RESUMO
A common outcome of antibiotic exposure in patients and in vitro is the evolution of a hypermutator phenotype that enables rapid adaptation by pathogens. While hypermutation is a robust mechanism for rapid adaptation, it requires trade-offs between the adaptive mutations and the more common "hitchhiker" mutations that accumulate from the increased mutation rate. Using quantitative experimental evolution, we examined the role of hypermutation in driving the adaptation of Pseudomonas aeruginosa to colistin. Metagenomic deep sequencing revealed 2,657 mutations at ≥5% frequency in 1,197 genes and 761 mutations in 29 endpoint isolates. By combining genomic information, phylogenetic analyses, and statistical tests, we showed that evolutionary trajectories leading to resistance could be reliably discerned. In addition to known alleles such as pmrB, hypermutation allowed identification of additional adaptive alleles with epistatic relationships. Although hypermutation provided a short-term fitness benefit, it was detrimental to overall fitness. Alarmingly, a small fraction of the colistin-adapted population remained colistin susceptible and escaped hypermutation. In a clinical population, such cells could play a role in reestablishing infection upon withdrawal of colistin. We present here a framework for evaluating the complex evolutionary trajectories of hypermutators that applies to both current and emerging pathogen populations.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Antibacterianos/farmacologia , Mutação/efeitos dos fármacos , Adaptação Fisiológica/genética , Alelos , Proteínas de Bactérias/genética , Colistina/farmacologia , Evolução Molecular , Genoma Bacteriano/genética , Mutação/genética , Taxa de Mutação , Fenótipo , Filogenia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genéticaRESUMO
Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization.
Assuntos
DNA Complementar/genética , Ebolavirus/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus de RNA/isolamento & purificação , RNA/genética , Nanoporos , Fatores de TempoRESUMO
Effective microbial forensic analysis of materials used in a potential biological attack requires robust methods of morphological and genetic characterization of the attack materials in order to enable the attribution of the materials to potential sources and to exclude other potential sources. The genetic homogeneity and potential intersample variability of many of the category A to C bioterrorism agents offer a particular challenge to the generation of attributive signatures, potentially requiring whole-genome or proteomic approaches to be utilized. Currently, irradiation of mail is standard practice at several government facilities judged to be at particularly high risk. Thus, initial forensic signatures would need to be recovered from inactivated (nonviable) material. In the study described in this report, we determined the effects of high-dose gamma irradiation on forensic markers of bacterial biothreat agent surrogate organisms with a particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis. While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we found that irradiated material could be utilized to generate accurate whole-genome sequence data on the Illumina and Roche 454 sequencing platforms.
Assuntos
Bactérias/efeitos da radiação , Armas Biológicas , Genoma Bacteriano/efeitos da radiação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Ciências Forenses , Raios gama , Análise de Sequência de DNARESUMO
Organ-on-a-chip platforms are utilized in global bioanalytical and toxicological studies as a way to reduce materials and increase throughput as compared to in vivo based experiments. These platforms bridge the infrastructure and regulatory gaps between in vivo animal work and human systems, with models that exemplify active biological pathways. In conjunction with the advent of increased capabilities associated with next generation sequencing and mass spectrometry based '-omic' technologies, organ-on-a-chip platforms provide an excellent opportunity to investigate the global changes at multiple biological levels, including the transcriptome, proteome and metabolome. When investigated concurrently, a complete profile of cellular and regulatory perturbations can be characterized following treatment with specific agonists. In this study, global effects were observed and analyzed following liver chip exposure to the chemical warfare agent, VX. Even though the primary mechanism of action of VX (i.e. acetylcholinesterase inhibition) is well characterized, recent in vivo studies suggest additional protein binding partners that are implicated in metabolism and cellular energetic pathways. In addition, secondary toxicity associated with peripheral organ systems, especially in human tissues, is not well defined. Our results demonstrate the potential of utilizing an organ-on-a-chip platform as a surrogate system to traditional in vivo studies. This is realized by specifically indicating significant dysregulation of several cellular processes in response to VX exposure including but not limited to amino acid synthesis, drug metabolism, and energetics pathways.
Assuntos
Substâncias para a Guerra Química , Animais , Humanos , Substâncias para a Guerra Química/toxicidade , Acetilcolinesterase , Sistemas Microfisiológicos , MultiômicaRESUMO
BACKGROUND: Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. RESULTS: PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. CONCLUSIONS: This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.
Assuntos
Burkholderia/classificação , Burkholderia/imunologia , Antígenos O/análise , Animais , Vias Biossintéticas/genética , Reações Cruzadas , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Humanos , Immunoblotting , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , SorotipagemRESUMO
A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army's Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology's Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.
RESUMO
Mutants of an attenuated Bacillus anthracis (ΔANR) strain conferring increasing levels of ciproï¬oxacin resistance have been described. Here, we report the draft genome sequences of the parent strain (ΔANR pXO1-, pXO2-) and its derivatives conferring low (step 1; 0.5 µg/ml), medium (step 2; 8 to 16 µg/ml), and high (step 3; 32 to 64 µg/ml) levels of ciprofloxacin resistance.
RESUMO
The whole genomes for six botulinum neurotoxin-producing clostridial strains were sequenced to provide references for under-represented toxin types, bivalent strains or unusual toxin complexes associated with a bont gene. The strains include three Clostridium botulinum Group I strains (CDC 297, CDC 1436, and Prevot 594), a Group II C. botulinum strain (Eklund 202F), a Group IV Clostridium argentinense strain (CDC 2741), and a Group V Clostridium baratii strain (Sullivan). Comparisons of the Group I genomic sequences revealed close relationships and conservation of toxin gene locations with previously published Group I C. botulinum genomes. The bont/F6 gene of strain Eklund 202F was determined to be a chimeric toxin gene composed of bont/F1 and bont/F2. The serotype G strain CDC 2741 remained unfinished in 20 contigs with the bont/G located within a 1.15Mb contig, indicating a possible chromosomal location for this toxin gene. Within the genome of C. baratii Sullivan strain, direct repeats of IS1182 insertion sequence (IS) elements were identified flanking the bont/F7 toxin complex that may be the mechanism of bont insertion into C. baratii. Highlights of the six strains are described and release of their genomic sequences will allow further study of unusual neurotoxin-producing clostridial strains.
Assuntos
Toxinas Botulínicas/genética , Clostridium/genética , Clostridium/patogenicidade , Transferência Genética Horizontal/genética , Genoma Bacteriano/genética , Infecções por Clostridium/microbiologia , DNA Bacteriano/genética , Microbiologia Ambiental , Microbiologia de Alimentos , Humanos , Família Multigênica/genética , Filogenia , Alinhamento de SequênciaRESUMO
The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.
Assuntos
Burkholderia pseudomallei/genética , Ordem dos Genes , Genes Bacterianos/genética , Genoma Bacteriano/genética , Algoritmos , Burkholderia pseudomallei/classificação , Burkholderia pseudomallei/isolamento & purificação , Evolução Molecular , Transferência Genética Horizontal , Variação Genética , Modelos Genéticos , Recombinação Genética , Especificidade da EspécieRESUMO
How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Bactérias/genética , DNA Nucleotidiltransferases/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Chaperonas Moleculares/genética , Mutação , Salmonella typhimurium/genética , Animais , Proteínas de Bactérias/metabolismo , DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Flagelos/genética , Flagelos/ultraestrutura , Genes Reguladores , Camundongos , Camundongos Endogâmicos BALB C , Chaperonas Moleculares/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Análise de Sequência de DNA , Inoculações SeriadasRESUMO
We describe here a strain of Yersinia pestis, G1670A, which exhibits a baseline mutation rate elevated 250-fold over wild-type Y. pestis. The responsible mutation, a C to T substitution in the mutS gene, results in the transition of a highly conserved leucine at position 689 to arginine (mutS(L689R)). When the MutSL 689R protein of G1670A was expressed in a ΔmutS derivative of Y. pestis strain EV76, mutation rates observed were equivalent to those observed in G1670A, consistent with a causal association between the mutS mutation and the mutator phenotype. The observation of a mutator allele in Yersinia pestis has potential implications for the study of evolution of this and other especially dangerous pathogens.
Assuntos
Mutação , Fenótipo , Yersinia pestis/genética , Yersinia pestis/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Expressão Gênica , Teste de Complementação Genética , Genoma Bacteriano , República da Geórgia , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Yersinia pestis/isolamento & purificaçãoRESUMO
Plague disease caused by the gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19(th) century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen.
Assuntos
Genoma Bacteriano/genética , Peste/genética , Yersinia pestis/isolamento & purificação , Marcadores Genéticos , Variação Genética , Genômica , New Mexico , Peste/microbiologia , Virulência/genética , Yersinia pestis/genéticaRESUMO
BACKGROUND: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). RESULTS: Archival strains and two "present day" type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the "military" isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of "military" isolates on lactate-containing media, and showed that the "military" strains exhibited a hypersporulating phenotype. CONCLUSIONS: Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.