Rare serotype c Haemophilus influenzae invasive isolate: characterization of the first case in Portugal.

We report for the first time in Portugal a serotype c Haemophilus influenzae isolated from an adult, with HIV-1 infection. Whole-genome sequencing characterized the isolate as clonal complex ST-7, albeit with a novel MLST (ST2754) due to a unique atpG profile. Integration of this genome with other available H. influenzae serotype c genomes from PubMLST revealed its overall genetic distinctiveness, with the closest related isolate being identified in France in 2020. This surveillance study, involving collaboration among hospitals and reference laboratory, successfully contributed to the identification and characterization of this rare serotype.

Sustained increase of paediatric invasive Streptococcus pyogenes infections dominated by M1UK and diverse emm12 isolates, Portugal, September 2022 to May 2023.

Since autumn 2022, observed numbers of paediatric invasive group A Streptococcus infections in Portugal (n = 89) were higher than in pre-COVID-19 seasons. Between September 2022 and May 2023, the dominant diagnoses were pneumonia (25/79), mostly with empyema (20/25), and sepsis (22/79). A number of cases required admission to intensive care (27/79) and surgery (35/79), and the case fatality rate was 5.1% (4/79). Genomic sequencing (n = 55) revealed multiple genetic lineages, dominated by the M1UK sublineage (26/55) and more diverse emm12 isolates (12/55).

In vitro activity of imipenem/relebactam against Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal (SUPERIOR and STEP studies).

OBJECTIVES: To study the in vitro activity of imipenem/relebactam and comparators and the imipenem/relebactam resistance mechanisms in a Pseudomonas aeruginosa collection from Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17) surveillance studies. METHODS: P. aeruginosa isolates (n = 474) were prospectively recovered from complicated urinary tract (cUTI), complicated intra-abdominal (cIAI) and lower respiratory tract (LRTI) infections in 11 Portuguese and 8 Spanish ICUs. MICs were determined (ISO broth microdilution). All imipenem/relebactam-resistant P. aeruginosa isolates (n = 30) and a subset of imipenem/relebactam-susceptible strains (n = 32) were characterized by WGS. RESULTS: Imipenem/relebactam (93.7% susceptible), ceftazidime/avibactam (93.5% susceptible) and ceftolozane/tazobactam (93.2% susceptible) displayed comparable activity. The imipenem/relebactam resistance rate was 6.3% (Portugal 5.8%; Spain 8.9%). Relebactam restored imipenem susceptibility to 76.9% (103/134) of imipenem-resistant isolates, including MDR (82.1%; 32/39), XDR (68.8%; 53/77) and difficult-to-treat (DTR) isolates (67.2%; 45/67). Among sequenced strains, differences in population structure were detected depending on the country: clonal complex (CC)175 and CC309 in Spain and CC235, CC244, CC348 and CC253 in Portugal. Different carbapenemase gene distributions were also found: VIM-20 (n = 3), VIM-1 (n = 2), VIM-2 (n = 1) and VIM-36 (n = 1) in Spain and GES-13 (n = 13), VIM-2 (n = 3) and KPC-3 (n = 2) in Portugal. GES-13-CC235 (n = 13) and VIM type-CC175 (n = 5) associations were predominant in Portugal and Spain, respectively. Imipenem/relebactam showed activity against KPC-3 strains (2/2), but was inactive against all GES-13 producers and most of the VIM producers (8/10). Mutations in genes affecting porin inactivation, efflux pump overexpression and LPS modification might also be involved in imipenem/relebactam resistance. CONCLUSIONS: Microbiological results reinforce imipenem/relebactam as a potential option to treat cUTI, cIAI and LRTI caused by MDR/XDR P. aeruginosa isolates, except for GES-13 and VIM producers.

Seroprevalence of anti-SARS-CoV-2 antibodies in COVID-19 patients and healthy volunteers up to 6 months post disease onset.

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.

Distinct epidemiology and resistance mechanisms affecting ceftolozane/tazobactam in Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal depicted by WGS.

OBJECTIVES: To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17). METHODS: P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS. RESULTS: Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains. CONCLUSIONS: GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved.

Heterogeneity of penicillin-non-susceptible group B streptococci isolated from a single patient in Germany.

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 ß-lactam-resistant GBS, with two PRGBS variants being isolated from one patient.

Streptococcus pneumoniae Serotype 3 in Mexico (1994 to 2017): Decrease of the Unusual Clonal Complex 4909 Lineage following PCV13 Introduction.

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 < 0.001) following PCV13 introduction (2012 to 2017). The CC4909 isolates were represented mostly by ST1119 (n = 40), seemingly having a restricted geographic origin, with isolates in the PubMLST database having been recovered only in Mexico, the United States, and Germany. A genomic analysis of publicly available genomes showed that ST1119 isolates have less than 32% similarity with ST180 isolates, indicating that these lineages are more separated than revealed by traditional multilocus sequence typing. Considering the suggestions of a lower efficacy of the 13-valent pneumococcal conjugate vaccine against serotype 3, the different dynamics of the two major serotype 3 lineages in Mexico following the introduction of PCV13 should be closely monitored.

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

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

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

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

Susceptibility patterns of Staphylococcus aureus biofilms in diabetic foot infections.

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 ß-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.

Molecular typing, virulence traits and antimicrobial resistance of diabetic foot staphylococci.

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.

Comparing Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Phenotypic and Molecular Methods for Identification of Species within the Streptococcus anginosus Group.

The heterogeneity of members of the Streptococcus anginosus group (SAG) has traditionally hampered their correct identification. Recently, the group was subdivided into 6 taxa whose prevalence among human infections is poorly described. We evaluated the accuracy of the Rapid ID32 Strep test, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and a PCR multiplex method to identify 212 SAG isolates recovered from human infections to the species and subspecies level by using multilocus sequence analysis (MLSA) as the gold standard. We also determined the antimicrobial susceptibilities of the isolates. Representatives of all SAG taxa were found among our collection. MALDI-TOF MS and the Rapid ID32 Strep test correctly identified 92% and 68% of the isolates to the species level, respectively, but showed poor performance at the subspecies level, and the latter was responsible for major identification errors. The multiplex PCR method results were in complete agreement with the MLSA identifications but failed to distinguish the subspecies Streptococcus constellatus subsp. pharyngis and S. constellatus subsp. viborgensis. A total of 145 MLSA sequence types were present in our collection, indicating that within each taxon a number of different lineages are capable of causing infection. Significant antibiotic resistance was observed only to tetracycline, erythromycin, and clindamycin and was present in most taxa. MALDI-TOF MS is a reliable method for routine SAG species identification, while the need for identification to the subspecies level is not clearly established.

Impact of efflux in the development of multidrug resistance phenotypes in Staphylococcus aureus.

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.

Export of the pneumococcal phage SV1 lysin requires choline-containing teichoic acids and is holin-independent.

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.

Methaemoglobin and COHb in patients with malaria.

BACKGROUND: Haemolytic conditions may contribute to disease pathogenesis and severe clinical manifestations through the liberation of free haemoglobin (Hb) and production of toxic free haem. Thus, free Hb and haem should be associated with altered MetHb and COHb levels in malaria as in other conditions. METHODS: This study comprises data collected at three different sites: (i) a retrospective analysis of the first arterial blood gas result (ABGS) of any patient during 2010 at the University Hospital in Lisbon; (ii) a retrospective analysis of ABGS from patients with severe malaria admitted to the intensive care unit in Berlin, Germany; and (iii) a prospective study of non-invasive MetHb measurements in children with and without malaria in Lambaréné, Gabon. RESULTS: In Lisbon, the mean MetHb level was 1.4% (SD: 0.5) in a total of 17,834 ABGS. Only 11 of 98 samples with a MetHb level of >3.0 referred to infections. COHb levels showed no particular association with clinical conditions, including sepsis. In 13 patients with severe malaria in Berlin, the mean MetHb levels on admission was 1.29%, with 1.36% for cerebral malaria and 1.14% for non-cerebral malaria (P > 0.05). All COHb measurements were below 2.3%. In Lambaréné, Gabon, 132 healthy children had a mean MetHb level of 1.57%, as compared to 150 children with malaria, with a value of 1.77% and 2.05% in uncomplicated and complicated cases, respectively (P < 0.01). CONCLUSIONS: The data appears consistent with the methaemoglobin/haem hypothesis in malaria and sepsis pathogenesis. However, although MetHb was significantly different between healthy controls and children with malaria in Africa, the difference was rather small, also when compared to previous studies. Still, non-invasive bedside MetHb testing may warrant further evaluation as it could be a simple adjuvant tool for prognosis in resource poor settings.

CC180 clade dynamics do not universally explain Streptococcus pneumoniae serotype 3 persistence post-vaccine: a global comparative population genomics study.

Background Clonal complex 180 (CC180) is currently the major clone of serotype 3 Streptococcus pneumoniae (Spn). The 13-valent pneumococcal conjugate vaccine (PCV13) does not have significant efficacy against serotype 3 despite polysaccharide inclusion in the vaccine. It was hypothesized that PCV13 may effectively control Clade I of CC180 but that Clades III and IV are resistant, provoking a population shift that enables serotype 3 persistence. This has been observed in the United States, England, and Wales but not Spain. We tested this hypothesis further utilizing a dataset from Portugal. Methods We whole-genome sequenced (WGS) 501 serotype 3 strains from Portugal isolated from patients with pneumococcal infections between 1999-2020. The draft genomes underwent phylogenetic analyses, pangenome profiling, and a genome-wide association study (GWAS). We also completed antibiotic susceptibility testing and compiled over 2,600 serotype 3 multilocus sequence type 180 (MLST180) WGSs to perform global comparative genomics. Findings CC180 Clades I, II, III, IV, and VI distributions were similar when comparing non-invasive pneumonia isolates and invasive disease isolates (Fisher's exact test, P=0.29), and adult and pediatric cases (Fisher's exact test, P=0.074). The serotype 3 CCs shifted post-PCV13 (Fisher's exact test, P<0.0001) and Clade I became dominant. Clade I is largely antibiotic-sensitive and carries the phiOXC141 prophage but the pangenome is heterogenous. Strains from Portugal and Spain, where Clade I remains dominant post-PCV13, have larger pangenomes and are associated with the presence of two genes encoding hypothetical proteins. Interpretation Clade I became dominant in Portugal post-PCV13, despite the burden of the prophage and antibiotic sensitivity. The accessory genome content may mitigate these fitness costs. Regional differences in Clade I prevalence and pangenome heterogeneity suggest that clade dynamics is not a generalizable approach to understanding serotype 3 vaccine escape. Funding National Institute of Child Health and Human Development, Pfizer, and Merck Sharp & Dohme.

Rebound of pediatric invasive pneumococcal disease in Portugal after the COVID-19 pandemic was not associated with significant serotype changes.

OBJECTIVES: The COVID-19 pandemic led to the institution of public health measures in many countries which reduced respiratory infections. We aimed to identify and characterize changes in pediatric (<18 years) invasive pneumococcal disease (pIPD) in Portugal in 2018-2023. METHODS: pIPD cases were identified by culture and molecular methods and stratified by age and serotype. When available the susceptibility of the isolates to antimicrobials was evaluated. RESULTS: pIPD cases were markedly reduced in the last trimester of 2019-2020 and the entire 2020-2021 season. While 2021-2022 was in line with pre-pandemic seasons, in 2022-2023, the number of pIPD cases exceeded those found pre-pandemic. Molecular tests were responsible for identifying and serotyping 30% of cases, highlighting their importance in evaluating pIPD. Among the 316 pIPD cases, 37 different serotypes were detected, of which serotypes 3 (n = 85, 26.9%), 8 (n = 25, 7.9%), 10A (n = 21, 6.6%) and 24F (n = 20, 6.3%) were the most frequent. The post-pandemic serotype distribution reflected mostly pre-pandemic trends and the rebound was not driven by particular serotypes. We identified many vaccine failures, most (n = 37) representing serotype 3 infections. Penicillin non-susceptibility increased from 14% pre-pandemic to 29%, with serotype 24F becoming particularly significant. CONCLUSIONS: The higher number of cases of pIPD post-COVID-19 in Portugal raises the possibility of a higher burden of pneumococcal disease in Europe post-pandemic. The relatively stable serotype distribution and the current availability of the higher valency conjugate vaccines PCV15 and PCV20, potentially preventing a large proportion of pIPD (43% and 67%, respectively), offer an opportunity to control this increase.

Extensively drug-resistant Pseudomonas aeruginosa: clinical features and treatment with ceftazidime/avibactam and ceftolozane/tazobactam in a tertiary care university hospital center in Portugal - A cross-sectional and retrospective observational study.

Introduction: Extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) is a growing concern due to its increasing incidence, limited therapeutic options, limited data on the optimal treatment, and high mortality rates. The study aimed to characterize the population, the outcome and the microbiological characteristics of XDR-PA identified in a Portuguese university hospital center. Methods: All XDR-PA isolates between January 2019 and December 2021 were identified. XDR-PA was defined as resistance to piperacillin-tazobactam, third and fourth generation cephalosporins, carbapenems, aminoglycosides and fluoroquinolones. A retrospective analysis of the medical records was performed. Results: One hundred seventy-eight individual episodes among 130 patients with XDR-PA detection were identified. The most common sources of infection were respiratory (32%) and urinary tracts (30%), although skin and soft tissue infections (18%) and primary bacteremia (14%) were also prevalent. Colonization was admitted in 64 cases. Several patients had risk factors for complicated infections, most notably immunosuppression, structural lung abnormalities, major surgery, hemodialysis or foreign intravascular or urinary devices. XDR-PA identification was more frequent in male patients with an average age of 64.3 ± 17.5 years. One non-susceptibility to colistin was reported. Only 12.4% were susceptible to aztreonam. Ceftazidime-avibactam (CZA) was susceptible in 71.5% of the tested isolates. Ceftolozane-tazobactam (C/T) was susceptible in 77.5% of the tested isolates. Antibiotic regimens with XDR-PA coverage were reserved for patients with declared infection, except to cystic fibrosis. The most frequently administered antibiotics were colistin (41 cases), CZA (39 cases), and C/T (16 cases). When combination therapy was used, CZA plus colistin was preferred. The global mortality rate among infected patients was 35.1%, significantly higher in those with hematologic malignancy (50.0%, p < 0.05), followed by the ones with bacteremia (44.4%, p < 0.05) and those medicated with colistin (39.0%, p < 0.05), especially the ones with respiratory infections (60.0%). Among patients treated with CZA or C/T, the mortality rate seemed to be lower. Discussion: XDR-PA infections can be severe and difficult to treat, with a high mortality rate. Even though colistin seems to be a viable option, it is likely less safe and efficient than CZA and C/T. To the best of the authors' knowledge, this is the first description of the clinical infection characteristics and treatment of XDR-PA in Portugal.

Changes in Streptococcus pyogenes causing invasive disease in Portugal: evidence for superantigen gene loss and acquisition.

The emergence of highly virulent and successful Streptococcus pyogenes (group A streptococci - GAS) clones has been attributed to the exchange of virulence factors by lateral gene transfer mechanisms, which strongly contribute to genomic diversity. We characterized a collection of 191 GAS isolates recovered from normally sterile sites in Portugal during 2006-2009 and compared them to invasive isolates obtained during 2000-2005. Antimicrobial resistance rates did not change significantly between the two periods and were generally low. In 2006-2009, emm1, emm89, emm3, and emm6 represented 60% of the isolates. The chromosomally encoded superantigen (SAg) genes speG and smeZ were present in the majority (>90%) of the isolates, while speJ was found in only 45%. The phage encoded SAgs varied greatly in prevalence (2-53%). The distribution of emm types, pulsed-field gel electrophoresis profiling (PFGE) clusters, and SAg profiles changed significantly between the periods, although there were no statistically supported changes in the prevalence of individual types. While the macrolide susceptible clone emm1-T1-ST28 remained dominant (28%), there was a significant decrease in clonal diversity as indicated by both PFGE profiling and emm typing. This was accompanied by intra-clonal divergence of SAg profiles, which was statistically confirmed for isolates representing emm1, emm28, and emm44. This diversification was associated with the loss and acquisition of SAg genes, carried by phages and of chromosomal origin. These data suggest an ongoing genomic diversification of GAS invasive isolates in Portugal that may contribute to the persistence of clones with improved fitness or virulence.