployment of a serogroup B meningococcal vaccine that was not yet licensed in the USA. This study investigated the use of whole-genome sequencing(WGS) to examine the genetic profile of the disease-causing outbreak isolates at each university. Comparative WGS revealed differences in evolutionary patterns between the two disease outbreaks. The University A outbreak isolates were very closely related, with differences primarily attributed to single nucleotide polymorphisms/insertion-deletion (SNP/indel) events. In contrast, the University B outbreak isolates segregated into two phylogenetic clades, differing in large part due to recombination events covering extensive regions (>30?kb) of the genome including virulence factors. This high-resolution comparison of two meningococcal disease outbreaks further demonstrates the genetic complexity of meningococcal bacteria as related to evolution and disease virulence.

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September 22, 2019

Genomic analysis of a pan-resistant isolate of Klebsiella pneumoniae, United States 2016.

Antimicrobial resistance is a threat to public health globally and leads to an estimated 23,000 deaths annually in the United States alone. Here, we report the genomic characterization of an unusualKlebsiella pneumoniae, nonsusceptible to all 26 antibiotics tested, that was isolated from a U.S.The isolate harbored four known beta-lactamase genes, including plasmid-mediatedblaNDM-1andblaCMY-6, as well as chromosomalblaCTX-M-15andblaSHV-28, which accounted for resistance to all beta-lactams tested. In addition, sequence analysis identified mechanisms that could explain all other reported nonsusceptibility results, including nonsusceptibility to colistin, tigecycline, and chloramphenicol. Two plasmids, IncA/C2 and IncFIB, were closely related to mobile elements described previously and isolated from Gram-negative bacteria from China, Nepal, India, the United States, and Kenya, suggesting possible origins of the isolate and plasmids. This is one of the firstK. pneumoniaeisolates in the United States to have been reported to the Centers for Disease Control and Prevention (CDC) as nonsusceptible to all drugs tested, including all beta-lactams, colistin, and tigecycline. IMPORTANCE Antimicrobial resistance is a major public health threat worldwide. Bacteria that are nonsusceptible or resistant to all antimicrobials available are of major concern to patients and the public because of lack of treatment options and potential for spread. AKlebsiella pneumoniaestrain that was nonsusceptible to all tested antibiotics was isolated from a U.S.Mechanisms that could explain all observed phenotypic antimicrobial resistance phenotypes, including resistance to colistin and beta-lactams, were identified through whole-genome sequencing. The large variety of resistance determinants identified demonstrates the usefulness of whole-genome sequencing for detecting these genes in an outbreak response. Sequencing of isolates with rare and unusual phenotypes can provide information on how these extremely resistant isolates develop, including whether resistance is acquired on mobile elements or accumulated through chromosomal mutations. Moreover, this provides further insight into not only detecting these highly resistant organisms but also preventing their spread.

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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

Strain-level genetic diversity of Methylophaga nitratireducenticrescens confers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm.

The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO[Formula: see text]) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59.The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO[Formula: see text], nitrite (NO[Formula: see text]) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures.During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2-3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO[Formula: see text] under oxic culture conditions, and has a 24-h lag time before growth and NO[Formula: see text] reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO[Formula: see text] as it carries a gene encoding a NirK-type NO[Formula: see text] reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process.These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.

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September 22, 2019

The complete replicons of 16 Ensifer meliloti strains offer insights into intra- and inter-replicon gene transfer, transposon-associated loci, and repeat elements.

Ensifer meliloti (formerly Rhizobium meliloti and Sinorhizobium meliloti) is a model bacterium for understanding legume-rhizobial symbioses. The tripartite genome of E. meliloti consists of a chromosome, pSymA and pSymB, and in some instances strain-specific accessory plasmids. The majority of previous sequencing studies have relied on the use of assemblies generated from short read sequencing, which leads to gaps and assembly errors. Here we used PacBio-based, long-read assemblies and were able to assemble, de novo, complete circular replicons. In this study, we sequenced, de novo-assembled and analysed 10 E. meliloti strains. Sequence comparisons were also done with data from six previously published genomes. We identified genome differences between the replicons, including mol% G+C and gene content, nucleotide repeats, and transposon-associated loci. Additionally, genomic rearrangements both within and between replicons were identified, providing insight into evolutionary processes at the structural level. There were few cases of inter-replicon gene transfer of core genes between the main replicons. Accessory plasmids were more similar to pSymA than to either pSymB or the chromosome, with respect to gene content, transposon content and G+C content. In our population, the accessory plasmids appeared to share an open genome with pSymA, which contains many nodulation- and nitrogen fixation-related genes. This may explain previous observations that horizontal gene transfer has a greater effect on the content of pSymA than pSymB, or the chromosome, and why some rhizobia show unstable nodulation phenotypes on legume hosts.

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September 22, 2019

Spread of plasmid-encoded NDM-1 and GES-5 carbapenemases among extensively drug-resistant and pandrug-resistant clinical Enterobacteriaceae in Durban, South Africa.

Whole-genome sequence analyses revealed the presence of blaNDM-1 (n = 31), blaGES-5 (n = 8), blaOXA-232 (n = 1), or blaNDM-5 (n = 1) in extensively drug-resistant and pandrug-resistant Enterobacteriaceae organisms isolated from in-patients in 10 private hospitals (2012 to 2013) in Durban, South Africa. Two novel NDM-1-encoding plasmids from Klebsiella pneumoniae were circularized by PacBio sequencing. In p19-10_01 [IncFIB(K); 223.434 bp], blaNDM-1 was part of a Tn1548-like structure (16.276 bp) delineated by IS26 The multireplicon plasmid p18-43_01 [IncR_1/IncFIB(pB171)/IncFII(Yp); 212.326 bp] shared an 80-kb region with p19-10_01, not including the blaNDM-1-containing region. The two plasmids were used as references for tracing NDM-1-encoding plasmids in the other genome assemblies. The p19-10_01 sequence was detected in K. pneumoniae (n = 7) only, whereas p18-43_01 was tracked to K. pneumoniae (n = 4), Klebsiella michiganensis (n = 1), Serratia marcescens (n = 11), Enterobacter spp. (n = 7), and Citrobacter freundii (n = 1), revealing horizontal spread of this blaNDM-1-bearing plasmid structure. Global phylogeny showed clustering of the K. pneumoniae (18/20) isolates together with closely related carbapenemase-negative ST101 isolates from other geographical origins. The South African isolates were divided into three phylogenetic subbranches, where each group had distinct resistance and replicon profiles, carrying either p19-10_01, p18-10_01, or pCHE-A1 (8,201 bp). The latter plasmid carried blaGES-5 and aacA4 within an integron mobilization unit. Our findings imply independent plasmid acquisition followed by local dissemination. Additionally, we detected blaOXA-232 carried by pPKPN4 in K. pneumoniae (ST14) and blaNDM-5 contained by a pNDM-MGR194-like genetic structure in Escherichia coli (ST167), adding even more complexity to the multilayer molecular mechanisms behind nosocomial spread of carbapenem-resistant Enterobacteriaceae in Durban, South Africa. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Genomic characterization of nonclonal mcr-1-positive multidrug-resistant Klebsiella pneumoniae from clinical samples in Thailand.

Multidrug-resistant Klebsiella pneumoniae strains are one of the most prevalent causes of nosocomial infections and pose an increasingly dangerous public health threat. The lack of remaining treatment options has resulted in the utilization of older drug classes, including colistin. As a drug of last resort, the discovery of plasmid-mediated colistin resistance by mcr-1 denotes the potential development of pandrug-resistant bacterial pathogens. To address the emergence of the mcr-1 gene, 118 gram-negative Enterobacteriaceae isolated from clinical samples collected at Queen Sirikit Naval Hospital in Chonburi, Thailand were screened for colistin resistance using automated antimicrobial susceptibility testing and conventional PCR screening. Two K. pneumoniae strains, QS17-0029 and QS17-0161, were positive for mcr-1, and both isolates were sequenced to closure using short- and long-read whole-genome sequencing. QS17-0029 carried 16 antibiotic resistance genes in addition to mcr-1, including 2 carbapenemases, blaNDM-1 and blaOXA-232. QS17-0161 carried 13 antibiotic resistance genes in addition to mcr-1, including the extended-spectrum ß-lactamase blaCTX-M-55. Both isolates carried multiple plasmids, but mcr-1 was located alone on highly similar 33.9?Kb IncX4 plasmids in both isolates. The IncX4 plasmid shared considerable homology to other mcr-1-containing IncX4 plasmids. This is the first report of a clinical K. pneumoniae strain from Thailand carrying mcr-1 as well as the first strain to simultaneously carry mcr-1 and multiple carbapenemase genes (QS17-0029). The identification and characterization of these isolates serves to highlight the urgent need for continued surveillance and intervention in Southeast Asia, where extensively drug-resistant pathogens are being increasingly identified in hospital-associated infections.

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September 22, 2019

Novel haloarchaeon Natrinema thermophila having the highest growth temperature among haloarchaea with a large genome size.

Environmental temperature is one of the most important factors for the growth and survival of microorganisms. Here we describe a novel extremely halophilic archaeon (haloarchaea) designated as strain CBA1119T isolated from solar salt. Strain CBA1119T had the highest maximum and optimal growth temperatures (66?°C and 55?°C, respectively) and one of the largest genome sizes among haloarchaea (5.1?Mb). It also had the largest number of strain-specific pan-genome orthologous groups and unique pathways among members of the genus Natrinema in the class Halobacteria. A dendrogram based on the presence/absence of genes and a phylogenetic tree constructed based on OrthoANI values highlighted the particularities of strain CBA1119T as compared to other Natrinema species and other haloarchaea members. The large genome of strain CBA1119T may provide information on genes that confer tolerance to extreme environmental conditions, which may lead to the discovery of other thermophilic strains with potential applications in industrial biotechnology.

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September 22, 2019

Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria.

Plasmodium falciparum, the most virulent agent of human malaria, shares a recent common ancestor with the gorilla parasite Plasmodium praefalciparum. Little is known about the other gorilla- and chimpanzee-infecting species in the same (Laverania) subgenus as P. falciparum, but none of them are capable of establishing repeated infection and transmission in humans. To elucidate underlying mechanisms and the evolutionary history of this subgenus, we have generated multiple genomes from all known Laverania species. The completeness of our dataset allows us to conclude that interspecific gene transfers, as well as convergent evolution, were important in the evolution of these species. Striking copy number and structural variations were observed within gene families and one, stevor, shows a host-specific sequence pattern. The complete genome sequence of the closest ancestor of P. falciparum enables us to estimate the timing of the beginning of speciation to be 40,000-60,000 years ago followed by a population bottleneck around 4,000-6,000 years ago. Our data allow us also to search in detail for the features of P. falciparum that made it the only member of the Laverania able to infect and spread in humans.

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September 22, 2019

Mutant phenotypes for thousands of bacterial genes of unknown function.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.

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September 22, 2019

In silico exploration of Red Sea Bacillus genomes for natural product biosynthetic gene clusters.

The increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions.We report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species.B. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems.

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September 22, 2019

Genome-wide analysis of Mycoplasma bovirhinis GS01 reveals potential virulence factors and phylogenetic relationships.

Mycoplasma bovirhinis is a significant etiology in bovine pneumonia and mastitis, but our knowledge about the genetic and pathogenic mechanisms of M. bovirhinis is very limited. In this study, we sequenced the complete genome of M. bovirhinis strain GS01 isolated from the nasal swab of pneumonic calves in Gansu, China, and we found that its genome forms a 847,985 bp single circular chromosome with a GC content of 27.57% and with 707 protein-coding genes. The putative virulence determinants of M. bovirhinis were then analyzed. Results showed that three genomic islands and 16 putative virulence genes, including one adhesion gene enolase, seven surface lipoproteins, proteins involved in glycerol metabolism, and cation transporters, might be potential virulence factors. Glycerol and pyruvate metabolic pathways were defective. Comparative analysis revealed remarkable genome variations between GS01 and a recently reported HAZ141_2 strain, and extremely low homology with others mycoplasma species. Phylogenetic analysis demonstrated that M. bovirhinis was most genetically close to M. canis, distant from other bovine Mycoplasma species. Genomic dissection may provide useful information on the pathogenic mechanisms and genetics of M. bovirhinis. Copyright © 2018 Chen et al.

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September 22, 2019

The FBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia.

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages FC31 and FBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the FBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on FBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a FBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that FBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

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