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September 22, 2019  |  

Whole-genome-sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374.

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

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September 22, 2019  |  

Screening and genomic characterization of filamentous hemagglutinin-deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Candidatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome.

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota.CandidatusNitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome ofCa.N. cavascurensis harbors anamoAXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although abona fidegene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism.Ca.N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

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September 22, 2019  |  

Using experimental evolution to identify druggable targets that could inhibit the evolution of antimicrobial resistance.

With multi-drug and pan-drug-resistant bacteria becoming increasingly common in hospitals, antibiotic resistance has threatened to return us to a pre-antibiotic era that would completely undermine modern medicine. There is an urgent need to develop new antibiotics and strategies to combat resistance that are substantially different from earlier drug discovery efforts. One such strategy that would complement current and future antibiotics would be a class of co-drugs that target the evolution of resistance and thereby extend the efficacy of specific classes of antibiotics. A critical step in the development of such strategies lies in understanding the critical evolutionary trajectories responsible for resistance and which proteins or biochemical pathways within those trajectories would be good candidates for co-drug discovery. We identify the most important steps in the evolution of resistance for a specific pathogen and antibiotic combination by evolving highly polymorphic populations of pathogens to resistance in a novel bioreactor that favors biofilm development. As the populations evolve to increasing drug concentrations, we use deep sequencing to elucidate the network of genetic changes responsible for resistance and subsequent in vitro biochemistry and often structure determination to determine how the adaptive mutations produce resistance. Importantly, the identification of the molecular steps, their frequency within the populations and their chronology within the evolutionary trajectory toward resistance is critical to assessing their relative importance. In this work, we discuss findings from the evolution of the ESKAPE pathogen, Pseudomonas aeruginosa to the drug of last resort, colistin to illustrate the power of this approach.

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September 22, 2019  |  

Multidrug-resistant Escherichia albertii: Co-occurrence of ß-lactamase and MCR-1 encoding genes.

Escherichia albertii is an emerging member of the Enterobacteriaceae causing human and animal enteric infections. Antimicrobial resistance among enteropathogens has been reported to be increasing in the past years. The purpose of this study was to investigate antibiotic resistance and resistance genes in E. albertii isolated from Zigong city, Sichuan province, China. The susceptibility to 21 antimicrobial agents was determined by Kirby-Bauer disk diffusion method. The highest prevalence was tetracycline resistance with a rate of 62.7%, followed by resistance to nalidixic acid and streptomycin with a rate of 56.9 and 51.0%, respectively. All isolates were sensitive or intermediate susceptible to imipenem, meropenem, amoxicillin-clavulanic acid, and levofloxacin. Among 51 E. albertii isolates, 15 were extended-spectrum ß-lactamase-producing as confirmed by the double disk test. The main ß-lactamase gene groups, i.e., blaTEM, blaSHV, and blaCTX-M, were detected in17, 20, and 22 isolates, respectively. Furthermore, four colistin-resistant isolates with minimum inhibitory concentrations of 8 mg/L were identified. The colistin-resistant isolates all harbored mcr-1 and blaCTX-M-55. Genome sequencing showed that E. albertii strain SP140150 carried mcr-1 and blaCTX-M-55 in two different plasmids. This study provided significant information regarding antibiotic resistance profiles and identified the co-occurrence of ß-lactamase and MCR-1 encoding genes in E. albertii isolates.

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September 22, 2019  |  

A validation approach of an end-to-end whole genome sequencing workflow for source tracking of Listeria monocytogenes and Salmonella enterica.

Whole genome sequencing (WGS), using high throughput sequencing technology, reveals the complete sequence of the bacterial genome in a few days. WGS is increasingly being used for source tracking, pathogen surveillance and outbreak investigation due to its high discriminatory power. In the food industry, WGS used for source tracking is beneficial to support contamination investigations. Despite its increased use, no standards or guidelines are available today for the use of WGS in outbreak and/or trace-back investigations. Here we present a validation of our complete (end-to-end) WGS workflow for Listeria monocytogenes and Salmonella enterica including: subculture of isolates, DNA extraction, sequencing and bioinformatics analysis. This end-to-end WGS workflow was evaluated according to the following performance criteria: stability, repeatability, reproducibility, discriminatory power, and epidemiological concordance. The current study showed that few single nucleotide polymorphism (SNPs) were observed for L. monocytogenes and S. enterica when comparing genome sequences from five independent colonies from the first subculture and five independent colonies after the tenth subculture. Consequently, the stability of the WGS workflow for L. monocytogenes and S. enterica was demonstrated despite the few genomic variations that can occur during subculturing steps. Repeatability and reproducibility were also demonstrated. The WGS workflow was shown to have a high discriminatory power and has the ability to show genetic relatedness. Additionally, the WGS workflow was able to reproduce published outbreak investigation results, illustrating its capability of showing epidemiological concordance. The current study proposes a validation approach comprising all steps of a WGS workflow and demonstrates that the workflow can be applied to L. monocytogenes or S. enterica.

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September 22, 2019  |  

Stress-adaptive responses associated with high-level carbapenem resistance in KPC-producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacteriaceae (CRE) organisms have emerged to become a major global public health threat among antimicrobial resistant bacterial human pathogens. Little is known about how CREs emerge. One characteristic phenotype of CREs is heteroresistance, which is clinically associated with treatment failure in patients given a carbapenem. Through in vitro whole-transcriptome analysis we tracked gene expression over time in two different strains (BR7, BR21) of heteroresistant KPC-producing Klebsiella pneumoniae, first exposed to a bactericidal concentration of imipenem followed by growth in drug-free medium. In both strains, the immediate response was dominated by a shift in expression of genes involved in glycolysis toward those involved in catabolic pathways. This response was followed by global dampening of transcriptional changes involving protein translation, folding and transport, and decreased expression of genes encoding critical junctures of lipopolysaccharide biosynthesis. The emerged high-level carbapenem-resistant BR21 subpopulation had a prophage (IS1) disrupting ompK36 associated with irreversible OmpK36 porin loss. On the other hand, OmpK36 loss in BR7 was reversible. The acquisition of high-level carbapenem resistance by the two heteroresistant strains was associated with distinct and shared stepwise transcriptional programs. Carbapenem heteroresistance may emerge from the most adaptive subpopulation among a population of cells undergoing a complex set of stress-adaptive responses.

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September 22, 2019  |  

Challenges of Francisella classification exemplified by an atypical clinical isolate.

The accumulation of sequenced Francisella strains has made it increasingly apparent that the 16S rRNA gene alone is not enough to stratify the Francisella genus into precise and clinically useful classifications. Continued whole-genome sequencing of isolates will provide a larger base of knowledge for targeted approaches with broad applicability. Additionally, examination of genomic information on a case-by-case basis will help resolve outstanding questions regarding strain stratification. We report the complete genome sequence of a clinical isolate, designated here as F. novicida-like strain TCH2015, acquired from the lymph node of a 6-year-old male. Two features were atypical for F. novicida: exhibition of functional oxidase activity and additional gene content, including proposed virulence determinants. These differences, which could potentially impact virulence and clinical diagnosis, emphasize the need for more comprehensive methods to profile Francisella isolates. This study highlights the value of whole-genome sequencing, which will lead to a more robust database of environmental and clinical genomes and inform strategies to improve detection and classification of Francisella strains. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Comparative genome analysis reveals a complex population structure of Legionella pneumophila subspecies.

The majority of Legionnaires’ disease (LD) cases are caused by Legionella pneumophila, a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila, fraseri and pascullei. During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei. We used partial sequence based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei. Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli. This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies. Published by Elsevier B.V.

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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  |  

Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype.

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and seven Stx2 subtypes have been described in E. coli, which differed in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated Stx2h in E. coli strains isolated from wild marmots in the Qinghai-Tibetan plateau, China. Stx2h shares 91.9% nucleic acid sequence identity and 92.9% amino acid identity to the nearest Stx2 subtype. The expression of Stx2h in type strain STEC299 was inducible by mitomycin C, and culture supernatant from STEC299 was cytotoxic to Vero cells. The Stx2h converting prophage was unique in terms of insertion site and genetic composition. Whole genome-based phylo- and patho-genomic analysis revealed STEC299 was closer to other pathotypes of E. coli than STEC, and possesses virulence factors from other pathotypes. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of E. coli strains. As the emergence of new Shiga toxin genotypes and new Stx-producing pathotypes pose a great threat to the public health, Stx2h should be further included in E. coli molecular typing, and in epidemiological surveillance of E. coli infections.

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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  |  

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  |  

Sequence analysis of IncA/C and IncI1 plasmids isolated from multidrug-resistant Salmonella Newport using Single-Molecule Real-Time Sequencing.

Multidrug-resistant (MDR) plasmids play an important role in disseminating antimicrobial resistance genes. To elucidate the antimicrobial resistance gene compositions in A/C incompatibility complex (IncA/C) plasmids carried by animal-derived MDR Salmonella Newport, and to investigate the spread mechanism of IncA/C plasmids, this study characterizes the complete nucleotide sequences of IncA/C plasmids by comparative analysis. Complete nucleotide sequencing of plasmids and chromosomes of six MDR Salmonella Newport strains was performed using PacBio RSII. Open reading frames were assigned using prokaryotic genome annotation pipeline (PGAP). To understand genomic diversity and evolutionary relationships among Salmonella Newport IncA/C plasmids, we included three complete IncA/C plasmid sequences with similar backbones from Salmonella Newport and Escherichia coli: pSN254, pAM04528, and peH4H, and additional 200 draft chromosomes. With the exception of canine isolate CVM22462, which contained an additional IncI1 plasmid, each of the six MDR Salmonella Newport strains contained only the IncA/C plasmid. These IncA/C plasmids (including references) ranged in size from 80.1 (pCVM21538) to 176.5?kb (pSN254) and carried various resistance genes. Resistance genes floR, tetA, tetR, strA, strB, sul, and mer were identified in all IncA/C plasmids. Additionally, blaCMY-2 and sugE were present in all IncA/C plasmids, excepting pCVM21538. Plasmid pCVM22462 was capable of being transferred by conjugation. The IncI1 plasmid pCVM22462b in CVM22462 carried blaCMY-2 and sugE. Our data showed that MDR Salmonella Newport strains carrying similar IncA/C plasmids clustered together in the phylogenetic tree using chromosome sequences and the IncA/C plasmids from animal-derived Salmonella Newport contained diverse resistance genes. In the current study, we analyzed genomic diversities and phylogenetic relationships among MDR Salmonella Newport using complete plasmids and chromosome sequences and provided possible spread mechanism of IncA/C plasmids in Salmonella Newport Lineage II.

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