Oropharyngeal microbial ecosystem perturbations influence the risk for acute respiratory infections in common variable immunodeficiency.

Background: The respiratory tract microbiome is essential for human health and well-being and is determined by genetic, lifestyle, and environmental factors. Patients with Common Variable Immunodeficiency (CVID) suffer from respiratory and intestinal tract infections, leading to chronic diseases and increased mortality rates. While CVID patients' gut microbiota have been analyzed, data on the respiratory microbiome ecosystem are limited. Objective: This study aims to analyze the bacterial composition of the oropharynx of adults with CVID and its link with clinical and immunological features and risk for respiratory acute infections. Methods: Oropharyngeal samples from 72 CVID adults and 26 controls were collected in a 12-month prospective study. The samples were analyzed by metagenomic bacterial 16S ribosomal RNA sequencing and processed using the Quantitative Insights Into Microbial Ecology (QIME) pipeline. Differentially abundant species were identified and used to build a dysbiosis index. A machine learning model trained on microbial abundance data was used to test the power of microbiome alterations to distinguish between healthy individuals and CVID patients. Results: Compared to controls, the oropharyngeal microbiome of CVID patients showed lower alpha- and beta-diversity, with a relatively increased abundance of the order Lactobacillales, including the family Streptococcaceae. Intra-CVID analysis identified age >45 years, COPD, lack of IgA, and low residual IgM as associated with a reduced alpha diversity. Expansion of Haemophilus and Streptococcus genera was observed in patients with undetectable IgA and COPD, independent from recent antibiotic use. Patients receiving azithromycin as antibiotic prophylaxis had a higher dysbiosis score. Expansion of Haemophilus and Anoxybacillus was associated with acute respiratory infections within six months. Conclusions: CVID patients showed a perturbed oropharynx microbiota enriched with potentially pathogenic bacteria and decreased protective species. Low residual levels of IgA/IgM, chronic lung damage, anti antibiotic prophylaxis contributed to respiratory dysbiosis.

Nasopharyngeal carriage of Haemophilus influenzae among adults with co-morbidities.

After the widespread use of Haemophilus influenzae type b (Hib) vaccine, H. influenzae invasive disease is now commonly due to non-encapsulated (NTHi), affecting mostly the youngest and the elderly. The objective of this study was to investigate H. influenzae nasopharyngeal carriage rate in adults with co-morbidities and possible associated risk factors. METHODS: Patients aged >50 years with co-morbidities attending medical centres were examined. A nasopharyngeal swab was analysed for H. influenzae presence by cultural and molecular methods (RT-PCR). Univariable and multivariable analysis of risk factors for H. influenzae carriage were performed. Serotype of isolates was determined by PCR capsular genotyping. Minimum inhibitory concentration (MIC) was determined by MIC gradient test and ß-lactamase production was detected by the nitrocephin test. Genotyping was performed by Multilocus sequence typing (MLST). Phylogenetic relationships among carriage and invasive NTHi strains were assessed. RESULTS: Among 248 enrolled patients (median age: 73 years), the carriage rate was 5.6% and 10.5% by cultural method or RT-PCR, respectively. Colonization with H. influenzae was significantly associated with the presence of acute respiratory symptoms (adjusted OR = 12.16, 95% CI: 3.05-48.58, p < 0.001). All colonizing isolates were NTHi. Three isolates (3/14, 21.4%) were resistant to ampicillin and beta-lactamase positive. MLST revealed a high degree of genetic diversity, with 11 different STs from 14 isolates. Eight out of the 11 (72.7%) STs were shared among carriage and invasive isolates. CONCLUSIONS: Adults ≥50 years old with co-morbidities are occasionally colonized by H. influenzae, even if the presence of co-morbidities is not a risk factor for colonization. The presence of acute respiratory symptoms is the only factor associated with H. influenzae colonization. Colonizing H. influenzae are all NTHi. Colonizing H. influenzae often belong to the same STs of invasive disease isolates.

Changes in Invasive Pneumococcal Disease Caused by Streptococcus pneumoniae Serotype 1 Following Introduction of PCV10 and PCV13: Findings from the PSERENADE Project.

Streptococcus pneumoniae serotype 1 (ST1) was an important cause of invasive pneumococcal disease (IPD) globally before the introduction of pneumococcal conjugate vaccines (PCVs) containing ST1 antigen. The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project gathered ST1 IPD surveillance data from sites globally and aimed to estimate PCV10/13 impact on ST1 IPD incidence. We estimated ST1 IPD incidence rate ratios (IRRs) comparing the pre-PCV10/13 period to each post-PCV10/13 year by site using a Bayesian multi-level, mixed-effects Poisson regression and all-site IRRs using a linear mixed-effects regression (N = 45 sites). Following PCV10/13 introduction, the incidence rate (IR) of ST1 IPD declined among all ages. After six years of PCV10/13 use, the all-site IRR was 0.05 (95% credibility interval 0.04-0.06) for all ages, 0.05 (0.04-0.05) for <5 years of age, 0.08 (0.06-0.09) for 5-17 years, 0.06 (0.05-0.08) for 18-49 years, 0.06 (0.05-0.07) for 50-64 years, and 0.05 (0.04-0.06) for ≥65 years. PCV10/13 use in infant immunization programs was followed by a 95% reduction in ST1 IPD in all ages after approximately 6 years. Limited data availability from the highest ST1 disease burden countries using a 3+0 schedule constrains generalizability and data from these settings are needed.

Global Landscape Review of Serotype-Specific Invasive Pneumococcal Disease Surveillance among Countries Using PCV10/13: The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) Project.

Serotype-specific surveillance for invasive pneumococcal disease (IPD) is essential for assessing the impact of 10- and 13-valent pneumococcal conjugate vaccines (PCV10/13). The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project aimed to evaluate the global evidence to estimate the impact of PCV10/13 by age, product, schedule, and syndrome. Here we systematically characterize and summarize the global landscape of routine serotype-specific IPD surveillance in PCV10/13-using countries and describe the subset that are included in PSERENADE. Of 138 countries using PCV10/13 as of 2018, we identified 109 with IPD surveillance systems, 76 of which met PSERENADE data collection eligibility criteria. PSERENADE received data from most (n = 63, 82.9%), yielding 240,639 post-PCV10/13 introduction IPD cases. Pediatric and adult surveillance was represented from all geographic regions but was limited from lower income and high-burden countries. In PSERENADE, 18 sites evaluated PCV10, 42 PCV13, and 17 both; 17 sites used a 3 + 0 schedule, 38 used 2 + 1, 13 used 3 + 1, and 9 used mixed schedules. With such a sizeable and generally representative dataset, PSERENADE will be able to conduct robust analyses to estimate PCV impact and inform policy at national and global levels regarding adult immunization, schedule, and product choice, including for higher valency PCVs on the horizon.

Serotype Distribution of Remaining Pneumococcal Meningitis in the Mature PCV10/13 Period: Findings from the PSERENADE Project.

Pneumococcal conjugate vaccine (PCV) introduction has reduced pneumococcal meningitis incidence. The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project described the serotype distribution of remaining pneumococcal meningitis in countries using PCV10/13 for least 5-7 years with primary series uptake above 70%. The distribution was estimated using a multinomial Dirichlet regression model, stratified by PCV product and age. In PCV10-using sites (N = 8; cases = 1141), PCV10 types caused 5% of cases <5 years of age and 15% among ≥5 years; the top serotypes were 19A, 6C, and 3, together causing 42% of cases <5 years and 37% ≥5 years. In PCV13-using sites (N = 32; cases = 4503), PCV13 types caused 14% in <5 and 26% in ≥5 years; 4% and 13%, respectively, were serotype 3. Among the top serotypes are five (15BC, 8, 12F, 10A, and 22F) included in higher-valency PCVs under evaluation. Other top serotypes (24F, 23B, and 23A) are not in any known investigational product. In countries with mature vaccination programs, the proportion of pneumococcal meningitis caused by vaccine-in-use serotypes is lower (≤26% across all ages) than pre-PCV (≥70% in children). Higher-valency PCVs under evaluation target over half of remaining pneumococcal meningitis cases, but questions remain regarding generalizability to the African meningitis belt where additional data are needed.

The changing epidemiology of carbapenemase-producing Klebsiella pneumoniae in Italy: toward polyclonal evolution with emergence of high-risk lineages.

BACKGROUND: Previous studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates. METHODS: From March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome. RESULTS: Twenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395. CONCLUSIONS: This WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.

Differential Responses of Human Dendritic Cells to Live or Inactivated Staphylococcus aureus: Impact on Cytokine Production and T Helper Expansion.

Understanding Staphylococcus aureus (S. aureus)-host immune system interaction is crucial to meet the tremendous medical need associated with this life-threatening bacterial infection. Given the crucial role of dendritic cells (DC) in dictating immune responses upon microbial challenge, we investigated how the bacterial viability and the conservation of structural integrity influence the response of human DC to S. aureus. To this end, human primary DC were stimulated with the methicillin-resistant S. aureus USA300 live strain, USA300 inactivated by heat (HI), ultraviolet irradiation (UVI), or paraformaldehyde treatment (PFAI) and subsequently analyzed for cell phenotype and immune-modulatory properties. Although no differences in terms of DC viability and maturation were observed when DC were stimulated with live or inactivated bacteria, the production of IL-12, IL-23, and other cytokines differed significantly. The Th1 and Th17 expansion was also more pronounced in response to live vs. inactivated S. aureus. Interestingly, cytokine production in DC treated with live and inactivated USA300 required phagocytosis, whereas blocking endosomal Toll-like receptor signaling mainly reduced the cytokine release by live and HI USA300. A further analysis of IFN-ß signaling revealed the induction of a cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING)-independent and IRF3-dependent signaling pathway(s) in UVI-stimulated DC. This study underscores the capacity of human DC to discriminate between live and inactivated S. aureus and, further, indicates that DC may represent a valuable experimental setting to test different inactivation methods with regard to the retention of S. aureus immunoregulatory properties. These and further insights may be useful for the development of novel therapeutic and prophylactic anti-S. aureus vaccine strategies.

Pneumococcal carriage among adults aged 50 years and older with co-morbidities attending medical practices in Rome, Italy.

BACKGROUND: Data on Streptococcus pneumoniae carriage in adults with co-morbidities are limited. In this study we estimated the pneumococcal carriage among adults with co-morbidities and evaluated socio-demographic and clinical risk factors. The potential coverage of the current pneumococcal vaccines recommended for adults (PCV13 and PPV23) was also investigated. METHODS: A cross-sectional study on S. pneumoniae carriage among unvaccinated adults ≥50 years with co-morbidities, presenting with or without acute respiratory symptoms at general practitioners in Rome, Italy, between October 2015 and July 2016 was conducted. Pneumococcal carriage was investigated by both cultural and molecular methods. Socio-demographic variables and co-morbidities were evaluated by logistic models as possible risk factors for pneumococcal carriage. RESULTS: Out of 248 patients (median age: 73 yrs; IQR: 65-79), 12 (4.8%) and 83 (33.5%) individuals were found colonized using cultural or molecular methods, respectively. Potential risk factors for pneumococcal colonization as ascertained by molecular methods were: low level of education (adjusted OR = 3.71, 95% CI: 1.62-9.40), winter months (December-March vs other months, adjusted OR = 2.56, 95% CI: 1.29-5.14), and presence of chronic lung diseases (adjusted OR = 2.18, 95% CI: 1.15-4.16). The combination of serotype-specific multiplex RT-PCR and conventional PCR allowed to identify 22 serotypes/group of serotypes, of which the most common were: 24F/24A/24B, 12F/12A/12B/44/46, 6A/6B, 14, 15B/15C, and 22F/22A. Prevalence of pneumococcal carriage due to PCV13 serotypes and non-PCV13 serotypes was 23.6% and 67.3%, respectively. Prevalence of colonization due to PPV23 serotypes was estimated to be 54.6%. CONCLUSIONS: A high prevalence of S. pneumoniae carriage was observed among adults with co-morbidities, especially among individuals affected by chronic lung diseases. These results support vaccine strategies based on the sequential administration of PCV13 and PPV23 to control potentially invasive pneumococcal strains in adults, especially in subjects with co-morbidities.

Staphylococcus aureus Esx Factors Control Human Dendritic Cell Functions Conditioning Th1/Th17 Response.

The opportunistic pathogen Staphylococcus aureus (S. aureus) is a major cause of nosocomial- and community-acquired infections. In addition, many antibiotic-resistant strains are emerging worldwide, thus, there is an urgent unmet need to pinpoint novel therapeutic and prophylactic strategies. In the present study, we characterized the impact of infection with the pandemic methicillin-resistant USA300 S. aureus strain on human primary dendritic cells (DC), key initiators and regulators of immune responses. In particular, among staphylococcal virulence factors, the function of EsxA and EsxB, two small acidic dimeric proteins secreted by the type VII-like secretion system Ess (ESAT-6-like secretion system), was investigated in human DC setting. A comparative analysis of bacterial entry, replication rate as well as DC maturation, apoptosis, signaling pathway activation and cytokine production was performed by using wild type (wt) USA300 and three isogenic mutants carrying the deletion of esxA (ΔesxA), esxB (ΔesxB), or both genes (ΔesxAB). The S. aureus mutant lacking only the EsxA protein (ΔesxA) stimulated a stronger pro-apoptotic phenotype in infected DC as compared to wt USA300, ΔesxAB, and ΔesxB strains. When the mutant carrying the esxB deletion (ΔesxB) was analyzed, a higher production of both regulatory and pro-inflammatory mediators was found in the infected DC with respect to those challenged with the wt counterpart and the other esx mutants. In accordance with these data, supernatant derived from ΔesxB-infected DC promoted a stronger release of both IFN-γ and IL-17 from CD4+ T cells as compared with those conditioned with supernatants derived from wild type USA300-, ΔesxAB-, and ΔesxA-infected cultures. Although, the interaction of S. aureus with human DC is not yet fully understood, our data suggest that both cytokine production and apoptotic process are modulated by Esx factors, thus indicating a possible role of these proteins in the modulation of DC-mediated immunity to S. aureus.

Impact of pneumococcal conjugate vaccine (PCV7 and PCV13) on pneumococcal invasive diseases in Italian children and insight into evolution of pneumococcal population structure.

BACKGROUND: The use of PCV7 for children immunization was gradually implemented in the Italian regions starting from 2006 and was replaced by PCV13 in 2010-2011. In this study we aimed to assess the PCV impact on invasive pneumococcal diseases (IPD) incidence, serotype distribution and antibiotic resistance in Italian children under 5years old. METHODS: All IPD cases in children from 5 Italian regions (Emilia-Romagna, Lombardia, A. P. Bolzano, A. P. Trento, and Piemonte) reported through the nationwide surveillance system during 2008-2014 were included in this study. Pneumococcal isolates were subjected to serotyping, antibiotic susceptibility testing, and clonal analysis according to standard methods. RESULTS: During the study period overall IPD incidence decreased from 7.8 cases/100,000 inhabitants in 2008 to 3.0 cases/100,000 in 2014 (61% decrease, P<0.001). In particular, from 2008 to 2014, PCV7-type IPD decreased from 2.92 to 0.13 cases/100,000 inhabitants (95% decrease, P<0.001) while PCV13-non-PCV7 type IPD decreased from 3.2 to 0.89 cases/100,000 inhabitants (72% decrease, P=0.008). Conversely, non-vaccine serotype (NVS) IPD increased overtime, becoming more common than PCV13 serotype IPD in 2013-2014. Emergent NVS 24F and 12F were the most prevalent in 2014. Antibiotic resistance testing revealed an overall increasing trend in penicillin resistance, from 14% in 2008 to 23% in 2014. Erythromycin resistance showed a downward trend, from 38% in 2008 to 27% in 2014. While in 2008 PCV13 serotypes were the major responsible for antibiotic resistance, during the following years antimicrobial resistance due to NVS increased, mainly as a result of expansion of pre-existing clones. CONCLUSIONS: Both PCVs led to a substantial decrease in vaccine-related IPD incidence in the children population. However NVS-related IPD increased, becoming the most prevalent in the last two-years period. Continuous surveillance is an essential tool to monitor evolution of pneumococcal population causing IPD in children.

ICESpy009, a Conjugative Genetic Element Carrying mef(E) in Streptococcus pyogenes.

Efflux-mediated macrolide resistance due to mef(E) and mel, carried by the mega element, is common in Streptococcus pneumoniae, for which it was originally characterized, but it is rare in Streptococcus pyogenes In S. pyogenes, mega was previously found to be enclosed in Tn2009, a composite genetic element of the Tn916 family containing tet(M) and conferring erythromycin and tetracycline resistance. In this study, S. pyogenes isolates containing mef(E), apparently not associated with other resistance determinants, were examined to characterize the genetic context of mega. By whole-genome sequencing of one isolate, MB56Spyo009, we identified a novel composite integrative and conjugative element (ICE) carrying mega, designated ICESpy009, belonging to the ICESa2603 family. ICESpy009 was 55 kb long, contained 61 putative open reading frames (ORFs), and was found to be integrated into hylA, a novel integration site for the ICESa2603 family. The modular organization of the ICE was similar to that of members of the ICESa2603 family carried by different streptococcal species. In addition, a novel cluster of accessory resistance genes was found inside a region that encloses mega. PCR mapping targeting ICESpy009 revealed the presence of a similar ICE in five other isolates under study. While in three isolates the integration site was the same as that of ICESpy009, in two isolates the ICE was integrated into rplL, the typical integration site of the ICESa2603 family. ICESpy009 was able to transfer macrolide resistance by conjugation to both S. pyogenes and S. pneumoniae, showing the first evidence of the transferability of mega from S. pyogenes.

Invasive pneumococcal disease in children and adults in seven Italian regions after the introduction of the conjugate vaccine, 2008-2014.

OBJECTIVE: To describe the trend of invasive pneumococcal disease in the years 2008-2014; to verify the impact of the conjugate vaccine and monitor the occurrence of serotype replacement. DESIGN: Prospective observational study based on data from the national surveillance for invasive bacterial diseases coordinated by the Istituto superiore di sanità. SETTING AND PARTICIPANTS: Seven Italian regions (A.P. Bolzano, A.P. Trento, Emilia-Romagna, Friuli-Venezia Giulia, Lombardia, Piemonte, Veneto), accounting for 43% of the national population. MAIN OUTCOME MEASURES: Number of cases and incidence of invasive pneumococcal diseases: global, stratified by age groups and by serotypes included or not in the PCV13. RESULTS: In 2008-2014, in the 0-4 age group IPD incidence for all serotypes decreased from 7.1 to 2.9/100,000; incidence for vaccine serotypes (VT) decreased from 5.5 to 1.1/100,000, while incidence for non-vaccine serotypes (NVT) increased from 1.6 to 2.0/100,000 (2.5 in 2013). In the >64 age group, IPD incidence increased from 5.3 to 7.5/100,000; VT incidence decreased from 3.9 to 3.2 (4.9 in 2010 and 4.3 in 2013), whereas NVT incidence increased from 1.4 to 4.4/100,000. CONCLUSION: Use of the conjugate vaccine has reduced the number of cases of IPD by VT in children; the increase in IPD by NVT, above all in older age groups, suggests a serotype replacement.

Carriage of Haemophilus influenzae is associated with pneumococcal vaccination in Italian children.

BACKGROUND: The pneumococcal population changes observed after the implementation of children immunization with pneumococcal conjugative vaccines (PCV) might have affected the composition of the microbial flora inhabiting the same ecological niche of Streptococcus pneumoniae. The aim of this study was to investigate the effect of PCV immunization, (PCV7 or PCV13), on S. pneumoniae and Haemophilus influenzae colonization in young children in Italy. METHODS: Nasopharyngeal swabs were obtained from 301 children under 6 years of age (vaccinated or unvaccinated with PCV) during the period January-April 2012. Presence of S. pneumoniae and H. influenzae was investigated using conventional cultural methods. S. pneumoniae isolates were serotyped by the Quellung reaction; capsular type of H. influenzae isolates was determined by PCR. The pattern of associations between the two species and potential risk factors were investigated by a Structural Equation Modelling (SEM) analysis. RESULTS: The prevalence of carriage was 31.56% and 43.18% for S. pneumoniae and H. influenzae, respectively. The majority of S. pneumoniae isolates belonged to non vaccine serotypes (non PCV13-types 81.1%) while H. influenzae isolates were all non-typeable. SEM analysis revealed a synergistic association between S. pneumoniae and H. influenzae colonization (rho: 0.27; 95%CI: 0.09-0.46; p=0.004). In addition, children vaccinated with PCV, either with PCV7 (coef 0.43; 95%CI: 0.07-0.79; p=0.021) or with PCV13 (coef: 0.45; 95%CI: 0.08-0.82; p=0.018), were more likely to be colonized by H. influenzae. CONCLUSIONS: Pneumococcal vaccination increased H. influenzae nasopharyngeal carriage in children. This result highlights that an indirect effect of PCV vaccination can be perturbation of the nasopharyngeal flora. In the era of higher-valent pneumococcal vaccines, surveillance of carriage is crucial to monitor alterations in the bacterial ecosystem, thus preventing possible clinical problems.

Characterization of Streptococcus pneumoniae clones from paediatric patients with cystic fibrosis.

The role of Streptococcus pneumoniae in cystic fibrosis (CF) is poorly understood. The pneumococcal population has changed over time after the introduction of the heptavalent conjugate vaccine (PCV7) and, more recently, the 13-valent conjugate vaccine (PCV13). Although serotypes and clones causing invasive pneumococcal disease or colonizing healthy children have been extensively analysed, little is known so far on the serotypes and clones of pneumococci in CF patients. The aim of this work was to investigate serotypes, antibiotic susceptibilities, genotypes and biofilm production of CF pneumococcal isolates. Overall, 44 S. pneumoniae strains collected from 32 paediatric CF patients from January 2010 to May 2012 in a large Italian CF Centre were tested for antimicrobial susceptibility testing by Etest, serotyped by the Quellung reaction and genotyped by a combination of different molecular typing methods, including pbp gene restriction profiling, pspA restriction profiling and sequencing, PFGE and multilocus sequence typing. Biofilm production by pneumococcal strains was also assessed. Penicillin non-susceptibility was 16 %. High resistance rates (>56 %) were observed for erythromycin, clindamycin and tetracycline. The most frequent serotype recovered was serotype 3 (31.8 %). The coverage of PCV7 and PCV13 was 6.8 and 47.7 %, respectively. More than 80 % of CF strains belonged to Pneumococcal Molecular Epidemiology Network (PMEN) reference clones, the most common being Netherlands(3)-ST180 (28.2 %), and Greece(21)-30/ST193 (15.4 %). All strains produced biofilm in vitro, although with large variability in biofilm formation efficiency. No correlation was found between biofilm levels and serotype, clone or antibiotic resistance. The high isolation rate of antibiotic-resistant serotype 3 pneumococci from CF patients suggests that PCV13 could increase protection from pneumococcal colonization and infection.

Identification of Streptococcus pneumoniae serotype 11E, serovariant 11Av and mixed populations by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and flow cytometric serotyping assay (FCSA).

BACKGROUND: Recent studies have identified Streptococcus pneumoniae serotype 11E and serovariant 11Av among isolates previously typed as 11A by classical serotyping methods. Serotype 11E and serovariant 11Av differ from serotype 11A by having totally or partially inactive wcjE, a gene in cps locus coding for an O-acetyl transferase. Serotype 11E is rare among carriage isolates but common among invasive isolates suggesting that it survives better during invasion. Aim of this work was to investigate the epidemiology of serotype 11A in a pneumococcal collection using a new serotyping approach based on High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy to distinguish serotypes 11A and 11E. METHODS: A collection of 48 (34 invasive and 14 carriage) S. pneumoniae isolates from Italy, previously identified as serotype 11A by the Quellung reaction, were investigated by wcjE sequencing, HR-MAS NMR spectroscopy and the reference flow cytometric serotyping assay (FCSA) based on monoclonal antibodies. RESULTS: HR-MAS NMR spectra from serotypes 11A and 11E showed different NMR peaks indicating that HR-MAS NMR could be used to distinguish these serotypes, although HR-MAS NMR could not distinguish serotype 11Av from serotype 11E unambiguously. Thirty-eight isolates were confirmed to be serotype 11A, 8 isolates with a mutated wcjE were serotype 11E, 1 isolate belonged to serovariant 11Av, and 1 isolate was a mixed population 11A/11Av. All 11E isolates were identified among invasive isolates. CONCLUSIONS: We proved that HR-MAS NMR can be of potential use for pneumococcal serotyping. The detection of serotype 11E among invasive isolates in our collection, supports previous epidemiological studies suggesting that mutations in wcjE can represent a mechanism promoting pneumococcal survival during invasion. The discovery of a spectrum of immunochemical diversity within established serotypes should stimulate efforts to develop new serotyping approaches.

Pneumococcal carriage in young children one year after introduction of the 13-valent conjugate vaccine in Italy.

BACKGROUND: In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced. METHODS: Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST). RESULTS: Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively. CONCLUSIONS: Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.

Increase of pneumococcal serotype 19A in Italy is due to expansion of the piliated clone ST416/CC199.

The emergence of Streptococcus pneumoniae serotype 19A, following use of the heptavalent pneumococcal conjugate vaccine (PCV7), has been favoured by multiple antibiotic resistance of this serotype and by other unknown factors. The aim of this study was to examine 19A isolates from invasive pneumococcal diseases (IPD) obtained before and after PCV7 implementation to ascertain which characteristics, including the presence of pili, might have favoured the emergence of this serotype in Italy. All S. pneumoniae isolates from IPD collected at the Italian National Institute of Health in the years 2001-2003 and 2006-2009 were serotyped. The 19A isolates were submitted to antimicrobial susceptibility testing by Etest and were genotyped by a combination of pulsed field gel electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). The presence of the pilus islets PI-1 and PI-2 was detected by PCR assays targeting a marker gene in each islet. The proportion of 19A isolates from IPD significantly increased from 4 % in 2001-2003 to 12 % in 2006-2009. This was largely due to the expansion of a clone characterized by sequence type (ST) 416, clonal complex (CC) 199, already present in Italy before PCV7 implementation. This clone included isolates susceptible to penicillin and containing PI-1 genes. Other CCs contributed to the emergence of serotype 19A: CC63 and CC193, already present in 2001-2003, and new-emerging CCs or clones such as CC230, CC320 and ST5204, that include drug-resistant and/or pilus-positive isolates. The expansion of serotype 19A in Italy might have been favoured not only by antibiotic resistance, but also by other bacterial factors such as the presence of pili.

Point mutations in wchA are responsible for the non-typability of two invasive Streptococcus pneumoniae isolates.

Non-typable Streptococcus pneumoniae (NTPn) strains are typically isolated from nasopharyngeal carriage or from conjunctivitis. Since the isolation of NTPn from invasive disease is rare, we characterized the genetic basis of the non-typability of two isolates obtained in Italy from two cases of bacteraemic pneumonia. MLST revealed that both NTPn belonged to ST191, which, according to the MLST database, is associated with serotype 7F. Sequencing of the capsular locus (cps) confirmed the presence of a 7F cps in both strains and revealed the existence of distinct single point mutations in the wchA gene (a glycosyltransferase), both leading to the translation of proteins truncated at the C terminus. To verify that these mutations were responsible for the non-typability of the isolates, a functional 7F WchA was overexpressed in both NTPn. The two NTPn along with their WchA-overexpressing derivatives were analysed by transmission electron microscopy and by high-resolution magic angle spinning NMR spectroscopy. Both NTPn were devoid of a polysaccharide capsule, and WchA overexpression was sufficient to restore the assembly of a serotype 7F capsule on the surface of the two NTPn. In conclusion, we identified two new naturally occurring point mutations that lead to non-typability in the pneumococcus, and demonstrated that WchA is essential for the biosynthesis of the serotype 7F capsule.

Genetic resistance elements carrying mef subclasses other than mef(A) in Streptococcus pyogenes.

In Streptococcus pyogenes, efflux-mediated erythromycin resistance is associated with the mef gene, represented mostly by mef(A), although a small portion of strains carry different mef subclasses. We characterized the composite genetic elements, including mef subclasses other than mef(A), associated with other resistance genes in S. pyogenes isolates. Determination of the genetic elements was performed by PCR mapping. The strains carrying mosaic mef(A/E), in which the 5' region was identical to mef(A) and the 3' region was identical to mef(E), also carried tet(O). The two genes were found enclosed in an element similar to S. pyogenes prophage Φm46.1, designated the Φm46.1-like element. In S. pyogenes strains carrying mef(E) and tet(M), mef(E) was included in a typical mega element, and in some strains, it was physically associated with tet(M) in the composite element Tn2009. S. pyogenes strains carrying mef(I) also carried catQ; the two genes were linked in a fragment representing a portion of the 5216IQ complex of Streptococcus pneumoniae, designated the defective IQ element. In the only isolate carrying a novel mef gene, this was associated with catQ and tet(M) in a genetic element similar to the 5216IQ complex of S. pneumoniae (5216IQ-like complex), suggesting that the novel mef is in fact a variant of mef(I). This study demonstrates that the composite elements containing mef are shared between S. pyogenes and S. pneumoniae and suggests that it is important to distinguish the mef subclass on the basis of the genetic element containing it.