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1.
Emerg Infect Dis ; 29(3): 467-476, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36823096

RESUMO

Molecular methods can enable rapid identification of Bartonella spp. infections, which are difficult to diagnose by using culture or serology. We analyzed clinical test results of PCR that targeted bacterial 16S rRNA hypervariable V1-V2 regions only or in parallel with PCR of Bartonella-specific ribC gene. We identified 430 clinical specimens infected with Bartonella spp. from 420 patients in the United States. Median patient age was 37 (range 1-79) years; 62% were male. We identified B. henselae in 77%, B. quintana in 13%, B. clarridgeiae in 1%, B. vinsonii in 1%, and B. washoensis in 1% of specimens. B. quintana was detected in 83% of cardiac specimens; B. henselae was detected in 34% of lymph node specimens. We detected novel or uncommon Bartonella spp. in 9 patients. Molecular diagnostic testing can identify Bartonella spp. infections, including uncommon and undescribed species, and might be particularly useful for patients who have culture-negative endocarditis or lymphadenitis.


Assuntos
Infecções por Bartonella , Bartonella henselae , Bartonella , Humanos , Masculino , Estados Unidos , Lactente , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Feminino , RNA Ribossômico 16S/genética , Infecções por Bartonella/microbiologia , Reação em Cadeia da Polimerase/métodos , Técnicas de Amplificação de Ácido Nucleico , Bartonella henselae/genética
2.
J Clin Microbiol ; 61(10): e0034723, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37787542

RESUMO

Whole-genome sequencing (WGS) provides greater resolution than other molecular epidemiology strategies and is emerging as a new gold standard approach for microbial strain typing. The Bruker IR Biotyper is designed as a screening tool to identify bacterial isolates that require WGS to establish accurate relationships, but its performance and utility in nosocomial outbreak investigations have not been thoroughly investigated. Here, we evaluated the IR Biotyper by retrospectively examining isolates tested by WGS during investigations of potential nosocomial transmission events or outbreaks. Ninety-eight clinical isolates from 14 different outbreak investigations were examined: three collections of Acinetobacter baumannii (n = 2, n = 9, n = 5 isolates in each collection), one of Escherichia coli (n = 16), two of Pseudomonas aeruginosa (n = 2 and n = 5), two of Serratia marcescens (n = 9 and n = 7), five of Staphylococcus aureus (n = 8, n = 4, n = 3, n = 3, n = 17), and one of Stenotrophomonas maltophilia (n = 8). Linear regression demonstrated a weak, positive correlation between the number of pairwise genome-wide single-nucleotide polymorphisms (SNPs) and IR Biotyper spectral distance values for Gram-positive (r = 0.43, P ≤ 0.0001), Gram-negative (r = 0.1554, P = 0.0639), and all organisms combined (r = 0.342, P ≤ 0.0001). Overall, the IR Biotyper had a positive predictive value (PPV) of 55.81% for identifying strains that were closely related by genomic identity, but a negative predictive value (NPV) of 86.79% for identifying unrelated isolates. When experimentally adjusted cut-offs were applied to A. baumannii, P. aeruginosa, and E. coli, the PPV was 62% for identifying strains that were closely related and the NPV was 100% for identifying unrelated isolates. Implementation of the IR Biotyper as a screening tool in this cohort would have reduced the number of Gram-negative isolates requiring further WGS analysis by 50% and would reduce the number of S. aureus isolates needing WGS resolution by 48%.


Assuntos
Infecção Hospitalar , Escherichia coli , Humanos , Escherichia coli/genética , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Estudos Retrospectivos , Espectroscopia de Infravermelho com Transformada de Fourier , Análise de Fourier , Staphylococcus aureus/genética , Genoma Bacteriano/genética , Surtos de Doenças
3.
J Clin Microbiol ; 61(8): e0184222, 2023 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-37428072

RESUMO

Identification and analysis of clinically relevant strains of bacteria increasingly relies on whole-genome sequencing. The downstream bioinformatics steps necessary for calling variants from short-read sequences are well-established but seldom validated against haploid genomes. We devised an in silico workflow to introduce single nucleotide polymorphisms (SNP) and indels into bacterial reference genomes, and computationally generate sequencing reads based on the mutated genomes. We then applied the method to Mycobacterium tuberculosis H37Rv, Staphylococcus aureus NCTC 8325, and Klebsiella pneumoniae HS11286, and used the synthetic reads as truth sets for evaluating several popular variant callers. Insertions proved especially challenging for most variant callers to correctly identify, relative to deletions and single nucleotide polymorphisms. With adequate read depth, however, variant callers that use high quality soft-clipped reads and base mismatches to perform local realignment consistently had the highest precision and recall in identifying insertions and deletions ranging from1 to 50 bp. The remaining variant callers had lower recall values associated with identification of insertions greater than 20 bp.


Assuntos
Biologia Computacional , Software , Humanos , Biologia Computacional/métodos , Sequenciamento Completo do Genoma , Genoma , Polimorfismo de Nucleotídeo Único , Bactérias , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos
4.
J Clin Microbiol ; 59(11): e0095521, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34406798

RESUMO

Broad-range fungal PCR is a powerful tool for identifying pathogens directly from patient specimens; however, reported estimates of clinical utility vary and costs discourage universal testing. We investigated the diagnostic and clinical utility of broad-range fungal PCR by examining 9 years of results from sinonasal specimens, hypothesizing that this anatomic location would identify immunocompromised patients at high risk for invasive fungal disease. We retrospectively identified 644 PCRs and 1,446 fungal cultures from sinus sites. To determine the relative performance of each testing modality, we performed chart review on 52 patients having specimens submitted for culture and PCR on the same day. Positivity rates were significantly higher for PCR (37.1%) than culture (13.7%) but similar for formalin-fixed and fresh tissues (42.3% versus 34.6%). Relative to culture, PCR had significantly faster turnaround time to both preliminary (94.5 versus 108.8 h) and final positive (137.9 versus 278.5 h) results. Among chart-reviewed patients, 88% were immunocompromised, 65% had proven or probable fungal disease, and testing sensitivities for culture and PCR (67.5% and 85.0%) were not statistically different. Nevertheless, PCR identified pathogens not recovered by culture in 14.9% of cases and informed clinical decision-making in 16.7% of all reviewed cases, and sensitivity of PCR combined with culture (90.0%) was higher than that of culture alone. We conclude that broad-range fungal PCR is frequently informative for patients at risk of serious fungal disease and is complementary to and has faster turnaround time than culture. Formalin-fixed tissue does not adversely affect diagnostic yield, but anatomic site may impact assay positivity rates.


Assuntos
Micoses , Sinusite , DNA Fúngico/genética , Humanos , Micoses/diagnóstico , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Sensibilidade e Especificidade , Sinusite/diagnóstico
5.
Vet Pathol ; 58(4): 624-642, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33357072

RESUMO

Coxiella burnetii, a highly adapted obligate intracellular bacterial pathogen and the cause of the zoonosis Q fever, is a reemerging public health threat. C. burnetii employs a Type IV secretion system (T4SS) to establish and maintain its intracellular niche and modulate host immune responses including the inhibition of apoptosis. Interactions between C. burnetii and caspase-1-mediated inflammasomes are not fully elucidated. This study confirms that C. burnetii does not activate caspase-1 during infection of mouse macrophages in vitro. C. burnetii-infected cells did not develop NLRP3 and ASC foci indicating its ability to avoid cytosolic detection. C. burnetii is unable to inhibit the pyroptosis and IL-1ß secretion that is induced by potent inflammasome stimuli but rather enhances these caspase-1-mediated effects. We found that C. burnetii upregulates pro-IL-1ß and robustly primes NLRP3 inflammasomes via TLR2 and MyD88 signaling. As for wildtype C. burnetii, T4SS-deficient mutants primed and potentiated NLRP3 inflammasomes. An in vivo model of pulmonary infection in C57BL/6 mice was developed. Mice deficient in NLRP3 or caspase-1 were like wildtype mice in the development and resolution of splenomegaly due to red pulp hyperplasia, and histologic lesions and macrophage kinetics, but had slightly higher pulmonary bacterial burdens at the greatest measured time point. Together these findings indicate that C. burnetii primes but avoids cytosolic detection by NLRP3 inflammasomes, which are not required for the clinical resistance of C57BL/6 mice. Determining mechanisms employed by C. burnetii to avoid cytosolic detection via NLRP3 inflammasomes will be beneficial to the development of preventative and interventional therapies for Q fever.


Assuntos
Coxiella burnetii , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Febre Q , Animais , Camundongos , Camundongos Endogâmicos C57BL , Febre Q/imunologia
6.
Infect Immun ; 89(1)2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33046510

RESUMO

Immunocompromised patients are more susceptible to recurrent nontyphoidal Salmonella (NTS) bacteremia. A key manifestation of HIV infection is the loss of CD4 T cells, which are crucial for immunity to Salmonella infection. We characterized the consequences of CD4 T cell depletion in mice where virulent Salmonella establish chronic infection, similar to chronic NTS disease in humans. Salmonella-infected, CD4-depleted 129X1/SvJ mice remained chronically colonized for at least 5 weeks, displaying increased splenomegaly and more severe splenitis than infected mice with CD4 T cells. Mature erythrocytes, immature erythroid cells, and phagocytes accounted for the largest increase in splenic cellularity. Anemia, which is associated with increased mortality in Salmonella-infected humans, was exacerbated by CD4 depletion in infected mice and was accompanied by increased splenic sequestration of erythrocytes and fewer erythropoietic elements in the bone marrow, despite significantly elevated levels of circulating erythropoietin. Splenic sequestration of red blood cells, the appearance of circulating poikilocytes, and elevated proinflammatory cytokines suggest inflammation-induced damage to erythrocytes contributes to anemia and splenic retention of damaged cells in infected animals. Depleting CD4 T cells led to increased myeloid cells in peripheral blood, spleen, and bone marrow, as well as expansion of CD8 T cells, which has been observed in CD4-depleted humans. This work describes a mouse model of Salmonella infection that recapitulates several aspects of human disease and will allow us to investigate the interplay of innate and adaptive immune functions with chronic inflammation, anemia, and susceptibility to Salmonella infection.


Assuntos
Anemia/etiologia , Linfócitos T CD4-Positivos/imunologia , Hospedeiro Imunocomprometido , Mielopoese/imunologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Anemia/diagnóstico , Animais , Medula Óssea/patologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunidade Celular , Camundongos , Infecções por Salmonella/complicações , Infecções por Salmonella/diagnóstico , Salmonella typhimurium/imunologia , Índice de Gravidade de Doença , Esplenomegalia/patologia
7.
Clin Infect Dis ; 71(8): 1896-1904, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31665255

RESUMO

BACKGROUND: Campylobacter species are among the most common causes of enteric bacterial infections worldwide. Men who have sex with men (MSM) are at increased risk for sexually transmitted enteric infections, including globally distributed strains of multidrug-resistant Shigella species. METHODS: This was a retrospective study of MSM-associated Campylobacter in Seattle, Washington and Montréal, Québec with phenotypic antimicrobial resistance profiles and whole genome sequencing (WGS). RESULTS: We report the isolation of 2 clonal lineages of multidrug-resistant Campylobacter coli from MSM in Seattle and Montréal. WGS revealed nearly identical strains obtained from the 2 regions over a 4-year period. Comparison with the National Center for Biotechnology Information's Pathogen Detection database revealed extensive Campylobacter species clusters carrying multiple drug resistance genes that segregated with these isolates. Examination of the genetic basis of antimicrobial resistance revealed multiple macrolide resistance determinants including a novel ribosomal RNA methyltransferase situated in a CRISPR (clustered regularly interspaced short palindromic repeats) array locus in a C. coli isolate. CONCLUSIONS: As previously reported for Shigella, specific multidrug-resistant strains of Campylobacter are circulating by sexual transmission in MSM populations across diverse geographic locations, suggesting a need to incorporate sexual behavior in the investigation of clusters of foodborne pathogens revealed by WGS data.


Assuntos
Infecções por Campylobacter , Campylobacter coli , Minorias Sexuais e de Gênero , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/epidemiologia , Campylobacter coli/genética , Farmacorresistência Bacteriana , Homossexualidade Masculina , Humanos , Macrolídeos , Masculino , Testes de Sensibilidade Microbiana , Quebeque/epidemiologia , Estudos Retrospectivos , Washington/epidemiologia
8.
J Clin Microbiol ; 58(12)2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33028602

RESUMO

The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture ("16S Capture"). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.


Assuntos
Metagenômica , DNA Bacteriano/genética , Genes de RNAr , Humanos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
10.
J Clin Microbiol ; 57(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31434720

RESUMO

Enterobacteriaceae represent a diverse and medically important family of bacteria that are difficult to identify to the species level using the standard molecular method of 16S rRNA gene sequencing. Prior work has demonstrated the value of dnaJ gene sequence analysis in resolving different members of the family. However, existing protocols are not optimized for clinical use and exhibit several limitations in practice. Here, we describe an improved assay for dnaJ-based identification of Enterobacteriaceae which boasts increased broad-range specificity across genera, shorter amplicon sizes that are suitable for use with formalin-fixed or direct patient specimens, and enhanced amplification efficiency and assay sensitivity through the incorporation of locked nucleic acid chemistries. Sequence analysis of public databases indicates that the partial dnaJ sequence interrogated by this design retains high discriminatory power among Enterobacteriaceae genera and species, with only particular lineages of Shigella sp. and Escherichia coli proving unresolvable. Limits of detection studies using 8 disparate species indicated that amplification was consistently achievable across organisms and allowed robust dideoxynucleotide chain terminator sequencing from as little as 10 genome equivalents of template, depending on the species interrogated. Retrospective application of the dnaJ assay to patient specimens enabled unambiguous classification of Enterobacteriaceae to the species level in 22 of 27 (81.5%) positive specimens examined, with most remaining cases representing unresolvable calls between closely related Escherichia coli and Shigella species. We expect that this assay will facilitate the accurate molecular identification of species from the Enterobacteriaceae family in a variety of clinical specimens and diagnostic contexts.


Assuntos
Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/classificação , Proteínas de Choque Térmico HSP40/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Primers do DNA/genética , DNA Bacteriano/análise , Infecções por Enterobacteriaceae/diagnóstico , Proteínas de Escherichia coli/genética , Genótipo , Humanos , Limite de Detecção , Oligonucleotídeos/genética , Filogenia , RNA Ribossômico 16S/genética
12.
Clin Infect Dis ; 67(11): 1688-1696, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29697761

RESUMO

Background: Molecular syndromic diagnostic panels can enhance pathogen identification in the approximately 2-4 billion episodes of acute gastroenteritis that occur annually worldwide. However, the clinical utility of these panels has not been established. Methods: We conducted a prospective, multi-center study to investigate the impact of the BioFire FilmArray Gastrointestinal polymerase chain reaction panel on clinical diagnosis and decision-making, and compared the clinical acuity of patients with positive results obtained exclusively with the FilmArray with those detected by conventional stool culture. A total of 1887 consecutive fecal specimens were tested in parallel by FilmArray and stool culture. Laboratory and medical records were reviewed to determine rates of detection, turnaround times, clinical features, and the nature and timing of clinical decisions. Results: FilmArray detected pathogens in 35.3% of specimens, compared to 6.0% for culture. Median time from collection to result was 18 hours for FilmArray and 47 hours for culture. Median time from collection to initiation of antimicrobial therapy was 22 hours for FilmArray and 72 hours for culture. Patients diagnosed by FilmArray were more likely to receive targeted rather than empirical therapy, compared to those diagnosed by culture (P = .0148). Positive Shiga-like toxin-producing E. coli results were reported 47 hours faster with FilmArray and facilitated discontinuation of empirical antimicrobials. Patients diagnosed exclusively by FilmArray had clinical characteristics similar to those identified by culture. Conclusions: FilmArray markedly improved clinical sensitivity in patients with acute diarrhea, identified cases with clinical acuity comparable to those identified by culture, and enabled clinicians to make more timely and targeted therapeutic decisions.


Assuntos
Tomada de Decisão Clínica , Escherichia coli/isolamento & purificação , Gastroenterite/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Fezes/microbiologia , Feminino , Gastroenterite/microbiologia , Humanos , Lactente , Masculino , Técnicas Microbiológicas , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Adulto Jovem
13.
Genome Res ; 25(1): 119-28, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25373147

RESUMO

Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole-genome sequencing and de novo assembly of 312 blood- or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pan-genome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different body sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one subclone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a 3-yr period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole-genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kind of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting.


Assuntos
Escherichia coli/genética , Genoma Bacteriano , Análise de Sequência de DNA/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Feminino , Biblioteca Gênica , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Modelos Logísticos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fenótipo , Filogenia , Infecções Urinárias/microbiologia , Fatores de Virulência/genética , Adulto Jovem
14.
PLoS Genet ; 11(7): e1005413, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26230489

RESUMO

Bacterial whole genome sequencing holds promise as a disruptive technology in clinical microbiology, but it has not yet been applied systematically or comprehensively within a clinical context. Here, over the course of one year, we performed prospective collection and whole genome sequencing of nearly all bacterial isolates obtained from a tertiary care hospital's intensive care units (ICUs). This unbiased collection of 1,229 bacterial genomes from 391 patients enables detailed exploration of several features of clinical pathogens. A sizable fraction of isolates identified as clinically relevant corresponded to previously undescribed species: 12% of isolates assigned a species-level classification by conventional methods actually qualified as distinct, novel genomospecies on the basis of genomic similarity. Pan-genome analysis of the most frequently encountered pathogens in the collection revealed substantial variation in pan-genome size (1,420 to 20,432 genes) and the rate of gene discovery (1 to 152 genes per isolate sequenced). Surprisingly, although potential nosocomial transmission of actively surveilled pathogens was rare, 8.7% of isolates belonged to genomically related clonal lineages that were present among multiple patients, usually with overlapping hospital admissions, and were associated with clinically significant infection in 62% of patients from which they were recovered. Multi-patient clonal lineages were particularly evident in the neonatal care unit, where seven separate Staphylococcus epidermidis clonal lineages were identified, including one lineage associated with bacteremia in 5/9 neonates. Our study highlights key differences in the information made available by conventional microbiological practices versus whole genome sequencing, and motivates the further integration of microbial genome sequencing into routine clinical care.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/transmissão , Genoma Bacteriano/genética , Unidades de Terapia Intensiva , Microbiota/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bactérias/classificação , Bactérias/genética , Infecções Bacterianas/microbiologia , Técnicas de Tipagem Bacteriana , Biodiversidade , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , DNA Bacteriano/genética , Feminino , Variação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Estudos Prospectivos , Centros de Atenção Terciária , Adulto Jovem
15.
Clin Chem ; 62(11): 1465-1473, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27624135

RESUMO

BACKGROUND: Humans suffer from infections caused by single species or more complex polymicrobial communities. Identification of infectious bacteria commonly employs microbiological culture, which depends upon the in vitro propagation and isolation of viable organisms. In contrast, detection of bacterial DNA using next generation sequencing (NGS) allows culture-independent microbial profiling, potentially providing important new insights into the microbiota in clinical specimens. METHODS: NGS 16S rRNA gene sequencing (NGS16S) was compared with culture using (a) synthetic polymicrobial samples for which the identity and abundance of organisms present were precisely defined and (b) primary clinical specimens. RESULTS: Complex mixtures of at least 20 organisms were well resolved by NGS16S with excellent reproducibility. In mixed bacterial suspensions (107 total genomes), we observed linear detection of a target organism over a 4-log concentration range (500-3 × 106 genomes). NGS16S analysis more accurately recapitulated the known composition of synthetic samples than standard microbiological culture using nonselective media, which distorted the relative abundance of organisms and frequently failed to identify low-abundance pathogens. However, extended quantitative culture using selective media for each of the component species recovered the expected organisms at the proper abundance, validating NGS16S results. In an analysis of sputa from cystic fibrosis patients, NGS16S identified more clinically relevant pathogens than standard culture. CONCLUSIONS: Biases in standard, nonselective microbiological culture lead to a distorted characterization of polymicrobial mixtures. NGS16S demonstrates enhanced reproducibility, quantification, and classification accuracy compared with standard culture, providing a more comprehensive, accurate, and culture-free analysis of clinical specimens.


Assuntos
Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , DNA Bacteriano/genética , Técnicas Microbiológicas/normas , Análise de Sequência de DNA/tendências , Humanos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/normas
17.
Emerg Infect Dis ; 21(1): 95-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25529016

RESUMO

Metronidazole- and carbapenem-resistant Bacteroides fragilis are rare in the United States. We isolated a multidrug-resistant anaerobe from the bloodstream and intraabdominal abscesses of a patient who had traveled to India. Whole-genome sequencing identified the organism as a novel Bacteroides genomospecies. Physicians should be aware of the possibility for concomitant carbapenem- and metronidazole-resistant Bacteroides infections.


Assuntos
Infecções por Bacteroides/microbiologia , Bacteroides/efeitos dos fármacos , Adenocarcinoma/sangue , Adenocarcinoma/microbiologia , Adenocarcinoma/secundário , Idoso , Antibacterianos/farmacologia , Bacteroides/genética , Bacteroides/isolamento & purificação , Infecções por Bacteroides/sangue , Neoplasias do Colo/sangue , Neoplasias do Colo/microbiologia , Neoplasias do Colo/patologia , Farmacorresistência Bacteriana Múltipla , Genoma Bacteriano , Humanos , Masculino , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA
18.
Mol Microbiol ; 94(6): 1272-84, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25315056

RESUMO

Bistable flagellar and virulence gene expression generates specialized Salmonella subpopulations with distinct functions. Repressing flagellar genes allows Salmonella to evade caspase-1 mediated host defenses and enhances systemic colonization. By definition, bistability arises when intermediate states of gene expression are rendered unstable by the underlying genetic circuitry. We demonstrate sustained bistable fliC expression in virulent Salmonella 14028 and document dynamic control of the distribution, or single-cell census, of flagellar gene expression by the mutually repressing repressors YdiV and FliZ. YdiV partitions cells into the fliC-OFF subpopulation, while FliZ partitions cells into the fliC-HIGH subpopulation at late time points during growth. Bistability of ΔfliZ populations and ydiV-independent FliZ control of flagellar gene expression provide evidence that the YdiV-FliZ mutually repressing repressor circuit is not required for bistability. Repression and activation by YdiV and FliZ (respectively) can shape the census of fliC expression independently, and bistability collapses into a predominantly intermediate population in the absence of both regulators. Metered expression of YdiV and FliZ reveals variable sensitivity to these regulators and defines conditions where expression of FliZ enhances fliC expression and where FliZ does not alter the fliC census. Thus, this evolved genetic circuitry coordinates multiple layers of regulatory heterogeneity into a binary response.


Assuntos
Proteínas de Bactérias/metabolismo , Salmonella typhimurium/patogenicidade , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
19.
J Clin Microbiol ; 53(4): 1072-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25631811

RESUMO

Nosocomial infections pose a significant threat to patient health; however, the gold standard laboratory method for determining bacterial relatedness (pulsed-field gel electrophoresis [PFGE]) remains essentially unchanged 20 years after its introduction. Here, we explored bacterial whole-genome sequencing (WGS) as an alternative approach for molecular strain typing. We compared WGS to PFGE for investigating presumptive outbreaks involving three important pathogens: vancomycin-resistant Enterococcus faecium (n=19), methicillin-resistant Staphylococcus aureus (n=17), and Acinetobacter baumannii (n=15). WGS was highly reproducible (average≤0.39 differences between technical replicates), which enabled a functional, quantitative definition for determining clonality. Strain relatedness data determined by PFGE and WGS roughly correlated, but the resolution of WGS was superior (P=5.6×10(-8) to 0.016). Several discordant results were noted between the methods. A total of 28.9% of isolates which were indistinguishable by PFGE were nonclonal by WGS. For A. baumannii, a species known to undergo rapid horizontal gene transfer, 16.2% of isolate pairs considered nonidentical by PFGE were clonal by WGS. Sequencing whole bacterial genomes with single-nucleotide resolution demonstrates that PFGE is prone to false-positive and false-negative results and suggests the need for a new gold standard approach for molecular epidemiological strain typing.


Assuntos
Genoma Bacteriano/genética , Epidemiologia Molecular/métodos , Tipagem Molecular/métodos , Análise de Sequência de DNA/métodos , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Infecção Hospitalar/microbiologia , DNA Bacteriano/análise , DNA Bacteriano/genética , Surtos de Doenças , Humanos
20.
J Clin Microbiol ; 53(8): 2773-6, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26063867

RESUMO

A man with newly diagnosed AIDS presented with months of back pain and fever. Computed tomography (CT) results demonstrated aortitis with periaortic tissue thickening. DNA amplification of biopsy tissue revealed Bartonella quintana, and Bartonella serologies were subsequently noted to be positive. The patient improved with prolonged doxycycline and rifabutin treatment. This case illustrates how molecular techniques are increasingly important in diagnosing Bartonella infections.


Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Aortite/diagnóstico , Aortite/patologia , Bartonella quintana/isolamento & purificação , Febre das Trincheiras/diagnóstico , Febre das Trincheiras/patologia , Antibacterianos/uso terapêutico , Anticorpos Antibacterianos/sangue , Aortite/tratamento farmacológico , Biópsia por Agulha , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Doxiciclina/uso terapêutico , Genes de RNAr , Histocitoquímica , Humanos , Masculino , Microscopia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Rifabutina/uso terapêutico , Análise de Sequência de DNA , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Febre das Trincheiras/tratamento farmacológico
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