Mário Ramirez

PHYLOViZ: phylogenetic inference and data visualization for sequence based typing methods

BackgroundWith the decrease of DNA sequencing costs, sequence-based typing methods are rapidly becoming the gold standard for epidemiological surveillance. These methods provide reproducible and comparable results needed for a global scale bacterial population analysis, while retaining their usefulness for local epidemiological surveys. Online databases that collect the generated allelic profiles and associated epidemiological data are available but this wealth of data remains underused and are frequently poorly annotated since no user-friendly tool exists to analyze and explore it.ResultsPHYLOViZ is platform independent Java software that allows the integrated analysis of sequence-based typing methods, including SNP data generated from whole genome sequence approaches, and associated epidemiological data. goeBURST and its Minimum Spanning Tree expansion are used for visualizing the possible evolutionary relationships between isolates. The results can be displayed as an annotated graph overlaying the query results of any other epidemiological data available.ConclusionsPHYLOViZ is a user-friendly software that allows the combined analysis of multiple data sources for microbial epidemiological and population studies. It is freely available at http://www.phyloviz.net.

Invasive infections due to Streptococcus pyogenes: seasonal variation of severity and clinical characteristics, Iceland, 1975 to 2012.

Epidemiology and clinical characteristics of invasive Group A streptococcal infections (IGASI) are highly variable. Long-term studies are needed to understand the interplay between epidemiology and virulence. In a population-based study of IGASI in Iceland from 1975 to 2012, 288 cases were identified by positive cultures from normally sterile body sites. Charts were reviewed retrospectively and emm-types of viable Streptococcus pyogenes isolates (n=226) determined. Comparing the first and last decade of the study period, IGASI incidence increased from 1.09 to 3.96 cases per 100,000 inhabitants per year. The most common were emm types 1 (25%), 28 (11%) and 89 (11%); emm1 strains were most likely to cause severe infections. Infections in adults were significantly more likely to be severe during the seasonal peak from January to April (risk ratio: 2.36, 95% confidence interval: 1.34–4.15). Significant seasonal variability in severity was noted among patients with diagnosis of sepsis, respiratory infection and cellulitis, with 38% of severe infections in January to April compared with 16% in other months (p<0.01). A seasonal increase in severity of IGASI suggested that generalised seasonal increase in host susceptibility, rather than introduction of more virulent strains may play a role in the pathogenesis of these potentially fatal infections.

Superantigen gene complement of Streptococcus pyogenes—relationship with other typing methods and short-term stability

The profiling of the superantigen (SAg) encoding genes has been frequently used as a complementary typing method for group A streptococci (GAS), but a confusing gene nomenclature and a large diversity of primers used in screening has led to some conflicting results. The aim of this work was to develop a polymerase chain reaction (PCR) method capable of efficiently amplifying all the known allelic variants of these genes, and to evaluate the congruence of this methodology with other commonly used molecular typing methods. The presence of the 11 known SAg genes and two other exotoxin-encoding genes (speB and speF) was tested in a collection of 480 clinical GAS isolates, using two multiplex PCR reactions. The SAg gene profile was compared with other typing methods. Four naturally occurring deletions involving the genes speB, speF, and rgg were characterized, two of which were found among invasive isolates. The absence of the chromosomally encoded genes speG and smeZ was supported by Southern blot hybridization and associated with specific GAS lineages, while the presence of phage-encoded genes was more variable. Positive associations between SAg genes or between SAg profiles and emm types or pulsed-field gel electrophoresis (PFGE) clusters were observed. The results suggest that the SAg profile diversifies faster than other properties commonly used for molecular typing, such as emm type and multilocus sequence typing (MLST) sequence types (STs), and can be a useful complement in GAS molecular epidemiology. Still, the short-term stability of the SAg gene profile among prevalent genetic lineages may largely explain the observed associations between SAg genes.

The Viriato Study

AbstractBackground: The Viriato study is a prospective, multicentre laboratory-based surveillance study of antimicrobial susceptibility in which 30 microbiology laboratories throughout Portugal are asked to isolate identify and submit to a central laboratory for testing Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis responsible for community-acquired lower respiratory tract infections and Streptococcus pyogenes from tonsillitis.n Objective: To monitor changes in antimicrobial resistance patterns of these frequent respiratory pathogens.n Methods: Susceptibility was determined by disk diffusion (Kirby-Bauer) or using Etest strips following the Clinical and Laboratory Standards Institute guidelines.n Results: From 1999 to 2007 over 13 900 isolates were analysed. Among S. pneumoniae penicillin non-susceptibility decreased from 25% in 1999 to 18% in 2007 (p = 0.002) but resistance to macrolides showed a steady increase, reaching 20% in the last 6 years. Resistance to amoxicillin and the quinolones remained stable and very low (1–2%) throughout the study period. Antimicrobial resistance among H. influenzae and M. catarrhalis remained stable. The most significant resistance was to ampicillin, of 10–12% and greater than 80%, respectively, as a result of the production of β-lactamases. Macrolide resistance among S. pyogenes was stable during 1999–2003 (20–23%) but after 2003 there was a steady decline in resistance, which in 2007 reached 10%.n Conclusion: The Viriato surveillance study showed that penicillin remains the most active antimicrobial agent against S. pyogenes causing tonsillitis, and amoxicillin-clavulanate and the quinolones are the most active in vitro simultaneously against S. pneumoniae, H. influenzae and M. catarrhalis responsible for community-acquired lower respiratory tract infections in Portugal.

Pherotype influences biofilm growth and recombination in Streptococcus pneumoniae.

In Streptococcus pneumoniae the competence-stimulating peptide (CSP), encoded by the comC gene, controls competence development and influences biofilm growth. We explored the influence of pherotype, defined by the two major comC allelic variants (comC1 and comC2), on biofilm development and recombination efficiency. Among isolates recovered from human infections those presenting comC1 show a higher capacity to form in vitro biofilms. The influence of pherotype on biofilm growth was confirmed by experiments with isogenic strains differing in their comC alleles. Biofilm architecture evaluated by confocal laser scanning microscopy showed that strains carrying comC1 form biofilms that are denser and thicker than those carrying the comC2 allele. Isogenic strains carrying the comC1 allele yielded more transformants than those carrying the comC2 allele in both planktonic and biofilm growth. Transformation assays with comC knockout strains show that ComD1 needs lower doses of the signaling peptide to reach the same biological outcomes. In contrast to mixed planktonic growth, within mixed biofilms inter-pherotype genetic exchange is less frequent than that occurring between bacteria of the same pherotype. Since biofilms are a major bacterial lifestyle, these observations may explain the genetic differentiation between populations with different pherotypes reported previously. Considering that biofilms have been associated with colonization our results suggest that strains carrying the comC1 allele may be more transmissible and more efficient at persisting in carriage. Both effects may help explain the higher prevalence of the comC1 allele in the pneumococcal population.

Epidemiological Surveillance and Typing Methods to Track Antibiotic Resistant Strains Using High Throughput Sequencing

High-Throughput Sequencing (HTS) technologies transformed the microbial typing and molecular epidemiology field by providing the cost-effective ability for researchers to probe draft genomes, not only for epidemiological markers but also for antibiotic resistance and virulence determinants. In this chapter, we provide protocols for the analysis of HTS data for the determination of multilocus sequence typing (MLST) information and for determining presence or absence of antibiotic resistance genes.

An ontology and a REST API for sequence based microbial typing data

In the Microbial typing field, the need to have a common understanding of the concepts described and the ability to share results within the community is an increasingly important requisite for the continued development of portable and accurate sequence-based typing methods. These methods are used for bacterial strain identification and are fundamental tools in Clinical Microbiology and Bacterial Population Genetics studies. In this article we propose an ontology designed for the microbial typing field, focusing on the widely used Multi Locus Sequence Typing methodology, and a RESTful API for accessing information systems based on the proposed ontology. This constitutes an important first step to accurately describe, analyze, curate, and manage information for microbial typing methodologies based on sequence based typing methodologies, and allows for the future integration with data analysis Web services.

Chapter 86 – Streptococcus pneumoniae

Research on Streptococcus pneumoniae has revealed key aspects in the study of infectious diseases and pathogen evolution, but pneumococci remain a major cause of morbidity and mortality. New insights into the pathogenic mechanisms of this species confirmed the key role of the capsular polysaccharide but have also identified the important functions played by proteins in the interaction with the host. The capacity of pneumococci to exchange DNA is central in the adaptation to human-imposed selective pressures and our understanding of the mechanisms underlying competence is raising new questions about the evolution of these bacteria. A greater knowledge of the biology of pneumococci resulted in new diagnostic tests that together with a revision of the breakpoints defining resistance to penicillin and the advent of conjugate vaccines, are changing our understanding of the burden of pneumococcal disease and our approaches to prevent and treat infections by this important pathogen.