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Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P

Abstract The Two Weeks in the World research project has resulted in a dataset of 3087 clinically relevant bacterial genomes with pertaining metadata, collected from 59 diagnostic units in 35 countries around the world during 2020. A relational database is available with metadata and summary data from selected bioinformatic analysis, such as species prediction and identification of acquired resistance genes.

IntroductionBacteremia is a life-threatening condition that can progress to sepsis and septic shock, leading to significant mortality in the emergency department (ED). The standard diagnostic method, blood culture, is time-consuming and prone to false positives and false negatives. Although not widely accepted, several clinical and artificial intelligence-based algorithms have been recently developed to predict bacteremia. However, these strategies require further identification of new variables to improve their diagnostic accuracy. This study proposes a novel strategy to predict positive blood cultures by assessing sepsis-induced immunosuppression status through endotoxin tolerance assessment.MethodsOptimal assay conditions have been explored and tested in sepsis-suspected patients meeting the Sepsis-3 criteria. Blood samples were collected at ED admission, and endotoxin (lipopolysaccharide, LPS) challenge was performed to evaluate the innate immune response through cytokine profiling.ResultsClinical variables, immune cell population biomarkers, and cytokine levels (tumor necrosis factor [TNFα], IL-1β, IL-6, IL-8, and IL-10) were measured. Patients with positive blood cultures exhibited significantly lower TNFα production after LPS challenge than did those with negative blood cultures. The study also included a validation cohort to confirm that the response was consistent.DiscussionThe results of this study highlight the innate immune system immunosuppression state as a critical parameter for sepsis diagnosis. Notably, the present study identified a reduction in monocyte populations and specific cytokine profiles as potential predictive markers. This study showed that the LPS challenge can be used to effectively distinguish between patients with bloodstream infection leading to sepsis and those whose blood cultures are negative, providinga rapid and reliable diagnostic tool to predict positive blood cultures. The potential applicability of these findings could enhance clinical practice in terms of the accuracy and promptness of sepsis diagnosis in the ED, improving patient outcomes through timely and appropriate treatment.

ABSTRACT Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as Haemophilus influenzae, an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of H. influenzae in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of Haemophilus parainfluenzae as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones.

Summary: Background: During early stages of COVID-19 pandemic, antimicrobials were commonly prescribed. Aim: To describe clinical, microbiological and antimicrobial use changes in bloodstream infections (BSI) of ICU patients during the first wave of COVID-19 pandemic compared to pre-COVID-19 era. Methods: Observational cohort study of patients admitted to ICU of Bellvitge University Hospital was conducted during the COVID-19 pandemic (March-June 2020) and before COVID-19 pandemic (March-June 2019). Differences in clinical characteristics, antimicrobial consumption and incidence and aetiology of BSI were measured. Findings: COVID-19 patients had significantly less comorbidities with obesity the only risk factor that increased in frequency. COVID-19 patients more frequently required invasive supportive care measures, had longer median ICU stay and higher mortality rates. The incidence of BSIs was higher in COVID-19 period (RR 3.2 [95%CI 2.2–4.7]), occurred in patients who showed prolonged median ICU stay (21days) and was associated with high mortality rate (47%). The highest increases in the aetiological agents were observed for AmpC-producing bacteria (RR 11.1 [95%CI 2.6–47.9]) and non-fermenting rods (RR 7.0 [95%CI 1.5–31.4]). The emergence of bacteraemia caused by Gram-negative rods resistant to amoxicillin-clavulanate, which was used as empirical therapy during early stages of the pandemic, led to an escalation towards broader-spectrum antimicrobials such as meropenem and colistin which was also associated with the emergence of resistant isolates. Conclusions: The epidemiological shift towards resistant phenotypes in critically ill COVID-19 patients was associated with the selective use of antimicrobials. Our study provides evidence of the impact of empirical therapy on the selection of bacteria and their consequences on BSI over the subsequent months.

Tetracycline resistance in streptococci is mainly due to ribosomal protection mediated by the tet(M) gene that is usually located in the integrative and conjugative elements (ICEs) of the Tn916-family. In this study, we analyzed the genes involved in tetracycline resistance and the associated mobile genetic elements (MGEs) in Streptococcus dysgalactiae subsp. equisimilis (SDSE) causing invasive disease. SDSE resistant to tetracycline collected from 2012 to 2019 in a single hospital and from 2018 in three other hospitals were analyzed by whole genome sequencing. Out of a total of 84 SDSE isolates, 24 (28.5%) were resistant to tetracycline due to the presence of tet(M) (n = 22), tet(W) (n = 1), or tet(L) plus tet(W) (n = 1). The tet(M) genes were found in the ICEs of the Tn916-family (n = 10) and in a new integrative and mobilizable element (IME; n = 12). Phylogenetic analysis showed a higher genetic diversity among the strains carrying Tn916 than those having the new IME, which were closely related, and all belonged to CC15. In conclusion, tetracycline resistance in SDSE is mostly due to the tet(M) gene associated with ICEs belonging to the Tn916-family and a new IME. This new IME is a major cause of tetracycline resistance in invasive Streptococcus dysgalactiae subsp. equisimilis in our settings.

Methicillin-resistant Staphylococcus aureus bloodstream infections (MRSA-BSI) are a significant cause of mortality. We analysed the evolution of the molecular and clinical epidemiology of MRSA-BSI (n = 784) in adult patients (Barcelona, 1990–2019). Isolates were tested for antimicrobial susceptibility and genotyped (PFGE), and a selection was sequenced (WGS) to characterise the pangenome and mechanisms underlying antimicrobial resistance. Increases in patient age (60 to 71 years), comorbidities (Charlson’s index > 2, 10% to 94%), community-onset healthcare-associated acquisition (9% to 60%), and 30-day mortality (28% to 36%) were observed during the 1990–1995 and 2014–2019 periods. The proportion of catheter-related BSIs fell from 57% to 20%. Current MRSA-BSIs are caused by CC5-IV and an upward trend of CC8-IV and CC22-IV clones. CC5 and CC8 had the lowest core genome proportions. Antimicrobial resistance rates fell, and only ciprofloxacin, tobramycin, and erythromycin remained high (>50%) due to GyrA/GrlA changes, the presence of aminoglycoside-modifying enzymes (AAC(6′)-Ie-APH(2″)-Ia and ANT(4′)-Ia), and mph(C)/msr(A) or erm (C) genes. Two CC22-IV strains showed daptomycin resistance (MprF substitutions). MRSA-BSI has become healthcare-associated, affecting elderly patients with comorbidities and causing high mortality rates. Clonal replacement with CC5-IV and CC8-IV clones resulted in lower antimicrobial resistance rates. The increased frequency of the successful CC22-IV, associated with daptomycin resistance, should be monitored.

Streptococcal infections are usually treated with beta-lactam antibiotics, but, in case of allergic patients or reduced antibiotic susceptibility, macrolides and fluoroquinolones are the main alternatives. This work focuses on studying macrolide resistance rates, genetic associated determinants and antibiotic consumption data in Spain, Europe and also on a global scale. Macrolide resistance (MR) determinants, such as ribosomal methylases (erm(B), erm(TR), erm(T)) or active antibiotic efflux pumps and ribosomal protectors (mef(A/E)-mrs(D)), are differently distributed worldwide and associated with different clonal lineages and mobile genetic elements. MR rates vary together depending on clonal dynamics and on antibiotic consumption applying selective pressure. Among Streptococcus, higher MR rates are found in the viridans group, Streptococcus pneumoniae and Streptococcus agalactiae, and lower MR rates are described in Streptococcus pyogenes. When considering different geographic areas, higher resistance rates are usually found in East-Asian countries and milder or lower in the US and Europe. Unfortunately, the availability of data varies also between countries; it is scarce in low- and middle- income countries from Africa and South America. Thus, surveillance studies of macrolide resistance rates and the resistance determinants involved should be promoted to complete global knowledge among macrolide resistance dynamics.

Introduction Methicillin-susceptible Staphylococcus aureus (MSSA) bacteraemia is a frequent condition, with high mortality rates. There is a growing interest in identifying new therapeutic regimens able to reduce therapeutic failure and mortality observed with the standard of care of beta-lactam monotherapy. In vitro and small-scale studies have found synergy between cloxacillin and fosfomycin against S. aureus. Our aim is to test the hypothesis that cloxacillin plus fosfomycin achieves higher treatment success than cloxacillin alone in patients with MSSA bacteraemia.Methods We will perform a superiority, randomised, open-label, phase IV–III, two-armed parallel group (1:1) clinical trial at 20 Spanish tertiary hospitals. Adults (≥18 years) with isolation of MSSA from at least one blood culture ≤72 hours before inclusion with evidence of infection, will be randomly allocated to receive either cloxacillin 2 g/4-hour intravenous plus fosfomycin 3 g/6-hour intravenous or cloxacillin 2 g/4-hour intravenous alone for 7 days. After the first week, sequential treatment and total duration of antibiotic therapy will be determined according to clinical criteria by the attending physician.Primary endpoints: (1) Treatment success at day 7, a composite endpoint comprising all the following criteria: patient alive, stable or with improved quick-Sequential Organ Failure Assessment score, afebrile and with negative blood cultures for MSSA at day 7. (2) Treatment success at test of cure (TOC) visit: patient alive and no isolation of MSSA in blood culture or at another sterile site from day 8 until TOC (12 weeks after randomisation).We assume a rate of treatment success of 74% in the cloxacillin group. Accepting alpha risk of 0.05 and beta risk of 0.2 in a two-sided test, 183 subjects will be required in each of the control and experimental groups to obtain statistically significant difference of 12% (considered clinically significant).Ethics and dissemination Ethical approval has been obtained from the Ethics Committee of Bellvitge University Hospital (AC069/18) and from the Spanish Medicines and Healthcare Product Regulatory Agency (AEMPS, AC069/18), and is valid for all participating centres under existing Spanish legislation. The results will be presented at international meetings and will be made available to patients and funders.Trial registration number The protocol has been approved by AEMPS with the Trial Registration Number EudraCT 2018-001207-37. ClinicalTrials.gov Identifier: NCT03959345; Pre-results.

The prevalence of a new hypervirulent and hypermucoviscous K. pneumoniae phenotype (Hmv) is increasing worldwide, mainly linked to serotypes K1 and K2. Since capsular thickness can directly affect the capability to form biofilms, we aimed to evaluate the association between the Hmv phenotype with adhesion and biofilm formation in a collection of clinical K. pneumoniae isolates. We selected 38 Hmv clinical isolates [15 serotype K1; 9 serotype K2; 3 non-K1/K2 (rmpA+); 11 non-K1/K2 (rmpA-)] and 7 non-Hmv clinical isolates. The Hmv phenotype was assessed through the mucoviscosity test. Serum resistance was determined by bacterial viability tests in pooled human serum. Adhesion was evaluated with the Biofilm Ring Test®, and biofilm formation was identified by crystal violet staining (Solid-Liquid, SLI-biofilm) or visual examination (Air-Liquid, ALI-biofilm). This study linked for the first time the formation of robust ALI-biofilm plugs by K. pneumoniae to the capsular serotype K1, a group of hypervirulent strains which are generally highly susceptible to the antimicrobial agents. Among all the studied isolates, the capsular serotype K1 presented lower initial adhesion despite having the adhesins mrkD and fimH but higher ALI-biofilm formation than isolates with other capsular serotypes (K2 or non-K1/K2). This structure might confer increased resistance to a group of hypervirulent K. pneumoniae serotype K1.