RESUMEN
BACKGROUND: Cambodia introduced the 13-valent pneumococcal conjugate vaccine (PCV13) in January 2015 using a 3 + 0 dosing schedule and no catch-up campaign. We investigated the effects of this introduction on pneumococcal colonization and invasive disease in children aged <5 years. METHODS: There were 6 colonization surveys done between January 2014 and January 2018 in children attending the outpatient department of a nongovernmental pediatric hospital in Siem Reap. Nasopharyngeal swabs were analyzed by phenotypic and genotypic methods to detect pneumococcal serotypes and antimicrobial resistance. Invasive pneumococcal disease (IPD) data for January 2012-December 2018 were retrieved from hospital databases. Pre-PCV IPD data and pre-/post-PCV colonization data were modelled to estimate vaccine effectiveness (VE). RESULTS: Comparing 2014 with 2016-2018, and using adjusted prevalence ratios, VE estimates for colonization were 16.6% (95% confidence interval [CI] 10.6-21.8) for all pneumococci and 39.2% (95% CI 26.7-46.1) for vaccine serotype (VT) pneumococci. There was a 26.0% (95% CI 17.7-33.0) decrease in multidrug-resistant pneumococcal colonization. The IPD incidence was estimated to have declined by 26.4% (95% CI 14.4-35.8) by 2018, with a decrease of 36.3% (95% CI 23.8-46.9) for VT IPD and an increase of 101.4% (95% CI 62.0-145.4) for non-VT IPD. CONCLUSIONS: Following PCV13 introduction into the Cambodian immunization schedule, there have been declines in VT pneumococcal colonization and disease in children aged <5 years. Modelling of dominant serotype colonization data produced plausible VE estimates.
Asunto(s)
Infecciones Neumocócicas , Vacunas Neumococicas , Pueblo Asiatico , Cambodia/epidemiología , Niño , Preescolar , Vacuna Neumocócica Conjugada Heptavalente , Humanos , Lactante , Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/prevención & control , Serogrupo , Vacunas ConjugadasRESUMEN
UNLABELLED: Nasopharyngeal colonization is important for Streptococcus pneumoniae evolution, providing the opportunity for horizontal gene transfer when multiple strains co-occur. Although colonization with more than one strain of pneumococcus is common, the factors that influence the ability of strains to coexist are not known. A highly variable blp (bacteriocin-like peptide) locus has been identified in all sequenced strains of S. pneumoniae This locus controls the regulation and secretion of bacteriocins, small peptides that target other bacteria. In this study, we analyzed a series of cocolonizing isolates to evaluate the impact of the blp locus on human colonization to determine whether competitive phenotypes of bacteriocin secretion restrict cocolonization. We identified a collection of 135 nasopharyngeal samples cocolonized with two or more strains, totaling 285 isolates. The blp locus of all strains was characterized genetically with regard to pheromone type, bacteriocin/immunity content, and potential for locus functionality. Inhibitory phenotypes of bacteriocin secretion and locus activity were assessed through overlay assays. Isolates from single colonizations (n = 298) were characterized for comparison. Cocolonizing strains had a high diversity of blp cassettes; approximately one-third displayed an inhibitory phenotype in vitro Despite in vitro evidence of competition, pneumococci cocolonized the subjects independently of blp pheromone type (P = 0.577), bacteriocin/immunity content, blp locus activity (P = 0.798), and inhibitory phenotype (P = 0.716). In addition, no significant differences were observed when single and cocolonizing strains were compared. Despite clear evidence of blp-mediated competition in experimental models, the results of our study suggest that the blp locus plays a limited role in restricting pneumococcal cocolonization in humans. IMPORTANCE: Nasopharyngeal colonization with Streptococcus pneumoniae (pneumococcus) is important for pneumococcal evolution, as the nasopharynx represents the major site for horizontal gene transfer when multiple strains co-occur, a phenomenon known as cocolonization. Understanding how pneumococcal strains interact within the competitive environment of the nasopharynx is of chief importance in the context of pneumococcal ecology. In this study, we used an unbiased collection of naturally co-occurring pneumococcal strains and showed that a biological process frequently used by bacteria for competition-bacteriocin production-is not decisive in the coexistence of pneumococci in the host, in contrast to what has been shown in experimental models.
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Proteínas Bacterianas/metabolismo , Nasofaringe/microbiología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
Streptococcus pneumoniae of serotype 3 possess a mucoid capsule and cause disease associated with high mortality rates relative to other pneumococci. Phylogenetic analysis of a complete reference genome and 81 draft sequences from clonal complex 180, the predominant serotype 3 clone in much of the world, found most sampled isolates belonged to a clade affected by few diversifying recombinations. However, other isolates indicate significant genetic variation has accumulated over the clonal complex's entire history. Two closely related genomes, one from the blood and another from the cerebrospinal fluid, were obtained from a patient with meningitis. The pair differed in their behaviour in a mouse model of disease and in their susceptibility to antimicrobials, with at least some of these changes attributable to a mutation that up-regulated the patAB efflux pump. This indicates clinically important phenotypic variation can accumulate rapidly through small alterations to the genotype.
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Genoma Bacteriano , Mutación , Filogenia , Streptococcus pneumoniae/genética , Animales , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Meningitis/sangre , Meningitis/líquido cefalorraquídeo , Meningitis/microbiología , Ratones , Serotipificación , Streptococcus pneumoniae/patogenicidadRESUMEN
The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.
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Técnicas Bacteriológicas/métodos , Tuberculosis Extensivamente Resistente a Drogas/diagnóstico , Genoma Bacteriano , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Análisis de Secuencia de ADN/métodos , Genes Bacterianos , Genotipo , Hospitales de Enseñanza , Humanos , Londres , Mutación , Mycobacterium tuberculosis/aislamiento & purificación , Factores de TiempoRESUMEN
BACKGROUND: Carriage of either single or multiple pneumococcal serotypes (multiple carriage) is a prerequisite for developing invasive pneumococcal disease. However, despite the reported high rates of pneumococcal carriage in Malawi, no data on carriage of multiple serotypes has been reported previously. Our study provides the first description of the prevalence of multiple pneumococcal carriage in Malawi. METHODS: The study was conducted in Blantyre and Karonga districts in Malawi, from 2008 to 2012. We recruited 116 children aged 0-13 years. These children were either HIV-infected (N = 44) or uninfected (N = 72). Nasopharyngeal samples were collected using sterile swabs. Pneumococcal serotypes in the samples were identified by microarray. Strains that could not be typed by microarray were sequenced to characterise possible genetic alterations within the capsular polysaccharide (CPS) locus. RESULTS: The microarray identified 179 pneumococcal strains (from 116 subjects), encompassing 43 distinct serotypes and non-typeable (NT) strains. Forty per cent (46/116) of children carried multiple serotypes. Carriage of vaccine type (VT) strains was higher (p = 0.028) in younger (0-2 years) children (71 %, 40/56) compared to older (3-13 years) children (50 %, 30/60). Genetic variations within the CPS locus of known serotypes were observed in 19 % (34/179) of the strains identified. The variants included 13-valent pneumococcal conjugate vaccine (PCV13) serotypes 6B and 19A, and the polysaccharide vaccine serotype 20. Serotype 6B variants were the most frequently isolated (47 %, 16/34). Unlike the wild type, the CPS locus of the 6B variants contained an insertion of the licD-family phosphotransferase gene. The CPS locus of 19A- and 20-variants contained an inversion in the sugar-biosynthesis (rmlD) gene and a 717 bp deletion within the transferase (whaF) gene, respectively. CONCLUSIONS: The high multiple carriage in Malawian children provides opportunities for genetic exchange through horizontal gene transfer. This may potentially lead to CPS locus variants and vaccine escape. Variants reported here occurred naturally, however, PCV13 introduction could exacerbate the CPS genetic variations. Further studies are therefore recommended to assess the invasive potential of these variants and establish whether PCV13 would offer cross-protection. We have shown that younger children (0-2 years) are a reservoir of VT serotypes, which makes them an ideal target for vaccination.
Asunto(s)
Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/genética , Adolescente , Cápsulas Bacterianas/genética , Técnicas Bacteriológicas , Niño , Preescolar , Protección Cruzada/inmunología , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Femenino , Variación Genética , Infecciones por VIH/complicaciones , Infecciones por VIH/diagnóstico , Humanos , Lactante , Recién Nacido , Malaui , Masculino , Nasofaringe/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Infecciones Neumocócicas/complicaciones , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/inmunología , Análisis de Secuencia de ADN , Serogrupo , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
BACKGROUND: The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur. RESULTS: Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater. CONCLUSIONS: We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.
Asunto(s)
Adhesión Bacteriana , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Competencia de la Transformación por ADN , Mutación Puntual , Streptococcus pneumoniae/fisiología , Proteínas Bacterianas/genética , Línea Celular , Análisis Mutacional de ADN , Células Epiteliales/microbiología , Genoma Bacteriano , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nasofaringe/microbiología , Infecciones Neumocócicas/microbiología , Análisis de Secuencia de ADN , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/aislamiento & purificación , Transformación GenéticaRESUMEN
BACKGROUND: Pneumococci could evade pneumococcal conjugate vaccines (PCV) by modifying, mutating, or deleting vaccine-serotype capsule genes or by downregulating capsule production. We sought to assess whether pneumococci that are nontypeable (NT) by the Quellung reaction truly lack capsule genes or are failing to produce capsule in vitro. METHODS: We applied multilocus sequence typing and a microarray for detection of pneumococcal polysaccharide capsule biosynthesis genes to NT carriage (children aged <5 years; years 1997-2000, 2006-2008) and NT invasive disease (IPD) (all ages; years 1994-2007) isolates from Native American communities. RESULTS: Twenty-seven of 28 (96.4%) NT IPD isolates had sequence types (STs) typically found among typeable IPD isolates and contained whole or fragments of capsule genes that matched known serotypes; 1 NT-IPD isolate had a profile resembling NT carriage isolates. Forty-nine of 76 (64.5%) NT carriage isolates had STs that typically lack capsule genes and were similar to NT carriage isolates found globally. CONCLUSIONS: This is the first documentation of IPD from an NT strain confirmed to lack all known capsule genes. Most NT IPD isolates have or had the capacity to produce capsule, whereas a majority of NT carriage isolates lack this capacity. We found no evidence of pneumococcal adaptation to PCV7 via downregulation or deletion of vaccine-serotype capsule genes.
Asunto(s)
Indígenas Norteamericanos , Infecciones Neumocócicas/etnología , Infecciones Neumocócicas/virología , Streptococcus pneumoniae/clasificación , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Preescolar , Humanos , Tipificación de Secuencias Multilocus/métodos , Serotipificación/métodos , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
Although the major food-borne pathogen Campylobacter jejuni has been isolated from diverse animal, human and environmental sources, our knowledge of genomic diversity in C. jejuni is based exclusively on human or human food-chain-associated isolates. Studies employing multilocus sequence typing have indicated that some clonal complexes are more commonly associated with particular sources. Using comparative genomic hybridization on a collection of 80 isolates representing diverse sources and clonal complexes, we identified a separate clade comprising a group of water/wildlife isolates of C. jejuni with multilocus sequence types uncharacteristic of human food-chain-associated isolates. By genome sequencing one representative of this diverse group (C. jejuni 1336), and a representative of the bank-vole niche specialist ST-3704 (C. jejuni 414), we identified deletions of genomic regions normally carried by human food-chain-associated C. jejuni. Several of the deleted regions included genes implicated in chicken colonization or in virulence. Novel genomic insertions contributing to the accessory genomes of strains 1336 and 414 were identified. Comparative analysis using PCR assays indicated that novel regions were common but not ubiquitous among the water/wildlife group of isolates, indicating further genomic diversity among this group, whereas all ST-3704 isolates carried the same novel accessory regions. While strain 1336 was able to colonize chicks, strain 414 was not, suggesting that regions specifically absent from the genome of strain 414 may play an important role in this common route of Campylobacter infection of humans. We suggest that the genomic divergence observed constitutes evidence of adaptation leading to niche specialization.
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Animales Salvajes/microbiología , Campylobacter jejuni/genética , Variación Genética , Microbiología del Agua , Animales , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Campylobacter jejuni/clasificación , Campylobacter jejuni/aislamiento & purificación , Mapeo Cromosómico , Hibridación Genómica Comparativa , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
Previous studies have suggested that the pneumococcal niche changes from the nasopharynx to the oral cavity with age. We use an Experimental Human Pneumococcal Challenge model to investigate pneumococcal colonisation in different anatomical niches with age. Healthy adults (n = 112) were intranasally inoculated with Streptococcus pneumoniae serotype 6B (Spn6B) and were categorised as young 18-55 years (n = 57) or older > 55 years (n = 55). Colonisation status (frequency and density) was determined by multiplex qPCR targeting the lytA and cpsA-6A/B genes in both raw and culture-enriched nasal wash and oropharyngeal swab samples collected at 2-, 7- and 14-days post-exposure. For older adults, raw and culture-enriched saliva samples were also assessed. 64% of NW samples and 54% of OPS samples were positive for Spn6B in young adults, compared to 35% of NW samples, 24% of OPS samples and 6% of saliva samples in older adults. Many colonisation events were only detected in culture-enriched samples. Experimental colonisation was detected in 72% of young adults by NW and 63% by OPS. In older adults, this was 51% by NW, 36% by OPS and 9% by saliva. The nose, as assessed by nasal wash, is the best niche for detection of experimental pneumococcal colonisation in both young and older adults.
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Líquido del Lavado Nasal/microbiología , Nariz/microbiología , Infecciones Neumocócicas/diagnóstico , Streptococcus pneumoniae , Adolescente , Adulto , Factores de Edad , Anciano , ADN Bacteriano/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Infecciones Neumocócicas/microbiología , Saliva/microbiología , Streptococcus pneumoniae/genética , Adulto JovenRESUMEN
The importance of Mn(2+) for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn(2+) is required are yet to be fully elucidated. Here, we analyzed the effect of Mn(2+) limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn(2+) transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn(2+)-dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn(2+)-SodA and virulence-associated genes pcpA and prtA. We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC, nmlR, and czcD, in response to Mn(2+) stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn(2+)-replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn(2+) having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn(2+) transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn(2+) stress.
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Adhesinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico , Lipoproteínas/metabolismo , Manganeso/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/fisiología , Adhesinas Bacterianas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Medios de Cultivo , Electroforesis en Gel Bidimensional , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Lipoproteínas/genética , Manganeso/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Proteoma , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrolloRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) infections are complicated by the ability of the organism to grow in surface-adhered biofilms on a multitude of abiotic and biological surfaces. These multicellular communities are notoriously difficult to eradicate with antimicrobial therapy. Cells within the biofilm may be exposed to a sublethal concentration of the antimicrobial due to the metabolic and phenotypic diversity of the biofilm-associated cells or the protection offered by the biofilm structure. In the present study, the influence of a sublethal concentration of tigecycline on biofilms formed by an epidemic MRSA-16 isolate was investigated by transcriptome analysis. In the presence of the drug, 309 genes were upregulated and 213 genes were downregulated by more than twofold in comparison to the levels of gene regulation detected for the controls not grown in the presence of the drug. Microarray data were validated by real-time reverse transcription-PCR and phenotypic assays. Tigecycline altered the expression of a number of genes encoding proteins considered to be crucial for the virulence of S. aureus. These included the reduced expression of icaC, which is involved in polysaccharide intercellular adhesin production and biofilm development; the upregulation of fnbA, clfB, and cna, which encode adhesins which attach to human proteins; and the downregulation of the cap genes, which mediate the synthesis of the capsule polysaccharide. The expression of tst, which encodes toxic shock syndrome toxin 1 (TSST-1), was also significantly reduced; and an assay performed to quantify TSST-1 showed that the level of toxin production by cells treated with tigecycline decreased by 10-fold (P < 0.001) compared to the level of production by untreated control cells. This study suggests that tigecycline may reduce the expression of important virulence factors in S. aureus and supports further investigation to determine whether it could be a useful adjunct to therapy for the treatment of biofilm-mediated infections.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Minociclina/análogos & derivados , Factores de Virulencia/biosíntesis , Adhesinas Bacterianas/biosíntesis , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/biosíntesis , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Minociclina/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Bacteriano/biosíntesis , ARN Bacteriano/aislamiento & purificación , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TigeciclinaRESUMEN
Streptococcus pneumoniae (the pneumococcus) carriage is commonly used to measure effects of pneumococcal vaccines. Based on findings from culture-based studies, the World Health Organization recommends both nasopharyngeal (NP) and oropharyngeal (OP) sampling for detecting adult carriage. Given evidence of potential confounding by other streptococci, we evaluated molecular methods for pneumococcal identification and serotyping from 250 OP samples collected from adults in Fiji, using paired NP samples for comparison. Samples were screened using lytA quantitative PCR (qPCR), as well as pneumococcal identification and serotyping conducted by DNA microarray. A subset of OP samples were characterized by latex sweep agglutination and multiplex PCR. Alternate qPCR assays (piaB and bguR) for pneumococcal identification were evaluated. The lytA qPCR was less specific and had poor positive predictive value (PPV) in OP samples (88% and 26%, respectively) compared with NP samples (95% and 64%, respectively). Using additional targets piaB and/or bguR improved qPCR specificity in OP, although the PPV (42 to 53%) was still poor. Using microarray, we found that 102/107 (95%) of OP samples contained nonpneumococcal streptococci with partial or divergent complements of pneumococcal capsule genes. We explored 91 colonies isolated from 11 OP samples using various techniques, including multiplex PCR, latex agglutination, and microarray. We found that nonpneumococcal streptococci contribute to false positives in pneumococcal serotyping and may also contribute to spurious identification by qPCR. Our results highlight that molecular approaches should include multiple loci to minimize false-positive results when testing OP samples. Regardless of method, pneumococcal identification and serotyping results from OP samples should be interpreted with caution.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is a significant global pathogen. Accurate identification and serotyping are vital. In contrast with World Health Organization recommendations based on culture methods, we demonstrate that pneumococcal identification and serotyping with molecular methods are affected by sample type. Results from oropharyngeal samples from adults were often inaccurate. This is particularly important for assessment of vaccine impact using carriage studies, particularly in low- and middle-income countries where there are significant barriers for disease surveillance.
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Portador Sano/diagnóstico , Portador Sano/microbiología , Orofaringe/microbiología , Infecciones Neumocócicas/diagnóstico , Streptococcus pneumoniae/aislamiento & purificación , Adulto , Reacciones Falso Positivas , Femenino , Humanos , Masculino , Análisis por Micromatrices , Técnicas de Diagnóstico Molecular , Infecciones Neumocócicas/microbiología , Valor Predictivo de las Pruebas , Reacción en Cadena en Tiempo Real de la Polimerasa , Serotipificación , Manejo de Especímenes , Streptococcus pneumoniae/clasificaciónRESUMEN
Quinazoline-2,4-diones, such as PD 0305970, are new DNA gyrase and topoisomerase IV (topo IV) inhibitors with potent activity against gram-positive pathogens, including quinolone-resistant isolates. The mechanistic basis of dione activity vis-à-vis quinolones is not understood. We present evidence for Streptococcus pneumoniae gyrase and topo IV that PD 0305970 and quinolones interact differently with the enzyme breakage-reunion and Toprim domains, DNA, and Mg2+-four components that are juxtaposed in the topoisomerase cleavage complex to effect DNA scission. First, PD 0305970 targets primarily gyrase in Streptococcus pneumoniae. However, unlike quinolones, which select predominantly for gyrA (or topo IV parC) mutations in the breakage-reunion domain, unusually the dione selected for novel mutants with alterations that map to a region of the Toprim domain of GyrB (R456H and E474A or E474D) or ParE (D435H and E475A). This "dione resistance-determining region" overlaps the GyrB quinolone resistance-determining region and the region that binds essential Mg2+ ions, each function involving conserved EGDSA and PLRGK motifs. Second, dione-resistant gyrase and topo IV were inhibited by ciprofloxacin, whereas quinolone-resistant enzymes (GyrA S81F and ParC S79F) remained susceptible to PD 0305970. Third, dione-promoted DNA cleavage by gyrase occurred at a distinct repertoire of sites, implying that structural differences with quinolones are sensed at the DNA level. Fourth, unlike the situation with quinolones, the Mg2+ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg2+-dependent DNA unwinding. It appears that PD 0305970 interacts uniquely to stabilize the cleavage complex of gyrase/topo IV perhaps via an altered orientation directed by the bidentate 3-amino-2,4-dione moiety.
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Topoisomerasa de ADN IV/antagonistas & inhibidores , Pirrolidinas/farmacología , Quinazolinonas/farmacología , Quinolonas/farmacología , Streptococcus pneumoniae/enzimología , Inhibidores de Topoisomerasa II , Ciprofloxacina/química , Ciprofloxacina/farmacología , Estructura Molecular , Mutación , Reacción en Cadena de la Polimerasa , Pirrolidinas/química , Quinazolinonas/química , Análisis de Secuencia de ADN , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genéticaRESUMEN
Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones.
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Técnicas de Tipificación Bacteriana , Infecciones Comunitarias Adquiridas/microbiología , Electroforesis en Gel de Campo Pulsado , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/genética , Infecciones Estafilocócicas/microbiología , Adulto , Anciano , Análisis por Conglomerados , Infecciones Comunitarias Adquiridas/epidemiología , ADN Bacteriano/genética , Genotipo , Humanos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Epidemiología Molecular , Infecciones Estafilocócicas/epidemiologíaRESUMEN
Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.
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ADN Bacteriano/genética , Genoma Bacteriano , Análisis por Micromatrices , Mycobacterium avium subsp. paratuberculosis/clasificación , Mycobacterium avium subsp. paratuberculosis/genética , Polimorfismo Genético , Animales , Composición de Base , Duplicación de Gen , Orden Génico , Islas Genómicas , Genotipo , Humanos , Mutación INDEL , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , SinteníaRESUMEN
Pneumococcal carriage is a prerequisite for disease, and underpins herd protection provided by pneumococcal conjugate vaccines (PCVs). There are few data on the impact of PCVs in lower income settings, particularly in Asia. In 2013, the Lao People's Democratic Republic (Lao PDR) introduced 13-valent PCV (PCV13) as a 3â¯+â¯0 schedule (doses at 6, 10 and 14â¯weeks of age) with limited catch-up vaccination. We conducted two cross-sectional carriage surveys (pre- and two years post-PCV) to assess the impact of PCV13 on nasopharyngeal pneumococcal carriage in 5-8â¯week old infants (nâ¯=â¯1000) and 12-23â¯month old children (nâ¯=â¯1010). Pneumococci were detected by quantitative real-time PCR, and molecular serotyping was performed using DNA microarray. Post PCV13, there was a 23% relative reduction in PCV13-type carriage in children aged 12-23â¯months (adjusted prevalence ratio [aPR] 0.77 [0.61-0.96]), and no significant change in non-PCV13 serotype carriage (aPR 1.11 [0.89-1.38]). In infants too young to be vaccinated, there was no significant change in carriage of PCV13 serotypes (aPR 0.74 [0.43-1.27]) or non-PCV13 serotypes (aPR 1.29 [0.85-1.96]), although trends were suggestive of indirect effects. Over 70% of pneumococcal-positive samples contained at least one antimicrobial resistance gene, which were more common in PCV13 serotypes (pâ¯<â¯0.001). In 12-23â¯month old children, pneumococcal density of both PCV13 serotypes and non-PCV13 serotypes was higher in PCV13-vaccinated compared with undervaccinated children (pâ¯=â¯0.004 and pâ¯<â¯0.001, respectively). This study provides evidence of PCV13 impact on carriage in a population without prior PCV7 utilisation, and provides important data from a lower-middle income setting in Asia. The reductions in PCV13 serotype carriage in vaccine-eligible children are likely to result in reductions in pneumococcal transmission and disease in Lao PDR.
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Portador Sano/epidemiología , Infecciones Neumocócicas/epidemiología , Vacunas Neumococicas/administración & dosificación , Streptococcus pneumoniae/aislamiento & purificación , Portador Sano/inmunología , Estudios Transversales , Femenino , Humanos , Inmunidad Colectiva , Lactante , Laos/epidemiología , Masculino , Nasofaringe/microbiología , Infecciones Neumocócicas/prevención & control , Reacción en Cadena de la Polimerasa , Prevalencia , Serogrupo , Serotipificación , Vacunación , Vacunas Conjugadas/administración & dosificaciónRESUMEN
The emergence of drug resistance threatens to destroy tuberculosis control programs worldwide, with resistance to all first-line drugs and most second-line drugs detected. Drug tolerance (or phenotypic drug resistance) is also likely to be clinically relevant over the 6-month long standard treatment for drug-sensitive tuberculosis. Transcriptional profiling the response of Mycobacterium tuberculosis to antimicrobial drugs offers a novel interpretation of drug efficacy and mycobacterial drug-susceptibility that likely varies in dynamic microenvironments, such as the lung. This chapter describes the noninvasive sampling of tuberculous sputa and techniques for mRNA profiling M. tb bacilli during patient therapy to characterize real-world drug actions.
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Perfilación de la Expresión Génica , Mycobacterium tuberculosis/genética , Esputo/microbiología , Transcriptoma , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/microbiología , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica , Humanos , Mycobacterium tuberculosis/aislamiento & purificaciónRESUMEN
INTRODUCTION: Pneumococcal multiple serotype carriage is important for evolution of the species and to understand how the pneumococcal population is changing with vaccination. We aimed to determine the impact of the 13-valent pneumococcal conjugate vaccine (PCV13) on multiple serotype carriage. METHODS AND MATERIALS: Nasopharyngeal samples from fully vaccinated pneumococcal carriers (4 doses of PCV13, n=141, aged 18-72months) or from non-vaccinated pneumococcal carriers (0 doses of any PCV, n=140, same age group) were analyzed. Multiple serotype carriage was evaluated by DNA hybridization with a molecular serotyping microarray that detects all known serotypes. RESULTS: Vaccinated children had a lower prevalence of multiple serotype carriage than the non-vaccinated group (20.6% vs 29.3%, p=0.097), and a significantly lower proportion of PCV13 serotypes (6.4% vs 38.5%, p=0.0001). PCV13 serotypes found among vaccinated children were mostly detected as a minor serotype in co-colonization with a more abundant non-vaccine serotype. Vaccinated children were colonized by a significantly higher proportion of commensal non-pneumococcal Streptococcus spp. (58.2% vs 42.8%, p=0.012). In vaccinated children there were significantly less non-vaccine type (NVT) co-colonization events than expected based on the distribution of these serotypes in non-vaccinated children. CONCLUSIONS: The results suggest that vaccinated children have lower pneumococcal multiple serotype carriage prevalence due to higher competitive abilities of non-vaccine serotypes expanding after PCV13 use. This might represent an additional benefit of PCV13, as decreased co-colonization rates translate into decreased opportunities for horizontal gene transfer and might have implications for the evolution and virulence of pneumococci.
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Portador Sano/epidemiología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/administración & dosificación , Portador Sano/microbiología , Niño , Preescolar , Humanos , Lactante , Nasofaringe/microbiología , Vacunas Neumococicas/uso terapéutico , Portugal , Serogrupo , Streptococcus pneumoniae/clasificaciónRESUMEN
OBJECTIVES: We describe the first published cluster of extensively drug resistant Tuberculosis (XDR-TB) in the UK and show how early whole genome sequencing (WGS) of Mtb can assist in case management and contact investigations. METHODS: We describe the contact tracing investigation undertaken after the presentation of an adult with XDR-TB. Active cases were treated with an XDR-TB drug regimen and contacts underwent a programme of follow-up for 2 years. All isolates of Mycobacterium tuberculosis (Mtb) were assessed early using whole genome sequencing (WGS) as well as routine drug susceptibility testing (DST). RESULTS: Thirty-three contacts were screened. In the first year one confirmed and one probable case were identified through contact tracing. A further possible case was identified through epidemiological links. Two confirmed cases were identified through WGS 2 years later. Twenty-five (80%) contacts without evidence of tuberculosis were adherent to 1 year of follow-up and 14 (45%) were adherent to 2 years of follow-up. WGS of Mtb was used to guide drug choices, rapidly identify transmission events, and alter public health management. CONCLUSION: WGS of Mtb enabled rapid effective individualized treatment and facilitated public health interventions by early identification of transmission events.
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Manejo de Caso , Trazado de Contacto , Tuberculosis Extensivamente Resistente a Drogas/epidemiología , Tuberculosis Extensivamente Resistente a Drogas/transmisión , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Adulto , Antituberculosos/uso terapéutico , Niño , Brotes de Enfermedades , Tuberculosis Extensivamente Resistente a Drogas/tratamiento farmacológico , Tuberculosis Extensivamente Resistente a Drogas/prevención & control , Femenino , Humanos , Londres/epidemiología , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49.5%) of samples with more than one serotype being found in 67/297 (20.2%) of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44.4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.