Showing papers by "Mário Ramirez published in 2018"

chewBBACA: A complete suite for gene-by-gene schema creation and strain identification.

Abstract: Gene-by-gene approaches are becoming increasingly popular in bacterial genomic epidemiology and outbreak detection. However, there is a lack of open-source scalable software for schema definition and allele calling for these methodologies. The chewBBACA suite was designed to assist users in the creation and evaluation of novel whole-genome or core-genome gene-by-gene typing schemas and subsequent allele calling in bacterial strains of interest. chewBBACA performs the schema creation and allele calls on complete or draft genomes resulting from de novo assemblers. The chewBBACA software uses Python 3.4 or higher and can run on a laptop or in high performance clusters making it useful for both small laboratories and large reference centers. ChewBBACA is available at https://github.com/B-UMMI/chewBBACA.

Pediatric Complicated Pneumonia Caused by Streptococcus pneumoniae Serotype 3 in 13-Valent Pneumococcal Conjugate Vaccinees, Portugal, 2010-2015.

Abstract: Despite use of 7-valent pneumococcal conjugate vaccine, incidence of pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]) is reportedly increasing globally. We cultured and performed PCR on 152 pleural fluid samples recovered from pediatric patients in Portugal during 2010-2015 to identify and serotype Streptococcus pneumoniae. We identified only 17 cases by culture, but molecular methods identified S. pneumoniae in 68% (92/135) of culture-negative samples. The most frequent serotypes were 3, 1, and 19A, together accounting for 62% (68/109) of cases. Nineteen cases attributable to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes (mostly serotype 3) were detected among 22 children age-appropriately vaccinated with PCV13. The dominance of the additional serotypes included in PCV13 among PCPP cases in Portugal continues, even with PCV13 available on the private market (without reimbursement) since 2010 and with average annual coverage of 61% among age-eligible children. Our data suggest reduced effectiveness of PCV13 against serotype 3 PCPP.

Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

Abstract: High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.

Increasing macrolide resistance among Streptococcus agalactiae causing invasive disease in non-pregnant adults was driven by a single capsular-transformed lineage, Portugal, 2009 to 2015.

Abstract: We characterised Lancefield group B streptococcal (GBS) isolates causing invasive disease among non-pregnant adults in Portugal between 2009 and 2015. All isolates (n = 555) were serotyped, assigned to clonal complexes (CCs) by multilocus sequence typing and characterised by surface protein and pilus island gene profiling. Antimicrobial susceptibility was tested by disk diffusion and resistance genotypes identified by PCR. Overall, serotype Ia was most frequent in the population (31%), followed by serotypes Ib (24%) and V (18%). Serotype Ib increased significantly throughout the study period (p < 0.001) to become the most frequent serotype after 2013. More than 40% of isolates clustered in the CC1/alp3/PI-1+PI-2a genetic lineage, including most isolates of serotypes Ib (n = 110) and V (n = 65). Erythromycin and clindamycin resistance rates were 35% and 34%, respectively, both increasing from 2009 to 2015 (p < 0.010) and associated with CC1 and serotype Ib (p < 0.001). The Ib/CC1 lineage probably resulted from acquisition of the type Ib capsular operon in a single recombination event by a representative of the V/CC1 macrolide-resistant lineage. Expansion of the new serotype Ib/CC1 lineage resulted in increased macrolide resistance in GBS, causing invasive disease among adults in Portugal. The presence of this clone elsewhere may predict more widespread increase in resistance.

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS.

Abstract: Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB.

Conjugate vaccine serotypes persist as major causes of non-invasive pneumococcal pneumonia in Portugal despite declines in serotypes 3 and 19A (2012-2015).

Abstract: Non-invasive pneumococcal pneumonia (NIPP) is a frequent cause of morbidity and mortality worldwide. The 13-valent pneumococcal conjugate vaccine (PCV13) was included in the national immunization program of children living in Portugal in 2015. Until then, PCV7 (since late 2001) and PCV13 (since early 2010) were given through the private market. We determined the serotype distribution and antimicrobial susceptibility of isolates causing adult NIPP in 2012-2015 and compared the results with previously published data (2007-2011). There were 50 serotypes among the 1435 isolates. The most common were serotypes: 3 (14%), 11A (8%), 19F (6%), 23A (5%), 6C (5%), 19A (4%), 23B (4%), 9N (4%) and non-typable isolates (4%). When considering data since the availability of PCV13 for children in the private market, the proportion of PCV13 serotypes declined from 44.0% in 2010 to 29.7% in 2015 (p < 0.001), mainly due to early decreases in the proportions of serotypes 3 and 19A. In contrast, during the same period, PCV7 serotypes (11.9% in 2012-2015) and the serotypes exclusive of the 23-valent polysaccharide vaccine (26.0% in 2012-2015), remained relatively stable, while non-vaccine types increased from 27.0% in 2010 to 41.9% in 2015 (p<0.001). According to the Clinical and Laboratory Standards Institute (CLSI) breakpoints, penicillin non-susceptible and erythromycin resistant isolates accounted for 1% and 21.7%, respectively, of the isolates recovered in 2012-2015, with no significant changes seen since 2007. Comparison of NIPP serotypes with contemporary invasive disease serotypes identified associations of 19 serotypes with either disease presentation. The introduction of PCV13 in the national immunization program for children from 2015 onwards may lead to reductions in the proportion of NIPP due to vaccine serotypes but continued NIPP surveillance is essential due to a different serotype distribution from invasive disease.