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Background: Invasive pneumococcal disease due to serotype 3 (S3-IPD) is associated with high mortality rates and long-term adverse effects. The introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the Spanish paediatric immunisation programme has not led to a decrease in the adult S3-IPD. We aimed to analyse the incidence, clinical characteristics and genomics of S3-IPD in adults in Spain. Methods: Adult IPD episodes hospitalized in a Southern Barcelona hospital were prospectively collected (1994-2020). For genomic comparison, S3-IPD isolates from six Spanish hospitals (2008-2020) and historical isolates (1989-1993) were analysed by WGS (Illumina and/or MinION). Findings: From 1994 to 2020, 270 S3-IPD episodes were detected. When comparing pre-PCV (1994-2001) and late-PCV13 (2016-2020) periods, only modest changes in S3-IPD were observed (from 1.58 to 1.28 episodes per 100,000 inhabitants year). In this period, the incidence of the two main lineages shifted from 0.38 to 0.67 (CC180-GPSC12) and from 1.18 to 0.55 (CC260-GPSC83). The overall 30-day mortality remained high (24.1%), though a decrease was observed between the pre-PCV (32.4%; 95.0% CI, 22.0-45.0) and the late-PCV13 period (16.7%; 95.0% CI, 7.5-32.0) (p = 0.06). At the same time, comorbidities increased from 77.3% (95.0% CI, 65.0-86.0) to 85.7% (95.0% CI, 71.0-94.0) (p = 0.69). There were no differences in clinical characteristics or 30-day mortality between the two S3 lineages. Although both lineages were genetically homogeneous, the CC180-GPSC12 lineage presented a higher SNP density, a more open pan-genome, and a major presence of prophages and mobile genetic elements carrying resistance genes. Interpretation: Adult S3-IPD remained stable in our area over the study period despite PCV13 introduction in children. However, a clonal shift was observed. The decrease in mortality rates and the increase in comorbidities suggest a change in clinical management and overall population characteristics. The low genetic variability and absence of clinical differences between lineages highlight the role of the S3 capsule in the disease severity. Funding: This study has been funded by Instituto de Salud Carlos III (ISCIII) "PI18/00339", "PI21/01000", "INT22/00096", "FI22/00279", CIBER "CIBERES-CB06/06/0037", "CIBERINFEC-CB21/13/00009" and MSD grant "IISP 60168".
ABSTRACT
OBJECTIVES: To analyse the clonal dynamics and clinical characteristics of adult invasive pneumococcal disease (IPD) caused by MDR and penicillin-non-susceptible (PNS) pneumococci in Spain. METHODS: All adult IPD episodes were prospectively collected (1994-2018). Streptococcus pneumoniae isolates were serotyped, genotyped and tested for antimicrobial susceptibility. Changes in the incidence of IPD were analysed and risk factors contributing to MDR were assessed by logistic regression. RESULTS: Of 2095 IPD episodes, 635 (30.3%) were caused by MDR/PNS isolates. Over the study period, the incidence of MDR/PNS-IPD decreased (IRR 0.70; 95% CI 0.53-0.93) whereas that of susceptible isolates remained stable (IRR 0.96; 95% CI 0.80-1.16). A reduction of resistance rates to penicillin (-19.5%; 95% CI -37% to 2%) and cefotaxime (-44.5%; 95% CI -64% to -15%) was observed. Two clones, Spain9V-ST156 and Denmark14-ST230, accounted for 50% of current resistant disease. Among current MDR/PNS isolates, 45.8% expressed serotypes not covered by the upcoming PCV15/PCV20 vaccines. MDR/PNS episodes were associated with older patients with comorbidities, nosocomial acquisition and higher 30 day mortality. MDR/PNS pneumococci were not independently associated with 30 day mortality in multivariate analysis [OR 0.826 (0.648-1.054)]. CONCLUSIONS: Our study shows an overall reduction of MDR/PNS isolates in adults after the introduction of pneumococcal conjugate vaccines. However, a significant proportion of current resistant isolates are not covered by any of the upcoming PCV15/PCV20 vaccines. The burden of resistant disease is related to older patients with underlying conditions and caused by two major clones. Our data show that MDR is not a statistically significant factor related to increased mortality.
Subject(s)
Pneumococcal Infections , Pneumococcal Vaccines , Adult , Humans , Infant , Pneumococcal Infections/epidemiology , Serogroup , Serotyping , Spain/epidemiology , Streptococcus pneumoniae/geneticsABSTRACT
Automated antimicrobial susceptibility testing devices are widely implemented in clinical microbiology laboratories in Spain, mainly using EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published recommendations for including antimicrobial agents and selecting concentrations in these systems. Under the patronage of the Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish National Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this document. The main modifications from the previous version comprise the inclusion of new antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new resistance mechanisms. This proposal should be considered by different manufacturers and users when designing new panels or cards. In addition, recommendations for selective reporting are also included. With this approach, the implementation of EUCAST breakpoints will be easier, increasing the quality of antimicrobial susceptibility testing data and their microbiological interpretation. It will also benefit epidemiological surveillance studies as well as the clinical use of antimicrobials aligned with antimicrobial stewardship programs
Los sistemas automáticos utilizados en el estudio de la sensibilidad a los antimicrobianos están introducidos en la mayoría de los laboratorios de Microbiología Clínica en España, utilizando principalmente los puntos de corte del European Committee on Antimicrobial Susceptibility Testing (EUCAST). En 2007, un grupo de expertos publicó unas recomendaciones para incluir antimicrobianos y seleccionar concentraciones en estos sistemas. Bajo el auspicio del Comité Español del Antibiograma (COESANT) y del Grupo de Estudio de los Mecanismos de Acción y Resistencia a los Antimicrobianos (GEMARA) de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC) y alineado con el Plan Nacional frente a la Resistencia a los Antibióticos (PRAN), un grupo de expertos ha actualizado dicho documento. Las principales modificaciones realizadas sobre la versión anterior comprenden la inclusión de nuevos agentes antimicrobianos, la adaptación de los rangos de concentraciones para cubrir los puntos de corte clínicos y los puntos de corte epidemiológicos (ECOFF) definidos por el EUCAST, y para la inferencia de nuevos mecanismos de resistencia. Esta propuesta debería ser considerada por los diferentes fabricantes y los usuarios cuando se diseñen nuevos paneles o tarjetas. Además, se incluyen recomendaciones para realizar informes selectivos. Con este enfoque, la implementación de los puntos de corte del EUCAST será más fácil, aumentando la calidad de los datos del antibiograma y su interpretación microbiológica. También será de utilidad para los estudios de vigilancia epidemiológica, así como para el uso clínico de los antimicrobianos, de acuerdo con los programas de optimización de uso de antimicrobianos (PROA)
Subject(s)
Humans , Microbial Sensitivity Tests/methods , Automation , Professional Staff Committees , SpainABSTRACT
Automated antimicrobial susceptibility testing devices are widely implemented in clinical microbiology laboratories in Spain, mainly using EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published recommendations for including antimicrobial agents and selecting concentrations in these systems. Under the patronage of the Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish National Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this document. The main modifications from the previous version comprise the inclusion of new antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new resistance mechanisms. This proposal should be considered by different manufacturers and users when designing new panels or cards. In addition, recommendations for selective reporting are also included. With this approach, the implementation of EUCAST breakpoints will be easier, increasing the quality of antimicrobial susceptibility testing data and their microbiological interpretation. It will also benefit epidemiological surveillance studies as well as the clinical use of antimicrobials aligned with antimicrobial stewardship programs.
Subject(s)
Anti-Infective Agents , Microbial Sensitivity Tests/standards , Anti-Infective Agents/pharmacology , Automation, Laboratory , SpainABSTRACT
Serotypes/genotypes causing invasive pneumococcal disease (IPD) in adults are determined by vaccination strategies. The aim of this study was to assess the epidemiology of IPD in adults (≥18â¯years) after PCV13 introduction for children: serotypes, clonal complexes, antibiotic non-susceptibility and clinical presentations. We performed a prospective, clinical surveillance of hospitalized culture-confirmed IPDs in adults in nine Spanish hospitals (August 2010-June 2015). A total of 1087 culture-confirmed IPD episodes were included, of which 772 (71.0%) had bacteremic pneumonia (401 complicated/371 uncomplicated pneumonia), 122 (11.2%) meningitis, 102 (9.4%) non-focal bacteremia, 34 (3.1%) peritonitis and 57 (5.3%) others. The most common serotypes were: 3 (12.7%), 19A (8.5%), 8 (7.7%), 7F (6.3%), 1 (4.2%), 6C (4.2%), 11A (4.2%), 22F (4.2%) and 14 (4.0%). Vaccine types (PCV13â¯+â¯6C) caused 49.8% of IPD episodes, with a significant decrease over the 5-year period, and significant decreases in serotypes 6C and 7F. The most common genotypes were: CC180 (8.4%), CC191 (6.0%), and CC53 (5.0%). Vaccine types caused 53.9% (414/768) pneumonia episodes and 58.9% (235/399) complicated pneumonia, 53.4% IPD in adults <50â¯years (143/268), and 54.7% IPD in immunocompetent patients (337/616). Overall non-susceptibility was 25.9% to penicillin (1.1% for parenteral criteria), 24.9% to erythromycin and 2.7% to levofloxacin. CONCLUSIONS: Although the percentage of vaccine-types causing IPDs in adults significantly decreased, it remained high. Associations of vaccine types with pneumonia (with complicated pneumonia for specific serotypes), and immunocompetent patients point to the burden of IPD caused by PCV13 serotypes.
Subject(s)
Bacteremia/epidemiology , Epidemiological Monitoring , Pneumococcal Infections/epidemiology , Pneumococcal Vaccines/administration & dosage , Streptococcus pneumoniae/genetics , Adult , Aged , Female , Genotype , Hospitalization , Humans , Licensure , Male , Middle Aged , Pneumococcal Infections/microbiology , Pneumococcal Vaccines/therapeutic use , Prospective Studies , Serogroup , Serotyping , Spain/epidemiology , Streptococcus pneumoniae/isolation & purification , Young AdultABSTRACT
BACKGROUND: Streptococcus pneumoniae is an important pathogen in chronic obstructive pulmonary disease (COPD). We aimed at showing the recent changes in the epidemiology of pneumococcal diseases in patients with COPD, especially after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13). METHODS: From 2013 to 2016, strains causing invasive pneumococcal disease (IPD), non-bacteremic pneumococcal pneumonia (non-BPP), and acute exacerbation of COPD (AE-COPD) were prospectively included. Antimicrobial susceptibility testing, serotyping, and genotyping were analyzed. RESULTS: We collected 345 pneumococci from 286 COPD patients (57 IPD, 78 non-BPP, and 210 AE-COPD). The most frequent serotypes were serotypes 3 (14.0%), 8 (14.0%), and 12F (8.8%) in IPD; serotypes 3 (16.7%), 11A (9%), 9L/N (7.7%), and 23A (7.7%) in non-BPP; and serotypes 11A (11%), nontypeable (11%), and 6C (10%) in AE-COPD. Resistance rates were highest among AE-COPD strains. Penicillin resistance was associated with serotypes 11A (CC156) and 19A (CC320 and CC230). Compared with previous studies, fluoroquinolone resistance in AE-COPD increased (9.5%), PCV13 serotypes decreased (31.6%, 26.9%, and 16.7% for IPD, non-BPP, and AE-COPD, respectively), and serotype 11A-CC156 in AE-COPD and serotype 8 in IPD increased. CONCLUSION: The epidemiology of pneumococcal disease in COPD changed after the introduction of PCV13 in children. Increases in the highly invasive serotype 8 among patients with IPD and in serotype 11A-CC156 among patients with AE-COPD could compromise the ability of current PCVs to prevent diseases. Vaccines with a greater coverage could improve the benefits of adult vaccination.
ABSTRACT
Tracking bacterial evolution during chronic infection provides insights into how host selection pressures shape bacterial genomes. The human-restricted opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) infects the lower airways of patients suffering chronic obstructive pulmonary disease (COPD) and contributes to disease progression. To identify bacterial genetic variation associated with bacterial adaptation to the COPD lung, we sequenced the genomes of 92 isolates collected from the sputum of 13 COPD patients over 1 to 9 years. Individuals were colonized by distinct clonal types (CTs) over time, but the same CT was often reisolated at a later time or found in different patients. Although genomes from the same CT were nearly identical, intra-CT variation due to mutation and recombination occurred. Recurrent mutations in several genes were likely involved in COPD lung adaptation. Notably, nearly a third of CTs were polymorphic for null alleles of ompP1 (also called fadL), which encodes a bifunctional membrane protein that both binds the human carcinoembryonic antigen-related cell adhesion molecule 1 (hCEACAM1) receptor and imports long-chain fatty acids (LCFAs). Our computational studies provide plausible three-dimensional models for FadL's interaction with hCEACAM1 and LCFA binding. We show that recurrent fadL mutations are likely a case of antagonistic pleiotropy, since loss of FadL reduces NTHi's ability to infect epithelia but also increases its resistance to bactericidal LCFAs enriched within the COPD lung. Supporting this interpretation, truncated fadL alleles are common in publicly available NTHi genomes isolated from the lower airway tract but rare in others. These results shed light on molecular mechanisms of bacterial pathoadaptation and guide future research toward developing novel COPD therapeutics.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidate the bacterial pathways undergoing in vivo evolutionary adaptation, we compared bacterial genomes collected over time from 13 COPD patients and identified recurrent genetic changes arising in independent bacterial lineages colonizing different patients. Besides finding changes in phase-variable genes, we found recurrent loss-of-function mutations in the ompP1 (fadL) gene. We show that loss of OmpP1/FadL function reduces this bacterium's ability to infect cells via the hCEACAM1 epithelial receptor but also increases its resistance to bactericidal fatty acids enriched within the COPD lung, suggesting a case of antagonistic pleiotropy that restricts ΔfadL strains' niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways.
Subject(s)
Adaptation, Biological , Bacterial Outer Membrane Proteins/metabolism , Fatty Acid Transport Proteins/metabolism , Haemophilus Infections/microbiology , Haemophilus influenzae/genetics , Pneumonia, Bacterial/microbiology , Pulmonary Disease, Chronic Obstructive/microbiology , Aged , Aged, 80 and over , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Computational Biology , Fatty Acid Transport Proteins/chemistry , Fatty Acid Transport Proteins/genetics , Genetic Variation , Genome, Bacterial , Haemophilus influenzae/classification , Humans , Longitudinal Studies , Middle Aged , Molecular Docking Simulation , Mutation , Recombination, Genetic , Sequence Analysis, DNA , Sputum/microbiology , Whole Genome SequencingABSTRACT
Objectives: To analyse the epidemiology and genetic evolution of PMEN3 (Spain9V-156), a penicillin-non-susceptible clone of Streptococcus pneumoniae, causing invasive pneumococcal disease (IPD) in Barcelona during 1987-2016. Methods: WGS was performed on 46 representative isolates and the data were used to design additional molecular typing methods including partial MLST, PCR-RFLP and detection of surface-exposed proteins and prophages, to assign the remaining isolates to lineages. The isolates were also subjected to antimicrobial susceptibility testing. Results: Two hundred and twenty-seven adult cases of IPD caused by PMEN3 were identified. PMEN3 caused mainly pneumonia (84%) and the 30 day mortality rate was 23.1%. Evidence of recombination events was found, mostly in three regions, namely the capsular operon (associated with capsular switching) and adjacent regions containing pbp2x and pbp1a, the murM gene and the pbp2b-ddl region. Some of these genetic changes generated successful new variant serotype lineages, including one of serotype 11A that is not included in the current PCV13 vaccine. Other genetic changes led to increased MICs of ß-lactams. Notably, most isolates also harboured prophages coding for PblB-like proteins. Despite these adaptations, the ability of this clone to cause IPD remained unchanged over time, highlighting the importance of its core genetic background. Conclusions: Our study demonstrated successful adaptation of PMEN3 to persist over time despite the introduction of broader antibiotics and conjugate vaccines. In addition to enhancing understanding of the molecular evolution of PMEN3, these findings highlight the need for the development of non-serotype-based vaccines to fight pneumococcal infection.
Subject(s)
Anti-Bacterial Agents/pharmacology , Evolution, Molecular , Pneumococcal Infections/epidemiology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/genetics , Bacterial Typing Techniques , DNA, Bacterial/genetics , Genotype , Humans , Microbial Sensitivity Tests , Multilocus Sequence Typing , Operon , Pneumococcal Infections/mortality , Polymerase Chain Reaction , Prophages/genetics , Recombination, Genetic , Serogroup , Spain/epidemiology , Time Factors , Whole Genome SequencingABSTRACT
Little is known about the sensitivity of the BinaxNOW pneumococcal urinary antigen (PUA) test for adult pneumococcal pneumonia caused by different serotypes. In this study, we aimed to analyze the trends in the sensitivity of the PUA test over a 15-year period (2001 to 2015) and to analyze its sensitivity for pneumococcal pneumonia caused by different serotypes. In total, we analyzed 1,096 pneumococcal isolates from adults with pneumococcal pneumonia who had a PUA test performed at the onset of the episode. Three periods were analyzed: 2001 to 2005 (early use of the seven-valent pneumococcal conjugate vaccine [early PCV7]), 2006 to 2010 (late PCV7), and 2011 to 2015 (early PCV13). The sensitivity of the PUA test varied from 76.4% (95% confidence interval [CI], 70.5% to 82.4%) in the period from 2001 to 2005 to 77.9% in 2006 to 2010 (95% CI, 74.4% to 81.4%) and decreased to 60.5% (95% CI, 55.4% to 65.6%) in 2011 to 2015. This decrease was observed in 560 proven (83.2% in 2001 to 2005, 86.5% in 2006 to 2010, and 78.1%) and 536 probable (70.0% in 2001 to 2005, 68.7% in 2006 to 2010, and 41.5% in 2011 to 2015) episodes of pneumococcal pneumonia. Differences were observed in the sensitivity of the PUA test for diagnosing pneumonia caused by certain serotypes, being highest for the 9V (90.6%), 14 (86.8%), 18C (100%), and 20 (100%) serotypes and lowest for the 8 (55.2%), 9L/N (39.1%), 11A (48.8%), 23B (33.3%), and nontypeable (47.8%) serotypes. Comparing 2001 to 2005, 2006 to 2010, and 2011 to 2015, the prevalence of serotypes 9V (3.1%, 3.7%, and 1.7%, respectively) and 14 (7.2%, 5.1%, and 3.1%, respectively) decreased, while the prevalence of serotypes 23B (0%, 0.7%, and 1.4%, respectively), 9L/N (1.0%, 1.6%, and 3.4%, respectively), 11A (2.6%, 4.2%, and 3.7%, respectively), and 8 (1.5%, 1.5%, and 5.1%, respectively) increased. The PUA test sensitivity varied by pneumococcal pneumonia serotype, and these differences and the changes in serotype distribution were associated with an overall decrease in the sensitivity of the PUA test.
Subject(s)
Clinical Laboratory Techniques/methods , Pneumonia, Pneumococcal/microbiology , Serogroup , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/immunology , Adult , Aged , Aged, 80 and over , Antigens, Bacterial/urine , Heptavalent Pneumococcal Conjugate Vaccine/immunology , Hospitals, Teaching , Humans , Middle Aged , Pneumonia, Pneumococcal/epidemiology , Prevalence , Sensitivity and Specificity , Spain/epidemiology , Streptococcus pneumoniae/isolation & purification , Young AdultABSTRACT
Molecular epidemiology of Klebsiella pneumoniae bacteremic strains allows for a better understanding of preventive and therapeutic strategies. Clinical and microbiological characteristics of 348 K. pneumoniae bacteremia cases (2007-2009) were retrospectively characterized by multilocus sequence typing and extended-spectrum beta-lactamases (ESBL) production. Overall, 223 (64.08%) cases were nosocomial (NA), 58 (16.67%) healthcare associated, and 67 (19.25%) community acquired. The main infection origins were urinary tract (16.6%, 50.0%, and 43.3%), biliary tract (10.8%, 24.2%, and 31.3%), and catheter-related infection (39.9%, 5.2%, and 0%). The 30-day mortality rate was around 20%. The rates of resistance were around 45% the highest being among NA cases, and ESBL production was detected in 7.2% of cases. A total of 161 different sequence types were grouped into 13 clonal sets by e-burst analysis. No relationship could be established between clonal sets and the origin of infection or the healthcare-related settings. The high genetic variability among the isolates suggests their intrapatient endogenous origin.
Subject(s)
Bacteremia/microbiology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Aged , Biliary Tract Diseases/microbiology , Cross Infection/microbiology , Drug Resistance, Bacterial/genetics , Female , Genetic Variation/genetics , Humans , Male , Middle Aged , Molecular Epidemiology , Retrospective Studies , Urinary Tract Infections/microbiology , beta-Lactamases/geneticsABSTRACT
BACKGROUND: The bronchial mucosa is protected by a specialized immune system focused on the prevention of colonization and infection by potentially pathogenic microorganisms (PPMs). Immunoglobulin A (IgA) is the principal antibody involved in this mechanism. A defective immune barrier may facilitate the recurrent presence of PPMs in COPD. PURPOSE: The aim of this study was to determine IgA-mediated bronchial specific immune responses against Pseudomonas aeruginosa in stable patients with severe disease. METHODS: COPD patients with good-quality sputum samples obtained during stability were included and classified according to the presence or absence of chronic bronchial colonization by P. aeruginosa. Levels of specific IgA for P. aeruginosa in sputum were determined by ELISA and expressed as ratios, using the pooled level of 10 healthy subjects as reference (optical density450 patient/control). RESULTS: Thirty-six stable COPD patients were included, 15 of whom had chronic colonization by P. aeruginosa. Levels of specific IgA against P. aeruginosa in stable non-colonized patients were lower than those in healthy subjects (IgA ratio: median =0.15 [interquartile range {IQR} 0.05-0.36]). Colonized patients had higher levels, (1.56 [IQR 0.59-2.79]) (p<0.001, Mann-Whitney U test), with figures equivalent but not exceeding the reference value. CONCLUSION: IgA-based immune response against P. aeruginosa was low in severe COPD patients. Levels of specific IgA against this microorganism were higher in colonized patients, but did not attain clear-cut levels above the reference. An impaired local response against P. aeruginosa may favor chronic colonization and recurrent infections in severe COPD.
Subject(s)
Antibodies, Bacterial/analysis , Immunoglobulin A/analysis , Lung/immunology , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/immunology , Pulmonary Disease, Chronic Obstructive/immunology , Respiratory Tract Infections/immunology , Aged , Case-Control Studies , Cross-Sectional Studies , Female , Host-Pathogen Interactions , Humans , Immunity, Mucosal , Lung/microbiology , Male , Middle Aged , Pseudomonas Infections/diagnosis , Pseudomonas Infections/microbiology , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/microbiology , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/microbiology , Severity of Illness Index , Sputum/immunology , Sputum/microbiologyABSTRACT
A prospective laboratory-based multicenter study that collected all adult invasive pneumococcal disease (IPD) episodes from 6 Spanish hospitals before (2008-2009) and after (2012-2013). The 13-valent pneumococcal conjugate vaccine (PCV13) licensure was conducted in order to analyze the impact of PCV13 introduction for children on adult IPD. A total of 1558 IPD episodes were detected. The incidence of IPD decreased significantly in the second period by -33.9% (95% CI, -40.3% to -26.8%). IPD due to PCV7 serotypes (-52.7%; 95% CI, -64.2% to -37.5%) and to PCV13 additional serotypes (-55.0% 95% CI, -62.0% to -46.7%) significantly decreased whereas IPD due to non-PCV13 serotypes remained stable (1.0% 95% CI, -12.9% to 17.2%). IPD due to all PCV13 additional serotypes significantly declined with the exception of serotype 3 (-11.3%; 95%CI -35.0% to 21.1%). IPD due to two non-PCV13 serotypes varied: serotype 6C that rose (301.6%; 95%CI, 92.7% to 733.3%, p<0.001), related to the expansion of ST3866C, and serotype 8 that decreased (-34.9%, 95%CI, -57.1 to -1.2, p = 0.049), related to a decline of the ST638. The recombinant clone ST652111A (variant of ST1569V) increased in frequency. The decrease of serotype 19A IPD was linked to a fall in those antibiotic susceptible clones. In the last period, rates of penicillin- and cefotaxime-resistance remained under 10% and 4%, respectively. Adult IPD decreased after the PCV13 introduction in Spain due to herd protection. The spread of multidrug resistant clones (ST3866C, ST652111A) related to non-PCV13 serotypes needs further surveillance.
Subject(s)
Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/administration & dosage , Adolescent , Adult , Female , Humans , Male , Middle Aged , Prospective Studies , Spain , Young AdultABSTRACT
AIM: The bronchial microbiome of severe chronic obstructive pulmonary disease patients colonized by Pseudomonas aeruginosa was analyzed using 16S rRNA gene sequencing to identify differences related to biofilm-forming capacity. PATIENTS & METHODS: Patient sputum samples from 21 patients were studied. RESULTS: Statistically significant differences related to biofilm-forming capacity were only found for genera with relative abundances <1%, and Fusobacterium was over-represented when biofilm-forming capacity was high. Genera with relative abundances >50% which increased from baseline were observed in 10/14 exacerbations, but corresponded to Pseudomonas only in three episodes, while other pathogenic genera were identified in seven. CONCLUSION: The bronchial microbiome shows differences according with P. aeruginosa biofilm-forming capacity. Pathogenic microorganisms other than P. aeruginosa cause a significant part of the exacerbations in colonized chronic obstructive pulmonary disease patients.
Subject(s)
Biofilms/growth & development , Bronchi/microbiology , Microbiota , Pseudomonas Infections/pathology , Pseudomonas aeruginosa/physiology , Pulmonary Disease, Chronic Obstructive/microbiology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Humans , Pseudomonas Infections/microbiology , Pulmonary Disease, Chronic Obstructive/pathology , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sputum/microbiologyABSTRACT
The identification of commensal streptococci species is an everlasting problem due to their ability to genetically transform. A new challenge in this respect is the recent description of Streptococcus pseudopneumoniae as a new species, which was distinguished from closely related pathogenic S. pneumoniae and commensal S. mitis by a variety of physiological and molecular biological tests. Forty-one atypical S. pneumoniae isolates have been collected at the German National Reference Center for Streptococci (GNRCS). Multilocus sequence typing (MLST) confirmed 35 isolates as the species S. pseudopneumoniae A comparison with the pbp2x sequences from 120 commensal streptococci isolated from different continents revealed that pbp2x is distinct among penicillin-susceptible S. pseudopneumoniae isolates. Four penicillin-binding protein x (PBPx) alleles of penicillin-sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pseudopneumoniae, S. pneumoniae, and S. mitis, and S. infantis and S. oralis sequences were found in S. pneumoniae from Japan. PBP2x genes of the family of mosaic genes related to pbp2x in the S. pneumoniae clone Spain23F-1 were observed in S. oralis and S. infantis as well, confirming its global distribution. Thirty-eight sites were altered within the PBP2x transpeptidase domains of penicillin-resistant strains, excluding another 37 sites present in the reference genes of sensitive strains. Specific mutational patterns were detected depending on the parental sequence blocks, in agreement with distinct mutational pathways during the development of beta-lactam resistance. The majority of the mutations clustered around the active site, whereas others are likely to affect stability or interactions with the C-terminal domain or partner proteins.
Subject(s)
Penicillin Resistance/genetics , Penicillin-Binding Proteins/genetics , Streptococcus pneumoniae/genetics , Viridans Streptococci/classification , Viridans Streptococci/genetics , Alleles , Catalytic Domain/genetics , DNA, Bacterial/genetics , Genetic Variation/genetics , Genome, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Multilocus Sequence Typing , Mutation/genetics , Streptococcal Infections/drug therapy , Streptococcal Infections/pathology , Streptococcus pneumoniae/isolation & purification , Viridans Streptococci/isolation & purificationABSTRACT
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that mainly causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. However, the underlying mechanisms involved in this process are poorly elucidated. In this study, we used a transposon mutant library to identify bacterial genes involved in biofilm formation. The growth and biofilm formation of 4,172 transposon mutants were determined, and the involvement of the identified genes in biofilm formation was validated in in vitro experiments. Here, we present experimental data showing that increased bacterial lysis, through interference with peptidoglycan synthesis, results in elevated levels of extracellular DNA, which increased biofilm formation. Interestingly, similar results were obtained with subinhibitory concentrations of ß-lactam antibiotics, known to interfere with peptidoglycan synthesis, but such an effect does not appear with other classes of antibiotics. These results indicate that treatment with ß-lactam antibiotics, especially for ß-lactam-resistant NTHi isolates, might increase resistance to antibiotics by increasing biofilm formation. IMPORTANCE Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release of bacterial genomic DNA. Subinhibitory concentrations of ß-lactam antibiotics, which are often prescribed to treat H. influenzae infections, increase biofilm formation through a similar mechanism. Therefore, when ß-lactam antibiotics do not reach their MIC in vivo, they might not only drive selection for ß-lactam-resistant clones but also increase biofilm formation and resistance to other antimicrobial compounds.
ABSTRACT
The diverse pneumococcal diseases are associated with different pneumococcal lineages, or clonal complexes. Nevertheless, intra-clonal genomic variability, which influences pathogenicity, has been reported for surface virulence factors. These factors constitute the communication interface between the pathogen and its host and their corresponding genes are subjected to strong selective pressures affecting functionality and immunogenicity. First, the presence and allelic dispersion of 97 outer protein families were screened in 19 complete pneumococcal genomes. Seventeen families were deemed variable and were then examined in 216 draft genomes. This procedure allowed the generation of binary vectors with 17 positions and the classification of strains into surfotypes. They represent the outer protein subsets with the highest inter-strain discriminative power. A total of 116 non-redundant surfotypes were identified. Those sharing a critical number of common protein features were hierarchically clustered into 18 surfogroups. Most clonal complexes with comparable epidemiological characteristics belonged to the same or similar surfogroups. However, the very large CC156 clonal complex was dispersed over several surfogroups. In order to establish a relationship between surfogroup and pathogenicity, the surfotypes of 95 clinical isolates with different serogroup/serotype combinations were analyzed. We found a significant correlation between surfogroup and type of pathogenic behavior (primary invasive, opportunistic invasive, and non-invasive). We conclude that the virulent behavior of S. pneumoniae is related to the activity of collections of, rather than individual, surface virulence factors. Since surfotypes evolve faster than MLSTs and directly reflect virulence potential, this novel typing protocol is appropriate for the identification of emerging clones.
ABSTRACT
During surveillance studies we detected, among over 1500 presumptive pneumococci, 11 isolates displaying conflicting or novel results when characterized by widely accepted phenotypic (optochin susceptibility and bile solubility) and genotypic (lytA-BsaAI-RFLP and MLST) identification methods. We aimed to determine the genetic basis for the unexpected results given by lytA-BsaAI-RFLP and investigate the accuracy of the WHO recommended lytA real-time PCR assay to classify these 11 isolates. Three novel lytA-BsaAI-RFLP signatures were found (one in pneumococcus and two in S. mitis). In addition, one pneumococcus displayed the atypical lytA-BsaAI-RFLP signature characteristic of non-pneumococci and two S. pseudopneumoniae displayed the typical lytA-BsaAI-RFLP pattern characteristic of pneumococci. lytA real-time PCR misidentified these three isolates. In conclusion, identification of pneumococci by lytA real-time PCR, and other lytA-based methodologies, may lead to false results. This is of particular relevance in the increasingly frequent colonization studies relying solely on culture-independent methods.
Subject(s)
Bacterial Typing Techniques/methods , Diagnostic Errors , Molecular Diagnostic Techniques/methods , Pneumococcal Infections/diagnosis , Streptococcus pneumoniae/isolation & purification , Adult , Aged , Child, Preschool , Female , Humans , Male , Middle Aged , Pneumococcal Infections/microbiology , Real-Time Polymerase Chain Reaction/methodsABSTRACT
OBJECTIVES: Many outer multidomain proteins play fundamental virulent roles in an allele-dependent manner. We aimed to investigate the influence of the outer SP1992 protein, here renamed DiiA (Dimorphic invasion-involved A), in pneumococcal disease. METHODS: The presence and type of diiA allele was screened by PCR in 560 clinical isolates. Isogenic mutants carrying progressive diiA deletions were constructed and checked in mouse models of infection. DiiA binding to human molecules was carried out by surface plasmon resonance. RESULTS: The diiA gene is exclusive of Streptococcus pneumoniae and included in the core genome. DiiA variants contain one or two imperfect repeats (R1 and R2), an unstructured region and a cell-wall anchor domain. Clonal complexes carrying both repeats were associated with invasive disease, while those carrying R2 preferentially caused non-invasive syndromes in patients with underlying risk factors. Mutants lacking both repeats were less efficient in nasopharyngeal colonization and dissemination from lungs. Moreover, the ΔdiiA defective strain suffered a severe impairment in bacterial proliferation in blood. Purified DiiA bound to collagen and lactoferrin with high affinity. CONCLUSIONS: DiiA is a distinctive pneumococcal virulence factor contributing to colonization and long-term invasion in this pathogen.
Subject(s)
Bacterial Proteins/metabolism , Cell Wall/metabolism , Streptococcal Infections/microbiology , Streptococcal Infections/pathology , Streptococcus pneumoniae/pathogenicity , Virulence Factors/metabolism , Adult , Animals , Bacterial Proteins/genetics , Collagen/metabolism , Disease Models, Animal , Gene Deletion , Humans , Lactoferrin/metabolism , Male , Mice, Inbred C57BL , Polymerase Chain Reaction , Protein Binding , Surface Plasmon Resonance , Virulence Factors/geneticsABSTRACT
OBJECTIVE: To determine changes in mortality among adults with invasive pneumococcal disease (IPD) after introducing pneumococcal conjugate vaccines (PCVs) in children. METHODS: An active surveillance of adults with culture-proven IPD in Barcelona. Serotype-specific mortality and rates of disease and death were analysed in three periods: baseline (1994-2001), PCV7 (2002-2009) and PCV13 (2010-2013). RESULTS: Overall, IPD caused by PCV7 serotypes was associated with increased case fatality rate (24 percent). In patients 18-64 years (baseline vs. PCV7 vs. PCV13 periods), case fatality rate from IPD decreased (22 vs.14 vs. 12 percent), and it was associated with a decline in PCV7 serotypes (3.56 vs. 2.80 vs. 1.49 cases/10(5) person-years) and in PCV7 serotypes-specific death (0.74 vs. 0.53 vs. 0.09 deaths/10(5) person-years). In patients ≥65 years, case fatality rate did not change (24 vs. 22 vs. 24 percent); however, there was a decline in PCV7 serotypes-specific death (4.94 vs. 3.58 vs. 2.45 deaths/10(5) person-years), and an increase in non-PCV serotypes-specific death (2.55 vs. 3.70 vs. 4.09 deaths/10(5) person-years). CONCLUSIONS: The use of PCVs for children was associated with a reduction of mortality from IPD in adults 18-64 years, through the indirect effect of herd protection. In older adults, age-related factors could play a role in IPD mortality.
Subject(s)
Pneumococcal Infections/mortality , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/therapeutic use , Streptococcus pneumoniae/immunology , Adolescent , Adult , Aged , Child , Humans , Middle Aged , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Retrospective Studies , Spain/epidemiology , Young AdultABSTRACT
OBJECTIVES: The objectives of this study were to establish the frequency of Haemophilus haemolyticus in clinical samples, to determine the antimicrobial resistance rate and to identify the mechanisms of resistance to ß-lactams and quinolones. METHODS: An updated database was used to differentiate between MALDI-TOF MS results for Haemophilus influenzae and H. haemolyticus. Antimicrobial susceptibility was studied by microdilution, following EUCAST criteria. The ß-lactamase types were identified by PCR analysis of isolates that tested positive for nitrocefin hydrolysis. Mutations in the ftsI gene were identified in isolates with ampicillin MICs ≥0.25 mg/L. Mutations in the quinolone resistance-determining region (QRDR) were identified in isolates with ciprofloxacin MICs ≥0.5 mg/L. RESULTS: Overall, we identified 69 H. haemolyticus isolates from 1706 clinical isolates of Haemophilus spp. from respiratory, genital, invasive, and other infection sources. The frequency of H. haemolyticus was low in respiratory samples compared with that of H. influenzae, but in genital-related samples, the frequency was similar to that of H. influenzae. We found low antimicrobial resistance rates among H. haemolyticus isolates, with 8.7% for ampicillin, 8.7% for co-trimoxazole, 7.2% for tetracycline and 4.3% for ciprofloxacin. Mutations in the ftsI gene classified the isolates into four groups, including the newly described Group Hhae IV, which presents mutations in the ftsI gene not identified in H. influenzae and H. haemolyticus type strains. Three ciprofloxacin-resistant H. haemolyticus isolates with mutations affecting GyrA and ParC were identified. CONCLUSIONS: The frequency of H. haemolyticus was low, especially in respiratory samples, where H. influenzae is the main pathogen of this genus. Although antimicrobial resistance rates were low, three ciprofloxacin-resistant H. haemolyticus clinical isolates have been identified for the first time.