Enterobacteriaceae showing resistance to cephalosporins due to extended-spectrum b-lactamases (ESBLs) or plasmid-mediated AmpC enzymes, and those producing carbapenemases have spread worldwide during the last decades. Many of these isolates are also resistant to other first-line agents such as fluoroquinolones or aminoglycosides, leaving few available options for therapy. Thus, older drugs such as colistin and fosfomycin are being increasingly used. Infections caused by these bacteria are associated with increased morbidity and mortality compared with those caused by their susceptible counterparts. Most of the evidence supporting the present recommendations is from in vitro data, animal studies, and observational studies. While carbapenems are considered the drugs of choice for ESBL and AmpC producers, recent data suggest that certain alternatives may be suitable for some types of infections. Combined therapy seems superior to monotherapy in the treatment of invasive infections caused by carbapenemase-producing Enterobacteriaceae. Optimization of dosage according to pharmacokinetics/pharmacodynamics data is important for the treatment of infections caused by isolates with borderline minimum inhibitory concentration due to lowlevel resistance mechanisms. The increasing frequency and the rapid spread of multidrug resistance among the Enterobacteriaceae is a true and complex public health problem. IntroductionThe traditional first-line available options for treating serious infections caused by enterobacteria include penicillins, cephalosporins, monobactams, carbapenems, fluorquinolones, and in certain situations, aminoglycosides. The frequency of resistance to these first-line agents has been rising worldwide during the last decades [Paterson 2006;Rhomberg and Jones 2009;Houghton et al. 2010;Nordmann, et al. 2011] and now reach high proportions in many areas of the world.

Background Cefiderocol is a novel siderophore cephalosporin, developed for activity against MDR Gram-negative bacilli (MDR-GNB). Objectives To assess the in vitro antibacterial activity of cefiderocol against a collection of MDR-GNB clinical isolates from hospitals in southern Spain. Methods Two hundred and thirty-one isolates of successful clones were tested: 125 Enterobacterales (121 ESBL- and/or carbapenemase-producing Klebsiella pneumoniae and 4 carbapenemase-producing Enterobacter cloacae), 80 Acinetobacter baumannii, 6 Pseudomonas aeruginosa and 20 Stenotrophomonas maltophilia. Ceftolozane/tazobactam, ceftazidime, ceftazidime/avibactam, cefepime, aztreonam, meropenem, amikacin, ciprofloxacin, colistin and tigecycline were used as comparators against Enterobacterales, P. aeruginosa and A. baumannii. Minocycline, levofloxacin and trimethoprim/sulfamethoxazole were studied against S. maltophilia instead of aztreonam, ciprofloxacin and cefepime. MICs were determined by broth microdilution according to CLSI guidelines. MIC determination was performed in CAMHB for all antimicrobials except cefiderocol, where iron-depleted CAMHB was used. Results Cefiderocol showed potent in vitro activity against the isolates analysed. MIC50 and MIC90 values were in the ranges 0.125–8 mg/L and 0.5–8 mg/L, respectively, and 98% of isolates were inhibited at ≤4 mg/L. Only five isolates showed cefiderocol MICs of >4 mg/L: three ST2/OXA-24/40-producing A. baumannii, one ST114/VIM-1-producing E. cloacae and one ST114/VIM-1 + OXA-48-producing E. cloacae. All KPC-3-producing K. pneumoniae were susceptible to cefiderocol, even those resistant to ceftazidime/avibactam. P. aeruginosa isolates showed cefiderocol MICs of <4 mg/L, including those resistant to ceftolozane/tazobactam. S. maltophilia isolates displayed cefiderocol MICs of <4 mg/L, including those resistant to levofloxacin and/or trimethoprim/sulfamethoxazole. Conclusions Cefiderocol showed excellent activity against MDR-GNB, including carbapenem-resistant isolates, and was the most active antimicrobial tested against this collection.

Background More data are needed about the safety of antibiotic de-escalation in specific clinical situations as a strategy to reduce exposure to broad-spectrum antibiotics. The aims of this study were to investigate predictors of de-escalation and its impact on the outcome of patients with bloodstream infection due to Enterobacteriaceae (BSI-E). Methods A post hoc analysis was performed on a prospective, multicenter cohort of patients with BSI-E initially treated with ertapenem or antipseudomonal β-lactams. Logistic regression was used to analyze factors associated with early de-escalation (EDE) and Cox regression for the impact of EDE and late de-escalation (LDE) on 30-day all-cause mortality. A propensity score (PS) for EDE vs no de-escalation (NDE) was calculated. Failure at end of treatment and length of hospital stay were also analyzed. Results Overall, 516 patients were included. EDE was performed in 241 patients (46%), LDE in 95 (18%), and NDE in 180 (35%). Variables independently associated with a lower probability of EDE were multidrug-resistant isolates (odds ratio [OR], 0.50 [95% confidence interval {CI}, .30–.83]) and nosocomial infection empirically treated with imipenem or meropenem (OR, 0.35 [95% CI, .14–.87]). After controlling for confounders, EDE was not associated with increased risk of mortality; hazard ratios (HR) (95% CIs) were as follows: general model, 0.58 (.25–1.31); model with PS, 0.69 (.29–1.65); and PS-based matched pairs, 0.98 (.76–1.26). LDE was not associated with mortality. De-escalation was not associated with clinical failure or length of hospital stay. Conclusions De-escalation in patients with monomicrobial bacteremia due to Enterobacteriaceae was not associated with a detrimental impact on clinical outcome.

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

Although the failure of antibiotic treatment is normally attributed to resistance, tolerance and persistence display a significant role in the lack of response to antibiotics. Due to the fact that several nosocomial pathogens show a high level of tolerance and/or resistance to chlorhexidine, in this study we analyzed the molecular mechanisms associated with chlorhexidine adaptation in two clinical strains of Klebsiella pneumoniae by phenotypic and transcriptomic studies. These two strains belong to ST258-KPC3 (high-risk clone carrying β-lactamase KPC3) and ST846-OXA48 (low-risk clone carrying β-lactamase OXA48). Our results showed that the K. pneumoniae ST258-KPC3CA and ST846-OXA48CA strains exhibited a different behavior under chlorhexidine (CHLX) pressure, adapting to this biocide through resistance and tolerance mechanisms, respectively. Furthermore, the appearance of cross-resistance to colistin was observed in the ST846-OXA48CA strain (tolerant to CHLX), using the broth microdilution method. Interestingly, this ST846-OXA48CA isolate contained a plasmid that encodes a novel type II toxin/antitoxin (TA) system, PemI/PemK. We characterized this PemI/PemK TA system by cloning both genes into the IPTG-inducible pCA24N plasmid, and found their role in persistence and biofilm formation. Accordingly, the ST846-OXA48CA strain showed a persistence biphasic curve in the presence of a chlorhexidine-imipenem combination, and these results were confirmed by the enzymatic assay (WST-1).

CLSI and EUCAST methodologies showed low agreement for determining the MIC of amoxicillin/clavulanate. EUCAST-derived MICs seemed more predictive of failure than CLSI-derived ones. EUCAST-derived MICs >16/2 mg/L were independently associated with therapeutic failure.

ObjectivesCARB-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.MethodsIn 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.ResultsIn 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 blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–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).ConclusionThis 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.

Objectives To analyse by WGS the ceftolozane/tazobactam (C/T) resistance mechanisms in Escherichia coli and Klebsiella spp. isolates recovered from complicated intra-abdominal and urinary tract infections in patients from Spanish ICUs (SUPERIOR surveillance study, 2016–17). Methods The clonal relatedness, the resistome and the virulome of 45 E. coli and 43 Klebsiella spp. isolates with different C/T susceptibility profiles were characterized. Results In E. coli, two (C/T susceptible) carbapenemase producers (VIM-2-CC23, OXA-48-ST38) were detected. The most relevant clone was ST131-B2-O25:H4-H30 (17/45), particularly the CTX-M-15-ST131-H30-Rx sublineage (15/17). ST131 strains were mainly C/T susceptible (15/17) and showed an extensive virulome. In non-ST131 strains (28/45), CTX-M enzymes [CTX-M-14 (8/24); CTX-M-15 (6/24); CTX-M-1 (3/24); CTX-M-32 (2/24)] were found in different clones. C/T resistance was detected in non-clonal E. coli isolates (13%, 6/45) with ESBL (4/6) and non-ESBL (2/6) genotypes. Among Klebsiella spp., Klebsiella pneumoniae (42/43) and Klebsiella michiganensis (1/43) species were identified; 42% (18/43) were carbapenemase producers and 58% showed a C/T resistance phenotype (25/43). OXA-48-ST11 (12/18), OXA-48-ST392 (2/18), OXA-48-ST15 (2/18), NDM-1-ST101 (1/18) and OXA-48+VIM-2-ST15 (1/18) isolates were found, all C/T resistant. Correlation between carbapenemase detection and resistance to C/T was demonstrated (P < 0.001). In non-carbapenemase-producing K. pneumoniae (25/43), C/T resistance (28%, 7/25) was detected in ESBL (3/7) and AmpC (2/7) producers. Overall, an extensive virulome was found and was correlated with carbapenemase carriage (P < 0.001) and C/T resistance (P < 0.05), particularly in OXA-48-ST11 strains (P < 0.05). Conclusions Prediction of antimicrobial susceptibility profiles using WGS is challenging. Carbapenemase-encoding genes are associated with C/T resistance in K. pneumoniae, but other resistance mechanisms might be additionally involved.