Addressing common challenges of biotherapeutic protein peptide mapping using recombinant trypsin.

Peptide mapping is the key method for characterization of primary structure of biotherapeutic proteins. This method relies on digestion of proteins into peptides that are then analyzed for amino acid sequence and post-translational modifications. Owing to its high activity and cleavage specificity, trypsin is the protease of choice for peptide mapping. In this study, we investigated critical requirements of peptide mapping and how trypsin affects these requirements. We found that the commonly used MS-grade trypsins contained non-specific, chymotryptic-like cleavage activity causing generation of semi-tryptic peptides and degradation of tryptic-specific peptides. Furthermore, MS-grade trypsins contained pre-existing autoproteolytic peptides and, moreover, additional autoproteolytic peptides were resulting from prominent autoproteolysis during digestion. In our long-standing quest to improve trypsin performance, we developed novel recombinant trypsin and evaluated whether it could address major trypsin drawbacks in peptide mapping. The study showed that the novel trypsin was free of detectable non-specific cleavage activity, had negligible level of autoproteolysis and maintained high activity over the course of digestion reaction. Taking advantage of the novel trypsin advanced properties, especially high cleavage specificity, we established the application for use of large trypsin quantities to digest proteolytically resistant protein sites without negative side effects. We also tested trypsin/Lys-C mix comprising the novel trypsin and showed elimination of non-specific cleavages observed in the digests with the commonly used trypsins. In addition, the improved features of the novel trypsin allowed us to establish the method for accurate and efficient non-enzymatic PTM analysis in biotherapeutic proteins.

Corrigendum: CARB-ES-19 multicenter study of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli from all Spanish provinces reveals interregional spread of high-risk clones such as ST307/OXA-48 and ST512/KPC-3.

[This corrects the article DOI: 10.3389/fmicb.2022.918362.].

Recommendations of the Spanish Antibiogram Committee (COESANT) for in vitro susceptibility testing of antimicrobial agents by disk diffusion / Recomendaciones del Comité Español del Antibiograma (COESANT) para el estudio de sensibilidad in vitro a los antimicrobianos por difusión con discos

Disk diffusion is a well standardized method that provides reliable categorical results to guide antimicrobial therapy in numerous types of infections. Based on the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which are widely implemented in Spain, the Spanish Antibiogram Committee (COESANT) has drawn up recommendations for antimicrobial selection by the disk diffusion technique, including selective reporting and its use for the detection of resistance mechanisms. Factors affecting disk diffusion results, along with advantages and shortcomings of the method, are also discussed.(AU)

La difusión con discos es un método estandarizado que proporciona resultados fiables para guiar la terapia antimicrobiana en numerosos tipos de infecciones. En base a las directrices del European Committee on Antimicrobial Susceptibility Testing (EUCAST), ampliamente implantadas en España, el Comité Español del Antibiograma (COESANT) ha elaborado recomendaciones para la selección de antimicrobianos para ser estudiados mediante la técnica de difusión con discos, su notificación selectiva en el informe de sensibilidad y su uso para la detección de mecanismos de resistencia. También se discuten los factores que afectan los resultados obtenidos mediante la técnica de difusión con discos junto con las ventajas y desventajas del método.(AU)

The Interventions and Challenges of Antimicrobial Stewardship in the Emergency Department.

Over the last decades, we have witnessed a constant increase in infections caused by multi-drug-resistant strains in emergency departments. Despite the demonstrated effectiveness of antimicrobial stewardship programs in antibiotic consumption and minimizing multi-drug-resistant bacterium development, the characteristics of emergency departments pose a challenge to their implementation. The inclusion of rapid diagnostic tests, tracking microbiological results upon discharge, conducting audits with feedback, and implementing multimodal educational interventions have proven to be effective tools for optimizing antibiotic use in these units. Nevertheless, future multicenter studies are essential to determine the best way to proceed and measure outcomes in this scenario.

Significant increase of CTX-M-15-ST131 and emergence of CTX-M-27-ST131 Escherichia coli high-risk clones causing healthcare-associated bacteraemia of urinary origin in Spain (ITUBRAS-2 project).

OBJECTIVES: To assess the microbiological characteristics of Escherichia coli causing healthcare-associated bacteraemia of urinary origin (HCA-BUO) in Spain (ITUBRAS-2 project), with particular focus on ESBL producers and isolates belonging to ST131 high-risk clone (HiRC). Clinical characteristics and outcomes associated with ST131 infection were investigated. METHODS: A total of 222 E. coli blood isolates were prospectively collected from patients with HCA-BUO from 12 tertiary-care hospitals in Spain (2017-19). Antimicrobial susceptibility and ESBL/carbapenemase production were determined. ST131 subtyping was performed. A subset of 115 isolates were selected for WGS to determine population structure, resistome and virulome. Clinical charts were reviewed. RESULTS: ESBL-producing E. coli prevalence was 30.6% (68/222). ST131 represented 29.7% (66/222) of E. coli isolates and accounted for the majority of ESBL producers (46/68, 67.6%). The C2/H30-Rx subclone accounted for most ST131 isolates (44/66) and was associated with CTX-M-15 (37/44) and OXA-1 enzymes (27/44). Cluster C1-M27 was identified in 4/10 isolates belonging to subclade C1/H30-R1 and associated with CTX-M-27. Additionally, ST131 isolates showed a high content of other acquired resistance genes, and clade C/ST131 isolates carried characteristic QRDR mutations. They were categorized as uropathogenic E. coli and had higher aggregate virulence scores. ST131 infection was associated with more complex patients, prior use of cephalosporins and inadequate empirical treatment but was not associated with worse clinical outcomes. CONCLUSIONS: ST131 HiRC is the main driver of ESBL-producing E. coli causing HCA-BUO in Spain, mainly associated with the expansion of subclade CTX-M-15-C2/H30-Rx and the emergence of CTX-M-27-C1/H30-R1 (Cluster C1-M27).

Acute Bacterial Meningitis in Healthy Adult Patients: A Prospective Cohort Study.

Spontaneous bacterial meningitis (BM) is more common among patients with underlying conditions, but its characteristics in previously healthy patients are not well described. We analyzed the time trends of BM in terms of characteristics, and outcomes in patients without comorbidities. PATIENTS AND METHODS: Single-center, prospective observational cohort study of 328 adults with BM hospitalized in a tertiary university hospital in Barcelona (Spain). We compared the features of infections diagnosed in 1982-2000 and 2001-2019. The main outcome measure was in-hospital mortality. RESULTS: The median age of the patients increased from 37 to 45 years. Meningococcal meningitis significantly diminished (56% versus 31%, p < 0.000) whereas listerial meningitis increased (1.2% versus 8%, p = 0.004). Systemic complications were more common in the second period, although mortality did not vary significantly between periods (10.4% versus 9.2%). However, after adjusting for relevant variables, infection in the second period was associated with lower risk death. CONCLUSIONS: Adult patients without underlying comorbidities that developed BM in recent years were older and more likely to have pneumococcal or listerial infections and systemic complications. In-hospital death was less likely in the second period, after adjusting for risk factors of mortality.

Recommendations of the Spanish Antibiogram Committee (COESANT) for in vitro susceptibility testing of antimicrobial agents by disk diffusion.

Disk diffusion is a well standardized method that provides reliable categorical results to guide antimicrobial therapy in numerous types of infections. Based on the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which are widely implemented in Spain, the Spanish Antibiogram Committee (COESANT) has drawn up recommendations for antimicrobial selection by the disk diffusion technique, including selective reporting and its use for the detection of resistance mechanisms. Factors affecting disk diffusion results, along with advantages and shortcomings of the method, are also discussed.

Outcomes of octogenarians and nonagenarians with Pseudomonas aeruginosa bacteremia: a multicenter retrospective study.

BACKGROUND: P. aeruginosa bacteremia is a common and severe infection carrying high mortality in older adults. We aimed to evaluate outcomes of P. aeruginosa bacteremia among old adults (≥ 80 years). METHODS: We included the 464/2394 (19%) older adults from a retrospective multinational (9 countries, 25 centers) cohort study of individuals hospitalized with P. aeruginosa bacteremia. Bivariate and multivariable logistic regression models were used to evaluate risk factors for 30-day mortality among older adults. RESULTS: Among 464 adults aged ≥ 80 years, the mean age was 84.61 (SD 3.98) years, and 274 (59%) were men. Compared to younger patients, ≥ 80 years adults had lower Charlson score; were less likely to have nosocomial acquisition; and more likely to have urinary source. Thirty-day mortality was 30%, versus 27% among patients 65-79 years (n = 894) and 25% among patients < 65 years (n = 1036). Multivariate analysis for predictors of mortality among patients ≥ 80 years, demonstrated higher SOFA score (odds ratio [OR] 1.36, 95% confidence interval [CI] 1.23-1.51, p < 0.001), corticosteroid therapy (OR 3.15, 95% CI: 1.24-8.01, p = 0.016) and hospital acquired P. aeruginosa bacteremia (OR 2.30, 95% CI: 1.33-3.98, p = 0.003) as predictors. Appropriate empirical therapy within 24 h, type of definitive anti-pseudomonal drug, and type of regimen (monotherapy or combination) were not associated with 30-day mortality. CONCLUSIONS: In older adults with P. aeruginosa bacteremia, background conditions, place of acquisition, and disease severity are associated with mortality, rather than the antimicrobial regimen. In this regard, preventive efforts and early diagnosis before organ failure develops might be beneficial for improving outcomes.

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3.

Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were bla OXA-48 (263/377), bla KPC-3 (62/377), bla VIM-1 (28/377), and bla NDM-1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.

Clinical usefulness of quantifying microbial load from diabetic foot ulcers: A multicenter cohort study.

AIMS: To evaluate if microbial load from diabetic foot ulcers (DFUs) can help in predicting outcomes. METHODS: A multicenter prospective cohort study was performed in an outpatient setting (September 1, 2017-January 31, 2019) in diabetic patients with DFU.Quantitative cultures from DFU tissue biopsies at a baseline visit were obtained; high and low microbial loads were defined as ≥6logCFU/mL and <6logCFU/mL, respectively. Diagnosis of DFU infection was made and managed according to established guidelines. The outcome was evaluated at 6 month-visit as failure (persistence/new infection/amputation) or cure. RESULTS: Out of 65 patients, 52 (80%) had long-standing DFUs (≥4 weeks), with high microbial load in 19 (29%).DFU infection (n = 24, 37%) was not associated with high microbial load in all patients but those with shorter DFU duration.Treatment failure occurred in 20/57 (35%) patients; high DFU microbial load was associated with worse outcome (n = 9/20, 45% failure rate, adjusted OR4.69; 95% CI, 1.22-18.09; p = 0.03),mainly due to the subgroup of patients with high microbial load and long-stand DFUs. CONCLUSIONS: Since patients with high microbial load had a worse outcome, quantitative cultures from DFUs can identify patients who would benefit from antibiotic therapy.

Duration of Treatment for Pseudomonas aeruginosa Bacteremia: a Retrospective Study.

INTRODUCTION: There is no consensus regarding optimal duration of antibiotic therapy for Pseudomonas aeruginosa bacteremia. We aimed to evaluate the impact of short antibiotic course. METHODS: We present a retrospective multicenter study including patients with P. aeruginosa bacteremia during 2009-2015. We evaluated outcomes of patients treated with short (6-10 days) versus long (11-15 days) antibiotic courses. The primary outcome was a composite of 30-day mortality or bacteremia recurrence and/or persistence. Univariate and inverse probability treatment-weighted (IPTW) adjusted multivariate analysis for the primary outcome was performed. To avoid immortal time bias, the landmark method was used. RESULTS: We included 657 patients; 273 received a short antibiotic course and 384 a long course. There was no significant difference in baseline characteristics of patients. The composite primary outcome occurred in 61/384 patients in the long-treatment group (16%) versus 32/273 in the short-treatment group (12%) (p = 0.131). Mortality accounted for 41/384 (11%) versus 25/273 (9%) of cases, respectively. Length of hospital stay was significantly shorter in the short group [median 13 days, interquartile range (IQR) 9-21 days, versus median 15 days, IQR 11-26 days, p = 0.002]. Ten patients in the long group discontinued antibiotic therapy owing to adverse events, compared with none in the short group. On univariate and multivariate analyses, duration of therapy was not associated with the primary outcome. CONCLUSIONS: In this retrospective study, 6-10 days of antibiotic course for P. aeruginosa bacteremia were as effective as longer courses in terms of survival and recurrence. Shorter therapy was associated with reduced length of stay and less drug discontinuation.

Combination versus monotherapy as definitive treatment for Pseudomonas aeruginosa bacteraemia: a multicentre retrospective observational cohort study.

BACKGROUND: Pseudomonas aeruginosa bacteraemia is a common and serious infection. No consensus exists regarding whether definitive combination therapy is superior to monotherapy. We aimed to evaluate the impact of combination therapy on mortality. METHODS: This was a multicentre retrospective study (nine countries, 25 centres), including 1277 patients with P. aeruginosa bacteraemia during 2009-15. We evaluated the association between ß-lactam plus aminoglycoside or quinolone combination therapy versus ß-lactam monotherapy and mortality. The primary outcome was 30 day all-cause mortality. Univariate and multivariate Cox regression analyses were conducted, introducing combination as a time-dependent variable. Propensity score was conducted to adjust for confounding for choosing combination therapy over monotherapy. RESULTS: Of 1119 patients included, 843 received definitive monotherapy and 276 received combination therapy (59% aminoglycoside and 41% quinolone). Mortality at 30 days was 16.9% (189/1119) and was similar between combination (45/276; 16.3%) and monotherapy (144/843; 17.1%) groups (P = 0.765). In multivariate Cox regression, combination therapy was not associated with reduced mortality (HR 0.98, 95% CI 0.64-1.53). No advantage in terms of clinical failure, microbiological failure or recurrent/persistent bacteraemia was demonstrated using combination therapy. Likewise, adverse events and resistance development were similar for the two regimens. CONCLUSIONS: In this retrospective cohort, no mortality advantage was demonstrated using combination therapy over monotherapy for P. aeruginosa bacteraemia. Combination therapy did not improve clinical or microbiological failure rates, nor affect adverse events or resistance development. Our finding of no benefit with combination therapy needs confirmation in well-designed randomized controlled trials.

Intraoperative Bacterial Contamination and Activity of Different Antimicrobial Prophylaxis Regimens in Primary Knee and Hip Replacement.

Surgical antimicrobial prophylaxis (SAP) is important for the prevention of prosthetic joint infections (PJIs) and must be effective against the microorganisms most likely to contaminate the surgical site. Our aim was to compare different SAP regimens (cefazolin, cefuroxime, or vancomycin, alone or combined with gentamicin) in patients undergoing total knee (TKA) and hip (THA) arthroplasty. In this preclinical exploratory analysis, we analyzed the results of intraoperative sample cultures, the ratio of plasma antibiotic levels to the minimum inhibitory concentrations (MICs) for bacteria isolated at the surgical wound and ATCC strains, and serum bactericidal titers (SBT) against the same microorganisms. A total of 132 surgical procedures (68 TKA, 64 THA) in 128 patients were included. Cultures were positive in 57 (43.2%) procedures (mostly for coagulase-negative staphylococci and Cutibacterium spp.); the rate was lower in the group of patients receiving combination SAP (adjusted OR 0.475, CI95% 0.229-0.987). The SAP regimens evaluated achieved plasma levels above the MICs in almost all of intraoperative isolates (93/94, 98.9%) and showed bactericidal activity against all of them (SBT range 1:8-1:1024), although SBTs were higher in patients receiving cefazolin and gentamicin-containing regimens. The potential clinical relevance of these findings in the prevention of PJIs remains to be determined.

Do Prosthetic Joint Infections Worsen the Functional Ambulatory Outcome of Patients with Joint Replacements? A Retrospective Matched Cohort Study.

OBJECTIVES: To assess the effect on the functional ambulatory outcome of postoperative joint infection (PJI) cured at the first treatment attempt versus not developing PJI in patients with hip and knee prostheses. METHODS: In a single-hospital retrospectively matched cohort study, each patient with PJI between 2007 and 2016 was matched on age, sex, type of prosthesis and year of implantation with two other patients with uninfected arthroplasties. The definition of a PJI cure included infection eradication, no further surgical procedures, no PJI-related mortality and no suppressive antibiotics. Functional ambulatory status evaluated one year after the last surgery was classified into four simple categories: able to walk without assistance, able to walk with one crutch, able to walk with two crutches, and unable to walk. Patients with total hip arthroplasties (THA), total knee arthroplasties (TKA) and partial hip arthroplasty (PHA) were analysed separately. RESULTS: A total of 109 PJI patients (38 TKA, 41 THA, 30 PHA) and 218 non-PJI patients were included. In a model adjusted for clinically relevant variables, PJI was associated with a higher risk of needing an assistive device for ambulation (vs. walking without aid) among THA (adjusted odds ratio (OR) 3.10, 95% confidence interval (95% CI) 1.26-7.57; p = 0.014) and TKA patients (OR 5.40, 95% CI 2.12-13.67; p < 0.001), and with requiring two crutches to walk or being unable to walk (vs. walking unaided or with one crutch) among PHA patients (OR 3.05, 95% CI 1.01-9.20; p = 0.047). CONCLUSIONS: Ambulatory outcome in patients with hip and knee prostheses with postoperative PJI is worse than in patients who do not have PJI.

Pathogenesis of Staphylococcus epidermidis in prosthetic joint infections: Can identification of virulence genes differentiate between infecting and commensal strains?

BACKGROUND: Staphylococcus epidermidis is a commensal of human skin flora and a frequent causative microorganism in prosthetic joint infections (PJIs). To date, no single marker has been identified to distinguish infecting strains from commensal S. epidermidis populations. AIM: We aimed to find possible genetic markers to distinguish between the two populations. METHODS: We analyzed 50 S. epidermidis strains from patients with PJIs, 50 from skin of healthy individuals (commensal strains) and 17 from the surgical field of patients undergoing primary arthroplasty. In these three groups we studied the antimicrobial susceptibility profile, sequence type, biofilm formation, and virulence factors. Strains from the surgical field have not been compared previously with strains from the other two groups. FINDINGS: S. epidermidis strains from PJI patients were significantly more antibiotic resistant than commensal strains and surgical field strains. A wide variety of sequences types was found in commensal and surgical field strains. The predominant sequence type was ST2 and it was only present in PJI strains (44%). Differences in biofilm production did not differ between populations. Virulence genes sdrF and bhp, the complete ica operon, and the insertion sequence IS256 were significantly predominant in PJI strains. In contrast, embp and hld genes and the mobile element ACME were more prevalent in commensal strains. Surgical field strains could be a valid control group to discriminate between infecting and commensal strains. CONCLUSION: A combination of characteristic features can differentiate between infecting and commensal S. epidermidis strains in PJI, while a single marker cannot.

Efficacy of Ceftolozane-Tazobactam in Combination with Colistin against Extensively Drug-Resistant Pseudomonas aeruginosa, Including High-Risk Clones, in an In Vitro Pharmacodynamic Model.

Combination therapy is an attractive therapeutic option for extensively drug-resistant (XDR) Pseudomonas aeruginosa infections. Colistin has been the only treatment available for these infections for many years, but its results are suboptimal. Ceftolozane-tazobactam (C/T) is a newly available therapeutic option that has shown good antipseudomonal activity, even against a number of XDR P. aeruginosa strains. However, data about combinations containing C/T are scarce. The aim of this study was to analyze the activity of C/T and colistin alone and in combination against a collection of XDR P. aeruginosa strains containing 24 representative clinical isolates from a multicentre Spanish study. Twenty-four time-kill experiments performed over 24 h were conducted in duplicate to determine the effects of colistin and C/T alone and combined. An in vitro pharmacodynamic chemostat model then was used to validate this combination against three selected XDR P. aeruginosa ST175 isolates with different susceptibility levels to C/T. Static time-kill assays demonstrated superior synergistic or additive effect for C/T plus colistin against 21 of the 24 isolates studied. In the in vitro dynamic pharmacokinetic/pharmacodynamic (PK/PD) model, the C/T regimen of 2/1 g every 8 h with a steady-state concentration of 2 mg/liter colistin effectively suppressed the bacterial growth at 24 h. Additive or synergistic interactions were observed for C/T plus colistin against XDR P. aeruginosa strains and particularly against C/T-resistant strains. C/T plus colistin may be a useful treatment for XDR P. aeruginosa infections, including those caused by high risk-clones resistant to C/T.

Ceftazidime, Carbapenems, or Piperacillin-tazobactam as Single Definitive Therapy for Pseudomonas aeruginosa Bloodstream Infection: A Multisite Retrospective Study.

BACKGROUND: The optimal antibiotic regimen for Pseudomonas aeruginosa bacteremia is controversial. Although ß-lactam monotherapy is common, data to guide the choice between antibiotics are scarce. We aimed to compare ceftazidime, carbapenems, and piperacillin-tazobactam as definitive monotherapy. METHODS: A multinational retrospective study (9 countries, 25 centers) including 767 hospitalized patients with P. aeruginosa bacteremia treated with ß-lactam monotherapy during 2009-2015. The primary outcome was 30-day all-cause mortality. Univariate and multivariate, including propensity-adjusted, analyses were conducted introducing monotherapy type as an independent variable. RESULTS: Thirty-day mortality was 37/213 (17.4%), 42/210 (20%), and 55/344 (16%) in the ceftazidime, carbapenem, and piperacillin-tazobactam groups, respectively. Type of monotherapy was not significantly associated with mortality in either univariate, multivariate, or propensity-adjusted analyses (odds ratio [OR], 1.14; 95% confidence interval [CI], 0.52-2.46, for ceftazidime; OR, 1.3; 95% CI, 0.67-2.51, for piperacillin-tazobactam, with carbapenems as reference in propensity adjusted multivariate analysis; 542 patients). No significant difference between antibiotics was demonstrated for clinical failure, microbiological failure, or adverse events. Isolation of P. aeruginosa with new resistance to antipseudomonal drugs was significantly more frequent with carbapenems (36/206 [17.5%]) versus ceftazidime (25/201 [12.4%]) and piperacillin-tazobactam (28/332 [8.4%] (P = .007). CONCLUSIONS: No significant difference in mortality, clinical, and microbiological outcomes or adverse events was demonstrated between ceftazidime, carbapenems, and piperacillin-tazobactam as definitive treatment of P. aeruginosa bacteremia. Higher rates of resistant P. aeruginosa after patients were treated with carbapenems, along with the general preference for carbapenem-sparing regimens, suggests using ceftazidime or piperacillin-tazobactam for treating susceptible infection.

Risk factors for mortality among patients with Pseudomonas aeruginosa bacteraemia: a retrospective multicentre study.

This study aimed to evaluate risk factors for 30-day mortality among hospitalised patients with Pseudomonas aeruginosa bacteraemia, a highly fatal condition. A retrospective study was conducted between 1 January 2009 and 31 October 2015 in 25 centres (9 countries) including 2396 patients. Univariable and multivariable analyses of risk factors were conducted for the entire cohort and for patients surviving ≥48 h. A propensity score for predictors of appropriate empirical therapy was introduced into the analysis. Of the 2396 patients, 636 (26.5%) died within 30 days. Significant predictors (odds ratio and 95% confidence interval) of mortality in the multivariable analysis included patient-related factors: age (1.02, 1.01-1.03); female sex (1.34, 1.03-1.77); bedridden functional capacity (1.99, 1.24-3.21); recent hospitalisation (1.43, 1.07-1.92); concomitant corticosteroids (1.33, 1.02-1.73); and Charlson comorbidity index (1.05, 1.01-1.93). Infection-related factors were multidrug-resistant Pseudomonas (1.52, 1.15-2.1), non-urinary source (2.44, 1.54-3.85) and Sequential Organ Failure Assessment (SOFA) score (1.27, 1.18-1.36). Inappropriate empirical therapy was not associated with increased mortality (0.81, 0.49-1.33). Among 2135 patients surviving ≥48 h, hospital-acquired infection (1.59, 1.21-2.09), baseline endotracheal tube (1.63, 1.13-2.36) and ICU admission (1.53, 1.02-2.28) were additional risk factors. Risk factors for mortality among patients with P. aeruginosa were mostly irreversible. Early appropriate empirical therapy was not associated with reduced mortality. Further research should be conducted to explore subgroups that may not benefit from broad-spectrum antipseudomonal empirical therapy. Efforts should focus on prevention of infection, mainly hospital-acquired infection and multidrug-resistant pseudomonal infection.

Colistin plus meropenem combination is synergistic in vitro against extensively drug-resistant Pseudomonas aeruginosa, including high-risk clones.

BACKGROUND: Extensively drug-resistant (XDR) Pseudomonas aeruginosa (P. aeruginosa) and particularly P. aeruginosa high-risk clones, are of growing concern because treatment options are limited. For years, colistin monotherapy has been the only available treatment, but is well known that is not an optimal treatment. A combination of colistin with another antibiotic could be a possible therapeutic option. OBJECTIVES: This study aimed to investigate effective antibiotic combinations against 20 XDR P. aeruginosa isolates obtained in a Spanish multicentre study (2015). METHODS: Forty-five checkerboards with six antipseudomonal antibiotics (amikacin, aztreonam, ceftazidime, meropenem, colistin, and ceftolozane/tazobactam) were performed to determine whether combinations were synergic or additive by fractional inhibitory concentration indices. On average, 15 different regimens were evaluated in duplicate against the three most prevalent high-risk clones (ST175, ST235, ST111) by time-kill analyses over 24h. The combination showing synergism in the three high-risk clones was validated in all studied XDR isolates. RESULTS: In time-kill curves, the untreated control failed, as did each study regimen when administered alone. Two combinations were synergistic in the three high-risk clones that were initially studied: amikacin plus ceftazidime and colistin plus meropenem, with the second being the most effective combination. The efficacy of colistin plus meropenem was then tested in all 20 isolates. A synergistic bacterial density reduction for the duration of the study occurred in 80% of the entire XDR collection. CONCLUSIONS: These data suggest that colistin plus meropenem may be a useful combination for the treatment of infections due to XDR P. aeruginosa, including high-risk clones, which warrants evaluation in a clinical trial.

Molecular characterization of OXA-48 carbapenemase-producing Klebsiella pneumoniae strains after a carbapenem resistance increase in Catalonia / Caracterización molecular de las cepas de Klebsiella pneumoniae productoras de carbapenemasa OXA-48 tras un aumento de la resistencia a las carbapenemas en Cataluña

Introduction: To characterize OXA-48 carbapenemase-producing Klebsiella pneumoniae strains isolated after an increase in carbapenem resistance in Catalonia. Methodology: K. pneumoniae identification, antimicrobial susceptibility studies, the Modified Hodge Test method, amplification of antimicrobial resistance genes (against β-lactamases, quinolones and aminoglycosides), molecular typing (by PFGE and MLST), conjugation assays, plasmid characterization (PBRT-PCR and Southern blot), a description of mobile genetic elements and statistical analysis were done. Results: OXA-48 was the only carbapenemase detected, with a prevalence of 1.9%. The blaOXA-48 gene was located in an IncL conjugative plasmid of 62 kb and integrated into the transposons Tn1999.2 (91.7%) or Tn1999.1. Five PFGE profiles (A to E) were found, which exactly matched the MLST: ST101, ST17, ST1233, ST14 and ST405, respectively. ST1233 is described here for the first time. K. pneumoniae OXA-48-producing strains were also CTX-M-15 carriers, some producing OXA-1 and TEM-1 penicillinases. The acquired qnrB66 and qnrB1 and aac(3′)-IIa, aac(6′)-Ib genes were also identified. Conclusion: The K. pneumoniae ST405 clone has played an important role in the growing prevalence of OXA-48 in Catalonia. All clones described preserved the blaOXA-48 genetic environment and mobile genetic elements (Tn1999). Notably, the three strains with minor sequence types in this study are not multiresistant strains. These strains are expanding in elderly patients (average age of 76 years) with serious underlying diseases, mainly women (61.2%)

Introducción: El objetivo de este estudio fue caracterizar las cepas de Klebsiella pneumoniae productoras de carbapenemasa OXA-48 aisladas tras observar un aumento de estos aislados resistentes a los carbapenémicos en Cataluña. Métodos: Se realizó la identificación de K. pneumoniae, estudios de sensibilidad antimicrobiana, el test de Hodge modificado, amplificación de genes de resistencia antimicrobiana (contra β-lactamasas, quinolonas y aminoglucósidos), tipificación molecular (por PFGE y MLST), ensayos de conjugación, caracterización de plásmidos (PBRT-PCR y Southern blot), descripción de los elementos genéticos móviles y el análisis estadístico. Resultados: OXA-48 fue la única carbapenemasa presente, con una prevalencia del 1,9%. El gen blaOXA-48 se localizó en un plásmido conjugativo IncL de 62kb e integrado en los transposones Tn1999.2 (91,7%) o Tn1999.1. Se encontraron 5 perfiles diferentes de PFGE (A a E), que tenían una concordancia exacta con el MLST: ST101, ST17, ST1233, ST14 y ST405, respectivamente. El ST1233 se describe aquí por primera vez. Las cepas productoras de K. pneumoniae OXA-48 también fueron portadoras de CTX-M-15 y algunas de ellas productoras también de penicilinasas OXA-1 y TEM-1. Los genes adquiridos qnrB66 y qnrB1 y aac(3’)-IIa, aac(6’)-Ib también se identificaron. Conclusión: El clon K. pneumoniae ST405 tiene un papel importante en la creciente prevalencia de OXA-48 en Cataluña. Todos los clones descritos preservaron el entorno genético de blaOXA-48, así como los elementos genéticos móviles (Tn1999). Notablemente, las 3 cepas con tipos de secuencia menos prevalentes en este estudio no son cepas multirresistentes. Además, la expansión de estas cepas con blaOXA-48 se está produciendo en pacientes de edad avanzada (promedio de edad de 76 años), la mayoría mujeres (61,2%) con enfermedades subyacentes graves