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Purpose: to characterize the distribution and mechanisms involved in ceftolozane/tazobactam (C/T) resistance in Pseudomonas aeruginosa isolates recovered from intensive care units (ICUs) in Portugal as part of the STEP surveillance study. Materials and methods: a total of 226 P. aeruginosa isolates were collected from patients with low respiratory tract infections (LRTI) admitted to ICUs between June 2017 and July 2018. Susceptibility to antimicrobials including the recent C/T combination was determined by EUCAST-criteria. Whole genome sequencing (WGS) was performed in a subset of 17 isolates. Results: multidrug resistant (MDR) and extremely drug resistant (XDR) phenotypes accounted for 20.4% and 25.7% of cases, respectively. C/T showed the highest susceptibility rate in both MDR (100%) and XDR (93.1%) isolates, followed by amikacin (97.8% MDR and 79.3% XDR). bla KPC-3 (n  = 2) and bla GES-13 (n  = 1) carbapenemase genes were detected in 3 of the 17 sequenced isolates, but only the GES-13-producing isolate displayed resistance to C/T. Additionally, the C/T-resistant phenotype was also found in two non-carbapenemase producers that carried known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene. Conclusions: C/T was highly active against MDR/XDR- P. aeruginosa isolates causing LRTI in ICUs. Moreover, beyond carbapenemase-encoding genes, mutations in chromosomal PBP-encoding genes might also be involved in ceftolozane/tazobactam resistance in Portugal. [ABSTRACT FROM AUTHOR]

CrpP enzymes have been recently described as a novel ciprofloxacin-resistance mechanism. We investigated by whole genome sequencing the presence of crpP-genes and other mechanisms involved in quinolone resistance in MDR/XDR-Pseudomonas aeruginosa isolates (n = 55) with both ceftolozane-tazobactam susceptible or resistant profiles recovered from intensive care unit patients during the STEP (Portugal) and SUPERIOR (Spain) surveillance studies. Ciprofloxacin resistance was associated with mutations in the gyrA and parC genes. Additionally, plasmid-mediated genes (qnrS2 and aac(6′)-Ib-cr) were eventually detected. Ten chromosomal crpP-like genes contained in related pathogenicity genomic islands and 6 different CrpP (CrpP1-CrpP6) proteins were found in 65% (36/55) of the isolates. Dissemination of CrpP variants was observed among non-related clones of both countries, including the CC175 (Spain) high-risk clone and CC348 (Portugal) clone. Interestingly, 5 of 6 variants (CrpP1-CrpP5) carried missense mutations in an amino acid position (Gly7) previously defined as essential conferring ciprofloxacin resistance, and decreased ciprofloxacin susceptibility was only associated with the novel CrpP6 protein. In our collection, ciprofloxacin resistance was mainly due to chromosomal mutations in the gyrA and parC genes. However, crpP genes carrying mutations essential for protein function (G7, I26) and associated with a restored ciprofloxacin susceptibility were predominant. Despite the presence of crpP genes is not always associated with ciprofloxacin resistance, the risk of emergence of novel CrpP variants with a higher ability to affect quinolones is increasing. Furthermore, the spread of crpP genes in highly mobilizable genomic islands among related and non-related P. aeruginosa clones alert the dispersion of MDR pathogens in hospital settings. [ABSTRACT FROM AUTHOR]

Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) Mass Spectrometry (MS) is a reference method for microbial identification and it can be used to predict Antibiotic Resistance (AR) when combined with artificial intelligence methods. However, current solutions need time-costly preprocessing steps, are difficult to reproduce due to hyperparameter tuning, are hardly interpretable, and do not pay attention to epidemiological differences inherent to data coming from different centres, which can be critical. We propose using a multi-view heterogeneous Bayesian model (KSSHIBA) for the prediction of AR using MALDI-TOF MS data together with their epidemiological differences. KSSHIBA is the first model that removes the ad-hoc preprocessing steps that work with raw MALDI-TOF data. In addition, due to its Bayesian probabilistic nature, it does not require hyperparameter tuning, provides interpretable results, and allows exploiting local epidemiological differences between data sources. To test the proposal, we used data from 402 Klebsiella pneumoniae isolates coming from two different domains and 20 different hospitals located in Spain and Portugal. KSSHIBA outperforms current state-of-the-art approaches in antibiotic susceptibility prediction, obtaining a 0.78 AUC score in Wild Type classification and a 0.90 AUC score in Extended-Spectrum Beta-Lactamases (ESBL)+Carbapenemases (CP)-producers. The proposal consistently removes the need for ad-hoc preprocessing by working with raw MALDI-TOF data, which, in turn, reduces the time needed to obtain the results of the resistance mechanism in microbiological laboratories. The proposed model implementation as well as both data domains are publicly available. [Display omitted] • Antibiotic Resistance in K. pneumoniae is predicted by using KSSHIBA. • Raw MALDI-TOF MS data is used, getting rid of time-costly external preprocessing. • Double dimensionality reduction is performed by kernel methods and factor analysis. • Hyperparameter tuning is eliminated using a Bayesian model. • The bacteria epidemiological differences are tackled by a multiview model. [ABSTRACT FROM AUTHOR]

• KPC-3-producing K. pneumoniae and CTX-M-15-producing ST131-H30Rx- E. coli most common MDR-Enterobacterales in Portuguese ICU patients. • Greater diversity of ESBL- K. pneumoniae clones was observed during the STEP collection than in other studies in Portugal. • Carbapenemase-encoding genes are associated with absence of activity of ceftolozane-tazobactam in Klebsiella spp. • WGS is a powerful tool for typing characterization and study of the resistome and virulome of nosocomial pathogens. • The ability to establish genotype-to-phenotype relationships may still be limited. Ceftolozane-tazobactam (C/T) is frequently used for infections caused by multidrug-resistant (MDR)-Enterobacterales isolates. Whole-genome sequencing (WGS, Illumina-Hiseq 4000/NovaSeq 6000, OGC, UK) was used to study the population structure, the resistome and the virulome of C/T-susceptible and -resistant MDR Escherichia spp. (n=30) and Klebsiella spp. (n=78) isolates, recovered from lower respiratory, intra-abdominal and urinary tract infections of ICU patients from 11 Portuguese Hospitals (STEP study, 2017-2018). Minimum inhibitory concentrations (MICs) were determined (ISO-broth microdilution, breakpoints EUCAST-2020). In Escherichia spp. , a weak concordance between the phenotypic and the WGS method (P =0.051) was observed in the carbapenemase detection (3/30) [ bla VIM-2 (2/3), bla KPC-3 (1/3)]; VIM-2- Escherichia coli isolates were C/T-susceptible and only the KPC-3- Escherichia marmotae producer showed C/T-resistance. Overall, CTX-M-15- E. coli -ST131-O25:H4-H30-Rx (11/30) was the most frequent subclone, followed by CTX-M-27- E. coli- ST131-O25:H4-H30 (4/4). Moreover, a wide resistome and virulome were detected in all E. coli isolates. Among Klebsiella spp. isolates [ K. pneumoniae (67/78), K. aerogenes (7/78), K. oxytoca (2/78), K. variicola (2/78)], concordance (P <0.001) was observed between the phenotypic and the genomic carbapenemase detection (21/78) [ bla KPC-3 (14/21), bla OXA-48 (3/21), bla OXA-181 (3/21)]. A high correlation between C/T-resistance and carbapenemase detection was established (P <0.05). Overall, a high clonal diversity was observed, mainly in KPC-3-producing K. pneumoniae isolates. An extensive resistome was detected in Klebsiella spp. isolates, whereas virulence determinants were mostly identified in carbapenemase producers (P <0.001). WGS is a powerful tool for typing characterization and microbiological study of MDR-Enterobacterales pathogens. Furthermore, carbapenemase genes are associated with C/T-resistance in Klebsiella spp., but other mechanisms might also be involved. [ABSTRACT FROM AUTHOR]

• Ceftolozane-tazobactam was the most potent agent against Pseudomonas aeruginosa • Overall susceptibility of ceftolozane-tazobactam against Enterobacterales was 86.9% • Ceftolozane-tazobactam showed excellent activity against Escherichia coli (99.4%) • Ceftolozane-tazobactam activity was decreased in Klebsiella due to a high rate of beta-lactamase producers • The in vitro activity of ceftolozane-tazobactam enhances its use in ICUs The STEP surveillance study was designed to increase knowledge about distribution of multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa in Portugal, focusing on the intensive care unit (ICU). Antimicrobial susceptibility of common agents was also evaluated and compared with that of one of the latest therapeutic introductions, ceftolozane-tazobactam (C/T). Clinical isolates of Enterobacterales (n=426) and P. aeruginosa (n=396) from patients admitted in Portuguese ICUs were included. Activity of C/T and comparators was investigated using standard broth microdilution. Isolates were recovered from urinary tract (UTI, 36.9%), intra-abdominal (IAI, 24.2%) and lower respiratory tract (LRTI, 38.9%) infections. In P. aeruginosa , overall distribution of MDR/extremely-drug resistant (XDR)/pan-drug resistant (PDR) isolates accounted for 21.2%, 23.2% and 0.8%, respectively. C/T was the most potent agent tested against P. aeruginosa and MDR/XDR/PDR phenotypes. In Escherichia coli , extended-spectrum beta-lactamases (ESBL) and carbapenemase (CP) phenotypes accounted for 16.6% and 1.7%, respectively, whereas in Klebsiella spp., ESBL and CP-phenotypes represented 28.5% and 17.9%, respectively. Overall, susceptibility of C/T against Enterobacterales was 86.9%. C/T was the least affected agent in E. coli (99.4% susceptibility), whereas its activity was moderate in Klebsiella spp. (71.5%) and Enterobacter spp. (70.4%), due in part to a high rate of ESBL and CP-phenotypes. In Enterobacterales, bla KPC was the most prevalent CP gene (63.0%), followed by bla OXA-48 (33.3%) and bla VIM (3.7%). These microbiological results reinforce C/T as a therapeutic option in ICU patients with UTI, IAI or LRTI due to P. aeruginosa or Enterobacterales isolates, but not for CP producers. [ABSTRACT FROM AUTHOR]

Objectives: To study the in vitro activity of imipenem/relebactam and comparators and the imipenem/relebactam resistance mechanisms in a Pseudomonas aeruginosa collection from Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17) surveillance studies., Methods: P. aeruginosa isolates (n = 474) were prospectively recovered from complicated urinary tract (cUTI), complicated intra-abdominal (cIAI) and lower respiratory tract (LRTI) infections in 11 Portuguese and 8 Spanish ICUs. MICs were determined (ISO broth microdilution). All imipenem/relebactam-resistant P. aeruginosa isolates (n = 30) and a subset of imipenem/relebactam-susceptible strains (n = 32) were characterized by WGS., Results: Imipenem/relebactam (93.7% susceptible), ceftazidime/avibactam (93.5% susceptible) and ceftolozane/tazobactam (93.2% susceptible) displayed comparable activity. The imipenem/relebactam resistance rate was 6.3% (Portugal 5.8%; Spain 8.9%). Relebactam restored imipenem susceptibility to 76.9% (103/134) of imipenem-resistant isolates, including MDR (82.1%; 32/39), XDR (68.8%; 53/77) and difficult-to-treat (DTR) isolates (67.2%; 45/67). Among sequenced strains, differences in population structure were detected depending on the country: clonal complex (CC)175 and CC309 in Spain and CC235, CC244, CC348 and CC253 in Portugal. Different carbapenemase gene distributions were also found: VIM-20 (n = 3), VIM-1 (n = 2), VIM-2 (n = 1) and VIM-36 (n = 1) in Spain and GES-13 (n = 13), VIM-2 (n = 3) and KPC-3 (n = 2) in Portugal. GES-13-CC235 (n = 13) and VIM type-CC175 (n = 5) associations were predominant in Portugal and Spain, respectively. Imipenem/relebactam showed activity against KPC-3 strains (2/2), but was inactive against all GES-13 producers and most of the VIM producers (8/10). Mutations in genes affecting porin inactivation, efflux pump overexpression and LPS modification might also be involved in imipenem/relebactam resistance., Conclusions: Microbiological results reinforce imipenem/relebactam as a potential option to treat cUTI, cIAI and LRTI caused by MDR/XDR P. aeruginosa isolates, except for GES-13 and VIM producers., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Imipenem-relebactam is a novel β-lactam-β-lactamase inhibitor combination. We evaluated the in vitro activity of imipenem-relebactam and comparators against Enterobacterales clinical isolates recovered in 8 Spanish and 11 Portuguese intensive care units (ICUs) (SUPERIOR, 2016-2017; STEP, 2017-2018). Overall, 747 Enterobacterales isolates (378 Escherichia coli, 252 Klebsiella spp., 64 Enterobacter spp., and 53 other species) were prospectively collected from ICU patients with complicated intraabdominal (cIAI), complicated urinary tract (cUTI), and lower respiratory tract (LRTI) infections. MICs were determined (ISO-broth microdilution), and whole-genome sequencing (WGS) was performed in a subset of isolates displaying susceptible and resistant imipenem-relebactam MICs. Imipenem-relebactam (98.7% susceptible) showed similar activity to ceftazidime-avibactam (99.5% susceptible) and higher than ceftolozane-tazobactam (86.9% susceptible). Imipenem-relebactam was inactive against 1.3% (10/747) isolates, all of them due to carbapenemase production (9 K. pneumoniae and 1 E. cloacae). Imipenem-relebactam was active against 100% of extended-spectrum β-lactamase (ESBL)-E. coli and ESBL-Klebsiella spp. isolates and 80.4% of carbapenemase-Klebsiella spp. producers. Carbapenemase genes were confirmed by WGS in 41 Klebsiella spp.: OXA-48 (20/41), KPC-3 (14/41), OXA-181 (4/41), NDM-1 (1/41), OXA-48 + VIM-2 (1/41), and KPC-3 + VIM-2 (1/41). In Klebsiella spp. isolates, relebactam restored imipenem susceptibility in all KPC-3 producers, and resistant isolates (7/41) were mostly OXA-48 + CTX-M-15-K. pneumoniae high-risk clones (7/9). Intercountry differences were detected as follows: OXA-48 (17/21) was dominant in Spain, unlike KPC-3 (14/15) in Portugal. Imipenem-relebactam was 100% active against CTX-M-15-ST131-H30Rx-E. coli high-risk clone, predominant in both countries. Our results depict the potential role of imipenem-relebactam in ICU patients with cIAIs, cUTIs, and LRTIs due to wild-type ESBL- and carbapenemase-producing Enterobacterales , particularly KPC producers. IMPORTANCE We comparatively evaluate the in vitro activity of a drug combination consisting of a carbapenem (imipenem) and a novel inhibitor of beta-lactamases (relebactam), a mechanism that destroys beta-lactam antibiotics. We assess the activity against a collection of Enterobacterales clinical isolates recovered from difficult-to-treat infections in patients admitted to different intensive care units in Portugal and Spain. Imipenem-relebactam shows excellent activity in avoiding common resistance mechanisms in this setting, such as extended-spectrum beta-lactamases and carbapenemases widely distributed, including KPCs. We show few resistant isolates (<2%). Molecular characterization by whole-genome sequencing shows that most of the resistant isolates produced specific carbapenemase, such as OXA-48 or metalo-betalactamases. Our study updates the activity of imipenem-relebactam in light of current epidemiology in a hospital setting in which the use of this combination is needed due to the presence of infections due to multidrug-resistant isolates.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.

Objectives: To investigate the diversity of integrons and extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae from chickens and swine in Portugal and analyse the clonal relationships between Portuguese ESBL-producing isolates of animal and human origin., Methods: We analysed samples from faeces of healthy swine (HSF, n = 35), from uncooked chicken carcasses (CM, n = 20) and from faeces of healthy chickens (HCF, n = 20). Samples were plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). ESBLs were characterized by PCR and DNA sequencing. Bacterial identification, antibiotic susceptibility and conjugation assays were performed by standard procedures. Isolate clonal relatedness was established by PFGE and by RAPD for PFGE non-typeable isolates. Escherichia coli phylogenetic groups were identified by a multiplex PCR. Integron analysis was accomplished by PCR-RFLP and sequencing., Results: ESBL-producing Enterobacteriaceae were identified in 60% of CM, 10% of HCF and 5.7% of HSF samples, respectively, mostly corresponding to E. coli (phylogroups A, D and B1). TEM-52, SHV-2 and CTX-M-1 were detected from chicken and SHV-12 from swine samples. High clonal diversity was observed and most bla(ESBL) genes were transferable (67%). Class 1 and/or class 2 integrons were identified in 80% of CM, 10% of HCF and 63% of HSF samples, with class 1 integrons more common than class 2 integrons (36% versus 12% of the isolates recovered, respectively). Ten class 1 integron types are described, aadA1 and dfrA1-aadA1 being the most frequently found. Two class 1 integron types (aadA13-estX and dfrA14-aadA1-catB2) and one class 2 integron (aadA1) are first reported here., Conclusions: This study is the first report of ESBLs and integrons from chickens and swine in Portugal and highlights the antibiotic-resistant bacteria and/or resistance genes that might be acquired by humans through the food chain.

Objectives: To investigate the occurrence and the diversity of Ambler class A ESBLs among Enterobacteriaceae from different Portuguese clinical settings over a 2 year period (2002-04)., Methods: One hundred and nine extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolates from five geographically distant health institutions in Portugal were studied. ESBLs were characterized by isoelectric focussing, PCR and further sequencing. Antibiotic susceptibility testing, transfer of resistance genes and clonal diversity were determined by standard procedures. Plasmid relatedness was established by comparison of random amplified polymorphic DNA (RAPD) patterns., Results: ESBLs were identified as TEM (46%), SHV (30%), CTX-M (22%) and GES (2%) types; TEM-24, TEM-52, SHV-12 and CTX-M-15 enzymes being the most frequently found. Inter-hospital dissemination of epidemic strains harbouring the most prevalent ESBLs was detected, including the TEM-24-producing Enterobacter aerogenes European epidemic clone. Conjugative transfer of ESBLs was achieved for 67% of isolates and epidemic plasmids containing specific bla genes were detected (bla(CTX-M-15) and bla(TEM-24)). We describe two new ESBLs, SHV-90 (A187T, G238S and E240K) and SHV-91 (P20S and E240K), and a new TEM-type enzyme conferring a phenotype resembling that of a complex mutant TEM beta-lactamase, designated as TEM-154 (M69L and R164S). The broad-spectrum beta-lactamases SHV-26, SHV-36 and TEM-110 were first observed in our country., Conclusions: We describe a complex ESBL epidemiology in Portugal, including widespread dissemination of known strains and plasmids coding for TEM-24 and CTX-M-15 enzymes as observed in other European countries.