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Purpose: to characterize the distribution and mechanisms involved in ceftolozane/tazobactam (C/T) resistance in Pseudomonas aeruginosa isolates recovered from intensive care units (ICUs) in Portugal as part of the STEP surveillance study. Materials and methods: a total of 226 P. aeruginosa isolates were collected from patients with low respiratory tract infections (LRTI) admitted to ICUs between June 2017 and July 2018. Susceptibility to antimicrobials including the recent C/T combination was determined by EUCAST-criteria. Whole genome sequencing (WGS) was performed in a subset of 17 isolates. Results: multidrug resistant (MDR) and extremely drug resistant (XDR) phenotypes accounted for 20.4% and 25.7% of cases, respectively. C/T showed the highest susceptibility rate in both MDR (100%) and XDR (93.1%) isolates, followed by amikacin (97.8% MDR and 79.3% XDR). blaKPC-3 (n = 2) and blaGES-13 (n = 1) carbapenemase genes were detected in 3 of the 17 sequenced isolates, but only the GES-13-producing isolate displayed resistance to C/T. Additionally, the C/T-resistant phenotype was also found in two non-carbapenemase producers that carried known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene. Conclusions: C/T was highly active against MDR/XDR-P. aeruginosa isolates causing LRTI in ICUs. Moreover, beyond carbapenemase-encoding genes, mutations in chromosomal PBP-encoding genes might also be involved in ceftolozane/tazobactam resistance in Portugal.

Objectives Streptococcus agalactiae [group B streptococci (GBS)] have been considered uniformly susceptible to penicillin. However, increasing reports from Asia and North America are documenting penicillin-non-susceptible GBS (PRGBS) with mutations in pbp genes. Here we report, to the best of our knowledge, the first two PRGBS isolates recovered in Europe (AC-13238-1 and AC-13238-2), isolated from the same patient. Methods Two different colony morphologies of GBS were noted from a surgical abscess drainage sample. Both were serotyped and antimicrobial susceptibility testing was performed by different methodologies. High-throughput sequencing was done to compare the isolates at the genomic level, to identify their capsular type and ST, to evaluate mutations in the pbp genes and to compare the isolates with the genomes of other PRGBS isolates sharing the same serotype and ST. Results Isolates AC-13238-1 and AC-13238-2 presented MICs above the EUCAST and CLSI breakpoints for penicillin susceptibility. Both shared the capsular type Ia operon and ST23. Genomic analysis uncovered differences between the two isolates in seven genes, including altered pbp genes. Deduced amino acid sequences revealed critical substitutions in PBP2X in both isolates. Comparison with serotype Ia clonal complex 23 PRGBS from the USA reinforced the similarity between AC-13238-1 and AC-13238-2, and their divergence from the US strains. Conclusions Our results support the in-host evolution of β-lactam-resistant GBS, with two PRGBS variants being isolated from one patient.

© 2020 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., SARS-CoV-2 has emerged as a human pathogen, causing clinical signs, from fever to pneumonia-COVID-19-but may remain mild or asymptomatic. To understand the continuing spread of the virus, to detect those who are and were infected, and to follow the immune response longitudinally, reliable and robust assays for SARS-CoV-2 detection and immunological monitoring are needed. We quantified IgM, IgG, and IgA antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) or the Spike (S) protein over a period of 6 months following COVID-19 onset. We report the detailed setup to monitor the humoral immune response from over 300 COVID-19 hospital patients and healthcare workers, 2500 University staff, and 198 post-COVID-19 volunteers. Anti-SARS-CoV-2 antibody responses follow a classic pattern with a rapid increase within the first three weeks after symptoms. Although titres reduce subsequently, the ability to detect anti-SARS-CoV-2 IgG antibodies remained robust with confirmed neutralization activity for up to 6 months in a large proportion of previously virus-positive screened subjects. Our work provides detailed information for the assays used, facilitating further and longitudinal analysis of protective immunity to SARS-CoV-2. Importantly, it highlights a continued level of circulating neutralising antibodies in most people with confirmed SARS-CoV-2., We like to acknowledge the funding from the European Union H2020 ERA project (No 667824 – EXCELLtoINNOV) and the Fundação para a Ciência e a Tecnologia (FCT) to P.F‐C. (SFRH/BD/131605/2017), PTDC/MED‐IMU/28003/2017, and research4COVID19 (no. 231_596873172, Generating SARS‐CoV2 seroconversion assay and no. 729, High‐throughput SARS‐CoV2 neutralizing antibodies assessment), with additional support by Sociedade Francisco Manuel dos Santos.

SARS-CoV-2 has emerged as a novel human pathogen, causing clinical signs, from fever to pneumonia – COVID-19 – but may remain mild or even asymptomatic. To understand the continuing spread of the virus, to detect those who are and were infected, and to follow the immune response longitudinally, reliable and robust assays for SARS-CoV-2 detection and immunological monitoring are needed and have been setup around the world. We quantified immunoglobulin M (IgM), IgG and IgA antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) or the Spike (S) protein over a period of five months following COVID-19 disease onset or in previously SARS-CoV-2 PCR-positive volunteers. We report the detailed setup to monitor the humoral immune response from over 300 COVID-19 hospital patients and healthcare workers, 2500 University staff and 187 post-COVID19 volunteers, and assessing titres for IgM, IgG and IgA. Anti-SARS-CoV-2 antibody responses followed a classic pattern with a rapid increase within the first three weeks after symptoms. Although titres reduce from approximately four weeks, the ability to detect SARS-CoV-2 antibodies remained robust for five months in a large proportion of previously virus-positive screened subjects. Our work provides detailed information for the assays used, facilitating further and longitudinal analysis of protective immunity to SARS-CoV-2. Moreover, it highlights a continued level of circulating neutralising antibodies in most people with confirmed SARS-CoV-2, at least up to five months after infection.

Fluctuations in the clonal composition of Group A Streptococcus (GAS) have been associated with the emergence of successful lineages and with upsurges of invasive infections (iGAS). This study aimed at identifying changes in the clones causing iGAS in Portugal. Antimicrobial susceptibility testing, emm typing and superantigen (SAg) gene profiling were performed for 381 iGAS isolates from 2010–2015. Macrolide resistance decreased to 4%, accompanied by the disappearance of the M phenotype and an increase of the iMLSB phenotype. The dominant emm types were: emm1 (28%), emm89 (11%), emm3 (9%), emm12 (8%), and emm6 (7%). There were no significant changes in the prevalence of individual emm types, emm clusters, or SAg profiles when comparing to 2006–2009, although an overall increasing trend was recorded during 2000–2015 for emm1, emm75, and emm87. Short-term increases in the prevalence of emm3, emm6, and emm75 may have been driven by concomitant SAg profile changes observed within these emm types, or reflect the emergence of novel genomic variants of the same emm types carrying different SAgs.

The STEP surveillance study was designed to increase knowledge about distribution of multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa in Portugal, focusing on the intensive care unit (ICU). Antimicrobial susceptibility of common agents was also evaluated and compared with that of one of the latest therapeutic introductions, ceftolozane-tazobactam (C/T). Clinical isolates of Enterobacterales (n=426) and P. aeruginosa (n=396) from patients admitted in Portuguese ICUs were included. Activity of C/T and comparators was investigated using standard broth microdilution. Isolates were recovered from urinary tract (UTI, 36.9%), intra-abdominal (IAI, 24.2%) and lower respiratory tract (LRTI, 38.9%) infections. In P. aeruginosa, overall distribution of MDR/extremely-drug resistant (XDR)/pan-drug resistant (PDR) isolates accounted for 21.2%, 23.2% and 0.8%, respectively. C/T was the most potent agent tested against P. aeruginosa and MDR/XDR/PDR phenotypes. In Escherichia coli, extended-spectrum beta-lactamases (ESBL) and carbapenemase (CP) phenotypes accounted for 16.6% and 1.7%, respectively, whereas in Klebsiella spp., ESBL and CP-phenotypes represented 28.5% and 17.9%, respectively. Overall, susceptibility of C/T against Enterobacterales was 86.9%. C/T was the least affected agent in E. coli (99.4% susceptibility), whereas its activity was moderate in Klebsiella spp. (71.5%) and Enterobacter spp. (70.4%), due in part to a high rate of ESBL and CP-phenotypes. In Enterobacterales, blaKPC was the most prevalent CP gene (63.0%), followed by blaOXA-48 (33.3%) and blaVIM (3.7%). These microbiological results reinforce C/T as a therapeutic option in ICU patients with UTI, IAI or LRTI due to P. aeruginosa or Enterobacterales isolates, but not for CP producers.

Klebsiella pneumoniae is a clinically relevant pathogen and a frequent cause of hospital-acquired (HA) and community-acquired (CA) urinary tract infections (UTI). The increased resistance of this pathogen is leading to limited therapeutic options. To investigate the epidemiology, virulence, and antibiotic resistance profile of K. pneumoniae in urinary tract infections, we conducted a multicenter retrospective study for a total of 81 isolates (50 CA-UTI and 31 HA-UTI) in Portugal. The detection and characterization of resistance and virulence determinants were performed by molecular methods (PCR, PCR-based replicon typing, and multilocus sequence typing (MLST)). Out of 50 CA-UTI isolates, six (12.0%) carried &beta, lactamase enzymes, namely blaTEM-156 (n = 2), blaTEM-24 (n = 1), blaSHV-11 (n = 1), blaSHV-33 (n = 1), and blaCTX-M-15 (n = 1). All HA-UTI were extended-spectrum &beta, lactamase (ESBL) producers and had a multidrug resistant profile as compared to the CA-UTI isolates, which were mainly resistant to ciprofloxacin, levofloxacin, tigecycline, and fosfomycin. In conclusion, in contrast to community-acquired isolates, there is an overlap between virulence and multidrug resistance for hospital-acquired UTI K. pneumoniae pathogens. The study is the first to report different virulence characteristics for hospital and community K. pneumoniae pathogens, despite the production of &beta, lactamase and even with the presence of CTX-M-15 ESBL, a successful international ST15 clone, which were identified in both settings. This highlights that a focus on genomic surveillance should remain a priority in the hospital environment.

Research Areas: Microbiology ABSTRACT - Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence. info:eu-repo/semantics/publishedVersion

Streptococcus pneumoniae expressing serotype 3 has a high virulence and a high case fatality ratio. Most studies of serotype 3 pneumococci have focused on a single lineage, the widespread sequence type 180 (ST180). To evaluate the serotype 3 lineages causing infections in Mexico, we characterized 196 isolates recovered from 1994 to 2017. The isolates were mostly susceptible to all antimicrobials tested. A single meningitis isolate was resistant to penicillin, and the resistance to erythromycin was 5.2%. The isolates represented the widely disseminated clonal complex 180 (CC180; n = 140), the unusual CC4909 (n = 42), CC260 (n = 11), and a few singletons (n = 3). CC260 was less frequent among pneumococcal invasive disease isolates than CC180 and CC4909 (P = 0.015). There was a decrease of CC4909 (P

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. info:eu-repo/semantics/publishedVersion

© 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved., Introduction: The rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniae producing extended-spectrum -lactamases and carbapenemases in Klebsiella pneumoniae is one of the most significant threats to public health. However, questions and controversies regarding the interactions between resistance and virulence in multidrug-resistant K. pneumoniae isolates remain unclear. Methods: A retrospective cohort study was performed with 100 K. pneumoniae isolates recovered from a tertiary care university hospital centre in Lisbon over a 31-year period. Resistance and virulence determinants were screened using molecular methods (PCR, M13-PCR and MLST). Results: The predominant virulence profile (fimH, mrkDv1, khe) was shared by all isolates, indicative of an important role of type 1 and 3 fimbrial adhesins and haemolysin, regardless of the type of -lactamase produced. However, accumulation of virulence factors was identified in KPC-3-producers, with a higher frequency (p < 0.05) of capsular serotype K2 and iucC aerobactin when compared with non-KPC-3 - lactamases or carbapenemases. Additionally, 9 different virulence profiles were found, indicating that the KPC-3 carbapenemase producers seem to adapt successfully to the host environment and maintain virulence via several pathways. Conclusion: This study describes an overlapping of multidrug-resistance and virulence determinants in ST-14K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen., Introducción: La rápida y compleja evolución de los mecanismos de resistencia de Klebsiella pneumoniae productora de beta-lactamasas de espectro extendido y carbapenemasas en Klebsiella pneumoniae es una de las amenazas más importantes para la salud pública. Sin embargo, aun existe controversia sobre la interacción entre la resistencia y la virulencia en aislados de K. pneumoniae resistentes a múltiples antimicrobianos. Métodos: Se realizó un estudio de cohorte retrospectivo con 100 aislados de Klebsiella pneumoniae de un centro hospitalario universitario en Lisboa durante 31 anos. ˜ Los determinantes de la resistencia y virulencia se rastrearon utilizando métodos moleculares (PCR, M13-PCR y MLST). Resultados: Todos los aislados compartían un perfil de virulencia predominante (fimH, mrkDv1, khe), lo que indica un papel importante de las adhesinas fimbriales de tipo 1 y 3, y de la hemolisina, independientemente del tipo de -lactamasa producida. Sin embargo, la acumulación de factores de virulencia del serotipo capsular K2 y la aerobactina iucC se identificó con una mayor frecuencia en las cepas productoras de KPC-3 (p < 0,05) en comparación con las productoras de otras -lactamasas o carbapenemasas. Además, se encontraron 9 perfiles de virulencia diferentes, indicativos de que las cepas productoras de carbapenemasa KPC-3 parecen adaptarse con éxito al entorno y mantener la virulencia por varias vías. Conclusión: Este estudio describe la unión de resistencia a múltiples antimicrobianos junto con determinantes de virulencia en aislados clínicos de K. pneumoniae ST-14K2 KPC-3 lo que puede suponer un desafío adicional en el tratamiento de infecciones causadas por este patógeno., This work was supported by the Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)., Herein, we describe a case report of carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae isolates that were identified from the same patient at a Tertiary University Hospital Centre in Portugal. Antimicrobial susceptibility and the molecular characterization of resistance and virulence determinants were performed. PCR screening identified the presence of the resistance genes blaKPC-3, blaTEM-1 and blaSHV-1 in both isolates. The KPC-3 K. pneumoniae isolate belonged to the ST-14 high risk clone and accumulated an uncommon resistance and virulence profile additional to a horizontal dissemination capacity. In conclusion, the molecular screening led to the first identification of the A. baumannii KPC-3 producer in Portugal with a full antimicrobial resistance profile including tigecycline and colistin.

Although penicillin remains the first-choice treatment for Streptococcus pyogenes infection, macrolides are important alternatives for allergic patients and lincosamides are recommended together with β-lactams in invasive infections. S. pyogenes may exhibit macrolide resistance because of active efflux (mef genes) or target modification (erm genes), the latter conferring cross resistance to lincosamides and streptogramin B. Worldwide, resistance is restricted to a limited number of genetic lineages, despite resistance genes being encoded on mobile genetic elements. For reasons that are not completely clear, resistance and the associated phenotypes are highly variable across countries. Although resistance remains high in several countries, particularly in Asia, an overall decreasing trend of resistance has been noted in recent years, mostly in Europe. This decrease is not always accompanied by declines in macrolide consumption, suggesting significant roles of other factors in determining the dynamics of macrolide-resistant clones. Continued surveillance is needed to obtain further insights into the forces governing macrolide resistance in S. pyogenes.

© 2015 John Wiley & Sons Ltd, Streptococcosis is a disease with major health and economic impacts on the tilapia industry worldwide (Mian et al. 2009). In 1997, more than US$150 million of annual losses representing treatment, morbidity and mortality were recorded and this value increased to US$250 million by 2008 (Klesius, Shoemaker & Evans 2008). In Colombia, Streptococcus agalactiae (Lancefield group B streptococci; GBS) is the main bacterial pathogen of tilapia (Jiménez et al. 2007), causing heavy but not yet quantified losses., P.B. was supported by a grant from COLCIENCIAS, Scholarship to doctoral studies ‘Generación Bicentenario’ No. 121731 and DIB grant to research project No. 14357. E.R.M. was supported by a grant from Fundação para a Ciência e a Tecnologia (SFRH/BPD/80038/2011).

Copyright © 2017 Martins, Pedroso-Roussado, Melo-Cristino, Ramirez and The Portuguese Group for the Study of Streptococcal Infections. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., The molecular characterization of 218 GBS isolates recovered from neonatal invasive infections in Portugal in 2005-2015 revealed the existence of a small number of genetically distinct lineages that were present over a significant time-span. Serotypes III and Ia were dominant in the population, together accounting for >80% of the isolates. Clonal complex 17 included 50% of all isolates, highlighting the importance of the hypervirulent genetic lineage represented by serotype III ST17/rib/PI-1+PI-2b. Serotype Ia was represented mainly by ST23, previously reported as dominant among invasive disease in non-pregnant adults in Portugal, but also by ST24, showing an increased frequency among late-onset disease. Overall erythromycin resistance was 16%, increasing during the study period (p < 0.001). Macrolide resistance was overrepresented among CC1 and CC19 isolates (p < 0.001 and p = 0.008, respectively). While representatives of the hypervirulent CC17 lineage were mostly susceptible to macrolides, we identified for the first time in Europe a recently emerging sublineage characterized by the loss of PI-1 (CC17/PI-2b), simultaneously resistant to macrolides, lincosamides, and tetracycline, also exhibiting high-level resistance to streptomycin and kanamycin. The stability and dominance of CC17 among neonatal invasive infections in the past decades indicates that it is extremely well adapted to its niche; however emerging resistance in this genetic background may have significant implications for the prevention and management of GBS disease., EM was supported by a grant from Fundação para a Ciência e a Tecnologia (SFRH/BPD/80038/2011). This work was partially funded by a grant from the governments of Iceland, Lichtenstein and Norway (EEA-PT06).

Although serogroup 6 was among the first to be recognized among Streptococcus pneumoniae, several new serotypes were identified since the introduction of pneumococcal conjugate vaccines (PCVs). A decrease of the 6B-2 variant among invasive pneumococcal disease (IPD), but not 6B-1, was noted post conjugate vaccine introduction, underpinned by a decrease of CC273 isolates. Serotype 6C was associated with adult IPD and increased in this age group representing two lineages (CC315 and CC395), while the same lineages expressed other serogroup 6 serotypes in children. Taken together, these findings suggest a potential cross-protection of PCVs against serotype 6C IPD among vaccinated children but not among adults. Serotype 6A became the most important serogroup 6 serotype in children but it decreased in adult IPD. No other serogroup 6 serotypes were detected, so available phenotypic or simple genotypic assays remain adequate for distinguishing serotypes within serogroup 6 isolates.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) can be severely pathogenic in humans and is increasingly isolated from horses with respiratory, reproductive or other diseases, although it is often considered a commensal bacterium. Here a PCR protocol is described for identifying SDSE recovered from humans. A multiplex PCR targeting the 16S rRNA and the streptokinase precursor gene has been optimized for differentiating between SDSE strains isolated from humans and those isolated from horses. Previously, the sequence of the streptokinase precursor gene of SDSE recovered from horses has been found in two human cases of pneumonia in Japan. Although further evaluation is required, the findings of this study suggest that SDSE strains are host-specific and this multiplex PCR protocol can be useful in further epidemiological studies and for investigating the zoonotic potential of SDSE.

Although rarely isolated from cystic fibrosis (CF) patients, Burkholderia dolosa is associated with accelerated lung function decline. During 18 years of epidemiological surveillance in the major Portuguese CF centre in Lisbon, only one patient was infected with B. dolosa. Pulmonary deterioration, associated with the evolution of forced expiratory volume in 1 s, occurred during 5.5 years of colonization with this B. dolosa clone (with the new sequence type ST-668). Transient co-colonization with Burkholderia cenocepacia and other bacterial and fungal pathogens occurred, but B. dolosa prevailed until the patient’s death. The systematic assessment of relevant phenotypes for the sequential clonal isolates examined in this retrospective study (14 of B. dolosa and four of B. cenocepacia) showed that they were variants, although in general no isolation time-dependent pattern of alteration was identified. However, the first B. dolosa isolate retrieved was more susceptible to gentamicin, imipenem and tobramycin, and exhibited a higher swarming motility compared with most of the isolates obtained during the later stages of disease progression and antimicrobial therapy.

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of infection in the community (CA-MRSA), but in spite of its relevance, no data exist concerning its epidemiology in Portugal. In this study, we aimed to evaluate the prevalence, population structure, and origin of MRSA in the Portuguese community. A total of 527 isolates, both methicillin-susceptible S. aureus (MSSA) and MRSA, were collected from individuals with no healthcare-related risk factors attending 16 healthcare institutions in Portugal. Isolates were characterized for the presence of mecA, Panton-Valentine leukocidin (PVL), and arginine catabolic mobile element (ACME), and by staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa, and multilocus sequence typing (MLST). Susceptibility to a panel of 13 antibiotics was tested. Isolates relatedness was analyzed by goeBURST and BURP. We found a high frequency (21.6%) of MRSA in the community. However, only 11.4% of the isolates belonged to typical CA-MRSA epidemic clones (USA300, USA400, USA700, Southwest Pacific, European, and ST398). The remaining isolates, which constituted the great majority (88.6%), belonged to hospital-associated MRSA (HA-MRSA) epidemic clones, namely, to the EMRSA-15 clone (77.2%). PVL was rare and carried by 17 isolates only (five MRSA and 12 MSSA). In the whole collection, some MRSA and MSSA were highly related. The high frequency of MRSA in the community in Portugal seems to result mainly from dissemination from the hospital. They might also have emerged from an extant MSSA population, by SCCmec acquisition, or MRSA clonal introduction from abroad.

Streptococcus pneumoniae bacteriophages (phages) rely on a holin-lysin system to accomplish host lysis. Due to the lack of lysin export signals, it is assumed that holin disruption of the cytoplasmic membrane allows endolysin access to the peptidoglycan. We investigated the lysis mechanism of pneumococcal phage SV1, by using lysogens without holin activity. Upon phage induction in a holin deficient background, phage lysin was gradually targeted to the cell wall, in spite of lacking any obvious signal sequence. Our data indicate that export of the phage lysin requires the presence of choline in the teichoic acids, an unusual characteristic of pneumococci. At the bacterial surface, the exolysin remains bound to choline residues without inducing lysis, but is readily activated by the collapse of the membrane potential. Additionally, the activation of the major autolysin LytA, which also participates in phage-mediated lysis, is equally related to perturbations of the membrane proton motive force. These results indicate that collapse of the membrane potential by holins is sufficient to trigger bacterial lysis. We found that the lysin of phage SV1 reaches the peptidoglycan through a novel holin-independent pathway and propose that the same mechanism could be used by other pneumococcal phages.

Since 2010 the 13-valent pneumococcal conjugate vaccine (PCV13) replaced the 7-valent vaccine (PCV7) as the leading pneumococcal vaccine used in children through the private sector. Although, neither of the PCVs were used significantly in adults, changes in adult invasive pneumococcal disease (IPD) were expected due to herd protection. We characterized n = 1163 isolates recovered from IPD in adults in 2012-2014 with the goal of documenting possible changes in serotype prevalence and antimicrobial resistance. Among the 54 different serotypes detected, the most frequent, accounting for half of all IPD, were serotypes: 3 (14%), 8 (11%), 19A (7%), 22F (7%), 14 (6%), and 7F (5%). The proportion of IPD caused by PCV7 serotypes remained stable during the study period (14%), but was smaller than in the previous period (19% in 2009-2011, p = 0.003). The proportion of IPD caused by PCV13 serotypes decreased from 51% in 2012 to 38% in 2014 (p < 0.001), mainly due to decreases in serotypes 7F and 19A. However, PCV13 serotype 3 remained relatively stable and the most frequent cause of adult IPD. Non-PCV13 serotypes continued the increase initiated in the late post-PCV7 period, with serotypes 8 and 22F being the most important emerging serotypes. Serotype 15A increased in 2012-2014 (0.7% to 3.5%, p = 0.011) and was strongly associated with antimicrobial resistance. However, the decreases in resistant isolates among serotypes 14 and 19A led to an overall decrease in penicillin non-susceptibility (from 17 to 13%, p = 0.174) and erythromycin resistance (from 19 to 13%, p = 0.034). Introduction of PCV13 in the NIP for children, as well as its availability for adults may further alter the serotypes causing IPD in adults in Portugal and lead to changes in the proportion of resistant isolates. info:eu-repo/semantics/publishedVersion

BACKGROUND: Foot infections are a major cause of morbidity in people with diabetes and the most common cause of diabetes-related hospitalization and lower extremity amputation. Staphylococcus aureus is by far the most frequent species isolated from these infections. In particular, methicillin-resistant S. aureus (MRSA) has emerged as a major clinical and epidemiological problem in hospitals. MRSA strains have the ability to be resistant to most β-lactam antibiotics, but also to a wide range of other antimicrobials, making infections difficult to manage and very costly to treat. To date, there are two fifth-generation cephalosporins generally efficacious against MRSA, ceftaroline and ceftobripole, sharing a similar spectrum. Biofilm formation is one of the most important virulence traits of S. aureus. Biofilm growth plays an important role during infection by providing defence against several antagonistic mechanisms. In this study, we analysed the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains isolated from diabetic foot infections. The antibiotic minimum inhibitory concentration (MIC) was determined for ten antimicrobial compounds, along with the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC), followed by PCR identification of genetic determinants of biofilm production and antimicrobial resistance. RESULTS: Results demonstrate that very high concentrations of the most used antibiotics in treating diabetic foot infections (DFI) are required to inhibit S. aureus biofilms in vitro, which may explain why monotherapy with these agents frequently fails to eradicate biofilm infections. In fact, biofilms were resistant to antibiotics at concentrations 10-1000 times greater than the ones required to kill free-living or planktonic cells. The only antibiotics able to inhibit biofilm eradication on 50 % of isolates were ceftaroline and gentamicin. CONCLUSIONS: The results suggest that the antibiotic susceptibility patterns cannot be applied to biofilm established infections. Selection of antimicrobial therapy is a critical step in DFI and should aim at overcoming biofilm disease in order to optimize the outcomes of this complex pathology.

Background Diabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes are foot infections that may be colonized by pathogenic and antimicrobial resistant bacteria, harboring several virulence factors, that could impair its successful treatment. Staphylococcus aureus is one of the most prevalent isolate in diabetic foot infections, together with aerobes and anaerobes. Methods In this study, conducted in the Lisbon area, staphylococci isolated (n = 53) from diabetic foot ulcers were identified, genotyped and screened for virulence and antimicrobial resistance traits. Genetic relationship amongst isolates was evaluated by pulsed-field-gel-electrophoresis with further multilocus sequence typing of the identified pulsotypes. PCR was applied for detection of 12 virulence genes and e-test technique was performed to determine minimal inhibitory concentration of ten antibiotics. Results Among the 53 isolates included in this study, 41 Staphylococcus aureus were identified. Staphylococcal isolates were positive for intercellular adhesins icaA and icaD, negative for biofilm associated protein bap and pantone-valentine leucocidin pvl. S. aureus quorum sensing genes agrI and agrII were identified and only one isolate was positive for toxic shock syndrome toxin tst. 36 % of staphylococci tested were multiresistant and higher rates of resistance were obtained for ciprofloxacin and erythromycin. Clonality analysis revealed high genomic diversity and numerous S. aureus sequence types, both community- and hospital-acquired, belonging mostly to clonal complexes CC5 and C22, widely diffused in Portugal nowadays. Conclusions This study shows that diabetic foot ulcer staphylococci are genomically diverse, present resistance to medically important antibiotics and harbour virulence determinants. These properties suggest staphylococci can contribute to persistence and severity of these infections, leading to treatment failure and to the possibility of transmitting these features to other microorganisms sharing the same niche. In this context, diabetic patients may become a transmission vehicle for microorganisms’ clones between community and clinical environments.

Plasmids play a key role in the genetic plasticity and survival of Staphylococcus aureus in challenging environments. Although many S. aureus plasmids have been described, still few studies portray the plasmid content of a given S. aureus population. The aim of this work was to characterize the plasmids carried by a collection of 53 S. aureus isolates collected in a large hospital in Lisbon, Portugal, and investigate their role in conferring resistance to several antimicrobial agents. Plasmids were present in 44 out of the 53 isolates and were grouped into eleven AccI restriction profiles. Plasmid curing of representative strains and comparison of antimicrobial susceptibility profiles between pairs of isogenic strains proved to be a valuable guidance tool in the identification of plasmid-located resistance genes. The plasmids harbored several resistance genes, namely blaZ (resistance to β-lactams), erm(C) (resistance to macrolides, lincosamides, and streptogramin B), cadA (resistance to cadmium and zinc), cadD (resistance to cadmium), and qacA and smr (resistance to biocides and dyes). This study demonstrates the impact of plasmids on the resistance properties of S. aureus, highlighting their role in the dissemination of antibiotic, heavy metal, and biocide resistance genes, and survival of this major pathogen in the hospital environment.

The pathogenic role of beta-hemolytic Streptococcus dysgalactiae in the equine host is increasingly recognized. A collection of 108 Lancefield group C (n = 96) or L (n = 12) horse isolates recovered in the United States and in three European countries presented multilocus sequence typing (MLST) alleles, sequence types and emm types (only 56% of the isolates could be emm typed) that were, with few exceptions, distinct from those previously found in human Streptococcus dysgalactiae subsp. equisimilis. Characterization of a subset of horse isolates by multilocus sequence analysis (MLSA) and 16S rRNA gene sequence showed that most equine isolates could also be differentiated from S. dysgalactiae strains from other animal species, supporting the existence of a horse specific genomovar. Draft genome information confirms the distinctiveness of the horse genomovar and indicates the presence of potentially horse-specific virulence factors. While this genomovar represents most of the isolates recovered from horses, a smaller MLST and MLSA defined sub-population seems to be able to cause infections in horses, other animals and humans, indicating that transmission between hosts of strains belonging to this group may occur.

Among the 1660 isolates recovered from invasive pneumococcal disease (IPD) in adults (> = 18 yrs) in 2008–2011, a random sample of ≥50% of each serotype (n = 871) was chosen for MLST analysis and evaluation for the presence and type of pilus islands (PIs). The genetic diversity was high with 206 different sequence types (STs) detected, but it varied significantly between serotypes. The different STs represented 80 clonal complexes (CCs) according to goeBURST with the six more frequent accounting for more than half (50.6%) of the isolates—CC156 (serotypes 14, 9V and 23F), CC191 (serotype 7F), CC180 (serotype 3), CC306 (serotype 1), CC62 (serotypes 8 and 11A) and CC230 (serotype 19A). Most of the isolates (n = 587, 67.3%) were related to 29 Pneumococcal Molecular Epidemiology Network recognized clones. The overall proportion of isolates positive for any of the PIs was small (31.9%) and declined gradually during the study period (26.6% in 2011), mostly due to the significant decline of serotype 1 which is associated with PI-2. The changes in serotypes that occurred in adult IPD after the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) for children were mostly due to the expansion of previously circulating clones, while capsular switching was infrequent and not related to vaccine use. The reduction of IPD caused by PCV7 serotypes in the years following PCV7 implementation did not result in a decline of antimicrobial resistance in part due to the selection of resistant genotypes among serotypes 14 and 19A.

A steady decline in macrolide resistance among Streptococcus pyogenes (group A streptococci [GAS]) in Portugal was reported during 1999 to 2006. This was accompanied by alterations in the prevalence of macrolide resistance phenotypes and in the clonal composition of the population. In order to test whether changes in the macrolide-resistant population reflected the same changing patterns of the overall population, we characterized both macrolide-susceptible and -resistant GAS associated with a diagnosis of tonsillo-pharyngitis recovered in the period from 2000 to 2005 in Portugal. Pulsed-field gel electrophoresis (PFGE) profiling was the best predictor of emm type and the only typing method that could discriminate clones associated with macrolide resistance and susceptibility within each emm type. Six PFGE clusters were significantly associated with macrolide susceptibility: T3-emm3-ST406, T4-emm4-ST39, T1-emm1-ST28, T6-emm6-ST382, B3264-emm89-ST101/ST408, and T2-emm2-ST55. Four PFGE clusters were associated with macrolide resistance: T4-emm4-ST39, T28-emm28-ST52, T12-emm22-ST46, and T1-emm1-ST28. We found no evidence for frequent ongoing horizontal transfer of macrolide resistance determinants. The diversity of the macrolide-resistant population was lower than that of susceptible isolates. The differences found between the two populations suggest that the macrolide-resistant population of GAS has its own dynamics, independent of the behavior of the susceptible population.

[Figure][1] A 27-year-old man developed high fever after returning from sub-Saharan Africa. Trying to overcome this febrile illness during his annual leave (winter season), he had medical home visits, but the fever was repeatedly misdiagnosed as a flulike illness and treated with paracetamol

Macrolide resistance among Streptococcus pyogenes (group A streptococci) in Portugal was stable during 1998–2003, but a rapid inversion in the dominant phenotypes was noted in the same period, with a sharp decrease in the proportion of isolates presenting the MLSB phenotype and a concomitant increase in isolates presenting the M phenotype. The characterization of group A streptococci recovered during 2004–2006, which is reported here, revealed that resistance was not stable during this period and that the decline in erythromycin resistance observed during 2004–2006 was due to a decrease in the prevalence of isolates presenting the M phenotype, while the proportion of isolates expressing the MLSB phenotype remained stable. Characterization by emm typing, T serotyping, pulsed-field gel electrophoresis (PFGE) profiling and multilocus sequence typing revealed a very diverse population. Several of the major PFGE clusters identified had already been found in the 1998–2003 study period, but others were found for the first time, e.g. T11/emm11/ST403, carrying the erm(B) gene, and T3/13/emm3/ST315, carrying the mef(A) gene. The clone defined as T12/emm12/ST36, previously found to be associated with mef(A), was now found to be predominantly associated with erm(B). The clonal dynamics of macrolide-resistant group A streptococci emphasizes the importance of considering factors other than antibiotic consumption in explaining the prevalence of resistant isolates.

The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia complex (Bcc) bacteria requires extensive and aggressive antibiotics therapy, exposing these bacteria to prolonged antibiotics-selective pressure. In the present study, we have compared the susceptibility patterns to 13 antimicrobials of 94 Bcc isolates obtained from 15 Portuguese CF patients in the course of chronic infection during a five-year survey. These isolates were previously genotyped and represent 11 different strains of the species B. cenocepacia (subgroups A and B), B. cepacia, B. multivorans, and B. stabilis. The results are consistent with the notion that CF Bcc isolates are resistant to the most clinically relevant antimicrobials and suggest an uneven distribution of resistance rates among the different species, with B. cenocepacia subgroup A isolates being the most resistant. Phenotypic variants exhibiting differences in the antimicrobial susceptibility patterns were obtained from the sputum samples of clinically deteriorated CF patients during chronic lung infection. The isolation of resistant variants coincided with periods of pulmonary exacerbation and antibiotics therapy.

The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria comprising around 20 related species. These bacteria are important opportunistic pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon, from 1995 to 2006, was re-examined in the present work. These isolates, formerly classified as Burkholderia cepacia (taxon K), are here reclassified as Burkholderia contaminans, including the former B. cepacia IST408, which was the focus of previous studies regarding the biosynthesis of the exopolysaccharide ‘cepacian’. The CF population examined has been previously described as having an exceptionally high representation of B. cepacia, presumably due to a contamination arising from saline solutions for nasal application. Twenty-one additional isolates, obtained from a chronically infected patient, from 2006 to 2010, were also identified as B. contaminans. This study also provides insight into the potential clinical impact of B. contaminans, a species that is rarely associated with CF infections. Isolates belonging to this species were shown to be involved in chronic and transient respiratory infections, and were associated with severe lung function deterioration and with a case of death with cepacia syndrome. However, since the patients were co-infected with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to prevail over B. cenocepacia isolates during co-infection of at least one chronically infected patient.

Objectives Macrolide resistance among Streptococcus pyogenes [group A streptococci (GAS)] in Portugal decreased between 1999 and 2006 and this decrease was accompanied by alterations in the prevalence of macrolide resistance phenotypes and clonal composition of the population. The aims of this study were to determine the macrolide resistance rate, resistance phenotypes and clones of GAS recovered from pharyngitis in 2007-13 in Portugal. Methods Antimicrobial susceptibility was tested by disc diffusion. Macrolide-resistant isolates were characterized by emm typing, T typing, PFGE profiling and MLST, and the presence of macrolide resistance determinants was determined by PCR. Results We found continuing changes in macrolide resistance phenotypes and a persistent decline in overall erythromycin resistance, from 10% in 2007 to 1% in 2013. During this period there was a marked increase in emm11-ST403 cMLSB isolates, the disappearance of the emm3-ST315 M lineage and changes in the prevalence of previously identified GAS clones. Unexpectedly, the decline in erythromycin resistance and the decreasing prevalence of the MLSB phenotype were accompanied by a high consumption of long-acting and intermediate-acting macrolides, known to select for resistance and particularly for the erm(B) gene. Conclusions The continuous decline in macrolide resistance detected since 2000, accompanied by a high clonal instability, emphasizes the importance of considering factors other than antibiotic consumption in explaining the prevalence of macrolide-resistant GAS.

Streptococcosis caused by Streptococcus agalactiae is one of the most important diseases in the tilapia aquaculture industry. The role of the capsule of Streptococcus agalactiae in adherence to fish surfaces has not been evaluated and the mechanism of capsular regulation during adhesion has not been described. The aim of this study was to evaluate the role of the capsule of S. agalactiae during adhesion to intestinal epithelium of tilapia (Oreochromis sp.) in an ex vivo infection model. We show that the capsule impairs the adhesion of bacteria to host intestinal epithelium. Wild type (WT) strain SaTiBe08-18 (S. agalactiae recovered from tilapia) had reduced adhesion (P < 0.0001) in comparison with its unencapsulated mutant of SaTiBe08-18 (Δcps). When WT was treated with sterile saline solution (pH 5) before infection of intestine explants, the adhesion was reached. The results suggest that the capsule impairs the adhesion of S. agalactiae to tilapia intestine and that the acidic milieu could regulate adherence of encapsulated strains. We found GlcNAc on the surface of adherent Δcps but not over the capsule in WT. This difference could be explained by the GlcNAc composition of Lancefield group B antigen and the peptidoglycan in GBS (Group B Streptococcus) and also may be related with better exposure of glycosylated adhesins in unencapsulated fish GBS. Understanding capsular regulation during adhesion of S. agalactiae may provide new leads to find a successful anti-adherence therapy to prevent streptococcosis in tilapia.

© 2015 Friães et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited., The emergence of clades within emm89 Streptococcus pyogenes isolates that rapidly became the dominant lineages expressing this emm type was recently reported in the United Kingdom and in a study that included isolates from the United States, Finland, and Iceland (United States/FI/IC). In the United Kingdom, the emerging clade was associated with the absence of the hasABC locus, responsible for the synthesis of the hyaluronic acid capsule. The study from the United States/FI/IC highlighted the strict association of the emerging clade with an nga promoter variant, also found in contemporary emm1 isolates, which results in increased expression of the nga locus. The study from the United Kingdom also examined this region and found that the nga-ifs-slo locus and surrounding sequences of the emerging clade shared 99% DNA identity with that of contemporary emm1 and emm12 strains, but the authors do not offer any information on the nga promoter (1). The acquisition of this region by emm1 isolates is currently considered the major molecular event triggering the success and enhanced virulence of this clone.

The etiological diagnosis of infections caused by Streptococcus pneumoniae has been expanded by non-culture methods, including the detection of pneumococcal DNA in biological samples and the identification of C polysaccharide in urine. The basis for these methods and their use and limitations in the recognition of infections of pneumococcal etiology are reviewed. The usefulness and applicability of traditional tests used for the identification of S. pneumoniae and the current and future usefulness of MALDI-TOF are discussed. The term clone is frequently used in molecular epidemiology, but it is used with different meanings by different authors. We discuss the terms clone, strain and isolate in the context of molecular epidemiology and of the increasing realization of the importance of lateral gene transfer in bacterial pathogen evolution. Of the multiple typing methods used to characterize pneumococci, serotyping, pulsed-field gel electrophoresis and multilocus sequence typing have had the widest acceptance and use. We discuss the major findings regarding disease epidemiology, population biology, and the impact of vaccination on pneumococci afforded by these methods. The advent of whole genome sequencing technologies has the potential to bring new insights onto pneumococcal population biology, evolution and epidemiology. Their impact on our current views and on key concepts in molecular epidemiology are presented.

Copyright © 2015, The Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/, To evaluate the importance of covRS and ropB mutations in invasive disease caused by Group A Streptococci (GAS), we determined the sequence of the covRS and ropB genes of 191 isolates from invasive infections and pharyngitis, comprising a diverse set of emm types and multilocus sequence types. The production of SpeB and the activity of NAD glycohydrolase (NADase) and streptolysin S (SLS) were evaluated. The results support the acquisition of null covS alleles (predicted to eliminate protein function), resulting in downregulation of SpeB and upregulation of NADase and SLS, as a mechanism possibly contributing to higher invasiveness. Among the isolates tested, this mechanism was found to be uncommon (10% of invasive isolates) and was not more prevalent among clones with enhanced invasiveness (including M1T1) but occurred in diverse genetic backgrounds. In lineages such as emm64, these changes did not result in upregulation of NADase and SLS, highlighting the diversity of regulatory pathways in GAS. Despite abrogating SpeB production, null alleles in ropB were not associated with invasive infection. The covRS and ropB genes are under stabilising selection and no expansion of isolates carrying null alleles has been observed, suggesting that the presence of these regulators is important for overall fitness.

Background Efflux has been recognized as a resistance mechanism to antimicrobials in Staphylococcus aureus; however its role on the development of clinically relevant resistance is still poorly characterized. This study aimed to examine the impact of efflux on development of resistance to fluoroquinolones and other antimicrobials in S. aureus strains representing relevant phenotypes in terms of antibiotic susceptibility and efflux activity. Methods Two closely related methicillin- and ciprofloxacin-resistant Staphylococcus aureus clinical strains, with different efflux capacity and the pan-susceptible strain ATCC25923 were exposed to constant concentrations of the efflux pump (EP) substrates ciprofloxacin, ethidium bromide and cetrimide. Parental and exposed strains were tested regarding their susceptibility towards antibiotics, biocides and ethidium bromide, efflux capacity and levels of EP gene expression. Occurrence of resistance-associated mutations was screened by sequencing. Results Multidrug resistance phenotypes emerged upon exposure, independently of the substrate or its concentration, which were correlated with increased efflux capacity of the exposed strains. The temporal pattern of EP gene expression disclosed an early-response with high expression of several genes, followed by a late-response, characterized by overexpression of specific genes. The overall cell response was more pronounced for strains with an initial basal efflux activity. Remarkably, detection of the IS256 element in the promoter regions of mgrA and norA, in some cases associated with increased gene expression, suggests that these genes may be hot spots for IS256 insertion events. The results obtained with exposure of ATCC25923 to ciprofloxacin were particularly striking, revealing a step-wise development of fluoroquinolone resistance, with a first efflux-mediated response, followed by the occurrence of a mutation in grlA that resulted in phenotypic resistance. Additionally, challenge by non-fluoroquinolone agents, particularly cetrimide, promoted cross resistance to fluoroquinolones, revealing the potential role of biocides as selective pressure for the emergence of resistance to these antibiotics. Conclusions This study reveals efflux as a significant component of S. aureus resistance to fluoroquinolones and biocides and as a primary mechanism to withstand stress imposed by antimicrobials. This efflux-mediated response can result in the emergence of multidrug resistance in healthcare environments and should be taken into account in the management of this major pathogen. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0572-8) contains supplementary material, which is available to authorized users.

Recently, two related chimeric genetic elements (Tn 1207.3 and Φ10394.4) were shown to carry the macrolide efflux gene mef in Streptococcus pyogenes (group A streptococci [GAS]). The dissemination of elements belonging to the Tn 1207.3 /Φ10394.4 family in recent isolates of GAS, Streptococcus dysgalactiae subsp. equisimilis , Streptococcus pneumoniae , and Streptococcus agalactiae recovered in Portugal was surveyed. In total, 149 GAS, 18 S. pneumoniae , 4 S. dysgalactiae subsp. equisimilis , and 5 S. agalactiae isolates from infections, presenting the M phenotype of macrolide resistance and containing the mef gene, were screened for the presence of Tn 1207.3 /Φ10394.4 by PCR targeting open reading frames (ORFs) specific for these related elements. All the GAS isolates tested and one of the S. dysgalactiae subsp. equisimilis isolates carried Tn 1207.3. However, neither of these elements was found in the isolates of the other streptococcal species. It was also noted that the DNAs of the isolates carrying Tn 1207.3 were resistant to cleavage by the endonuclease SmaI. Cloning and expression of ORF12 of Tn 1207.3 in Escherichia coli showed that it encoded a methyltransferase that rendered DNA refractory to cleavage by SmaI (M.Spy10394I). Using this characteristic as a marker for the presence of the Tn 1207.3 /Φ10394.4 family, we reviewed the literature and concluded that these genetic elements are widely distributed among tetracycline-susceptible GAS isolates presenting the M phenotype from diverse geographic origins and may have played an important role in the dissemination of macrolide resistance in this species.

Lancefield group G and group C streptococci (GGS and GCS, respectively) are pathogens responsible for a number of life-threatening infections. A collection of 116 recent (1998 to 2004) invasive ( n = 28) and noninvasive ( n = 88) GGS and GCS clinical isolates from Portugal were characterized. All isolates were identified as Streptococcus dysgalactiae subsp. equisimilis and characterized by emm typing and DNA macrorestriction profiling using pulsed-field gel electrophoresis (PFGE). emm typing revealed the presence of 22 distinct types, including 3 novel types. PFGE identified 14 clones with more than two isolates, but over half of the isolates were concentrated in 3 large clones. Individual clones and emm types showed a low level of association, since the majority of the clones included more than one emm type and the same emm type was found among diverse genetic backgrounds. Two emm types, stg2078 and stg10 , were significantly more frequent among invasive isolates, and another two, stg6792 and stg166b , were present only in noninvasive isolates, suggesting a correlation between emm type and invasive disease potential.

Resumo: O Estudo Viriato é um estudo nacional, prospectivo e multicêntrico, de vigilância da susceptibilidade aos antimicrobianos de bactérias frequentemente responsáveis por infecções do aparelho respiratório adquiridas na comunidade. Nos anos de 2003 e 2004 participaram 29 laboratórios de todo o país. Isolaram-se 2945 microrganismos que foram estudados num laboratório coordenador. Das 513 estirpes de Streptococcus pyogenes de doentes com amigdalo-faringite aguda, todas eram susceptíveis à penicilina e outros antibióticos beta-lactâmicos, mas 18,9% eram resistentes à eritromicina, claritromicina e azitromicina. Nas estirpes resistentes foi mais frequente o fenótipo M (67,0%) que confere resistência à eritromicina (CIM90=16 mg/L), claritromicina e azitromicina, mas susceptibilidade à clindamicina (CIM90=0,094 mg/L). De doentes com infecção do aparelho respiratório inferior estudaram-se 1300 estirpes de Streptococcus pneumoniae (pneumococos), 829 de Haemophilus influenzae e 303 de Moraxella catarrhalis. Em S. pneumoniae, 18,4% das estirpes eram resistentes à penicilina (3,5% com resistência elevada), 7,1% à cefuroxima, 0,5% à amoxicilina, 0,5% à amoxicilina/clavulanato, 18,8% à eritromicina, claritromicina e azitromicina, 14,5 % à tetraciclina, 16,5% ao cotrimoxazol e 0,4% à levofloxacina. Nas estirpes resistentes aos macrólidos, dominou o fenótipo MLSB (83,7%), caracterizado por resistência elevada (CIM90 >256 mg/L) à eritromicina, claritromicina, azitromicina e clindamicina. Produziam beta-lactamase 10,0% de H. influenzae e 96,4% de M. catarrhalis. Em H. influenzae demonstrou-se 5,5% de resistência à claritromicina e 13,4% ao cotrimoxazol. A quase totalidade das estirpes era susceptível à amoxicilina / clavulanato, cefuroxima, azitromicina, tetraciclina e ciprofloxacina. Em M. catarrhalis a resistência ao co-trimoxazol foi de 27,1% e à tetraciclina de 1,0%. Todas as estirpes eram susceptíveis à amoxicilina / clavulanato, cefuroxima, claritromicina, azitromicina e ciprofloxacina. De entre o conjunto de antibióticos ensaiado, a penicilina continua a ser o mais activo contra S. pyogenes e a amoxicilina / clavulanato e as quinolonas os mais activos simultaneamente contra S. pneumoniae, H. influenzae e M. catarrhalis.Rev Port Pneumol 2006; XII (1): 9-29 Abstract: The Viriato Study is a nationwide, prospective, multicenter surveillance study of the antimicrobial susceptibility of bacterial pathogens commonly associated with community-acquired respiratory tract infections in Portugal. In 2003 and 2004 a total of 2945 isolates was recovered in the 29 laboratories that participated in the study. Testing was undertaken in a central laboratory. Of the 513 Streptococcus pyogenes strains isolated from patients with acute tonsillitis all were susceptible to penicillin and other beta-lactams but 18.9% were resistant to erythromycin, clarithromycin and azithromycin. The M phenotype dominated (67%), conferring resistance to erythromycin (MIC90=16 mg/L), clarythromycin and azithromycin, but susceptibility to clindamycin (MIC90=0.094 mg/L). From patients with lower respiratory tract infection 1,300 strains of Streptococcus pneumoniae, 829 of Haemophilus influenzae, and 303 of Moraxella catarrhalis were studied. Among S. pneumoniae isolates 18.4% were resistant to penicillin (3.5% showing high-level resistance), 7.1% to cefuroxime, 0.5% to amoxicillin and amoxicillin/ clavulanate, 18.8% to erythromycin, clarithromycin and azithromycin, 14.9% to tetracycline, 16.5% to co-trimoxazol, and 0.4% to levofloxacin. Beta-lactamases were produced by 10.0% of H. influenzae and 96.4% of M. catarrhalis. In H. influenzae resistance to clarithromycin was 5.5% and to cotrimoxazole was 13.4%. Most strains were susceptible to amoxicillin/clavulanate, cefuroxime, azithromycin, tetracycline and ciprofloxacin. In M. catarrhalis resistance to co-trimoxazole was 27.1% and to tetracycline 1.0%. All strains were susceptible to amoxicillin/clavulanate, cefuroxime, clarithromycin, azithromycin and ciprofloxacin. Penicillin was the most active antimicrobial agent against S. pyogenes and amoxycillin / clavulanate and the quinolones the most active in vitro simultaneously against S. pneumoniae, H. influenza and M. catarrhalis.Rev Port Pneumol 2006; XII (1): 9-29 Palavras-chave: Portugal, Estudo Viriato, infecção respiratória, comunidade, 2003, 2004, susceptibilidade aos antimicrobianos, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Key-words: Portugal, Viriato Study, respiratory tract infections, community, 2003, 2004, antimicrobial resistance, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis

The Viriato Study is a nationwide, prospective, multicenter surveillance study of the antimicrobial susceptibility of bacterial pathogens commonly associated with community-acquired respiratory tract infections in Portugal. In 2003 and 2004 a total of 2945 isolates was recovered in the 29 laboratories that participated in the study. Testing was undertaken in a central laboratory. Of the 513 Streptococcus pyogenes strains isolated from patients with acute tonsillitis all were susceptible to penicillin and other beta-lactams but 18.9% were resistant to erythromycin, clarithromycin and azithromycin. The M phenotype dominated (67%), conferring resistance to erythromycin (MIC90 = 16 mg/L), clarythromycin and azithromycin, but susceptibility to clindamycin (MIC90 = 0.094 mg/L). From patients with lower respiratory tract infection 1,300 strains of Streptococcus pneumoniae, 829 of Haemophilus influenzae, and 303 of Moraxella catarrhalis were studied. Among S. pneumoniae isolates 18.4% were resistant to penicillin (3.5% showing high-level resistance), 7.1% to cefuroxime, 0.5% to amoxicillin and amoxicillin/clavulanate, 18.8% to erythromycin, clarithromycin and azithromycin, 14.9% to tetracycline, 16.5% to co-trimoxazol, and 0.4% to levofloxacin. Beta-lactamases were produced by 10.0% of H. influenzae and 96.4% of M. catarrhalis. In H. influenzae resistance to clarithromycin was 5.5% and to cotrimoxazole was 13.4%. Most strains were susceptible to amoxicillin/clavulanate, cefuroxime, azithromycin, tetracycline and ciprofloxacin. In M. catarrhalis resistance to co-trimoxazole was 27.1% and to tetracycline 1.0%. All strains were susceptible to amoxicillin/clavulanate, cefuroxime, clarithromycin, azithromycin and ciprofloxacin. Penicillin was the most active antimicrobial agent against S. pyogenes and amoxycillin / clavulanate and the quinolones the most active in vitro simultaneously against S. pneumoniae, H. influenza and M. catarrhalis. Resumo: O Estudo Viriato é um estudo nacional, prospectivo e multicêntrico, de vigilância da susceptibilidade aos antimicrobianos de bactérias frequentemente responsáveis por infecções do aparelho respiratório adquiridas na comunidade. Nos anos de 2003 e 2004 participaram 29 laboratórios de todo o país. Isolaram-se 2945 microrganismos que foram estudados num laboratório coordenador. Das 513 estirpes de Streptococcus pyogenes de doentes com amigdalo-faringite aguda, todas eram susceptíveis à penicilina e outros antibióticos beta-lactâmicos, mas 18,9% eram resistentes à eritromicina, claritromicina e azitromicina. Nas estirpes resistentes foi mais frequente o fenótipo M (67,0%) que confere resistência à eritromicina (CIM90 = 16 mg/L), claritromicina e azitromicina, mas susceptibilidade à clindamicina (CIM90 = 0,094 mg/L). De doentes com infecção do aparelho respiratório inferior estudaram-se 1300 estirpes de Streptococcus pneumoniae (pneumococos), 829 de Haemophilus influenzae e 303 de Moraxella catarrhalis. Em S. pneumoniae, 18,4% das estirpes eram resistentes à penicilina (3,5% com resistência elevada), 7,1% à cefuroxima, 0,5% à amoxicilina, 0,5% à amoxicilina/clavulanato, 18,8% à eritromicina, claritromicina e azitromicina, 14,5% à tetraciclina, 16,5% ao cotrimoxazol e 0,4% à levofloxacina. Nas estirpes resistentes aos macrólidos, dominou o fenótipo MLSB (83,7%), caracterizado por resistência elevada (CIM90>256 mg/L) à eritromicina, claritromicina, azitromicina e clindamicina. Produziam beta--lactamase 10,0% de H. influenzae e 96,4% de M. catarrhalis. Em H. influenzae demonstrou-se 5,5% de resistência à claritromicina e 13,4% ao cotrimoxazol. A quase totalidade das estirpes era susceptível à amoxicilina / clavulanato, cefuroxima, azitromicina, tetraciclina e ciprofloxacina. Em M. catarrhalis a resistência ao co-trimoxazol foi de 27,1% e à tetraciclina de 1,0%. Todas as estirpes eram susceptíveis à amoxicilina / clavulanato, cefuroxima, claritromicina, azitromicina e ciprofloxacina. De entre o conjunto de antibióticos ensaiado, a penicilina continua a ser o mais activo contra S. pyogenes e a amoxicilina / clavulanato e as quinolonas os mais activos simultaneamente contra S. pneumoniae, H. influenzae e M. catarrhalis. Key-words: Portugal, Viriato Study, respiratory tract infections, community, 2003, 2004, antimicrobial resistance, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Palavras-chave: Portugal, Estudo Viriato, infecção respiratória, comunidade, 2003, 2004, susceptibilidade aos antimicrobianos, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis

The availability of a conjugate vaccine has the potential to reduce the disease burden of pneumococci and to alter the serotype frequency in the disease-causing population through immunoselection. These changes will probably be reflected in the distributions of individual genetic lineages within the population. We present a characterization of a collection of recent (1999 to 2002) invasive isolates from Portugal ( n = 465) by macrorestriction profiling with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. During this time, serotypes 14, 1, 3, 4, 8, 9V, 23F, 7F, 19A, and 12B were the 10 most prevalent overall by decreasing rank order. By combining the PFGE data with the sequence types (STs) of 104 isolates, we were able to identify the genetic lineages of the majority of the isolates. We found 66 STs, including 20 novel STs, corresponding to 47 different lineages by e-BURST analysis. We found in our collection a number of previously identified internationally disseminated lineages, especially among macrolide-resistant and penicillin-resistant isolates, and these accounted for most of the isolates. Most of the major lineages (17 of 25) were identified in all years of the study, suggesting that the pneumococcal population associated with invasive disease was stable. This study provides a characterization of the pneumococcal population associated with invasive disease that will be useful for detecting potential selective effects of the novel conjugate vaccine.

Six clinical isolates of Enterobacter cloacae isolated in a Portuguese hospital, between April 1999 and November 2000, demonstrated resistance to almost all broad-spectrum cephalosporins, except to cefepime. These isolates were susceptible to quinolones and to aminoglycosides. lsoelectric focusing demonstrated production of beta-lactamases with pIs 8.0 and by all six isolates, exhibiting a cephalosporinase phenotype. The results of pulsed field gel electrophoresis revealed that these isolates were genetically unrelated. The amino acid sequence of six AmpC beta-lactamases (EclolFF, Eclo6FF, Eclo9FF, Eclo1OFF, Eclo11FF and Eclol5FF) shared 97-99% homology with the chromosomal AmpC beta-lactamase from E. cloacae P99 and 86-87% homology with those of two plasmid-mediated AmpC beta-lactamases, MIR-1 and ACT-1. This is the first report of chromosomal AmpC beta-lactamase production by E cloacae isolates in a Portuguese hospital. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

© Mary Ann Liebert, Inc., publishers. All rights reserved, USA and worldwide., A nationwide multicenter study (including 31 laboratories) of the antimicrobial susceptibility of 1210 Streptococcus pneumoniae isolates from patients with community-acquired lower respiratory tract infections (LRTI) was carried out over 3 years (1999-2001) in Portugal. Testing of all isolates was undertaken in a central laboratory. Overall macrolide resistance was 13.1%. Decreased susceptibility to penicillin was 24.5% (15.5% low-level and 9.0% high-level resistance). Taken into consideration, the resistance rates reported in a previous surveillance study of 1989-1993, a six-fold increase of erythromycin resistance in the last decade was documented. Resistance to erythromycin, clarithromycin, and azithromycin was higher in pediatric patients than in adults. The overwhelming majority (82.3%) of macrolide-resistant isolates were multidrug resistant, although 44.9% were fully susceptible to penicillin. Most macrolide-resistant isolates (80.4%) showed the MLSB phenotype (76.6% MLSB-constitutive resistance, and 3.8% MLSB-inducible resistance) and were also resistant to clindamycin, tetracycline, and co-trimoxazole. The M phenotype was seen in 19.6% isolates and these had MIC90 values of 8 mg/L for erythromycin and clarithromycin, and of 12 mg/L for azithromycin. The clinical significance of macrolide resistance in the management of LRTI is discussed. Because of the specific situation concerning macrolide resistance described in S. pneumoniae, careful use of macrolide antibiotics in therapy and cautious monitoring of macrolide resistance should be continued in Portugal.

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development., SCOPUS: ar.j, info:eu-repo/semantics/published