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IntroductionNon-invasive pneumococcal pneumonia (NIPP) is possibly the most frequent infection by Streptococcus pneumoniae in adults. However, the herd effect of vaccinating children in adult NIPP (aNIPP) remains poorly characterized.MethodsWe determined the serotype distribution and antimicrobial susceptibility of isolates causing aNIPP (>18 years) in 2016–2018 in Portugal; 3 years with near universal vaccination of children with the 13-valent conjugate vaccine (PCV13), following over a decade of significant PCV use in children in the private market.Results and discussionAmong the 1,149 aNIPP isolates, the most frequent serotypes detected were: 3 (n = 168, 14.6%), 11A (n = 102, 8.9%), 19F (n = 70, 6.1%), 23A and 23B (n = 62, 5.4% each), 9N (n = 60, 5.2%), 8 and 29/35B (n = 43, 3.7% each); together accounting for 53% of all isolates. The serotype distribution causing aNIPP was stable in 2016–2018, with the serotypes included in PCV7 still being important causes of disease and serotype 3, a PCV13 serotype, remaining the leading cause of aNIPP. There was an increase in penicillin non-susceptibility from 17% in 2016 to 24% in 2018 (p = 0.018). Some PCV13 serotypes, such as 14, 19A and 19F were associated to resistance, which may have contributed to their persistence. The fact that close to 20% of aNIPP is caused by four non-vaccine serotypes (23A, 23B, 9N, and 29/35B) and that there were significant differences in serotype distribution relative to invasive disease, stress the importance of maintaining the surveillance of these infections. The lack of a continued herd effect from vaccinating children and the significant fraction of aNIPP potentially preventable by PCV13 (30%), PCV15 (34%), PCV20 (53%) and the 23-valent polysaccharide vaccine (61%) underscore the importance of considering the broader use of pneumococcal vaccines in adults.

Abstract Klebsiella pneumoniae (Kp) bacteria are an increasing threat to public health and represent one of the most concerning pathogens involved in life-threatening infections and antimicrobial resistance (AMR). To understand the epidemiology of AMR of Kp in Portugal, we analysed whole genome sequencing, susceptibility testing and other meta data on 509 isolates collected nationwide from 16 hospitals and environmental settings between years 1980 and 2019. Predominant sequence types (STs) included ST15 (n = 161, 32%), ST147 (n = 36, 7%), ST14 (n = 26, 5%) or ST13 (n = 26, 5%), while 31% of isolates belonged to STs with fewer than 10 isolates. AMR testing revealed widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems. The most common carbapenemase gene was bla KPC-3 . Whilst the distribution of AMR linked plasmids appears uncorrelated with ST, their frequency has changed over time. Before year 2010, the dominant plasmid group was associated with the extended spectrum beta-lactamase gene bla CTX-M-15 , but this group appears to have been displaced by another carrying the bla KPC-3 gene. Co-carriage of bla CTX-M and bla KPC-3 was uncommon. Our results from the largest genomics study of Kp in Portugal highlight the active transmission of strains with AMR genes and provide a baseline set of variants for future resistance monitoring and epidemiological studies.

Abstract Vitamin D is a fundamental regulator of host defences by activating genes related to innate and adaptive immunity. Previous research shows a correlation between the levels of vitamin D in patients infected with SARS-CoV-2 and the degree of disease severity. This work investigates the impact of the genetic background related to vitamin D pathways on COVID-19 severity. For the first time, the Portuguese population was characterized regarding the prevalence of high impact variants in genes associated with the vitamin D pathways. This study enrolled 517 patients admitted to two tertiary Portuguese hospitals. The serum concentration of 25 (OH)D, was measured in the hospital at the time of patient admission. Genetic variants, 18 variants, in the genes AMDHD1, CYP2R1, CYP24A1, DHCR7, GC, SEC23A, and VDR were analysed. The results show that polymorphisms in the vitamin D binding protein encoded by the GC gene are related to the infection severity (p = 0.005). There is an association between vitamin D polygenic risk score and the serum concentration of 25 (OH)D (p = 0.04). There is an association between 25 (OH)D levels and the survival and fatal outcomes (p = 1.5e−4). The Portuguese population has a higher prevalence of the DHCR7 RS12785878 variant when compared with its prevalence in the European population (19% versus 10%). This study shows a genetic susceptibility for vitamin D deficiency that might explain higher severity degrees in COVID-19 patients. These results reinforce the relevance of personalized strategies in the context of viral diseases. Trial registration: NCT04370808.

ABSTRACT Imipenem-relebactam is a novel β-lactam-β-lactamase inhibitor combination. We evaluated the in vitro activity of imipenem-relebactam and comparators against Enterobacterales clinical isolates recovered in 8 Spanish and 11 Portuguese intensive care units (ICUs) (SUPERIOR, 2016–2017; STEP, 2017–2018). Overall, 747 Enterobacterales isolates (378 Escherichia coli, 252 Klebsiella spp., 64 Enterobacter spp., and 53 other species) were prospectively collected from ICU patients with complicated intraabdominal (cIAI), complicated urinary tract (cUTI), and lower respiratory tract (LRTI) infections. MICs were determined (ISO-broth microdilution), and whole-genome sequencing (WGS) was performed in a subset of isolates displaying susceptible and resistant imipenem-relebactam MICs. Imipenem-relebactam (98.7% susceptible) showed similar activity to ceftazidime-avibactam (99.5% susceptible) and higher than ceftolozane-tazobactam (86.9% susceptible). Imipenem-relebactam was inactive against 1.3% (10/747) isolates, all of them due to carbapenemase production (9 K. pneumoniae and 1 E. cloacae). Imipenem-relebactam was active against 100% of extended-spectrum β-lactamase (ESBL)-E. coli and ESBL-Klebsiella spp. isolates and 80.4% of carbapenemase-Klebsiella spp. producers. Carbapenemase genes were confirmed by WGS in 41 Klebsiella spp.: OXA-48 (20/41), KPC-3 (14/41), OXA-181 (4/41), NDM-1 (1/41), OXA-48 + VIM-2 (1/41), and KPC-3 + VIM-2 (1/41). In Klebsiella spp. isolates, relebactam restored imipenem susceptibility in all KPC-3 producers, and resistant isolates (7/41) were mostly OXA-48 + CTX-M-15-K. pneumoniae high-risk clones (7/9). Intercountry differences were detected as follows: OXA-48 (17/21) was dominant in Spain, unlike KPC-3 (14/15) in Portugal. Imipenem-relebactam was 100% active against CTX-M-15-ST131-H30Rx-E. coli high-risk clone, predominant in both countries. Our results depict the potential role of imipenem-relebactam in ICU patients with cIAIs, cUTIs, and LRTIs due to wild-type ESBL- and carbapenemase-producing Enterobacterales, particularly KPC producers. IMPORTANCE We comparatively evaluate the in vitro activity of a drug combination consisting of a carbapenem (imipenem) and a novel inhibitor of beta-lactamases (relebactam), a mechanism that destroys beta-lactam antibiotics. We assess the activity against a collection of Enterobacterales clinical isolates recovered from difficult-to-treat infections in patients admitted to different intensive care units in Portugal and Spain. Imipenem-relebactam shows excellent activity in avoiding common resistance mechanisms in this setting, such as extended-spectrum beta-lactamases and carbapenemases widely distributed, including KPCs. We show few resistant isolates (

ABSTRACT The prevalence and lineages of Streptococcus agalactiae (group B streptococci [GBS]) colonizing pregnant women are well studied, but less is known about colonization of nonpregnant adults. We characterized GBS colonization in adults as a potential reservoir for infections and tested for the presence of clones with a potentially higher invasive disease potential. We evaluated GBS gastrointestinal, genitourinary, and oral colonization among 336 nonpregnant adults in the community. We characterized the isolates by serotyping, multilocus sequence typing, profiling of surface protein genes and pili, and antimicrobial susceptibility and compared them with contemporary invasive isolates. The colonization rate (n = 107, 32%) among nonpregnant adults was like that of pregnant women. Colonization increased with age (~25% in the 18 to 29 and 30 to 44 years old groups and >42% in the ≥60 years old group), potentially explaining the higher incidence of disease with older age. Participants who were colonized at multiple sites (73%) were frequently carrying indistinguishable strains (93%), consistent with the existence of a single reservoir of colonization and transfer of GBS between sites within the same individual. The most frequent lineages found were serotype Ib/CC1 (n = 27), serotype V/CC1 (n = 19), serotype Ia/CC23 (n = 13), serotype III/ST17 (n = 13), and serotype Ib/CC10 (n = 11). Comparison with contemporary isolates causing invasive infections in Portugal did not reveal any lineage associated with either asymptomatic carriage or invasive disease. Asymptomatic colonization of nonpregnant adults is significant and could act as a reservoir for invasive disease, but in contrast to infant disease, we found no GBS lineages with an enhanced potential for causing invasive disease in adults. IMPORTANCE The increasing incidence of Streptococcus agalactiae (group B streptococci [GBS]) infections in adults and the inability of antimicrobial prophylaxis peripartum to control late-onset infections in infants motivate the study of the asymptomatic carrier state in nonpregnant adults. We found an overall carriage rate like that of pregnant women, increasing with age, potentially contributing to the higher incidence of GBS infections with age. Colonization of diabetic participants was not higher despite the higher number of infections in this group. Comparison between contemporary genetic lineages causing infections and found in asymptomatic carriers did not identify particularly virulent lineages. This means that any prophylactic approaches targeting colonization by particular lineages are expected to have a limited impact on GBS disease in adults.

The carbapenem-resistant Enterobacterales (CRE) strains have been identified by the World Health Organization as critical priority pathogens in research and development of diagnostics, treatments, and vaccines. However, recent molecular information about carbapenem-resistant K. pneumoniae (CRK) epidemiology in Portugal is still scarce. Thus, this study aimed to provide the molecular epidemiology, resistome, and virulome of CRK clinical strains recovered from a tertiary care hospital centre (2019–2021) using polymerase chain reaction (PCR) and the advanced molecular technique whole-genome sequencing (WGS). PCR amplification of carbapenemase genes was performed in 437 carbapenem-resistant K. pneumoniae strains. The most frequent carbapenemases were: KPC-3 (42%), followed by OXA-181 (20%), GES-5 (0.2%), and NDM-1 (0.2%). Additionally, 10 strains (2%) coproduced KPC-3 and OXA-181, and 1 strain coproduced KPC-3 and OXA-48 (0.2%). The genomic population structure of 68 strains characterized by WGS demonstrated the ongoing dissemination of four main high-risk clones: ST13, ST17, ST147, and ST307, while no clones belonging to the European predominant clonal groups (CG15 and CG258) were found. Moreover, we describe one K. pneumoniae ST39-KL62 that coproduced the NDM-1 carbapenemase and the extended-spectrum beta-lactamase CTX-M-15, and one K. pneumoniae ST29-KL54 GES-5 and BEL-1 coproducer. Furthermore, a high prevalence of iron siderophores were present in all CRK strains, with several strains presenting both colibactin and the hypermucoviscosity phenotype. Thus, the data presented here highlight an uncommon molecular epidemiology pattern in Portugal when compared with most European countries, further supporting the emergence and dissemination of nonclonal group 258 hypervirulent multidrug high-risk clones and the need to promote in-depth hospital molecular surveillance studies.

The combination of ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. Emerging cases caused by CZA-resistant strains that produce variants of KPC genes have already been reported worldwide. However, to the best of our knowledge, no CZA-resistant strains were reported in Portugal. In September 2019, a K. pneumoniae CZA-resistant strain was collected from ascitic fluid at a surgery ward of a tertiary University Hospital Center in Lisboa, Portugal. The strain was resistant to ceftazidime/avibactam, as well as to ceftazidime, cefoxitin, gentamicin, amoxicillin/clavulanic acid, and ertapenem, being susceptible to imipenem and tigecycline. A hypermucoviscosity phenotype was confirmed by string test. Whole-genome sequencing (WGS) analysis revealed the presence of an ST13 KPC70-producing K. pneumoniae, a KPC-3 variant, differing in two amino-acid substitutions (D179Y and T263A). The D179Y mutation in the KPC Ω-loop region is the most common amino-acid substitution in KPC-2 and KPC-3, further leading to CZA resistance. The second mutation causes a KPC-70 variant in which threonine replaces alanine (T263A). The CZA-resistant strain showed the capsular locus KL3 and antigen locus O1v2. Other important virulence factors were identified: fimbrial adhesins type 1 and type 3, as well as the cluster of iron uptake systems aerobactin, enterobactin, salmochelin, and yersiniabactin included in integrative conjugative element 10 (ICEKp10) with the genotoxin colibactin cluster. Herein, we report the molecular characterization of the first hypervirulent CZA-resistant ST13 KPC-70-producing K. pneumoniae strain in Portugal. The emergence of CZA-resistant strains might pose a serious threat to public health and suggests an urgent need for enhanced clinical awareness and epidemiologic surveillance.

The emergence of multidrug resistant Gram-negative pathogens, particularly carbapenemase producers, has forced clinicians to use last line antibiotics, such as colistin. Since colistin susceptibility testing presents several challenges, this study aimed at evaluating the performance of two alternative susceptibility methods for Klebsiella pneumoniae, namely, agar dilution (AD) and MIC test strips (MTS). These approaches were compared with the reference method, broth microdilution (BMD), and provide a quantitative description for the “skipped well” (SW) phenomenon. Colistin susceptibility was evaluated by BMD and AD in parallel and triplicate, using 141 K. pneumoniae clinical isolates while MTS performance was evaluated only for a subset (n = 121). Minimum inhibitory concentration analysis revealed that a substantial part (n = 26/141; 18.4%) of the initial isolates was deemed undetermined by BMD due to the following: discordance between replicates (1.4%); presence of multiple SWs (7.8%); and the combination of both events (9.2%). Both AD and MTS revealed a high number of false-susceptible strains (“very major errors”), 37.5% and 68.8%, respectively. However, AD agreement indices were reasonably high (EA = 71.3% and CA = 94.8%). For MTS these indices were lower, in particular EA (EA = 41.7% and CA = 89.6), but the approach enabled the detection of distinct sub-populations for four isolates. In conclusion, this study provides the most comprehensive study on the performance of AD and MTS for colistin susceptibility testing in K. pneumoniae, highlighting its limitations, and stressing the importance of sample size and composition. Further, this study highlights the impact of the SW phenomenon associated with the BMD method for K. pneumoniae.

The introduction of pneumococcal conjugate vaccines PCV7 and PCV13 led to decreases in incidence of pediatric invasive pneumococcal disease (pIPD) and changes in serotype distribution. We evaluated the consequences of higher vaccine uptake after the introduction of PCV13 in the National Immunization Plan (NIP) in 2015. Besides culture and conventional serotyping, the use of molecular methods to detect and serotype pneumococci in both pleural and cerebrospinal fluid samples contributed to 30% of all pIPD (n = 232) in 2015–2018. The most frequently detected serotypes were: 3 (n = 59, 26%), 10A (n = 17, 8%), 8 (n = 16, 7%) and 19A (n = 10, 4%). PCV13 serotypes still accounted for 46% of pIPD cases. Serotypes not included in any currently available conjugate vaccine (NVT) are becoming important causes of pIPD, with the increases in serotypes 8 and 33F being of particular concern given the importance of serotype 8 in adult IPD and the antimicrobial resistance of serotype 33F isolates. This study highlights the importance of using molecular methods in pIPD surveillance since these allowed a better case ascertainment and the identification of serotype 3 as the leading cause of pIPD. Even in a situation of vaccine uptake >95% for 3 years, PCV13 serotypes remain important causes of pIPD.

Increasing the uptake of the 13-valent pneumococcal conjugate vaccine (PCV13) in children is expected to alter the serotypes causing invasive pneumococcal disease (IPD) in adults due to herd protection. We characterized 2172 cases of adult IPD in 2015–2018 in Portugal after the introduction of PCV13 in the national immunization plan of 2015. Among the 58 detected serotypes, serotypes 8 (n = 413; 19%), 3 (n = 334; 15%), 22F (n = 148; 7%), 14 (n = 138; 6%), and 19A (n = 116; 5%) were the most frequent. Among PCV13 serotypes, 7F and 19A IPD decreased, but serotype 3 IPD remained stable. The non-PCV13 serotypes were a heterogeneous group, with serotypes 23A and 23B enriched among CSF cases; serotype 8 associated with younger patients; and serotypes 22F, 6C, and 31 associated with older patients. The continued increase of serotype 8 IPD was one of the drivers for the increased coverage of the 23-valent pneumococcal polysaccharide vaccine (PPV23; 80% in 2015–2018). Antimicrobial resistance was associated with older age and serotypes 6C, 11A, 14, 15A, 19A, and 19F. Three years after the introduction of PCV13 in the NIP with an uptake of >95%, the proportion of PCV13 serotypes causing IPD in adults stabilized in Portugal. The direct vaccination of adults may be important in preventing IPD in this age group.

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.

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.

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

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.

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 β-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 β-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 β-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.

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.

Resistance mediated by efflux has been recognized in Staphylococcus aureus in the last few decades, although its clinical relevance has only been recognized recently. The existence of only a few studies on the individual and overall contribution of efflux to resistance phenotypes associated with the need of well-established methods to assess efflux activity in clinical isolates contributes greatly to the lack of solid knowledge of this mechanism in S. aureus. This study aims to provide information on approaches useful to the assessment and characterization of efflux activity, as well as contributing to our understanding of the role of efflux to phenotypes of antibiotic resistance and biocide tolerance in S. aureus clinical isolates. The results described show that efflux is an important contributor to fluoroquinolone resistance in S. aureus and suggest it as a major mechanism in the early stages of resistance development. We also show that efflux plays an important role on the reduced susceptibility to biocides in S. aureus, strengthening the importance of this long neglected resistance mechanism to the persistence and proliferation of antibiotic/biocide-resistant S. aureus in the hospital environment.

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.

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.

There is limited information on the serotypes causing non-invasive pneumococcal pneumonia (NIPP). Our aim was to characterize pneumococci causing NIPP in adults to determine recent changes in serotype prevalence, the potential coverage of pneumococcal vaccines and changes in antimicrobial resistance. Serotypes and antimicrobial susceptibility profiles of a sample of 1300 isolates recovered from adult patients (≥18 yrs) between 1999 and 2011 (13 years) were determined. Serotype 3 was the most frequent cause of NIPP accounting for 18% of the isolates. The other most common serotypes were 11A (7%), 19F (7%), 19A (5%), 14 (4%), 22F (4%), 23F (4%) and 9N (4%). Between 1999 and 2011, there were significant changes in the proportion of isolates expressing vaccine serotypes, with a steady decline of the serotypes included in the 7-valent conjugate vaccine from 31% (1999-2003) to 11% (2011) (P

This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other biochemical analyses such as protein identification. This protocol can be easily adapted to different pneumococcal strains and growth conditions and it is well suited to isolate other proteins of interest.

According to the EARS-Net surveillance data, Portugal has the highest prevalence of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) in Europe, but the information on MRSA in the community is very scarce and the links between the hospital and community are not known. In this study we aimed to understand the events associated to the recent sharp increase in MRSA frequency in Portugal and to evaluate how this has shaped MRSA epidemiology in the community. With this purpose, 180 nosocomial MRSA isolates recovered from infection in two time periods and 14 MRSA isolates recovered from 89 samples of skin and soft tissue infections (SSTI) were analyzed by pulsed-field gel electrophoresis (PFGE), staphylococcal chromosome cassette mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). All isolates were also screened for the presence of Panton Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) by PCR. The results showed that ST22-IVh, accounting for 72% of the nosocomial isolates, was the major clone circulating in the hospital in 2010, having replaced two previous dominant clones in 1993, the Iberian (ST247-I) and Portuguese (ST239-III variant) clones. Moreover in 2010, three clones belonging to CC5 (ST105-II, ST125-IVc and ST5-IVc) accounted for 20% of the isolates and may represent the beginning of new waves of MRSA in this hospital. Interestingly, more than half of the MRSA isolates (8/14) causing SSTI in people attending healthcare centers in Portugal belonged to the most predominant clones found in the hospital, namely ST22-IVh (n = 4), ST5-IVc (n = 2) and ST105-II (n = 1). Other clones found included ST5-V (n = 6) and ST8-VI (n = 1). None of the MRSA isolates carried PVL and only five isolates (ST5-V-t179) carried ACME type II. The emergence and spread of EMRSA-15 may be associated to the observed increase in MRSA frequency in the hospital and the consequent spillover of MRSA into the community.

In Portugal, pneumococcal conjugate vaccines have been administered to children outside of the national immunization plan since 2001. We determined the serotype and antimicrobial susceptibility of 1265 isolates responsible for adult invasive pneumococcal infections (IPD) between 2009 and 2011 and compared the results with previously published data from 1999 to 2008. Serotypes 3 (12.6%), 7F (10.0%), 19A (9.1%), 14 (8.4%), 1 (6.9%) and 8 (6.2%) were the most frequent and together accounted for 53.2% of adult IPD. Serotypes 1 and 5 declined significantly while serotype 34, not included in any vaccine, increased. Taken together, the serotypes included in the 13-valent conjugate vaccine (PCV13) peaked among adult IPD isolates in 2008 (70.2%) and declined since then reaching 53.5% in 2011. The decline in the serotypes included in the 23-valent polysaccharide vaccine since 2007 was also significant but much more modest with 79.2% of the isolates causing IPD in 2011 expressing these serotypes. Since the changes in serotypes causing IPD in adults coincided with the 10-valent and PCV13 introduction in children, it is unlikely that vaccination triggered these changes although it may have accelerated them. The proportion of IPD caused by serotypes included in the 7-valent conjugate vaccine remained stable (19.0%). Both penicillin non-susceptibility and erythromycin resistance increased in the study period, with serotypes 14 and 19A accounting for the majority of resistant isolates.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen that can colonize and infect humans. Although most SDSE isolates possess the Lancefield group G carbohydrate, a significant minority have the group C carbohydrate. Isolates are further sub-typed on the basis of differences within the emm gene. To gain a better understanding of their molecular epidemiology and evolutionary relationships, multilocus sequence typing (MLST) analysis was performed on SDSE isolates collected from Australia, Europe and North America.The 178 SDSE isolates, representing 37 emm types, segregate into 80 distinct sequence types (STs) that form 17 clonal complexes (CCs). Eight STs recovered from all three continents account for >50% of the isolates. Thus, a small number of STs are highly prevalent and have a wide geographic distribution. Both ST and CC strongly correlate with group carbohydrate. In contrast, eleven STs were associated with >1 emm type, suggestive of recombinational replacements involving the emm gene; furthermore, 35% of the emm types are associated with genetically distant STs. Data also reveal a history of extensive inter- and intra-species recombination involving the housekeeping genes used for MLST. Sequence analysis of single locus variants identified through goeBURST indicates that genetic change mediated by recombination occurred approximately 4.4 times more frequently than by point mutation.A few genetic lineages with an intercontinental distribution dominate among SDSE causing infections in humans. The distinction between group C and G isolates reflects recent evolution, and no long-term genetic isolation between them was found. Lateral gene transfer and recombination involving housekeeping genes and the emm gene are important mechanisms driving genetic variability in the SDSE population.

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.

Very diverse research fields frequently deal with the analysis of multiple clustering results, which should imply an objective detection of overlaps and divergences between the formed groupings. The congruence between these multiple results can be quantified by clustering comparison measures such as the Wallace coefficient (W). Since the measured congruence is dependent on the particular sample taken from the population, there is variability in the estimated values relatively to those of the true population. In the present work we propose the use of a confidence interval (CI) to account for this variability when W is used. The CI analytical formula is derived assuming a Gaussian sampling distribution and recurring to the algebraic relationship between W and the Simpson's index of diversity. This relationship also allows the estimation of the expected Wallace value under the assumption of independence of classifications. We evaluated the CI performance using simulated and published microbial typing data sets. The simulations showed that the CI has the desired 95% coverage when the W is greater than 0.5. This behaviour is robust to changes in cluster number, cluster size distributions and sample size. The analysis of the published data sets demonstrated the usefulness of the new CI by objectively validating some of the previous interpretations, while showing that other conclusions lacked statistical support.