Diversity of pneumococcal surface protein A (PspA) among prevalent clones in Spain

<p>Abstract</p> <p>Background</p> <p>PspA is recognized as a major pneumococcal virulence factor and a possible vaccine candidate. The aim of this study was to analyze the PspA family and clade distribution among 112 Spanish pneumococci representatives of dominant clones among patients with invasive disease (n = 66) and nasopharyngeal healthy carriage in children (n = 46).</p> <p>Results</p> <p>PspA family 2 was predominant among invasive (63.6%) and carriage (54.3%) pneumococcal isolates. No PspA family 3 isolates were detected and only one strain was PspA negative. Although four clonal complexes contained strains of different clades, a clear association between clade and multi locus sequence typing results was found. Clades 1, 3 and 4 were associated with a wide variety of sequence types (ST) related to multiresistant and antibiotic-susceptible worldwide-disseminated clones. Clade 1 was associated with Spain^6B-ST90, Spain¹⁴-ST18, Colombia⁵-ST289, Sweden¹-ST306, Denmark¹⁴-ST230 and Sweden¹-ST304 clones. Clade 3 was associated with Spain^23F-ST81, Spain^9V-ST156, Tennessee¹⁴-ST67, Netherlands³-ST180 and Netherlands^7F-ST191 clones. Clade 4 was related to Sweden^15A-ST63, Netherlands^18C-ST113 and Greece²¹-ST193 clones. In contrast, PspA clade was not related to serotype, age or clinical origin of the isolates.</p> <p>Conclusion</p> <p>PspA clades were associated with genotypes. PspA family 2 and family 1 were dominant among major Spanish pneumococcal clones isolated from patients with invasive disease and nasopharyngeal carriage in children.</p

Oropharyngeal colonization by nontypeable Haemophilus influenzae among healthy children attending day care centers

Haemophilus influenzae colonizes the upper respiratory tract and can spread causing otitis and sinusitis. This work aimed to study the oropharyngeal carriage rate in healthy <5-year-old children attending day care centers in Oviedo, Spain in two consecutive years (January to March 2004-2005). The carriage rate was 42% (400/960) and highly variable among centers (range, 12% to 83%). Isolates were mainly identified as nontypeable H. influenzae (NTHi, 99%). Epidemiologically, 127 different genotypes were identified by PFGE with a minimum of two genotypes per center. One hundred fourteen children (12%) were included in both studies and none of them harbored the same strain over a period of time. The isolates only showed resistance to cotrimoxazol and ampicillin, presenting a shift in the level of ampicillin reduced susceptibility, showing a predominance of PBP3 mutations in 2004 and a predominance of β-lactamase production in 2005. This study proved the great genetic variability of NTHi isolates that present similar genotypic patterns in both years with no long-term carriage of the same strain

Emerging, non-PCV13 serotypes 11A and 35B of Streptococcus pneumoniae show high potential for biofilm formation in vitro

Background: Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS). Methodology/Principal Findings: We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed >45% of the biofilm produced by the non-encapsulated M11 strain. Conclusions/Significance This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance

Clonal spread of Klebsiella pneumoniae producing OXA-1 betalactamase in a Spanish hospital

Multi-drug resistant Klebsiella pneumoniae isolates are associated with nosocomial infections, in which colonized patients act as a reservoir and source of cross-infection for other patients. In this study, the antimicrobial susceptibility of K. pneumoniae was tested by microdilution using the commercial method MicroScan (Siemens). The genetic relatedness of K. pneumoniae strains was determined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PCR experiments were carried out to obtain primer sets and positive PCR products were purified and sequenced. From May 2007 until December 2009, 98 clonally related K. pneumoniae isolates were detected from clinical samples of 38 patients admitted to the University Hospital of Bellvitge, Barcelona, Spain, including 27 admitted to the intensive care unit (ICU). The most important sources of the isolates were: lower respiratory tract (n = 12), urine (n = 12), and blood (n = 11). The strains were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam, tobramycin, amikacin, and ciprofloxacin, and had diminished susceptibility to cefepime. All the isolates shared a common PFGE pattern related to sequence type 14 after MLST analysis. In K. pneumoniae isolates and their transconjugants, the blaOXA-1 gene was located in the variable region of a class I integron that also contains the aac(6&prime;)Ib-cr gene. Sequencing of the quinolone resistance determinant regions of gyrA and parC revealed a S83F change in GyrA and no changes in ParC. [Int Microbiol 2013; 16(4):227-233]Keywords: Klebsiella pneumoniae &middot; sequence type ST14 &middot; gene blaOXA-1 &middot; integrons &middot; nosocomial outbreak

An Overview of Macrolide Resistance in Streptococci: Prevalence, Mobile Elements and Dynamics

Streptococcal infections are usually treated with beta-lactam antibiotics, but, in case of allergic patients or reduced antibiotic susceptibility, macrolides and fluoroquinolones are the main alternatives. This work focuses on studying macrolide resistance rates, genetic associated determinants and antibiotic consumption data in Spain, Europe and also on a global scale. Macrolide resistance (MR) determinants, such as ribosomal methylases (erm(B), erm(TR), erm(T)) or active antibiotic efflux pumps and ribosomal protectors (mef(A/E)-mrs(D)), are differently distributed worldwide and associated with different clonal lineages and mobile genetic elements. MR rates vary together depending on clonal dynamics and on antibiotic consumption applying selective pressure. Among Streptococcus, higher MR rates are found in the viridans group, Streptococcus pneumoniae and Streptococcus agalactiae, and lower MR rates are described in Streptococcus pyogenes. When considering different geographic areas, higher resistance rates are usually found in East-Asian countries and milder or lower in the US and Europe. Unfortunately, the availability of data varies also between countries; it is scarce in low- and middle- income countries from Africa and South America. Thus, surveillance studies of macrolide resistance rates and the resistance determinants involved should be promoted to complete global knowledge among macrolide resistance dynamics

Molecular epidemiology of nontypeable haemophilus influenzae causing community-acquired pneumonia in adults

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen which causes a variety of respiratory infections. The objectives of the study were to determine its antimicrobial susceptibility, to characterize the b-lactam resistance, and to establish a genetic characterization of NTHi isolates. Ninety-five NTHi isolates were analyzed by pulsed field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). Antimicrobial susceptibility was determined by microdilution, and the ftsI gene (encoding penicillin-binding protein 3, PBP3) was PCR amplified and sequenced. Thirty (31.6%) isolates were non-susceptible to ampicillin (MIC$2 mg/L), with 10 of them producing b-lactamase type TEM-1 as a resistance mechanism. After ftsI sequencing, 39 (41.1%) isolates showed amino acid substitutions in PBP3, with Asn526->Lys being the most common (69.2%). Eighty-four patients were successfully treated with amoxicillin/clavulanic acid, ceftriaxone and levofloxacin. Eight patients died due either to aspiration or complication of their comorbidities. In conclusion, NTHi causing CAP in adults shows high genetic diversity and is associated with a high rate of reduced susceptibility to ampicillin due to alterations in PBP3. The analysis of treatment and outcomes demonstrated that NTHi strains with mutations in the ftsI gene could be successfully treated with ceftriaxone or fluoroquinolones

Clinical and molecular epidemiology of haemophilus influenzae causing invasive disease in adult patients

Objectives The epidemiology of invasive Haemophilus influenzae (Hi) has changed since the introduction of the Hi type b (Hib) vaccine. The aim of this study was to analyze the clinical and molecular epidemiology of Hi invasive disease in adults. Methods Clinical data of the 82 patients with Hi invasive infections were analyzed. Antimicrobial susceptibility, serotyping, and genotyping were studied (20082013). Results Men accounted for 63.4% of patients (whose mean age was 64.3 years). The most frequent comorbidities were immunosuppressive therapy (34.1%), malignancy (31.7%), diabetes, and COPD (both 22%). The 30-day mortality rate was 20.7%. The majority of the strains (84.3%) were nontypeable (NTHi) and serotype f was the most prevalent serotype in the capsulated strains. The highest antimicrobial resistance was for cotrimoxazole (27.1%) and ampicillin (14.3%). Twenty-three isolates (32.9%) had amino acid changes in the PBP3 involved in resistance. Capsulated strains were clonal and belonged to clonal complexes 6 (serotype b), 124 (serotype f), and 18 (serotype e), whereas NTHi were genetically diverse. Conclusions Invasive Hi disease occurred mainly in elderly and those with underlying conditions, and it was associated with a high mortality rate. NTHi were the most common cause of invasive disease and showed high genetic diversity

A Novel Typing Method for Streptococcus pneumoniae Using Selected Surface Proteins

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.This work was supported by a Miguel Servet contract from the Spanish Ministry of Health to AM, Plan Nacional de I+D+I of the Ministry of Science and Innovation (BIO2011-25343, BIO2014-555462-R, SAF2012-39444-C02), Fondo de Investigaciones Sanitarias de la Seguridad Social (PI11/00763) and Fondo Europeo de Desarrollo Regional (FEDER). CIBER Enfermedades Respiratorias is an initiative of the Instituto de Salud Carlos III.Peer reviewedPeer Reviewe

Molecular characterization of Streptococcus pneumoniae invasive serotype 19A isolates from adults in two Spanish regions (1994-2009)

From 1994 to 2009, the incidence of invasive serotype 19A pneumococci isolated from adults in Barcelona and San Sebastian almost doubled every 4 years. Genotyping of the 167 invasive isolates studied showed serotype 19A to be highly heterogeneous, with 35 different sequence types (STs) and a different clonal structure in each region and time period. Multiresistance, defined as non-susceptibility to three or more antimicrobials, was found in 86 (51.5%) isolates. The most frequent ST was the multidrug-resistant ST276 (n = 28), which is a single-locus variant of the Denmark14-ST230 global clone. The ST276 clone, only present in San Sebastian before 2001, was successfully disseminated from 2002 in both cities and was the main contributor to the overall increase of serotype 19A infections

A fresh look at polymicrobial bloodstream infection in cancer patients

Objectives: To assess the current incidence, clinical features, risk factors, aetiology, antimicrobial resistance and outcomes of polymicrobial bloodstream infection (PBSI) in patients with cancer. Methods: All prospectively collected episodes of PBSI in hospitalised patients were compared with episodes of monomicrobial bloodstream infection (MBSI) between 2006 and 2015. Results: We identified 194 (10.2%) episodes of PBSI and 1702 MBSI (89.8%). The presence of cholangitis, biliary stenting, neutropenia, corticosteroids, neutropenic enterocolitis and other abdominal infections were identified as risk factors for PBSI. Overall, Gram-negative organisms were the most frequent aetiology, but Enterococcus spp. were especially frequent causes of Gram-positive PBSI (30.8%). Multidrug-resistant (MDR) organisms were more commonly found in PBSI than in MBSI (20.6% vs 12.9%; p = 0.003). Compared to patients with MBSI, those with PBSI presented with higher early (15% vs 1.4%; p = 0.04) and overall (32% vs 20.9%; p<0.001) case-fatality rates. Risk factors for overall case-fatality were a high-risk MASCC (Multinational Association of Supportive Care in Cancer) index score, corticosteroid use, persistent bacteraemia and septic shock. Conclusions PBSI is a frequent complication in patients with cancer and is responsible for high mortality rates. Physicians should identify patients at risk for PBSI and provide empiric antibiotic therapy that covers the most frequent pathogens involved in these infections, including MDR strains