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1.
BMC Infect Dis ; 20(1): 279, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293324

RESUMO

BACKGROUND: The 13-valent Pneumococcal Conjugate Vaccine (PCV-13) was introduced in the National Immunization Programme (NIP) schedule in Russia in March 2014. Previously, the 7-valent Pneumococcal Conjugate Vaccine (PCV-7) was marketed in Russia in 2009 but has never been offered for mass vaccination. A carriage study was performed among children in Arkhangelsk in 2006. The objective was to determine the prevalence of carriage, serotype distribution, antimicrobial susceptibility and the molecular structure of Streptococcus pneumoniae strains before marketing and introduction of PCV-13. METHODS: A cross-sectional study was conducted on a cluster-randomized sample of children and a self-administrated questionnaire for parents/guardians.  Nasopharyngeal samples were collected from 438 children younger than 7 years attending nurseries and kindergartens in the Arkhangelsk region, Russia. Detailed demographic data, as well as information about the child's health, traveling, exposure to antimicrobials within the last 3 months and anthropometric measurements were collected for all study subjects. Variables extracted from the questionnaire were analysed using statistic regression models to estimate the risk of carriage. All pneumococcal  isolates were examined with susceptibility testing, serotyping and multilocus sequence typing. RESULTS: The overall prevalence of asymptomatic carriage was high and peaking at 36 months with a rate of 57%. PCV-13 covered 67.3% of the detected strains. High rates of non-susceptibility to penicillin, macrolides and multidrug resistance were associated with specific vaccine serotypes, pandemic clones, and local sequence types. Nine percent of isolates represented three globally disseminated disease-associated pandemic clones; penicillin- and macrolide-resistant clones NorwayNT-42 and Poland6B-20, as well as penicillin- and macrolide-susceptible clone Netherlands3-31. A high level of antimicrobial consumption was noted by the study. According to the parent's reports, 89.5% of the children used at least one antimicrobial regime since birth. None of the hypothesised predictors of S. pneumoniae carriage were statistically significant in univariable and multivariable logistic models. CONCLUSIONS: The study identified a high coverage of the PCV-13-vaccine, but serotype replacement and expansion of globally disseminated disease-associated clones with non-vaccine serotypes may be expected. Further surveillance of antimicrobial resistance and serotype distribution is therefore required.


Assuntos
Portador Sadio/epidemiologia , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Vacinas Conjugadas/imunologia , Antibacterianos/uso terapêutico , Portador Sadio/microbiologia , Criança , Pré-Escolar , Estudos Transversais , Farmacorresistência Bacteriana Múltipla , Feminino , Humanos , Lactente , Macrolídeos/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Nasofaringe/microbiologia , Penicilinas/uso terapêutico , Infecções Pneumocócicas/tratamento farmacológico , Prevalência , Federação Russa/epidemiologia , Sorogrupo , Sorotipagem , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/isolamento & purificação
2.
BMC Infect Dis ; 20(1): 168, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087697

RESUMO

BACKGROUND: Respiratory tract infection (RTI) in young children is a leading cause of morbidity and hospitalization worldwide. There are few studies assessing the performance for bronchoalveolar lavage fluid (BALF) versus oropharyngeal swab (OPS) specimens in microbiological findings for children with RTI. The primary purpose of this study was to compare the detection rates of OPS and paired BALF in detecting key respiratory pathogens using suspension microarray. METHODS: We collected paired OPS and BALF specimens from 76 hospitalized children with respiratory illness. The samples were tested simultaneously for 8 respiratory viruses and 5 bacteria by suspension microarray. RESULTS: Of 76 paired specimens, 62 patients (81.6%) had at least one pathogen. BALF and OPS identified respiratory pathogen infections in 57 (75%) and 49 (64.5%) patients, respectively (P > 0.05). The etiology analysis revealed that viruses were responsible for 53.7% of the patients, whereas bacteria accounted for 32.9% and Mycoplasma pneumoniae for 13.4%. The leading 5 pathogens identified were respiratory syncytial virus, Streptococcus pneumoniaee, Haemophilus influenzae, Mycoplasma pneumoniae and adenovirus, and they accounted for 74.2% of etiological fraction. For detection of any pathogen, the overall detection rate of BALF (81%) was marginally higher than that (69%) of OPS (p = 0.046). The differences in the frequency distribution and sensitivity for most pathogens detected by two sampling methods were not statistically significant. CONCLUSIONS: In this study, BALF and OPS had similar microbiological yields. Our results indicated the clinical value of OPS testing in pediatric patients with respiratory illness.


Assuntos
Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Criança Hospitalizada , Testes Diagnósticos de Rotina/métodos , Orofaringe/microbiologia , Orofaringe/virologia , Infecções Respiratórias/diagnóstico , Criança , Pré-Escolar , Feminino , Hospitalização , Humanos , Lactente , Recém-Nascido , Masculino , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/microbiologia , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação
3.
PLoS One ; 15(2): e0228799, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053640

RESUMO

BACKGROUND: Bangladesh introduced the 10-valent pneumococcal conjugate vaccine (PCV-10) in 2015. We measured population-based incidence of invasive pneumococcal disease (IPD) prior to introduction of PCV-10 to provide a benchmark against which the impact of PCV-10 can be assessed. METHODS: We conducted population, facility and laboratory-based surveillance in children 0-59 months of age in three rural sub-districts of Sylhet district of Bangladesh from January 2014 to June 2015. All children received two-monthly home visits with one week recall for morbidity and care seeking. Children attending the three Upazilla Health Complexes (UHC, sub-district hospitals) in the surveillance area were screened for suspected IPD. Blood samples were collected from suspected IPD cases for culture and additionally, cerebrospinal fluid (CSF) was collected from suspected meningitis cases for culture and molecular testing. Pneumococcal isolates were serotyped by Quellung. Serotyping of cases detected by molecular testing was done by sequential multiplex polymerase chain reaction. RESULTS: Children under surveillance contributed to 126,657 child years of observations. Sixty-three thousand three hundred eighty-four illness episodes were assessed in the UHCs. Blood specimens were collected from 8,668 suspected IPD cases and CSF from 177 suspected meningitis cases. Streptococcus pneumoniae was isolated from 46 cases; 32 (70%) were vaccine serotype. The population-based incidence of IPD was 36.3/100,000 child years of observations. About 80% of the cases occurred in children below two years of age. DISCUSSION: IPD was common in rural Bangladesh suggesting the potential benefit of an effective vaccine. Measurement of the burden of IPD requires multiple surveillance modalities.


Assuntos
Infecções Pneumocócicas/diagnóstico , Streptococcus pneumoniae/isolamento & purificação , Bangladesh/epidemiologia , Hemocultura , Pré-Escolar , DNA Bacteriano/metabolismo , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Meningite/diagnóstico , Meningite/epidemiologia , Meningite/microbiologia , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Sorogrupo , Streptococcus pneumoniae/genética
4.
PLoS One ; 15(2): e0229021, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032364

RESUMO

BACKGROUND: The nasopharynx can from time to time accommodate otherwise pathogenic bacteria. This phenomenon is called asymptomatic carriage. However, in case of decreased immunity, viral infection or any other enhancing factors, severe disease can develop. Our aim in this study was to survey the nasal carriage rates of four important respiratory pathogens in three different age groups of children attending nurseries, day-care centres and primary schools. This is the first study from Hungary about the asymptomatic carriage of H. influenzae and M. catarrhalis. METHODS: Altogether 580 asymptomatic children were screened in three Hungarian cities. Samples were collected from both nostrils with cotton swabs. The identification was based on both colony morphology and species-specific PCRs. Serotyping was performed for S. pneumoniae, H. influenzae and M. catarrhalis. Antibiotic susceptibility was determined with agar dilution, according to the EUCAST guidelines. Clonality was examined by PFGE. RESULTS AND CONCLUSIONS: Whereas the carriage rates of S. pneumoniae, H. influenzae and M. catarrhalis clearly decreased with age, that of S. aureus showed an opposite tendency. Multiple carriage was least prevalent if S. aureus was one of the participants. The negative association between this bacterium and the others was statistically significant. For pneumococcus, the overall carriage rate was lower compared to earlier years, and PCV13 serotypes were present in only 6.2% of the children. The majority of H. influenzae isolates was non-typeable and no type b was detected; serotype A was dominant among M. catarrhalis. All four bacteria were more sensitive to antibiotics compared to clinical isolates. No MRSAs were detected, but we found three mupirocin resistant strains. The positive effect of Hib- and PCV-vaccination is undoubted. Continued surveillance of these pathogens is required.


Assuntos
Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Haemophilus influenzae , Moraxella catarrhalis , Nasofaringe/microbiologia , Staphylococcus aureus , Streptococcus pneumoniae , Adolescente , Fatores Etários , Antibacterianos/farmacologia , Criança , Pré-Escolar , Coinfecção/epidemiologia , Coinfecção/microbiologia , Feminino , Infecções por Haemophilus/epidemiologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Hungria/epidemiologia , Lactente , Masculino , Testes de Sensibilidade Microbiana , Moraxella catarrhalis/classificação , Moraxella catarrhalis/efeitos dos fármacos , Moraxella catarrhalis/genética , Moraxella catarrhalis/isolamento & purificação , Infecções por Moraxellaceae/epidemiologia , Infecções por Moraxellaceae/microbiologia , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Vigilância em Saúde Pública , Fatores de Risco , Sorogrupo , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação
5.
Diagn Microbiol Infect Dis ; 96(1): 114914, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704066

RESUMO

Russia introduced PCV13 in 2014. We studied the serotype composition of S. pneumoniae isolated from the nasopharynx of healthy children younger than 6 years in St. Petersburg, Smolensk, Perm, Krasnoyarsk, Khanty-Mansiysk and Khabarovsk, between 2016 and 2018. 2.4% of children had completed a 3-dose course of PCV13, while 25.6% had received 1 or 2 doses. Pneumococcal DNA detection by PCR demonstrated S. pneumoniae in 37.2% of samples with regional variation between sites (27.3 to 56.9%). There was little difference between vaccinated, partially vaccinated and un-vaccinated children. Children who had received at least 1 dose of PCV13 had lower carriage rates of vaccine serotypes than their unvaccinated peers (49.9 vs. 61.4%; p < 0.001). Children who had received at least 1 dose of PCV13 showed increased carriage rates of non-vaccine serotypes (50 vs 38.6%; P < 0.001). Especially serogroup 15AF was more prevalent among fully immunized children than among their peers (12.5 vs 2.7%; P < 0.05).


Assuntos
Portador Sadio/microbiologia , Programas de Imunização , Nasofaringe/microbiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Streptococcus pneumoniae/classificação , Portador Sadio/epidemiologia , Criança , Pré-Escolar , Voluntários Saudáveis , Humanos , Lactente , Recém-Nascido , Infecções Pneumocócicas/epidemiologia , Prevalência , Federação Russa/epidemiologia , Sorogrupo , Streptococcus pneumoniae/genética
6.
Nat Commun ; 10(1): 5729, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844066

RESUMO

While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1-3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method's original applicability.


Assuntos
Elementos de DNA Transponíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/métodos , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , DNA Bacteriano/genética , Biblioteca Gênica , Genes Bacterianos/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Mutação , Análise de Célula Única/instrumentação , Streptococcus pneumoniae/genética
7.
Microbiol Spectr ; 7(6)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31858954

RESUMO

Streptococcus pneumoniae is a Gram-Positive pathogen that is a major causative agent of pneumonia, otitis media, sepsis and meningitis across the world. The World Health Organization estimates that globally over 500,000 children are killed each year by this pathogen. Vaccines offer the best protection against S. pneumoniae infections. The current polysaccharide conjugate vaccines have been very effective in reducing rates of invasive pneumococcal disease caused by vaccine type strains. However, the effectiveness of these vaccines have been somewhat diminished by the increasing numbers of cases of invasive disease caused by non-vaccine type strains, a phenomenon known as serotype replacement. Since, there are currently at least 98 known serotypes of S. pneumoniae, it may become cumbersome and expensive to add many additional serotypes to the current 13-valent vaccine, to circumvent the effect of serotype replacement. Hence, alternative serotype independent strategies, such as vaccination with highly cross-reactive pneumococcal protein antigens, should continue to be investigated to address this problem. This chapter provides a comprehensive discussion of pneumococcal vaccines past and present, protein antigens that are currently under investigation as vaccine candidates, and other alternatives, such as the pneumococcal whole cell vaccine, that may be successful in reducing current rates of disease caused by S. pneumoniae.


Assuntos
Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Streptococcus pneumoniae/imunologia , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Humanos , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/genética
8.
Nat Commun ; 10(1): 4852, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649284

RESUMO

Prophages (viral genomes integrated within a host bacterial genome) can confer various phenotypic traits to their hosts, such as enhanced pathogenicity. Here we analyse >1300 genomes of 70 different Streptococcus species and identify nearly 800 prophages and satellite prophages (prophages that do not encode their own structural components but rely on the bacterial host and another helper prophage for survival). We show that prophages and satellite prophages are widely distributed among streptococci in a structured manner, and constitute two distinct entities with little effective genetic exchange between them. Cross-species transmission of prophages is not uncommon. Furthermore, a satellite prophage is associated with virulence in a mouse model of Streptococcus pneumoniae infection. Our findings highlight the potential importance of prophages in streptococcal biology and pathogenesis.


Assuntos
Genoma Bacteriano/genética , Infecções Pneumocócicas/microbiologia , Prófagos/genética , Streptococcus pneumoniae/genética , Streptococcus/genética , Animais , Bacteriófagos/genética , Camundongos , Epidemiologia Molecular , Streptococcus/patogenicidade , Streptococcus/virologia , Streptococcus pneumoniae/patogenicidade , Streptococcus pneumoniae/virologia , Virulência/genética
9.
Genes (Basel) ; 10(9)2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31540216

RESUMO

A diverse set of mobile genetic elements (MGEs) transmit between Streptococcus pneumoniae cells, but many isolates remain uninfected. The best-characterised defences against horizontal transmission of MGEs are restriction-modification systems (RMSs), of which there are two phase-variable examples in S. pneumoniae. Additionally, the transformation machinery has been proposed to limit vertical transmission of chromosomally integrated MGEs. This work describes how these mechanisms can act in concert. Experimental data demonstrate RMS phase variation occurs at a sub-maximal rate. Simulations suggest this may be optimal if MGEs are sometimes vertically inherited, as it reduces the probability that an infected cell will switch between RMS variants while the MGE is invading the population, and thereby undermine the restriction barrier. Such vertically inherited MGEs can be deleted by transformation. The lack of between-strain transformation hotspots at known prophage att sites suggests transformation cannot remove an MGE from a strain in which it is fixed. However, simulations confirmed that transformation was nevertheless effective at preventing the spread of MGEs into a previously uninfected cell population, if a recombination barrier existed between co-colonising strains. Further simulations combining these effects of phase variable RMSs and transformation found they synergistically inhibited MGEs spreading, through limiting both vertical and horizontal transmission.


Assuntos
Enzimas de Restrição-Modificação do DNA/genética , Sequências Repetitivas Dispersas , Streptococcus pneumoniae/genética , Transferência Genética Horizontal , Streptococcus pneumoniae/enzimologia , Transformação Bacteriana
10.
Genes (Basel) ; 10(9)2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500179

RESUMO

We used whole genome sequencing (WGS) analysis to investigate the population structure of 877 Streptococcus pneumoniae isolates from five carriage studies from 2002 (N = 346), 2010 (N = 127), 2013 (N = 153), 2016 (N = 187) and 2018 (N = 64) in UK households which covers the period pre-PCV7 to post-PCV13 implementation. The genomic lineages seen in the population were determined using multi-locus sequence typing (MLST) and PopPUNK (Population Partitioning Using Nucleotide K-mers) which was used for local and global comparisons. A Roary core genome alignment of all the carriage genomes was used to investigate phylogenetic relationships between the lineages. The results showed an influx of previously undetected sequence types after vaccination associated with non-vaccine serotypes. A small number of lineages persisted throughout, associated with both non-vaccine and vaccine types (such as ST199), or that could be an example of serotype switching from vaccine to non-vaccine types (ST177). Serotype 3 persisted throughout the study years, represented by ST180 and Global Pneumococcal Sequencing Cluster (GPSC) 12; the local PopPUNK analysis and core genome maximum likelihood phylogeny separated them into two clades, one of which is only seen in later study years. The genomic data showed that serotype replacement in the carriage studies was mostly due to a change in genotype as well as serotype, but that some important genetic lineages, previously associated with vaccine types, persisted.


Assuntos
Portador Sadio/microbiologia , Genoma Bacteriano , Programas de Imunização/estatística & dados numéricos , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Adolescente , Adulto , Portador Sadio/epidemiologia , Criança , Pré-Escolar , Características da Família , Vacina Pneumocócica Conjugada Heptavalente/uso terapêutico , Humanos , Filogenia , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/prevenção & controle , Infecções Pneumocócicas/transmissão , Vacinas Pneumocócicas/uso terapêutico , Sorogrupo , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/isolamento & purificação , Reino Unido
11.
J Med Microbiol ; 68(10): 1466-1470, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31389781

RESUMO

Introduction. Streptococcus pneumoniae and Haemophilus influenzae are both commensals of the human nasopharynx with a high capacity to cause upper and lower respiratory tract infections.Aim. Molecular testing of nasopharyngeal samples from children at the primary care paediatric department presenting with acute respiratory tract infections (ARTIs).Methodology. From June 2016 to May 2017, 156 nasopharyngeal swabs from children diagnosed with ARTIs who had been admitted to or followed up as outpatients at the Department of Paediatrics, SANADOR Hospital (Bucharest, Romania) were tested for the presence of S. pneumoniae, H. influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Bordetella pertussis and Bordetella parapertussis DNA.Results. S. pneumoniae had the highest detection rate (53.8 %, n=84/156), followed by H. influenzae (41 %, n=64/156) and S. pneumoniae/H. influenzae co-detection (26.2 %, n=41/156).Conclusion. A definitive laboratory diagnosis of these micro-organisms can be made for invasive disease, but there are difficulties in establishing the aetiology for mucosal infection. Molecular detection tests could complement culture-based tests by strengthening their surveillance.


Assuntos
Infecções por Haemophilus/microbiologia , Haemophilus influenzae/isolamento & purificação , Nasofaringe/microbiologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/isolamento & purificação , Doença Aguda/terapia , Adolescente , Criança , Pré-Escolar , Feminino , Infecções por Haemophilus/diagnóstico , Infecções por Haemophilus/terapia , Haemophilus influenzae/genética , Hospitais/estatística & dados numéricos , Humanos , Lactente , Masculino , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/terapia , Romênia , Streptococcus pneumoniae/genética
14.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31451619

RESUMO

Streptococcus pneumoniae (pneumococcus) causes multiple infectious diseases. The pneumococcal competence system facilitates genetic transformation, spreads antibiotic resistance, and contributes to virulence. DNA-processing protein A (DprA) regulates the exit of pneumococcus from the competent state. Previously, we have shown that DprA is important in both bacteremia and pneumonia infections. Here, we examined the mechanisms of virulence attenuation in a ΔdprA mutant. Compared to the parental wild-type D39, the ΔdprA mutant enters the competent state when exposed to lower concentrations of the competence-stimulating peptide CSP1. The ΔdprA mutant overexpresses ComM, which delays cell separation after division. Additionally, the ΔdprA mutant overexpresses allolytic factors LytA, CbpD, and CibAB and is more susceptible to detergent-triggered lysis. Disabling of the competent-state-specific induction of ComM and allolytic factors compensated for the virulence loss in the ΔdprA mutant, suggesting that overexpression of these factors contributes to virulence attenuation. Finally, the ΔdprA mutant fails to downregulate the expression of multiple competence-regulated genes, leading to the excessive energy consumption. Collectively, these results indicate that an inability to properly exit the competent state disrupts multiple cellular processes that cause virulence attenuation in the ΔdprA mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Streptococcus pneumoniae/genética , Animais , Proteínas de Bactérias/genética , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Masculino , Proteínas de Membrana/genética , Camundongos , Nasofaringe/microbiologia , Pneumonia Pneumocócica/microbiologia
15.
PLoS Pathog ; 15(7): e1007987, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356624

RESUMO

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.


Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Lipoproteínas/fisiologia , Meningite Pneumocócica/microbiologia , Streptococcus pneumoniae/fisiologia , Streptococcus pneumoniae/patogenicidade , Fatores de Virulência/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Genes Bacterianos , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Lipoproteínas/deficiência , Lipoproteínas/genética , Masculino , Meningite Pneumocócica/líquido cefalorraquidiano , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteômica , Regulon , Streptococcus pneumoniae/genética , Virulência/genética , Virulência/fisiologia , Fatores de Virulência/genética
16.
Nucleic Acids Res ; 47(18): e112, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31361894

RESUMO

Covariance-based discovery of polymorphisms under co-selective pressure or epistasis has received considerable recent attention in population genomics. Both statistical modeling of the population level covariation of alleles across the chromosome and model-free testing of dependencies between pairs of polymorphisms have been shown to successfully uncover patterns of selection in bacterial populations. Here we introduce a model-free method, SpydrPick, whose computational efficiency enables analysis at the scale of pan-genomes of many bacteria. SpydrPick incorporates an efficient correction for population structure, which adjusts for the phylogenetic signal in the data without requiring an explicit phylogenetic tree. We also introduce a new type of visualization of the results similar to the Manhattan plots used in genome-wide association studies, which enables rapid exploration of the identified signals of co-evolution. Simulations demonstrate the usefulness of our method and give some insight to when this type of analysis is most likely to be successful. Application of the method to large population genomic datasets of two major human pathogens, Streptococcus pneumoniae and Neisseria meningitidis, revealed both previously identified and novel putative targets of co-selection related to virulence and antibiotic resistance, highlighting the potential of this approach to drive molecular discoveries, even in the absence of phenotypic data.


Assuntos
Biologia Computacional/métodos , Epistasia Genética , Genoma Bacteriano/genética , Genômica , Resistência Microbiana a Medicamentos/genética , Humanos , Metagenômica/métodos , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidade , Streptococcus pneumoniae/genética , Virulência/genética
17.
Eur J Med Chem ; 180: 321-339, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31323617

RESUMO

Macrolides are widely prescribed in clinic to treat various respiratory tract infections. However, due to their inappropriate use, the prevalence of macrolide-resistant strains among clinical isolates has become a concern for public health. Therefore, novel macrolides skeleton structures against resistant pathogens are badly needed. Thus, three series of novel 15-membered 11a-azahomoclarithromycin derivatives (series A-C) with the 1, 2, 3-triazole side chain were designed and synthesized through creatively opening the ring of clarithromycin (CAM), expanding the ring properly and introducing a suitable side chain of 1, 2, 3-triazole at the C12 and C13 positions, and evaluated for their antibacterial activity. The antibacterial results indicated that compounds 38b, 38l and 38v possessed strong antibacterial activity against Staphylococcus aureus ATCC25923 (0.25 µg/mL) and Bacillus subtilis ATCC9372 (0.25 µg/mL). Furthermore, compounds 9e and 38g were found to exhibit promising potent activity (8 µg/mL) against Streptococcus pneumonia AB11 expressing the ermB and mefA genes. In addition, the determination of minimum bactericidal concentration (MBC) indicated that the most promising compounds 38b, 38l, 38v, 9e and 38g were excellent bacteriostatic agents. The bactericidal curve showed that 9e exhibited antibacterial activity through bacteriostatic mechanism. Finally, 38b, 38l and 38v were confirmed to be non-toxic to MCF-7 breast cancer cells up to a concentration of 32 µg/mL in preliminary cytotoxicity assay. In summary, 38b, 38l, 38v, 9e and 38g can be served as lead compounds to provide a new perspective for further structural optimization.


Assuntos
Antibacterianos/farmacologia , Compostos Aza/farmacologia , Claritromicina/farmacologia , Desenho de Fármacos , Triazóis/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Compostos Aza/síntese química , Compostos Aza/química , Bacillus subtilis/efeitos dos fármacos , Claritromicina/síntese química , Claritromicina/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Relação Estrutura-Atividade , Triazóis/química
18.
BMC Infect Dis ; 19(1): 605, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291902

RESUMO

BACKGROUND: Streptococcus pneumoniae is a major cause of pneumonia, meningitis, and other serious infections among children in India. India introduced the 13-valent pneumococcal conjugate vaccine (PCV) in several states in 2017, and is expected to expand to nationwide coverage in the near future. To establish a baseline for measuring the impact of PCV in India, we assessed overall and serotype-specific nasopharyngeal carriage in two pediatric populations. METHODS: A cross-sectional study was conducted in Palwal District, Haryana, from December 2016 to July 2017, prior to vaccine introduction. Children 2-59 months of age with clinical pneumonia seeking healthcare and those in the community with no clear illness were targeted for enrollment. A nasopharyngeal swab was collected and tested for pneumococcus using conventional culture and sequential multiplex PCR. Isolates were tested for antimicrobial resistance using an E test. Children were considered colonized if pneumococcus was isolated by culture or PCR. The prevalence of pneumococcal and serotype-specific colonization was compared between groups of children using log-binomial regression. RESULTS: Among 601 children enrolled, 91 had clinical pneumonia and 510 were community children. The proportion colonized with S. pneumoniae was 74.7 and 54.5% among children with clinical pneumonia and community children, respectively (adjusted prevalence ratio: 1.38; 95% confidence interval: 1.19, 1.60). The prevalence of PCV13 vaccine-type colonization was similar between children with clinical pneumonia (31.9%) and community children (28.0%; p = 0.46). The most common colonizing serotypes were 6A, 6B, 14, 19A, 19F, and 23F, all of which are included in the PCV13 vaccine product. Antimicrobial resistance to at least one drug was similar between isolates from children with clinical pneumonia (66.1%) and community children (61.5%; p = 0.49); while resistance to at least two drugs was more common among isolates from children with clinical pneumonia (25.8% vs. 16.4%; p = 0.08). Resistance for all drugs was consistently higher for PCV13 vaccine-type serotypes compared to non-vaccine serotypes in both groups. CONCLUSION: This study provides baseline information on the prevalence of serotype-specific pneumococcal colonization among children prior to the introduction of PCV in India. Our results suggest a role for pneumococcal vaccines in reducing pneumococcal colonization and antimicrobial resistant isolates circulating in India.


Assuntos
Portador Sadio/microbiologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Antibacterianos/farmacologia , Portador Sadio/epidemiologia , Pré-Escolar , Estudos Transversais , Farmacorresistência Bacteriana , Feminino , Humanos , Índia/epidemiologia , Lactente , Masculino , Testes de Sensibilidade Microbiana , Nasofaringe/microbiologia , Infecções Pneumocócicas/epidemiologia , Prevalência , Sorogrupo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Vacinas Conjugadas
19.
J Med Microbiol ; 68(8): 1173-1188, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31268417

RESUMO

PURPOSE: Correct serotype identification of Streptococcus pneumoniae (pneumococcus) is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. Furthermore, correct identification and differentiation of the pathogenic S. pneumoniae from closely related commensal species of the mitis group of the genus Streptococcus are essential for correct serotype identification. METHODOLOGY: A new protocol for determining the existing 98 serotypes of pneumococcus was developed, applying two PCR amplifications and amplicon sequencing, using newly designed internal primers. The new protocol was validated using S. pneumoniae genome sequences, reference strains with confirmed serotypes and clinical isolates, and comparing the results with those from the traditional Quellung reaction or antiserum panel gel precipitation, in addition to real-time PCR analysis. The taxonomic identifications of 422 publicly available (GenBank) genome sequences of S. pneumoniae, Streptococcus pseudopneumoniae and Streptococcus mitis were assessed by whole-genome sequence average nucleotide identity based on blast (ANIb) analysis. RESULTS: The proposed sequetyping protocol generates a 1017 bp whole cpsB region sequence, increasing resolution for serotype identification in pneumococcus isolates. The identifications of all GenBank genome sequences of S. pneumoniae were confirmed, whereas most of the S. pseudopneumoniae and almost all of the S. mitis genome sequences did not fulfil the ANIb thresholds for species-level identification. The housekeeping biomarker gene, groEL, correctly identified S. pneumoniae but often misclassified S. pseudopneumoniae and S. mitis as S. pneumoniae. CONCLUSIONS: These studies affirm the importance of applying reliable identification protocols for S. pneumoniae before serotyping; our protocols provide reliable diagnostic tools, as well as an improved workflow, for serotype identification of pneumococcus and differentiation of serogroup 6 types.


Assuntos
Cápsulas Bacterianas/genética , Tipagem Molecular/métodos , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Genoma Bacteriano/genética , Humanos , Infecções Pneumocócicas/microbiologia , Proteínas Tirosina Fosfatases/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Sorogrupo , Sorotipagem/normas , Streptococcus/classificação , Streptococcus/genética , Streptococcus/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Fluxo de Trabalho
20.
J Med Microbiol ; 68(9): 1383-1390, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31347997

RESUMO

Introduction. In 2016-2017, there was an increase in the number of paediatric invasive pneumococcal disease (IPD) cases caused by Streptococcus pneumoniae serotype 12F in Chiba Prefecture, Japan. Serotype 12F is one of the major causative serotypes of IPD following the introduction of pneumococcal conjugate vaccine 13 (PCV13), and outbreaks of IPD caused by serotype 12F have recently been reported in several countries.Aim. Our goal here was to clarify the relationship among local outbreak strains and the outbreak strains in other countries, and for this we analysed clinical isolates of S. pneumoniae serotype 12F using several genetic identification methods.Methodology. All reported IPD cases caused by serotype 12F were reviewed and bacterial strains were collected and analysed. We also analysed S. pneumoniae serotype 12F strains isolated from other time periods, geographical areas, cases of adult IPD and respiratory specimens as control strains. Multi-locus sequence typing, PFGE and multi-locus variable number tandem repeat analysis (MLVA) were conducted on all isolates.Results. All 26 S. pneumoniae serotype 12F isolates, including control strains, belonged to a single sequence type (ST4846) that was the specific ST in Japan. All tested strains demonstrated five MLVA patterns and two PFGE patterns.Conclusion. We determined that the 2016-2017 outbreak of IPD in Chiba Prefecture was caused by clonally related isolates of serotype 12F. The continuous monitoring of IPD caused by serotype 12F is important for evaluating the impact of re-emerging pneumococcal serotypes following the PCV13 introduction era, and MLVA could be a useful tool for identification of outbreak strains.


Assuntos
Surtos de Doenças , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Sorogrupo , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/isolamento & purificação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Lactente , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Streptococcus pneumoniae/genética
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