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Background Although presurgical nasal decontamination with mupirocin (NDM) has been advocated as a measure for preventing postsurgical mediastinitis (PSM) due to Staphylococcus aureus, this strategy is not universally recommended due to lack of robust supporting evidence. We aimed to evaluate the role of preoperative NDM in the annual incidence of S. aureus PSM at our institution. Methods An interrupted time-series analysis, with an autoregressive error model, was applied to our single-center cohort by comparing preintervention (1990–2003) and postintervention (2005–2018) periods. Logistic regression was performed to analyze risk factors for S. aureus PSM. Results 12 236 sternotomy procedures were analyzed (6370 [52.1%] and 5866 [47.9%] in the pre- and postintervention periods, respectively). The mean annual percentage adherence to NDM estimated over the postintervention period was 90.2%. Only 4 of 127 total cases of S. aureus PSM occurred during the 14-year postintervention period (0.68/1000 sternotomies vs 19.31/1000 in the preintervention period; P Conclusions Our experience suggests the implementation of preoperative NDM significantly reduces the incidence of S. aureus PSM.

The population structure of a set of OXA-48-producing Klebsiella pneumoniae isolates belonging to sequence type 11 (ST11 Kp-OXA) and obtained from two hospitals in Madrid in the period from 2012 to 2015 was studied by genome sequencing. Overall, 97 ST11 Kp-OXA isolates were sequenced and their population structure and demography were studied by Bayesian phylodynamic analysis using core-genome SNVs. In total, 92 isolates were from Hospital La Paz, 57 of them from two selected units. The remaining five isolates were from different units of Hospital Doce de Octubre. Altogether, 96 out of the 97 ST11 Kp-OXA isolates could be ascribed to a single lineage that evolved into three sublineages. Demographic inference showed an expansion of the ST11 Kp-OXA in the first half of 2013 in agreement with the registered incidences. Dated phylogeny showed transmission clusters within hospital wards, between wards and between hospitals. The ST11 Kp-OXA outbreak in Hospital La Paz was largely due to the expansion of a single clone that was transmitted between different units and to Hospital Doce de Octubre. This clone diverged into three sub-lineages and spread out following a mixed mode of neutral core-genome evolution with some features of antibiotic selection, frequent large deletions and plasmid loss and gain events.

Introduction Levofloxacin and rifampicin are the preferred treatment for prosthetic joint infection (PJI) caused by Staphylococcus aureus, especially when managed with implant retention (DAIR). However, a significant variability of success has been reported, which could be related to intrinsic characteristics of the microorganism. Our aim was to evaluate the variability in the anti-biofilm response to levofloxacin and rifampicin in a clinical collection of S. aureus. Material and methods Eleven levofloxacin- and rifampicin-susceptible S. aureus isolates causing PJI managed with DAIR were included. Levofloxacin, rifampicin and levofloxacin + rifampicin were tested in an in vitro static biofilm model in microtitre plates, where 48 h biofilms were challenged with antimicrobials during 24 h. Additionally, two genetically similar strains were tested in the CDC Biofilm Reactor, where 48 h biofilms were treated during 56 h. Antimicrobial activity was assessed by viable biofilm-embedded cells recount, and by crystal violet staining. Results All antimicrobial regimens showed significant anti-biofilm activity, but a notable scattering in the response was observed across all strains (inter-strain coefficient of variation for levofloxacin, rifampicin and levofloxacin + rifampicin of 22.8%, 35.8% and 34.5%, respectively). This variability was tempered with the combination regimen when tested in the biofilm reactor. No correlation was observed between the minimal biofilm eradicative concentration and the antimicrobial activity. Recurrent S. aureus isolates exhibited higher biofilm-forming ability compared with strains from resolved infections (7.6 log10 cfu/cm2±0.50 versus 9.0 log10 cfu±0.07). Conclusions Significant variability may be expected in response to levofloxacin and rifampicin among biofilm-embedded S. aureus. A response in the lower range, together with other factors of bad prognosis, could be responsible of treatment failure.

Multidrug-resistant Pseudomonas aeruginosa (MDR-PSA) constitutes an emerging health problem. A predictive score of MDR-PSA infection would allow an early adaptation of empirical antibiotic therapy.We performed a single-centre case-control (1:2) retrospective study including 100 patients with MDR-PSA and 200 with a non-MDR-PSA infection. Cases and controls were matched by site of infection, clinical characteristics and immunosuppression. A point risk score for prediction of MDR-PSA infection was derived from a logistic regression model. Secondary outcomes (clinical improvement, complications and discharge) were also compared.Cases with MDR-PSA infection were younger than controls (67.5 vs. 73.0 y; P = 0.031) and have more frequent cirrhosis (9% vs. 2%; P = 0.005). Independent risk factors for MDR-PSA infection were prior antibiotic treatment (80% vs. 50.5%; P0.001), prior colonisation with MDR bacteria (41% vs. 13.5%; P0.001), hospital-acquired infection (63% vs. 47%; P = 0.009) and septic shock at diagnosis (33% vs. 14%; P0.001). Adequate therapy was less frequent in MDR-PSA infections (31% vs. 66.5% for empirical therapy; P0.001). The risk score included: previous MDR-PSA isolation (11 points), prior antibiotic use (3 points), hospital-acquired infection (2 points) and septic shock at diagnosis (2 points). It showed an area under the curve of 0.755 (95% CI: 0.70-0.81) and allowed to classify individual risk into various categories: 0-2 points (20%), 3-5 points (25%-45%), 7-11 points (55%-60%), 13-16 points (75%-87%) and a maximum of 18 points (93%).Infections due to MDR-PSA have a poorer prognosis than those produced by non-MDR-PSA. Our score could guide empirical therapy for MDR-PSA when P. aeruginosa is isolated.

Few studies have assessed the clinical and bacterial characteristics of Pseudomonas aeruginosa (PA) bacteraemic pneumonia (BP) episodes. This study analysed all non-duplicate PA-BP episodes from a tertiary hospital in 2013–2017. Epidemiology, clinical data, antimicrobial therapy and outcomes were recorded. Whole-genome sequencing was performed on PA blood isolates. The impact on early and late overall mortality of host, antimicrobial treatment and pathogen factors was assessed by multivariate logistic regression analysis. Of 55 PA-BP episodes, 32 (58.2%) were caused by extensively drug-resistant (XDR) PA. ST175 (32.7%) and ST235 (25.5%) were the most frequent high-risk clones. β-Lactamases/carbapenemases were detected in 29 isolates, including blaVIM-2 (27.2%) and blaGES type (25.5%) [blaGES-5 (20.0%), blaGES-1 (3.6%) and blaGES-20 (1.8%)]. The most prevalent O-antigen serotypes were O4 (34.5%) and O11 (30.9%). Overall, an extensive virulome was identified in all isolates. Early mortality (56.4%) was independently associated with severe neutropenia (aOR = 4.64, 95% CI 1.11–19.33; P = 0.035) and inappropriate empirical antimicrobial therapy (aOR = 5.71, 95% CI 1.41–22.98; P = 0.014). Additionally, late mortality (67.3%) was influenced by septic shock (aOR = 8.85, 95% CI 2.00–39.16; P = 0.004) and XDR phenotype (aOR = 5.46, 95% CI 1.25–23.85; P = 0.024). Moreover, specific genetic backgrounds [ST235, blaGES, gyrA (T83I), parC (S87L), exoU and O11 serotype] showed significant differences in patient outcomes. Our results confirm the high mortality associated with PA-BP. Besides relevant clinical characteristics and inappropriate empirical therapy, bacteria-specific genetics factors, such as XDR phenotype, adversely affect the outcome of PA-BP.

Background The positive-intraoperative-cultures-type prosthetic joint infection (PIOC-PJI) is considered when surgical cultures yield microorganisms in presumed aseptic arthroplasty revisions. Herein we assess the risk factors for failure in the largest cohort of PIOC-PJI patients reported to date. Methods A retrospective, observational, multicenter study was performed during 2007–2017. Surgeries leading to diagnose PIOC-PJI included only one-stage procedures with either complete or partial prosthesis revision. Failure was defined as recurrence caused by the same microorganism. Results 203 cases were included (age 72 years, 52% females). Coagulase-negative staphylococci (n = 125, 62%) was the main etiology, but some episodes were caused by virulent bacteria (n = 51, 25%). Prosthesis complete and partial revision was performed in 93 (46%) and 110 (54%) cases, respectively. After a median of 3.4 years, failure occurred in 17 episodes (8.4%, 95%CI 5.3–13.1). Partial revision was an independent predictor of failure (HR 3.63; 95%CI 1.03–12.8), adjusted for gram-negative bacilli (GNB) infection (HR 2.68; 95%CI 0.91–7.89) and chronic renal impairment (HR 2.40; 95%CI 0.90–6.44). Treatment with biofilm-active antibiotics (rifampin/fluoroquinolones) had a favorable impact on infections caused by staphylococci and GNB. Conclusion Overall prognosis of PIOC-PJI is good, but close follow-up is required in cases of partial revision and in infections caused by GNB.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Madrid, Spain, on 25 February 2020. It increased in frequency very fast and by the end of May more than 70,000 cases had been confirmed by reverse transcription-polymerase chain reaction (RT-PCR). To study the lineages and the diversity of the viral population during this first epidemic wave in Madrid we sequenced 224 SARS-CoV-2 viral genomes collected from three hospitals from February to May 2020. All the known major lineages were found in this set of samples, though B.1 and B.1.5 were the most frequent ones, accounting for more than 60% of the sequences. In parallel with the B lineages and sublineages, the D614G mutation in the Spike protein sequence was detected soon after the detection of the first coronavirus disease 19 (COVID-19) case in Madrid and in two weeks became dominant, being found in 80% of the samples and remaining at this level during all the study periods. The lineage composition of the viral population found in Madrid was more similar to the European population than to the publicly available Spanish data, underlining the role of Madrid as a national and international transport hub. In agreement with this, phylodynamic analysis suggested multiple independent entries before the national lockdown and air transportation restrictions., This research was partially supported through the European Commission Horizon 2020 Framework Programme (VIRUSCAN, FETPROACT-2016). E.D. was recipient of a Marie Skłodowska- Curie Individual Fellowship (Grant Agreement number 796084). E.V. is supported by the Subprograma Juan Rodés, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (JR18/00048). R.R. is supported by the Subprograma Río Hortega, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (CM19/00229).

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Background Staphylococcus aureus is the leading cause of prosthetic joint infection (PJI). Beyond the antibiogram, little attention has been paid to the influence of deep microbiological characteristics on patient prognosis. Our aim was to investigate whether microbiological genotypic and phenotypic features have a significant influence on infection pathogenesis and patient outcome. Methods A prospective multicenter study was performed, including all S. aureus PJIs (2016–2017). Clinical data and phenotypic (agr functionality, β-hemolysis, biofilm formation) and genotypic characteristics of the strains were collected. Biofilm susceptibility to antimicrobials was investigated (minimal biofilm eradication concentration [MBEC] assay). Results Eighty-eight patients (39.8% men, age 74.7 ± 14.1 years) were included. Forty-five had early postoperative infections (EPIs), 21 had chronic infections (CPIs), and 19 had hematogenous infections (HIs). Twenty (22.7%) were caused by methicillin-resistant S. aureus. High genotypic diversity was observed, including 16 clonal complexes (CCs), with CC5 being the most frequent (30.7%). agr activity was greater in EPI than CPI (55.6% vs 28.6%; P = .041). Strains causing EPI were phenotypically and genotypically similar, regardless of symptom duration. Treatment failure (36.5%) occurred less frequently among cases treated with implant removal. In cases treated with debridement and implant retention, there were fewer failures among those who received combination therapy with rifampin. No genotypic or phenotypic characteristics predicted failure, except vancomycin minimal inhibitory concentration ≥1.5 mg/L (23.1% failure vs 3.4%; P = .044). MBEC50 was >128 mg/L for all antibiotics tested and showed no association with prognosis. Conclusions S. aureus with different genotypic backgrounds is capable of causing PJI, showing slight differences in clinical presentation and pathogenesis. No major microbiological characteristics were observed to influence the outcome, including MBEC.

Whether multidrug resistance (MDR) is associated with mortality in patients with Pseudomonas aeruginosa bloodstream infections (BSI) remains controversial. Here, we explored the prognostic factors of P. aeruginosa BSI with emphasis on antimicrobial resistance and virulence. All P. aeruginosa BSI episodes in a 5-year period were retrospectively analyzed. The impact in early (5-day) and late (30-day) crude mortality of host, antibiotic treatment, and pathogen factors was assessed by multivariate logistic regression analysis. Of 243 episodes, 93 (38.3%) were caused by MDR-PA. Crude 5-day (20%) and 30-day (33%) mortality was more frequent in patients with MDR-PA (34.4% versus 11.3%, P < 0.001 and 52.7% versus 21.3%, P < 0.001, respectively). Early mortality was associated with neutropenia (adjusted odds ratio [aOR], 9.21; 95% confidence interval [CI], 3.40 to 24.9; P < 0.001), increased Pitt score (aOR, 2.42; 95% CI, 1.34 to 4.36; P = 0.003), respiratory source (aOR, 3.23; 95% CI,2.01 to 5.16; P < 0.001), inadequate empirical therapy (aOR, 4.57; 95% CI, 1.59 to 13.1; P = 0.005), shorter time to positivity of blood culture (aOR, 0.88; 95% CI, 0.80 to 0.97; P = 0.010), an exoU-positive genotype (aOR, 3.58; 95% CI, 1.31 to 9.79; P = 0.013), and the O11 serotype (aOR, 3.64; 95% CI, 1.20 to 11.1; P = 0.022). These risk factors were similarly identified for late mortality, along with an MDR phenotype (aOR, 2.18; 95% CI, 1.04 to 4.58; P = 0.040). Moreover, the O11 serotype (15.2%, 37/243) was common among MDR (78.4%, 29/37) and exoU-positive (89.2%, 33/37) strains. Besides relevant clinical variables and inadequate empirical therapy, pathogen-related factors such as an MDR phenotype, an exoU-positive genotype, and the O11 serotype adversely affect the outcome of P. aeruginosa BSI.

We have explored the relationship of phenotypic (antibiogram, β-haemolysis, agr functionality, biofilm formation) and genotypic characteristics on the prognosis of 18 cases of methicillin-resistant S. aureus prosthetic joint infection (2005-2015). All isolates belonged to CC5, and had agr type II. This pilot study suggests that phage-borne genes belonging to the immune evasion cluster (chp, sak and scn) were more frequent among episodes with treatment failure (80.0 vs. 37.5%).

Multidrug-resistant (MDR) Pseudomonas aeruginosa represents a major clinical concern. The interplay between antimicrobial resistance and virulence of P. aeruginosa was investigated in in vitro and in vivo studies. Thirty-eight well-characterized (21 MDR and 17 non-MDR) P. aeruginosa strains from patients with bacteraemia were analysed. Resistance phenotype, carbapenemase production, clonal relatedness, type III secretion system genotype, O-antigen serotype, cytotoxicity (ability to lyse cells) on A549 cells, and virulence (lethality in nematodes) in a Caenorhabditis elegans model were investigated. MDR strains showed lower cytotoxicity (35.4 ± 21.30% vs. 45.0 ± 18.78 %; P = 0.044) and virulence (66.7% vs. 100%; P = 0.011) than non-MDR strains. However, the pathogenicity of MDR high-risk clones varied broadly, with ST235 and ST175 clones being the most and least cytotoxic (51.8 ± 10.59% vs. 11.0 ± 1.25%; P < 0.0001) and virulent ([100% vs. 73.1; P = 0.075] and [0% vs. 93.9%; P < 0.0001], respectively). The pathogenicity of the ST235 clone was similar to that of non-MDR strains, and its ability to lyse cells and high virulence were related with the exoU-positive genotype. Furthermore, the O11 serotype was more frequent among the ST235 clone and exoU-positive genotype strains and was also essential for the pathogenicity of P. aeruginosa. Our data suggest that the pathogenicity of MDR high-risk clones is the result not only of the resistance phenotype but also of the virulence genotype. These findings have implications for the clinical management of patients and infection control programmes.

Background Staphylococcus aureus may invade and persist intracellularly in prosthetic joint infections (PJIs). Despite optimized treatments with levofloxacin plus rifampin, the intracellular reservoir may lead to infection relapse. This study assessed the intracellular activity of levofloxacin and rifampin in an in-vitro model of human osteoblastic infection. Methods Ten meticillin-susceptible S. aureus strains were used to infect osteoblastic MG63 cells. Osteoblasts were challenged with rifampin and levofloxacin at cortical and cancellous bone concentrations. Efficacy was measured as the intracellular counts of colony-forming units (log10CFU) compared with untreated controls. The emergence of small colony variants (SCVs) was determined, and the results were stratified according to the patient's prognosis (six cured and four with persistence/relapse). Results All regimes led to a significant decrease in CFU count compared with controls (1–2 log10CFU). Levofloxacin was the most effective treatment at both cortical and cancellous bone concentrations (-2.4 to -1.9 log10CFU, respectively). The addition of rifampin to levofloxacin did not improve performance (-1.9 log10CFU for cortical concentration and -1.8 log10 CFU for cancellous concentration). An increase in SCVs was observed in the presence of rifampin. The efficacy of antimicrobials was higher and the formation of SCVs was lower against strains belonging to PJIs with a favourable outcome. Conclusions Levofloxacin plus rifampin had good intracellular activity against S. aureus. However, from the intracellular perspective, the addition of rifampin to levofloxacin showed no benefit but could account for an increased number of SCVs.

Staphylococcus aureus bacteremia (SAB) is associated with high morbidity and mortality, which varies depending on the source of infection. Nevertheless, the global molecular epidemiology of SAB and its possible association with specific virulence factors remains unclear. Using DNA microarrays, a total of 833 S. aureus strains (785 SAB and 48 colonizing strains) collected in Spain over a period of 15 years (2002-2017) were characterized to determine clonal complex (CC), agr type and repertoire of resistance and virulence genes in order to provide an epidemiological overview of CCs causing bloodstream infection, and to analyze possible associations between virulence genes and the most common sources of bacteremia. The results were also analyzed by acquisition (healthcare-associated [HA] and community-acquired [CA]), methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains, and patient age (adults vs. children). Our results revealed high clonal diversity among SAB strains with up to 28 different CCs. The most prevalent CCs were CC5 (30.8%), CC30 (20.3%), CC45 (8.3%), CC8 (8.4%), CC15 (7.5%), and CC22 (5.9%), which together accounted for 80% of all cases. A higher proportion of CC5 was found among HA strains than CA strains (35.6 vs. 20.2%, p < 0.001). CC5 was associated with methicillin resistance (14.7 vs. 79.4%, p < 0.001), whereas CC30, CC45, and CC15 were correlated with MSSA strains (p < 0.001). Pathogen-related molecular markers significantly associated with a specific source of bacteremia included the presence of sea, undisrupted hlb and isaB genes with catheter-related bacteremia; sed, splE, and fib genes with endocarditis; undisrupted hlb with skin and soft tissue infections; and finally, CC5, msrA resistance gene and hla gene with osteoarticular source. Our study suggests an association between S. aureus genotype and place of acquisition, methicillin resistance and sources of bloodstream infection, and provides a valuable starting point for further research insights into intrinsic pathogenic mechanisms involved in the development of SAB., This work was supported by the Health Research Fund, Department of Health, Spain; Agency for Health Technology Assessment and Research (PI15/02013 and PI15/02125) and Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016-0002; 0003; 0004) and cofunded by the European Regional Development Fund (FEDER).

[Objective] We aimed to evaluate the impact of Staphylococcus aureus phenotype (vancomycin MIC) and genotype (agr group, clonal complex CC) on the prognosis and clinical characteristics of infective endocarditis (IE)., [Methods] We performed a multicentre, longitudinal, prospective, observational study (June 2013 to March 2016) in 15 Spanish hospitals. Two hundred and thirteen consecutive adults (≥18 years) with a definite diagnosis of S. aureus IE were included. Primary outcome was death during hospital stay. Main secondary end points were persistent bacteraemia, sepsis/septic shock, peripheral embolism and osteoarticular involvement., [Results] Overall in-hospital mortality was 37% (n = 72). Independent risk factors for death were age-adjusted Charlson co-morbidity index (OR 1.20; 95% CI 1.08–1.34), congestive heart failure (OR 3.60; 95% CI 1.72–7.50), symptomatic central nervous system complication (OR 3.17; 95% CI 1.41–7.11) and severe sepsis/septic shock (OR 4.41; 95% CI 2.18–8.96). In the subgroup of methicillin-susceptible S. aureus IE (n = 173), independent risk factors for death were the age-adjusted Charlson co-morbidity index (OR 1.17; 95% CI 1.03–1.31), congestive heart failure (OR 3.39; 95% CI 1.51–7.64), new conduction abnormality (OR 4.42; 95% CI 1.27–15.34), severe sepsis/septic shock (OR 5.76; 95% CI 2.57–12.89) and agr group III (OR 0.27; 0.10–0.75). Vancomycin MIC ≥1.5 mg/L was not independently associated with death during hospital nor was it related to secondary end points. No other genotype variables were independently associated with in-hospital death., [Conclusions] This is the first prospective study to assess the impact of S. aureus phenotype and genotype. Phenotype and genotype provided no additional predictive value beyond conventional clinical characteristics. No evidence was found to justify therapeutic decisions based on vancomycin MIC for either methicillin-resistant or methicillin-susceptible S. aureus., This study was supported by Fondo de Investigacion Sanitaria (FIS), Ministerio de Sanidad, Instituto de Salud Carlos III (PI12/01719 and PI12/01205) and Ministerio de Economia y Competitividad, Instituto de Salud Carlos III, co-financed by the European Development Regional Fund A way to achieve Europe ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015).

Objectives Septic arthritis is associated with significant case fatality and morbidity. Staphylococcus aureus is the most common cause of arthritis. We aimed to analyze the microbiological features of S. aureus causing native arthritis and to investigate their influence on the clinical outcome of the infection. Patients and methods We conducted a retrospective study including all episodes of S. aureus native arthritis between 2005–2015. Phenotypic (antimicrobial susceptibility, β-hemolysis, agr functionality, biofilm formation) and genotypic characteristics (pulsed-field gel electrophoresis, DNA microarrays) were investigated. The primary endpoint was microbiological failure of treatment, including infection relapse, persistence, or attributable death. Results Twenty-nine patients were included (65.5% of men, mean age: 59): seven (24.1%) patients presenting with methicillin-resistant S. aureus (MRSA) native arthritis and 19 with methicillin-susceptible S. aureus (MSSA) native arthritis. Treatment failure occurred in seven (26.9%) patients (4/7 patients [57.1%] among MRSA infections vs. 3/19 [15.8%] among MSSA infections). The persistence rate was similar in MRSA and MSSA infections (1/7 vs. 3/19). However, the case fatality was significantly higher in patients with MRSA infection (3/7 vs. 0/19). The most frequent clonal complex (CC) was CC5 (38.1%). MSSA showed higher genetic variability (nine CCs) versus MRSA (3 CCs). Conclusions Beyond methicillin resistance, we did not find phenotypic or genotypic factors associated with the poor outcome of S. aureus native arthritis. CC5 was the major CC, showing the higher genetic variability of MSSA versus MRSA.

Predictors of mortality and the impact of multidrug resistance and virulence on patients with Pseudomonas aeruginosa (PA) bacteraemia were evaluated. Patients with PA bacteraemia in a 12-month period were retrospectively analysed. Carbapenemase production, molecular typing and identification of virulence factor ExoU were carried out. The activity of ceftolozane-tazobactam and ceftazidime-avibactam was also investigated. The primary endpoint was 30-day crude mortality. Of 64 patients with bacteraemia, 24 (37.5%) were caused by extensively drug-resistant PA (XDR-PA): 10 (41.7%) cases involved the VIM-2 carbapenemase-producing ST175 clone, 11 (45.8%) the GES-5 carbapenemase-producing ST235 clone, and 3 (12.5%) were non-carbapenemase producers. The exoU genotype was detected in all ST235 strains and in 6 (15%) of the non-XDR isolates. Ceftazidime-avibactam (58.3%) showed greater activity than ceftolozane-tazobactam (12.5%) against XDR-PA isolates, particularly in GES-5 producers (100%). The 30-day crude mortality rate in patients with XDR-PA bacteraemia was higher than in cases caused by susceptible strains (62.5% vs. 30%; P=0.02). Multivariate analysis showed that independent risk factors associated with 30-day crude mortality were Pitt score ≥2 (OR, 42.31; 95% CI, 4.88-366.7; P=0.001) and respiratory source of bacteraemia (OR, 49.13; 95% CI 3.89-620.5; P=0.003). Stratified analysis adjusting for respiratory source revealed a non-significant trend towards higher mortality in patients with bacteraemia caused by the ST235 clone and exoU-producing isolates. These data support the notion that the XDR phenotype associated with the GES-5 carbapenemase-producing ST235 clone and the exoU-positive genotype adversely affects the outcome of patients with PA bacteraemia, particularly those with respiratory tract infections and a severe clinical presentation.

Here we report a retrospective clinical and molecular study conducted in a tertiary care facility in southern Madrid, Spain, from January 2009 to February 2014 to investigate the epidemiology of carbapenemase-producing Klebsiella pneumoniae (CPKp). Carbapenemase genes were identified in 97 non-duplicate K. pneumoniae isolates, including 59 harbouring blaOXA-48, 37 harbouring blaVIM-1 and 1 harbouring blaKPC-2. Pulsed-field gel electrophoresis (PFGE) analysis verified the presence of 20 different clonal types, whilst multilocus sequence typing (MLST) assigned the isolates to eight sequence types (STs). A gradual increase was noted in the number of CPKp isolated, ranging from 0.8% in 2009 to 4.3% in 2013. A large outbreak was also identified, initiated in 2013 owing to a blaOXA-48 and blaCTX-M-15 co-producing ST11 clone and involving a total of 44 patients. Whole-genome sequencing was used to characterise the resistome of a representative isolate from this outbreak. Bioinformatics analysis revealed the presence of 121 genes related to antibiotic and antiseptic resistance, mutations in the ompk35 and ompk36 genes, and the presence of the blaOXA-48 gene on a 62 811bp IncL/M-type plasmid as part of a Tn1999.2 composite transposon. These results portray the increasing trend in carbapenemase-producing isolates in this hospital and highlight the successful establishment of a blaOXA-48 and blaCTX-M-15 co-producing ST11 clone that has led to the displacement of previous circulating clones.

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis–multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% (n = 101) of the XDR isolates, distantly followed by ST244 (n = 16), ST253 (n = 12), ST235 (n = 8), and ST111 (n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital (n = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the β-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in β-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis–multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% ( n = 101) of the XDR isolates, distantly followed by ST244 ( n = 16), ST253 ( n = 12), ST235 ( n = 8), and ST111 ( n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital ( n = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the β-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in β-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

Carbapenemase producing Citrobacter freundii (CPCF) infections are still uncommon in European countries. Here we report a molecular study conducted in a tertiary care facility in southern Madrid, Spain, from 2009 to 2014 to investigate the epidemiology of CPCF. The blaIMP-1,blaIMP-2,blaKPC,blaNDM,blaOXA-48,blaVIM-1 and blaVIM-2 genes were screened by PCR. Molecular typing was carried out by Pulsed-field gel electrophoresis analysis (PFGE) and multilocus sequence typing (MLST). Whole genome sequencing (WGS) was performed to characterize the resistome and the mobile genetic elements associated with the carbapenems resistance of CPCF. A total of 11/521 (2.1%) isolates had reduced susceptibility to carbapenems. PCR amplification revealed the presence of blaVIM-1 in 10 isolates and blaKPC-2 in 2 isolates. One C. freundii isolate co-harbored blaVIM-1 and blaKPC-2 genes. PFGE and MLST assigned 10 different clonal, 4 previously reported (ST11, ST18, ST22 and ST64) and 6 new STs (ST89, ST90, ST91, ST92, ST92 and ST94). The blaVIM-1 gene was part of In624 (intI1-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1). In 3 of these isolates, plasmid-mediated quinolone resistance genes (qnrA1 and qnrB4) were present in its downstream region, taking part of a complex class 1 integron ([In624:ISCR1:qnrB4-blaDHA-1] and [In624:ISCR1:qnrA1]). On the other hand, the blaKPC-2 gene was associated with a Tn3-based transposon. The dissemination of the blaVIM-1 gene among various clones suggests a successful horizontal transfer of integron carrying elements that play a dominant role in the development of multidrug resistance in Enterobacteriaceae.

Even with appropriate clinical management, complicated methicillin-susceptible Staphylococcus aureus (MSSA) catheter-related bacteremia (CRB) is frequent. We investigated the influence of molecular characteristics of MSSA strains on the risk of complicated bacteremia (CB) in MSSA-CRB. A multicenter prospective study was conducted in Spain between 2011 and 2014 on MSSA-CRB. Optimized protocol-guided clinical management was required. CB included endocarditis, septic thrombophlebitis, persistent bacteremia and/or end-organ hematogenous spread. Molecular typing, agr functionality and DNA microarray analysis of virulence factors were performed in all MSSA isolates. Out of 83 MSSA-CRB episodes included, 26 (31.3%) developed CB. MSSA isolates belonged to 16 clonal complexes (CCs), with CC30 (32.5%), CC5 (15.7%) and CC45 (13.3) being the most common. Comparison between MSSA isolates in episodes with or without CB revealed no differences regarding agr type and functionality. However, our results showed that CC15 and the presence of genes like cna, chp and cap8 were associated with the development of CB. The multivariate analysis highlighted that the presence of cna (Hazard ratio 2.9; 95% CI 1.14-7.6) was associated with the development of CB. Our results suggest that particular CCs and specific genes may influence the outcome of MSSA-CRB.

Staphylococcus aureus is a major cause of bacteremia and, even with appropriate clinical management, causes high morbidity and mortality due to its involvement in endovascular complications and metastatic infections. Through different pathogenic in vivo and in vitro models we investigated the behavior of S. aureus most relevant clonal complexes (CCs) causing endovascular complications. We analyzed 14 S. aureus strains representing CC5, CC8, CC15, CC30 and CC45 that caused endovascular complications, including methicillin susceptible and resistant isolates and strains with different functionality of the agr global regulator. Their adherence to collagen, interaction with the endothelium, resistance to immune attack, capacity to form biofilm and virulence in the Galleria mellonella model were analyzed. CC30 and CC45 showed greater adhesion to collagen and CC8 showed a trend towards higher rate of intracellular persistence in endothelial cells. All CCs exhibited similar tolerance to neutrophil antimicrobial peptide hNP-1 and were capable of forming biofilms under static conditions. The virulence assay in the Galleria mellonella model demonstrated that CC15 and CC30 were the most and least virulent, respectively. The analysis of the genomic sequences of the most relevant virulence genes identified some CC15 specific gene patterns (absence of enterotoxins and sak gene) and variants (mainly in leucocidins and proteases), but did not reveal any gene or variant that could be responsible for the increased virulence detected for CC15 strains. Even though all the CCs were capable of causing endovascular complications, our results showed that different CCs are likely to produce these complications through different mechanisms which, if confirmed in more sophisticated models, would indicate the need to more specific management and therapeutic approaches.

Objectives: Limited data exist regarding the role of agr dysfunction in reducing susceptibility to vancomycin in methicillin-susceptibleStaphylococcus aureus (MSSA). This study investigated the clinical and molecular epidemiology of MSSA causing bacteraemia, with emphasis on the reduced susceptibility to vancomycin (RSV) phenotype (MIC ≥1.5 mg/L) and its relationship with agr dysfunction. Methods: All MSSA bloodstream isolates obtained at our hospital during 2010 were analysed. Antimicrobial susceptibility was determined and time –kill experiments were performed for oxacillin. Multilocus sequence type andagr genotype were determined and DNA microarray analysis of virulence factors was performed.agr dysfunction was assessed phenotypically and by RT–PCR quantification of RNAIII. Results: Of 84 MSSA, 55 (65.5%) exhibited the RSV phenotype, comprising 13 clonal complexes.agr II polymorphism was more prevalent in RSV than non-RSV isolates (41.8% versus 17.2%,P ¼ 0.023) and average levels of RNAIII gene expression were higher in RSV than non-RSV isolates (DCt 4.05+3.29 versus 1.5+2.11, P ¼ 0.005), implying greater agr dysfunction in RSV MSSA. Conclusions: We demonstrated a correlation between RSV phenotype in MSSA and reducedagr expression, particularly in association with theagr II genotype. These results may help to understand the role ofagr dysfunction in the increased mortality in MSSA infections.

Introduction The epidemiology of Burkholderia cepacia complex (Bcc) in cystic fibrosis (CF) is not widely known. Methods All CF patients with Bcc between 2002 and 2011 were reviewed, and a molecular analysis of isolates was performed. Results The prevalence of Bcc infection was 7.2% (18/250). Molecular analysis of 16 Bcc isolates showed 5 species (7 B. contaminans, 6 B. cepacia, 1 B. cenocepacia, 1 B. multivorans, and 1 B. stabilis) and 13 sequence types. There were no cases of cross-transmission. Conclusion A high diversity of Bcc species was found in infected CF patients.

Patients infected with these bacteria were more likely to have local endovascular complications., We investigated the prognostic role of high MICs for antistaphylococcal agents in patients with methicillin-sensitive Staphylococcus aureus catheter-related bloodstream infection (MSSA CRBSI). We prospectively reviewed 83 episodes from 5 centers in Spain during April 2011–June 2014 that had optimized clinical management and analyzed the relationship between E-test MICs for vancomycin, daptomycin, oxacillin, and linezolid and development of complicated bacteremia by using multivariate analysis. Complicated MSSA CRBSI occurred in 26 (31.3%) patients; MICs for vancomycin and daptomycin were higher in these patients (optimal cutoff values for predictive accuracy = 1.5 μg/mL and 0.5 μg/mL). High MICs for vancomycin (hazard ratio 2.4, 95% CI 1.2–5.5) and daptomycin (hazard ratio 2.4, 95% CI 1.1–5.9) were independent risk factors for development of complicated MSSA CRBSI. Our data suggest that patients with MSSA CRBSI caused by strains that have high MICs for vancomycin or daptomycin are at increased risk for complications.

A total of 183 patients were colonized or infected with multidrug-resistant Pseudomonas aeruginosa isolates at a hospital in Spain during 2007-2010; prevalence increased over this period from 2.8% to 15.3%. To characterize these isolates, we performed molecular epidemiologic and drug resistance analysis. Genotyping showed that 104 (56.8%) isolates belonged to a single major clone (clone B), which was identified by multilocus sequence typing as sequence type (ST) 175. This clone was initially isolated from 5 patients in 2008, and then isolated from 23 patients in 2009 and 76 patients in 2010. PCR analysis of clone B isolates identified the b/a(VIM-2) gene in all but 1 isolate, which harbored b/a(IMP-22). ST175 isolates were susceptible to only amikacin (75%) and colistin (100%). Emergence of the ST175 clone represents a major health problem because it compromises therapy for treatment of P aeruginosa nosocomial infections., This study was supported by Spanish Network for the Research in Infectious Diseases (RD06/0008) from the Instituto de Salud Carlos III, Spain.

The mechanisms responsible for the increasing prevalence of colistin-only-sensitive (COS) Pseudomonas aeruginosa isolates in a Spanish hospital were investigated. Pulsed-field gel electrophoresis revealed that 24 (50%) of the studied isolates belonged to the same clone, identified as the internationally spread sequence type 235 (ST235) through multilocus sequence typing. In addition to several mutational resistance mechanisms, an integron containing seven resistance determinants was detected. Remarkably, the extended-spectrum β-lactamase GES-1 and its Gly170Ser carbapenem-hydrolyzing derivative GES-5 were first documented to be encoded in a single integron. This work is the first to describe GES enzymes in Spain and adds them to the growing list of β-lactamases of concern (PER, VIM, and OXA) detected in ST235 clone isolates.

Chlorhexidine and mupirocin have been increasingly used in healthcare facilities to eradicate methicillin-resistant Staphylococcus aureus (MRSA) carriage. The aim of this study was to determine the prevalence and mechanisms of chlorhexidine and mupirocin resistance in MRSA from invasive infections and colonisation. MRSA isolates obtained from blood and nasal samples between 2012 and 2014 were analysed. Susceptibility to mupirocin was determined by disk diffusion and Etest and susceptibility to chlorhexidine by broth microdilution. The presence of mupA and qac (A/B and C) genes was investigated by PCR. Molecular typing was performed in high-level mupirocin-resistant (HLMR) isolates. Mupirocin resistance was identified in 15.6% of blood isolates (10.9% HLMR) and 15.1% of nasal isolates (12.0% HLMR). Presence of the mupA gene was confirmed in all HLMR isolates. For blood isolates, chlorhexidine minimum inhibitory concentrations (MICs) ranged from ≤0.125 to 4mg/L and minimum bactericidal concentrations (MBCs) from ≤0.125 to 8mg/L. In nasal isolates, chlorhexidine MICs and MBCs ranged from ≤0.125 to 2mg/L. The qacA/B gene was detected in 2.2% of MRSA isolates (chlorhexidine MIC range 0.25-2mg/L) and the qacC gene in 8.2% (chlorhexidine MIC range ≤0.125-1mg/L). The prevalence of qacC was 18.9% in HLMR isolates and 3.6% in mupirocin-susceptible isolates (P=0.009). Most of the HLMR isolates (97.1%) belonged to ST125 clone. These results suggest that chlorhexidine has a higher potential to prevent infections caused by MRSA. In contrast, mupirocin treatment should be used cautiously to avoid the spread of HLMR MRSA.

The annual incidence of Serratia marcescens bacteremia in a neonatal intensive care unit increased significantly between 2002 and 2010. Molecular epidemiology studies revealed that 8 clones were responsible for 85.2% of cases. Given that these infections are potentially preventable, even the appearance of 1 case of bacteremia should be an indicator for outbreak management.

We report the genome of colistin-only-susceptible Pseudomonas aeruginosa strain ST235 (PA_ST235). This isolate was obtained in the setting of an outbreak in a tertiary hospital in Spain. This clone was apparently associated with a significantly higher mortality rate. The ST235 clone also appears to be associated with greater virulence.

We present the draft genome sequence of a blood culture isolate of OXA-48-producing Klebsiella pneumoniae (sequence type 11 [ST11]) obtained in the course of a hospital outbreak in Spain. Sequence analysis showed 121 genes related to antibiotic and antiseptic resistance, including bla OXA-48 , which was located in an IncL/M plasmid.

Over a 6-year period (2007-2012), the emergence of Enterobacter cloacae isolates resistant to β-lactams and with reduced susceptibility to carbapenems was observed in Hospital Universitario 12 de Octubre (Madrid, Spain). To determine the possible role of metallo-β-lactamases (MBLs) in the resistance profile of these isolates, a molecular and clinical epidemiological study was performed, including determination of patients' clinical characteristics, genetic diversity of strains, resistance mechanisms to carbapenems, and the genetic environment of VIM-1. A total of 73 E. cloacae isolates showed resistance to extended-spectrum cephalosporins and reduced susceptibility to at least one carbapenem during 2007-2012. PCR amplification revealed the presence of bla(VIM-1) gene in 37 isolates, bla(VIM-2) in 1 isolate and bla(KPC) in 5 isolates. Molecular typing showed high clonal diversity of E. cloacae isolates carrying bla(VIM-1). The genetic environment of bla(VIM-1) was investigated and two integron structures were found: intI-bla(VIM-1)-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1 (In624); and intI-bla(VIM-1)-aacA4-aadA1-qacEΔ1/sul1 (In488). Isolates belonging to three clones (A, F and G) harboured different types of integron (In624 or In488) despite belonging to the same clone. Conjugal experiments showed an association with a conjugative plasmid of ca. 300 kb belonging to IncHI2 group, which is common in Spanish hospitals, suggesting that the widespread dissemination of bla(VIM-1) may be due to horizontal transfer of mobile genetic determinants rather than the result of spreading of a few clones. These results have implications for infection control programmes in the hospital.