RESUMO
BACKGROUND: Invasive meningococcal disease (IMD) is an acute, highly transmissible and potentially fatal disease caused by Neisseria meningitidis. Prompt antimicrobial therapy and prophylaxis are recommended, where penicillin or ciprofloxacin are the available choices. However, the emergence of resistant isolates of N. meningitidis poses a challenge for antimicrobial therapy. OBJECTIVES: To describe the clinical, epidemiological and biological characteristics of six penicillin- and ciprofloxacin-resistant, culture-confirmed IMD cases reported in El Salvador, Central America, between 2017 and 2019. METHODS: Following the detection of six patients presenting with IMD in El Salvador, clinical data were collected and epidemiological action plans conducted. Isolates were subjected to antimicrobial susceptibility testing by broth microdilution and WGS for genotyping and molecular characterization analysis, including phylogeny comparison with global sequences available from public databases. RESULTS: A total of six IMD cases caused by N. meningitidis serogroup Y, resistant to both penicillin (MIC >8.0 mg/L) and ciprofloxacin (MIC 0.125 mg/L), were detected from 2017 to 2019. Genomic analysis showed that penicillin resistance was mediated by the production of ß-lactamase ROB-1. Ciprofloxacin resistance was attributed to an amino acid substitution in DNA gyrase (T91I). All isolates were classified as ST3587, clonal complex 23, and were genetically highly similar, based on core-genome SNP analysis. CONCLUSIONS: To the best of our knowledge, we report the first cases of MDR N. meningitidis causing IMD in Latin America. Our findings highlight the emergence of this potential public health threat, with a profound impact on the efficacy of IMD treatment and prophylaxis protocols.
Assuntos
Infecções Meningocócicas , Neisseria meningitidis , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ciprofloxacina/farmacologia , El Salvador , Humanos , Infecções Meningocócicas/tratamento farmacológico , Infecções Meningocócicas/epidemiologia , Neisseria meningitidis/genética , SorogrupoRESUMO
We describe 2 human cases of infection with a new Neisseria species (putatively N. brasiliensis), 1 of which involved bacteremia. Genomic analyses found that both isolates were distinct strains of the same species, were closely related to N. iguanae, and contained a capsule synthesis operon similar to N. meningitidis.
Assuntos
Infecções Meningocócicas/diagnóstico , Neisseria/isolamento & purificação , Idoso , Brasil , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neisseria/genéticaRESUMO
This study presents a triplex real-time PCR assay that allows for the direct detection of Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in one reaction without DNA extraction, with similar sensitivity and specificity to singleplex assays. This approach saves time, specimen volume and reagents while achieving a higher testing throughput.
Assuntos
Haemophilus influenzae/genética , Meningites Bacterianas/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex , Neisseria meningitidis/genética , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus pneumoniae/genética , Genes Bacterianos/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/microbiologia , Neisseria meningitidis/isolamento & purificação , Sensibilidade e Especificidade , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
Detection of Neisseria meningitidis has become less time- and resource-intensive with a monoplex direct real-time PCR (drt-PCR) to amplify genes from clinical specimens without DNA extraction. To further improve efficiency, we evaluated two triplex drt-PCR assays for the detection of meningococcal serogroups AWX and BCY. The sensitivity and specificity of the triplex assays were assessed using 228 cerebrospinal fluid (CSF) specimens from meningitis patients and compared to the monoplex for six serogroups. The lower limit of detection range for six serogroup-specific drt-PCR assays was 178â»5264 CFU/mL by monoplex and 68â»2221 CFU/mL by triplex. The triplex and monoplex showed 100% agreement for six serogroups and the triplex assays achieved similar sensitivity and specificity estimates as the monoplex drt-PCR assays. Our triplex method reduces the time and cost of processing CSF specimens by characterizing six serogroups with only two assays, which is particularly important for testing large numbers of specimens for N. meningitidis surveillance.
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BACKGROUND: Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988-2006) for study (nâ=â372). METHODS: We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA. RESULTS: In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1. CONCLUSIONS: A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.
Assuntos
Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Neisseria meningitidis Sorogrupo B/genética , Técnicas de Tipagem Bacteriana , Sequência de Bases , Biodiversidade , Brasil/epidemiologia , Genes Bacterianos/genética , Variação Genética , Geografia , Humanos , Funções Verossimilhança , Infecções Meningocócicas/genética , Epidemiologia Molecular , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Neisseria meningitidis Sorogrupo B/classificação , Neisseria meningitidis Sorogrupo B/isolamento & purificação , Filogenia , Fatores de TempoRESUMO
Background: Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of SaËo Paulo (19882006) for study (n = 372). Methods: We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA. Results: In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the SaËo Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1. Conclusions: A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.
Assuntos
Epidemiologia , Epidemiologia Molecular , MeningiteRESUMO
Real-time (RT)-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF) was 100% (95% confidence limits, 96.0%-100%) for N. meningitidis, 97.8% (85.5%-99.9%) for S. pneumoniae, and 66.7% (9.4%-99.2%) for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by 52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0). RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil.
Assuntos
Meningites Bacterianas/microbiologia , Vigilância da População/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas de Tipagem Bacteriana , Brasil , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Contagem de Leucócitos , Masculino , Meningites Bacterianas/sangue , Meningites Bacterianas/líquido cefalorraquidiano , Pessoa de Meia-Idade , Análise Multivariada , Neisseria meningitidis/classificação , Neisseria meningitidis/genética , Reprodutibilidade dos Testes , Fatores de Risco , Sensibilidade e Especificidade , Adulto JovemRESUMO
OBJECTIVE: Neisseria meningitidis serogroup W135 has been associated with global outbreaks since the 2000 Hajj. Considering that N. meningitidis serogroup W135 is the third most prevalent serogroup isolated in Brazil in the last 10 years, and the possibility that the Hajj-related N. meningitidis serogroup W135 clone has been causing disease in Brazil, the present study characterized invasive N. meningitidis serogroup W135 isolates recovered in Brazil from 1990 to 2005. METHODS: The isolates were characterized by serotyping, PorA and PorB VR typing, FetA and 16S rRNA typing, multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). RESULTS: Based on MLST, 73% of the isolates were clustered in one major clone of ST-11 complex/ET37 complex. Strains of this clone had the same STs, serotypes and PorA VR types as found in Hajj-related N. meningitidis serogroup W135 clone. One of these strains had the Hajj-2000 outbreak strain genotype, including 16S rRNA gene sequence 31 and 84% relatedness by PFGE. CONCLUSION: Taken together, these data suggest that the Hajj-related N. meningitidis serogroup W135 clone is present in Brazil but has not yet caused a substantial number of infections. Given the emergence of N. meningitidis serogroup W135 globally and the unpredictability of meningococcal disease epidemiology, continued surveillance for this invasive N. meningitidis serogroup W135 clone is needed for control and prevention strategies.
Assuntos
Técnicas de Tipagem Bacteriana , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Neisseria meningitidis Sorogrupo W-135/classificação , Neisseria meningitidis Sorogrupo W-135/isolamento & purificação , Proteínas de Bactérias/genética , Brasil/epidemiologia , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Humanos , Epidemiologia Molecular , Neisseria meningitidis Sorogrupo W-135/genética , Neisseria meningitidis Sorogrupo W-135/fisiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Sorotipagem , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS: Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS: Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS: The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Variação Genética , Meningite Meningocócica/microbiologia , Vacinas Meningocócicas/genética , Neisseria meningitidis Sorogrupo B/genética , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Europa (Continente)/epidemiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Meningite Meningocócica/epidemiologia , Vacinas Meningocócicas/química , Vacinas Meningocócicas/metabolismo , Dados de Sequência Molecular , Neisseria meningitidis Sorogrupo B/imunologia , Neisseria meningitidis Sorogrupo B/metabolismo , Nova Zelândia/epidemiologia , África do Sul/epidemiologia , Estados Unidos/epidemiologiaRESUMO
The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Haemophilus influenzae/genética , Filogenia , Algoritmos , Haemophilus influenzae/classificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genéticaRESUMO
A molecular typing method based on the 16S rRNA sequence diversity was developed for Haemophilus influenzae isolates. A total of 330 H. influenzae isolates were analyzed, representing a diverse collection of U.S. isolates. We found a high level of 16S rRNA sequence heterogeneity (up to 2.73%) and observed an exclusive correlation between 16S types and serotypes (a to f); no 16S type was found in more than one serotype. Similarly, no multilocus sequence typing (MLST) sequence type (ST) was found in more than one serotype. Our 16S typing and MLST results are in agreement with those of previous studies showing that serotypable H. influenzae isolates behave as highly clonal populations and emphasize the lack of clonality of nontypable (NT) H. influenzae isolates. There was not a 1:1 correlation between 16S types and STs, but all H. influenzae serotypable isolates clustered similarly. This correlation was not observed for NT H. influenzae; the two methods clustered NT H. influenzae isolates differently. 16S rRNA gene sequencing alone provides a level of discrimination similar to that obtained with the analysis of seven genes for MLST. We demonstrated that 16S typing is an additional and complementary approach to MLST, particularly for NT H. influenzae isolates, and is potentially useful for outbreak investigation.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Haemophilus influenzae/classificação , RNA Ribossômico 16S/genética , Variação Genética , Haemophilus influenzae/genética , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , SorotipagemRESUMO
In 2000, a large international outbreak of meningococcal disease caused by Neisseria meningitidis serogroup W-135 was identified among pilgrims returning from the Hajj in Saudi Arabia. To assess ongoing risk, we evaluated N. meningitidis carriage among US travelers to the 2001 Hajj. Of 25 N. meningitidis isolates obtained, 15 (60%) were nongroupable and 8 (32%) were serogroup W-135 when tested by standard slide-agglutination techniques. Two additional nongroupable isolates were characterized as serogroup W-135 when tested by polymerase chain reaction. Nine of 10 serogroup W-135 isolates were indistinguishable from the Hajj-2000 clone. None of the departing, but 9 (1.3%) of the returning, pilgrims carried serogroup W-135 (P=.01); all carriers reported previous vaccination. Carriage of N. meningitidis serogroup W-135 increased significantly in pilgrims returning from the Hajj. Although the risk of disease to pilgrims appears to be low, the risk of spread to others of this pathogenic strain remains a concern.
Assuntos
Portador Sadio/epidemiologia , Infecções Meningocócicas/epidemiologia , Neisseria meningitidis Sorogrupo W-135/isolamento & purificação , Viagem , Adulto , Idoso , Portador Sadio/microbiologia , Feminino , Humanos , Islamismo , Masculino , Infecções Meningocócicas/microbiologia , Pessoa de Meia-Idade , Neisseria meningitidis Sorogrupo W-135/classificação , Faringe/microbiologia , Reação em Cadeia da Polimerase , Arábia Saudita , Sorotipagem , Estados UnidosRESUMO
Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.
Assuntos
Antraz , Antígenos de Bactérias , Bacillus cereus/genética , Bacillus cereus/patogenicidade , Toxinas Bacterianas/genética , Animais , Antraz/etiologia , Bacillus anthracis/classificação , Bacillus anthracis/citologia , Bacillus anthracis/genética , Bacillus cereus/classificação , Bacillus cereus/citologia , Genoma Bacteriano , Genômica , Humanos , Camundongos , Plasmídeos/genéticaRESUMO
Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia in children and young adults in the United States. Rapid and reliable identification of N. meningitidis serogroups is crucial for judicious and expedient response to cases of meningococcal disease, including decisions about vaccination campaigns. From 1997 to 2002, 1,298 N. meningitidis isolates, collected in the United States through the Active Bacterial Core surveillance (ABCs), were tested by slide agglutination serogrouping (SASG) at both the ABCs sites and the Centers for Disease Control and Prevention (CDC). For over 95% of isolates, SASG results were concordant, while discrepant results were reported for 58 isolates. To resolve these discrepancies, we repeated the SASG in a blinded fashion and employed ctrA and six serogroup-specific PCR assays (SGS-PCR) to determine the genetic capsule type. Seventy-eight percent of discrepancies were resolved, since results of the SGS-PCR and SASG blinded study agreed with each other and confirmed the SASG result at either state health laboratories or CDC. This study demonstrated the ability of SGS-PCR to efficiently resolve SASG discrepancies and identified the main cause of the discrepancies as overreporting of these isolates as nongroupable. It also reemphasized the importance of adherence to quality assurance procedures when performing SASG and prompted prospective monitoring for SASG discrepancies involving isolates collected through ABCs in the United States.
Assuntos
Testes de Aglutinação/métodos , Neisseria meningitidis/classificação , Reação em Cadeia da Polimerase/métodos , Cápsulas Bacterianas/genética , Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Humanos , Neisseria meningitidis/genética , Sensibilidade e Especificidade , Sorotipagem , Fatores de Transcrição/genéticaRESUMO
Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.
Assuntos
Técnicas de Tipagem Bacteriana , Burkholderia pseudomallei/classificação , Burkholderia/classificação , Genes de RNAr , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Animais , Burkholderia/genética , Infecções por Burkholderia/microbiologia , Burkholderia pseudomallei/genética , DNA Ribossômico/análise , Microbiologia Ambiental , Humanos , Melioidose/microbiologia , Dados de Sequência MolecularRESUMO
PorA protein is an important component of group B meningococcal protein-based vaccines. The goals of this study were: (i) to classify the non-serosubtypable strains recovered from vaccine failures and controls by porA variable region (VR) type; (ii) to investigate if point mutations of VRs of the porA gene are present in P1.19,15 strains recovered from vaccine failures and controls; (iii) to investigate if nucleotide sequence variation in the promoter region of porA gene is related to low expression of PorA protein. VR type P1.19,15 predominated in younger vaccine failures (3-47 months) compared to older failures (48-83 months). No changes in VRs of porA were observed in 46 P1.19,15 strains studied. A promoter spacer of 16bp and 10 guanidine residues in the polymeric G tract was detected in five of six strains with weak PorA expression. Overall, this study indicated that lack of antibody response was probably the major cause of low vaccine efficacy in young children.
Assuntos
Vacinas Bacterianas/imunologia , Região Variável de Imunoglobulina/genética , Meningite Meningocócica/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Porinas/genética , Regiões Promotoras Genéticas , Anticorpos Antibacterianos/imunologia , Criança , Pré-Escolar , Relação Dose-Resposta Imunológica , Eletroforese em Gel de Poliacrilamida , Humanos , Lactente , Neisseria meningitidis Sorogrupo B/classificação , Neisseria meningitidis Sorogrupo B/isolamento & purificação , Mutação Puntual , Porinas/biossíntese , Porinas/imunologia , VacinaçãoRESUMO
We investigated the diversity of the primary sequences of 16S rRNA genes among Neisseria meningitidis strains (Men) and evaluated the use of this approach as a molecular subtyping tool. We aligned and compared a 1,417-bp fragment of the 16S rRNA gene from 264 Men strains of serogroups A, B, C, and Y (MenA, MenB, MenC, and MenY, respectively) isolated throughout the world over a 30-year period. Thirty-one positions of difference were found among 49 16S types: differences between types ranged from 1 to 14 positions (0.07 to 0.95%). 16S types and serogroups were highly associated; only 3 out 49 16S types were shared by two or more serogroups. We have identified 16S types that are exclusively associated with strains of certain hypervirulent clones: 16S type 5 with MenA subgroup III, 16S type 4 with the MenB electrophoretic type 5 (ET-5) complex, and 16S types 12 and 13 with MenC of the ET-37 complex. For MenC strains, 16S sequencing provided the highest sensitivity and specificity and the best overall association with the outbreak-related versus sporadic isolates when compared with pulsed-field gel electrophoresis, multilocus enzyme electrophoresis, and multilocus sequence typing. We demonstrated for the first time an unexpected diversity among 16S rRNA genes of Men strains, identified 16S types associated with well-defined hypervirulent clones, and showed the potential of this approach to rapidly identify virulent strains associated with outbreaks and/or an increased incidence of sporadic disease.
Assuntos
Técnicas de Tipagem Bacteriana , Genes de RNAr/genética , Variação Genética , Neisseria meningitidis/classificação , Análise de Sequência de DNA , DNA Ribossômico/análise , Surtos de Doenças , Eletroforese/métodos , Humanos , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Dados de Sequência Molecular , Neisseria meningitidis/genética , RNA Ribossômico 16S/genética , SorotipagemAssuntos
Infecções Meningocócicas/microbiologia , Neisseria meningitidis/classificação , Adulto , Croácia/epidemiologia , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Infecções Meningocócicas/epidemiologia , Neisseria meningitidis/genética , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genéticaRESUMO
In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.
Assuntos
Antraz/diagnóstico , Antraz/microbiologia , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Bacillus thuringiensis/genética , Bacillus thuringiensis/isolamento & purificação , Genes Bacterianos/genética , Variação Genética/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Bacteriano/genéticaRESUMO
In 2000, >400 cases of disease caused by Neisseria meningitidis serogroup W135 (MenW135), the largest MenW135 outbreak reported to date, occurred worldwide among Hajj pilgrims and their contacts. To elucidate the origin of the outbreak strains and to investigate their relatedness to major clonal groups, genotypic and phenotypic subtyping was performed on 26 MenW135 outbreak-associated isolates and 50 MenW135 isolates collected worldwide from 1970 through 2000. All outbreak-associated isolates were members of a single clone of the hypervirulent electrophoretic type (ET)-37 complex, designated the "(W)ET-37 clone"; 19 additional MenW135 strains were also members of this clone, and the remaining 31 MenW135 strains were clearly distinct. The 2000 MenW135 outbreak was not caused by emergence of a new MenW135 strain but rather by expansion of the (W)ET-37 clone that has been in circulation at least since 1970; the strains most closely related to those causing the 2000 outbreak have been isolated in Algeria, Mali, and The Gambia in the 1990s.