Extended-spectrum β-lactamase producing Klebsiella spp. in chicken meat and humans: a comparison of typing methods

Recently, chicken meat was identified as a plausible source of extended-spectrum β-lactamase (ESBL) -producing Escherichia coli in humans. We investigated the relatedness of ESBL-producing Klebsiella spp. in chicken meat and humans. Furthermore, we tested the performance of SpectraCell RA^® (River Diagnostics), a new typing method based on Raman spectroscopy, in comparison with multilocus sequence typing (MLST) for Klebsiella pneumoniae. Twenty-seven phenotypically and genotypically confirmed ESBL-producing Klebsiella spp. isolates were typed with MLST and SpectraCell RA. The isolates derived from chicken meat, human rectal swabs and clinical blood cultures. In the 22 ESBL-producing K. pneumoniae isolates, CTX-M15 was the predominant genotype, found in five isolates of human origin and in one chicken meat isolate. With MLST, 16 different STs were found, including five new STs. Comparing the results of SpectraCell RA with MLST, we found a sensitivity of 70.0% and a specificity of 81.8% for the new SpectraCell RA typing method. Therefore, we conclude that SpectraCell RA is not a suitable typing method when evaluating relationships of ESBL-producing Klebsiella spp. at the population level. Although no clustering was found with isolates of chicken meat and human origin containing the same ESBL genes, MLST showed no clustering into distinctive clones of isolates from chicken meat and human origin. More studies are needed to elucidate the role of chicken meat in the rise of ESBL-producing Klebsiella spp. in humans.     

High invasiveness of pneumococcal serotypes included in the new generation of conjugate vaccines

The implementation of the seven-valent pneumococcal conjugate vaccine, PCV7, has resulted in significant changes in the pneumococcal population being carried and causing disease. We aimed to determine the invasive disease potential of serotypes causing invasive paediatric disease in the era of conjugate vaccines in Catalonia, Spain, and their potential coverage by the 13-valent pneumococcal conjugate vaccine, PCV13. As a secondary objective, we evaluated whether implementation of PCV7 had resulted in significant changes in the invasive disease potential of the most frequent serotypes circulating in the area. Two pneumococcal collections obtained from children admitted to the University Hospital Sant Joan de Déu (Barcelona, Spain) between 2007 and 2011 were compared: a first set of 159 invasive disease isolates, and a second set of 209 nasopharyngeal isolates recovered from healthy children admitted for minor surgery. The most common invasive serotypes were 1 (24.5%, n = 39), 19A (21.2%, n = 34), 5 (8.8%, n = 14), 7F (8.8%, n = 14) and 3 (5%, n = 8). The most common serotypes in carriage were 19A (10%, n = 21), 6C (9%, n = 19), 23B (8.1%, n = 17), 6A (7.6%, n = 16) and 19F (6.2%, n = 13). A significantly higher propensity to cause invasive disease was observed for serotypes 1, 3, 5, 7F and 19A, all of which are included in PCV13. After false-discovery-rate correction, the results were robust for serotypes 1, 5, 7F and 19A. Non-PCV13 serotypes had a low invasive disease potential. Our data reinforce the need for continuous surveillance and should encourage efforts to introduce universal vaccination with PCV13 in children in our region.     

Invasive pneumococcal pneumonia caused by 13-valent pneumococcal conjugate vaccine types in children with different schedules

Background

In Taiwan, the age group with the greatest incidence of invasive pneumococcal disease is 2–5 years of age, which is different from other countries. This study was conducted to identify risk factors and different 13-valent pneumococcal conjugate vaccine (PCV13) schedules associated with vaccine-type invasive pneumococcal pneumonia (IPP) despite prior vaccination.

Evaluation and Optimization of an ELISA Procedure to Quantify Antibodies Against Pneumococcal Polysaccharides Included in the 13-Valent Conjugate Vaccine

The 13-valent pneumococcal conjugate vaccine (PCV-13) is recommended for HIV-infected people, although its effectiveness in this population remains under evaluation. In this study, we describe the development, optimization, and analytical validation of an ELISA procedure to measure specific antibodies for the pneumococcal polysaccharide serotypes included in PCV13 vaccine, testing sera obtained from HIV-infected outpatients (n = 30) who received the vaccine. The protocol followed the last version of WHO guidelines, based on the new standard 007sp, with the modification of employing Statens Serum Institut (SSI) antigens.

We supplied the assay performance validation in terms of sensitivity, reproducibility, precision and accuracy. In addition we detailed optimal antigen-coating concentrations and ELISA conditions common to all 13 serotypes, suitable for laboratories performing these assays in order to standardize the method. Our procedure showed reproducibility and reliability, making it a valid alternative for evaluating the response to pneumococcal serotypes included in PCV13 vaccine.     

Molecular epidemiology of OXA-48-producing Klebsiella pneumoniae in France

We characterized 53 OXA-48-producing Klebsiella pneumoniae (OXA-48-Kp) isolated between 2011 and 2013 in 21 French hospitals. All the isolates were genotyped using MLST and PFGE and the population structure of the species was determined by a nucleotide-based analysis of the entire K. pneumoniae MLST database. Most of the OXA-48-Kp isolates also produced CTX-M-15 and remained susceptible to imipenem and meropenem. The isolates were distributed into 20 STs, of which five were dominant (ST15, ST101, ST147, ST395 and ST405). All the OXA-48-Kp clustered in the major clade of K. pneumoniae KpI.     

Detection of diverse SCCmec variants in methicillin-resistant Staphylococcus aureus and comparison of SCCmec typing methods

Non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 436), collected from four hospitals located in three Korean cities between 2001 and 2005, were investigated by SCCmec typing and multilocus sequence typing (MLST). Variations within SCCmec, especially type II, were detected in 165 (37.8%) isolates, and these variants were characterised using four different SCCmec typing methods. The predominant SCCmec type was a type II variant that differed from type II by the absence of a pUB110 insertion. MLST analysis showed that most of the isolates carrying SCCmec variants belonged to ST5.     

A bioinformatics pipeline for high-throughput microbial multilocus sequence typing (MLST) analyses

Multilocus sequence typing (MLST) analysis for semi-routine applications is hindered by the downstream, manually intensive steps of processing the raw sequence data files. This report describes the development of an MLST pipeline that automates DNA sequence editing and analysis in order to significantly reduce the time required for processing data. Validation using a pneumococcal dataset revealed complete agreement between the results generated by manual and automated workflows. The MLST pipeline was developed for both double-strand and single-strand sequencing.     

Experience with pneumococcal polysaccharide conjugate vaccine (conjugated to CRM197 carrier protein) in children and adults

Streptococcus pneumoniae-related infections are a major cause of morbidity and mortality in people of all ages worldwide. Pneumococcal vaccine development started in 1911 with a whole cell vaccine and more recently multivalent plain polysaccharide and polysaccharide conjugate vaccines have been developed. The recent vaccines rely on capsular polysaccharide antigens to induce serotype-specific immune responses. We summarize here the presentations on pneumococcal polysaccharide conjugate vaccine (conjugated to CRM197 carrier protein) given during the integrated symposium organized and funded by Pfizer International Operations during the 22nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) 31 March to 3 April 2012, London, UK. A dramatic reduction in the incidence of invasive pneumococcal diseases (IPD) due to vaccine serotypes (VST-IPD) has been reported since the introduction of a hepta-valent pneumococcal conjugate vaccine (PCV7). An indirect (herd) effect has been demonstrated to be associated with PCV7 infant vaccination programmes, with many studies reporting reductions in VST-IPD in populations that are not eligible for PCV7 vaccination. Since 2010, a 13-valent pneumococcal conjugate vaccine (PCV13) has been introduced into national immunization programmes and results from early surveillance suggest that this vaccine also has an impact on the serotypes unique to PCV13, as well as continuing to protect against the PCV7 serotypes. Data from a passive surveillance system in Europe in 2009, for instance, showed that the highest incidence of IPD remains in those aged >65 years and in children <5 years. PCV13 has now been licensed for vaccination of adults >50 years based on safety and immunogenicity data; an efficacy trial is being conducted. Regardless of previous pneumococcal vaccination status, if the use of 23-valent polysaccharide is considered appropriate, it is recommended to give PCV13 first. Novel immunization strategies remain the only practical means to reduce significantly the remaining global mortality and morbidity due to S. pneumoniae in adults.     

High-level fluoroquinolone resistance in a clinical Streptoccoccus pyogenes isolate in Germany

An isolate of Streptococcus pyogenes isolated from a 63-year-old woman with a serious wound infection was found to be highly resistant to fluoroquinolones (levofloxacin MIC > or = 32 mg/L). DNA amplification and sequencing revealed a serine-81 to phenylalanine substitution in gyrA and three substitutions in parC: serine-79 to phenylalanine, aspartic acid-91 to asparagine, and serine-140 to proline. To our knowledge, this is the first report from a European country of a clinical isolate of S. pyogenes with high-level fluoroquinolone resistance.     

Understanding the dynamics of imipenem-resistant Acinetobacter baumannii lineages within Portugal

A recent collection of 213 imipenem-resistant Acinetobacter baumannii (IRAB) clinical isolates was characterized for the presence of acquired carbapenem-hydrolysing class D β-lactamases (CHDLs) and clonality. A population structure analysis of IRAB was also conducted, with five molecular typing methods. Three main clusters, each one associated with a specific CHDL, were observed with multilocus sequence typing. Overall, our results suggest a switch in the dominant clone, with sequence type (ST) 92, carrying bla_OXA-23 (63.4%), replacing the closely related ST98, carrying bla_OXA-24/40 (22%). In addition, ST103, an independent lineage, was associated with bla_OXA-58-carrying isolates (14.6%).     

Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme

To further expand the limited multilocus sequence typing (MLST) database for Acinetobacter baumannii , 53 clinical isolates from various outbreaks in Europe and the USA, collected between 1991 and 2004, plus the A. baumannii reference strain ATCC 19606^T and 20 clinical Acinetobacter genomic species 13TU isolates from the same period, were analyzed using a new MLST scheme based on fragments of the gltA , gyrB , gdhB , recA , cpn60 , gpi and rpoD genes. Data were compared with typing results generated using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)-PCR. In total, 50 sequence types (STs) were distinguished among the A. baumannii isolates investigated, and the MLST data were in high concordance with the PFGE and RAPD-PCR results. Only five clonal complexes were identified by eBURST analysis, including the 21 STs listed in a previous study, suggesting high diversity among the A. baumannii isolates. With one exception, there was no relatedness among isolates from outbreaks in different countries (Europe) or regions (USA). No intercontinental spread was revealed. Acinetobacter genomic species 13TU isolates could also be analyzed using the A. baumannii MLST scheme (18 different STs) and could be distinguished from A. baumannii isolates according to characteristic sequences. It was concluded that the MLST scheme provides a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii and Acinetobacter genomic species 13TU.     

Comparison of multiple typing methods for Aspergillus fumigatus

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus . In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus , which requires a high discriminatory power.     

Longitudinal surveillance for meningitis by Acinetobacter in a large urban setting in Brazil

The study aim was to describe the emergence of carbapenem resistance and clonal complexes (CC), defined by multilocus sequence typing (MLST), in Acinetobacter baumannii in a surveillance system for meningitis. Starting in 1996 in an urban setting of Brazil, surveillance detected meningitis by Acinetobacter sp for the first time in 2002. Up to 2008, 35 isolates were saved. Carbapenem resistance emerged in 2006, reaching 70% of A. baumannii isolates in 2008, including one that was colistin resistant. A. baumannii belonged to CC113/79 (University of Oxford/Institute Pasteur schemes), CC235/162 and CC103/15. Dissemination of infections resistant to all antimicrobial agents may occur in the future.