Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

PURPOSE: To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. METHODS: CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. RESULTS: A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. CONCLUSIONS: The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Reporting antimicrobial susceptibilities and phenotypes of resistance to vancomycin in vancomycin-resistant Enterococcus spp. clinical isolates: A nationwide proficiency study / Informe de sensibilidad antimicrobiana y fenotipos de resistencia a vancomicina en aislados clínicos de Enterococcus spp. resistentes a vancomicina: estudio multicéntrico nacional

Introduction: The ability of Spanish microbiology laboratories to (a) determine antimicrobial susceptibility (AS), and (b) correctly detect the vancomycin resistance (VR) phenotype in vancomycin-resistant Enterococcus spp. (VRE) was evaluated. Methods: Three VRE isolates representing the VanA (E. faecium), VanB (E. faecium) and VanC (E. gallinarum) VR phenotypes were sent to 52 laboratories, which were asked for: (a) AS method used; (b) MICs of ampicillin, imipenem, vancomycin, teicoplanin, linezolid, daptomycin, ciprofloxacin, levofloxacin and quinupristin–dalfopristin, and high-level resistance to gentamicin and streptomycin; (c) VR phenotype. Results: (a) The most frequently used system was MicroScan; (b) according to the system, the highest percentage of discrepant MICs was found with gradient strips (21.3%). By antimicrobial, the highest rates of discrepant MICs ranged 16.7% (imipenem) to 0.7% (linezolid). No discrepant MICs were obtained with daptomycin or levofloxacin. Mayor errors (MEs) occurred with linezolid (1.1%/EUCAST) and ciprofloxacin (5.0%/CLSI), and very major errors (VMEs) with vancomycin (27.1%/EUCAST and 33.3%/CLSI) and teicoplanin (5.7%/EUCAST and 2.3%/CLSI). For linezolid, ciprofloxacin, and vancomycin, discrepant MICs were responsible for these errors, while for teicoplanin, errors were due to a misassignment of the clinical category. An unacceptable high percentage of VMEs was obtained using gradient strips (14.8%), especially with vancomycin, teicoplanin and daptomycin; (c) 86.4% of the centers identified VanA and VanB phenotypes correctly, and 95.0% the VanC phenotype. Conclusion: Most Spanish microbiology laboratories can reliably determine AS in VRE, but there is a significant percentage of inadequate interpretations (warning of false susceptibility) for teicoplanin in isolates with the VanB phenotype.(AU)

Introducción: Se evaluó la capacidad de los laboratorios de microbiología españoles para: (a) determinar la sensibilidad antimicrobiana (SA); y (b) detectar correctamente el fenotipo de resistencia a vancomicina (FRV) en Enterococcus spp. resistente a vancomicina (ERV). Métodos: Se enviaron 3 aislados de ERV (E. faecium/VanA, E. faecium/VanB y E. gallinarum/VanC) a 52 laboratorios, a los que se les solicitó: (a) método de SA; (b) CMI de ampicilina, imipenem, vancomicina, teicoplanina, linezolid, daptomicina, ciprofloxacino, levofloxacino y quinupristina-dalfopristina y resistencia de alto nivel a gentamicina y estreptomicina; y (c) fenotipo de resistencia a vancomicina. Resultados: (a) El sistema más utilizado fue MicroScan; y (b) el mayor porcentaje de CMI discrepantes se produjo con las tiras de gradiente (21,3%). Las tasas más elevadas de CMI discrepantes variaron entre el 16,7% (imipenem) y el 0,7% (linezolid). Se produjeron errores mayores con linezolid (1,1%/EUCAST) y ciprofloxacino (5,0%/CLSI) y errores máximos con vancomicina (27,1%/EUCAST y 33,3% CLSI) y teicoplanina (5,7%/EUCAST y 2,3%/CLSI). Para linezolid, ciprofloxacino y vancomicina las CMI discrepantes fueron las responsables de estos errores, mientras que para teicoplanina los errores se debieron a una asignación errónea de la categoría clínica. Se obtuvo un alto porcentaje de errores máximos utilizando tiras de gradiente (14,8%), especialmente con vancomicina, teicoplanina y daptomicina; y (c) el 86,4% de los centros identificaron correctamente los fenotipos VanA y VanB y el 95,0% el fenotipo VanC. Conclusión: La mayoría de los laboratorios de microbiología españoles determinan de forma fiable la SA en ERV, pero existe un porcentaje significativo de interpretaciones inadecuadas (falsa sensibilidad) para teicoplanina en aislados con fenotipo VanB.(AU)

Genomic analysis of the initial dissemination of carbapenem-resistant Klebsiella pneumoniae clones in a tertiary hospital.

Carbapenem-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections and the fastest-growing pathogen in Europe. Carbapenem resistance was detected at the Consorcio Hospital General Universitario de Valencia (CHGUV) in early 2015, and there has been a significant increase in carbapenem-resistant isolates since then. In this study, we collected carbapenem-resistant isolates from this hospital during the period of increase (from 2015 to 2019) and studied how K. pneumoniae carbapenem-resistant isolates emerged and spread in the hospital. A total of 225 isolates were subjected to whole-genome sequencing with Illumina NextSeq. We characterized the isolates by identifying lineages and antimicrobial resistance genes and plasmids, especially those related to reduced carbapenem susceptibility. Our findings show that the initial carbapenem resistance emergence and dissemination at the CHGUV occurred during a short period of 1 year. Furthermore, it was complex, involving six different lineages of types ST307, ST11, ST101 and ST437, different resistance-determinant factors, including OXA-48, NDM-1, NDM-23 and DHA-1, and different plasmids.

Reporting antimicrobial susceptibilities and phenotypes of resistance to vancomycin in vancomycin-resistant Enterococcus spp. clinical isolates: A nationwide proficiency study.

INTRODUCTION: The ability of Spanish microbiology laboratories to (a) determine antimicrobial susceptibility (AS), and (b) correctly detect the vancomycin resistance (VR) phenotype in vancomycin-resistant Enterococcus spp. (VRE) was evaluated. METHODS: Three VRE isolates representing the VanA (E. faecium), VanB (E. faecium) and VanC (E. gallinarum) VR phenotypes were sent to 52 laboratories, which were asked for: (a) AS method used; (b) MICs of ampicillin, imipenem, vancomycin, teicoplanin, linezolid, daptomycin, ciprofloxacin, levofloxacin and quinupristin-dalfopristin, and high-level resistance to gentamicin and streptomycin; (c) VR phenotype. RESULTS: (a) The most frequently used system was MicroScan; (b) according to the system, the highest percentage of discrepant MICs was found with gradient strips (21.3%). By antimicrobial, the highest rates of discrepant MICs ranged 16.7% (imipenem) to 0.7% (linezolid). No discrepant MICs were obtained with daptomycin or levofloxacin. Mayor errors (MEs) occurred with linezolid (1.1%/EUCAST) and ciprofloxacin (5.0%/CLSI), and very major errors (VMEs) with vancomycin (27.1%/EUCAST and 33.3%/CLSI) and teicoplanin (5.7%/EUCAST and 2.3%/CLSI). For linezolid, ciprofloxacin, and vancomycin, discrepant MICs were responsible for these errors, while for teicoplanin, errors were due to a misassignment of the clinical category. An unacceptable high percentage of VMEs was obtained using gradient strips (14.8%), especially with vancomycin, teicoplanin and daptomycin; (c) 86.4% of the centers identified VanA and VanB phenotypes correctly, and 95.0% the VanC phenotype. CONCLUSION: Most Spanish microbiology laboratories can reliably determine AS in VRE, but there is a significant percentage of inadequate interpretations (warning of false susceptibility) for teicoplanin in isolates with the VanB phenotype.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Tertiary Hospital from the Comunidad Valenciana (Spain).

To reduce the high rates of morbidity and mortality caused by methicillin-resistant Staphylococcus aureus (MRSA) strains, it is essential to prevent their transmission. This can be achieved through molecular surveillance of the infecting strains, for which the detection of the entry of new strains, the analysis of antimicrobial resistance, and their containment are essential. In this study, we have analyzed 190 MRSA isolates obtained at the Consorcio Hospital General Universitario de Valencia (Spain) from 2013 to 2018 with three approaches: Multilocus Sequence Typing, spa, and SCCmec typing. Although the incidence of S. aureus infections detected in the hospital increased in the study period, the frequency of MRSA isolates decreased from 33% to 18%. One hundred seventy-two MRSA isolates were resistant to three or more classes of antimicrobials, especially to fluoroquinolones. No relevant temporal trend in the distribution of antibiotic susceptibility was observed. The combination of the three typing schemes allowed the identification of 74 different clones, of which the combination ST125-t067-IV was the most abundant in the study (27 cases). Members of three clonal complexes, CC5, CC8, and CC22, comprised 91% of the isolates, and included 32 STs and 32 spa types. The emergence of low incidence strains throughout the study period and a large number of isolates resistant to different classes of antibiotics shows the need for epidemiological surveillance of this pathogen. Our study demonstrates that epidemiological and molecular surveillance is a powerful tool to detect the emergence of clinically important MRSA clones.

In vitro activity of imipenem/relebactam against Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal (SUPERIOR and STEP studies).

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.

Imipenem-Relebactam Susceptibility in Enterobacterales Isolates Recovered from ICU Patients from Spain and Portugal (SUPERIOR and STEP Studies).

Imipenem-relebactam is a novel ß-lactam-ß-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 ß-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.

Performance comparison of a flow cytometry immunoassay for intracellular cytokine staining and the QuantiFERON® SARS-CoV-2 test for detection and quantification of SARS-CoV-2-Spike-reactive-IFN-γ-producing T cells after COVID-19 vaccination.

PURPOSE: We compared the performance of an in-house-developed flow cytometry assay for intracellular cytokine staining (FC-ICS) and a commercially-available cytokine release assay (the QuantiFERON® SARS-CoV-2 Test [QF]) for detection and quantification of SARS-CoV-2-Spike (S)-reactive-IFN-γ-producing T cells after COVID-19 vaccination. PATIENTS AND METHODS: The sample included 141 individuals (all male; median age, 42 years; 20-72) who had been fully vaccinated with the Comirnaty® COVID-19 vaccine (at a median of 114 days; 34-145). Prior to vaccination, 91 were categorized as being SARS-CoV-2-naïve and 50 as SARS-CoV-2-experienced. A whole blood-based FC-ICS using 15-mer overlapping peptides encompassing the entire SARS-CoV-2 S protein was used for enumeration of virus-specific IFN-γ-producing CD4+ and CD8+ T cells. The QF test (Ag1 for CD4+ T cells and Ag2 for CD4+ and CD8+ T cells in combination) was carried out following the manufacturer's instructions. RESULTS: The FC-ICS and the QF assays returned significantly discordant qualitative results in both the entire cohort (P<0.001 with QF Ag1 and QF Ag2) and in SARS-CoV-2-naïve participants alone (P=0.005 and P=0.01, respectively). Discrepant results mostly involved FC-ICS positive/QF negative specimens. Overall, no correlation was found either between SARS-CoV-2 IFN-γ- CD4+ T-cell frequencies and IFN-γ levels measured in the QF Ag1 tube (P=0.78) or between the sum of SARS-CoV-2 IFN-γ CD4+ and CD8+ T-cell frequencies and IFN-γ levels quantified in the QF Ag2 tube. CONCLUSION: The data suggest a greater sensitivity for the FC-ICS assay than the QF test, and urge caution when comparing SARS-CoV-2 T-cell immune responses assessed using different analytical platforms.

Commercial Interferon-gamma release assay to assess the immune response to first and second doses of mRNA vaccine in previously COVID-19 infected versus uninfected individuals.

We analysed immunological response during vaccination by using quantitative anti-spike IgG antibodies (qAbs) and Interferon-gamma (IFNγ) production by SARS-CoV-2-specific CD4+ and CD8+ T cells (QuantiFERON® assay). Blood samples were collected at four time points: a day before the reception of first (T0) and second (T1) BNT162b2 doses, 14 (T2) and 28 days (T3) after second dose. Fifty individuals were included: 34 previously infected by SARS-CoV-2 (CoV2+) and 16 that were not (CoV2-). Among CoV2+, we only observed significant differences after the first dose in both qAbs and IFNγ+ T cells. CoV2- showed differences after each dose, and the response was lower than CoV2+. Older people presented a higher response in CoV2+, while in CoV2, young people responded best. Our results suggest that the second BNT162b2 vaccine dose is not a priority in people with previous COVID-19. QuantiFERON® is a good option to monitor T-cell immunity to SARS-CoV-2.

In Vitro Activity of Cefepime-Taniborbactam against Carbapenemase-Producing Enterobacterales and Pseudomonas aeruginosa Isolates Recovered in Spain.

Novel ß-lactam-ß-lactamase inhibitor combinations currently approved for clinical use are poorly active against metallo-ß-lactamase (MBL)-producing strains. We evaluated the in vitro activity of cefepime-taniborbactam (FTB [formerly cefepime-VNRX-5133]) and comparator agents against carbapenemase-producing Enterobacterales (n = 247) and carbapenem-resistant Pseudomonas species (n = 170) clinical isolates prospectively collected from different clinical origins in patients admitted to 8 Spanish hospitals. FTB was the most active agent in both Enterobacterales (97.6% MICFTB, ≤8/4 mg/L) and Pseudomonas (67.1% MICFTB, ≤8/4 mg/L) populations. The MICFTB was >8 mg/L in 6/247 (2.4%) Enterobacterales isolates (3 KPC-producing Klebsiella pneumoniae isolates, 1 VIM-producing Enterobacter cloacae isolate, 1 IMP-producing E. cloacae isolate, and 1 NDM-producing Escherichia coli isolate) and in 56/170 (32.9%) Pseudomonas isolates, 19 of them carbapenemase producers (15 producers of VIM, 2 of GES, 1 of GES+VIM, and 1 of GES+KPC). Against the Enterobacterales isolates with meropenem MICs of >2 mg/L (138/247), FTB was the most active agent against both serine-ß-lactamases (107/138) and MBL producers (31/138) (97.2 and 93.5% MICFTB, ≤8/4 mg/L, respectively), whereas the activity of comparators was reduced, particularly against the MBL producers (ceftazidime-avibactam, 94.4 and 12.9%, meropenem-vaborbactam, 85.0 and 64.5%, imipenem-relebactam, 76.6 and 9.7%, ceftolozane-tazobactam, 1.9 and 0%, and piperacillin-tazobactam, 0 and 0%, respectively). Among the meropenem-resistant Pseudomonas isolates (163/170; MIC, >2 mg/L), the activities of FTB against serine-ß-lactamase (35/163) and MBL (43/163) producers were 88.6 and 65.1%, respectively, whereas the susceptibilities of comparators were as follows: ceftazidime-avibactam, 88.5 and 16.0%, meropenem-vaborbactam, 8.5 and 7.0%, imipenem-relebactam, 2.9 and 2.3%, ceftolozane-tazobactam, 0 and 2.3%, and piperacillin-tazobactam, 0 and 0%, respectively. Microbiological results suggest FTB as a potential therapeutic option in patients infected with carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas isolates, including MBL producers.

Reporting antimicrobial susceptibilities and resistance phenotypes in Staphylococcus spp.: a nationwide proficiency study.

OBJECTIVES: To evaluate the proficiency of microbiology laboratories in Spain in antimicrobial susceptibility testing (AST) of Staphylococcus spp. MATERIALS AND METHODS: Eight Staphylococcus spp. with different resistance mechanisms were selected: six Staphylococcus aureus (CC-01/mecA, CC-02/mecC, CC-03/BORSA, CC-04/MLSBi, CC-06/blaZ and CC-07/linezolid resistant, cfr); one Staphylococcus epidermidis (CC-05/linezolid resistant, 23S rRNA mutation); and one Staphylococcus capitis (CC-08/daptomycin non-susceptible). Fifty-one laboratories were asked to report: (i) AST system used; (ii) antimicrobial MICs; (iii) breakpoints used (CLSI or EUCAST); and (iv) clinical category. Minor, major and very major errors (mEs, MEs and VMEs, respectively) were determined. RESULTS: The greatest MIC discrepancies found were: (i) by AST method: 19.4% (gradient diffusion); (ii) by antimicrobial agent: daptomycin (21.3%) and oxacillin (20.6%); and (iii) by isolate: CC-07/cfr (48.0%). The greatest error rates were: (i) by AST method: gradient diffusion (4.3% and 5.1% VMEs, using EUCAST and CLSI, respectively); (ii) by breakpoint: 3.8% EUCAST and 2.3% CLSI; (iii) by error type: mEs (0.8% EUCAST and 1.0% CLSI), MEs (1.8% EUCAST and 0.7% CLSI) and VMEs (1.2% EUCAST and 0.6% CLSI); (iii) by antimicrobial agent: VMEs (4.7% linezolid and 4.3% oxacillin using EUCAST); MEs (14.3% fosfomycin, 9.1% tobramycin and 5.7% gentamicin using EUCAST); and mEs (22.6% amikacin using EUCAST). CONCLUSIONS: Clinical microbiology laboratories should improve their ability to determine the susceptibility of Staphylococcus spp. to some antimicrobial agents to avoid reporting false-susceptible or false-resistant results. The greatest discrepancies and errors were associated with gradient diffusion, EUCAST breakpoints and some antimicrobials (mEs for aminoglycosides; MEs for fosfomycin, aminoglycosides and oxacillin; and VMEs for linezolid and oxacillin).

Distinct epidemiology and resistance mechanisms affecting ceftolozane/tazobactam in Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal depicted by WGS.

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.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures / Evaluación de la combinación de espectrometría de masas y el sistema Vitek 2 para la identificación rápida y el estudio de sensibilidad de Enterobacterales directamente desde frascos de hemocultivo positivos

OBJECTIVE: The aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. MATERIAL AND METHODS: Gram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. RESULTS: MALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n = 68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7 h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5 h. CONCLUSIONS: A simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients' clinical outcomes

OBJETIVO: Evaluar un método rápido de identificación y estudio de sensibilidad directamente desde hemocultivos positivos para bacilos gramnegativos del orden Enterobacterales. MATERIAL Y MÉTODOS: Los hemocultivos con bacilos gramnegativos fueron utilizados para identificación mediante MALDI-TOF, seleccionándose aquellos con Enterobacterales para estudio de sensibilidad con Vitek 2. Los resultados fueron comparados con el método estándar del laboratorio. RESULTADOS: MALDI-TOF identificó correctamente el 83% de las muestras obtenidas directamente de hemocultivos. En caso de Enterobacterales (n = 68), el 85% se identificaron a nivel de género y especie con un score > 1,7. El tiempo necesario para la obtención de CMIs fue de 7 horas, reduciéndose a 5 en amoxicilina-clavulánico, amicacina y ciprofloxacino, casi en el 50% de los casos. CONCLUSIONES: Un protocolo simplificado, con bajo coste y reducido tiempo de trabajo, combinando identificación y estudio de sensibilidad directamente desde hemocultivos, podría proporcionar beneficios en la elección y coste del tratamiento, y mejora clínica del paciente

Molecular epidemiology and drug-resistance mechanisms in carbapenem-resistant Klebsiella pneumoniae isolated in patients from a tertiary hospital in Valencia, Spain.

OBJECTIVES: The aim of this study has been to characterize carbapenem-resistant Klebsiella pneumoniae isolates and to determine the resistance mechanisms involved, the clonal relationship between strains and clinical and demographical data of the infected patients. METHODS: Clinical and demographical data from patients were collected and statistically analysed. Antimicrobial susceptibility testing was performed and resistance genes were detected both phenotypically and genotypically. Conjugation assays were performed to show horizontal transferability of resistance genes. Clonal relationship was also studied. Next-generation sequencing (NGS) was performed to obtain information regarding resistance genes, sequence types, virulence factors and plasmid types. RESULTS: Statistical significance was shown by the presence of an infection if there had been a previous hospital stay; urinary catheter carriage and chronic renal disease also indicated higher probabilities of being infected. More than 95% of the isolates were non-susceptible to third-generation cephalosporins, and more than 90% were non-susceptible to quinolones. Phenotypic and genotypic methods for resistance detection were concordant and later confirmed by NGS. This is the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area. No plasmid-mediated colistin resistance was found. Tetracycline, sulfonamides and aminoglycoside resistance genes were found in almost all the isolates studied. No virulence factors were detected. Multilocus sequence typing showed more than 15 different sequence types, with ST101, ST307 and ST11 being the most prevalent. CONCLUSIONS: This study is the first to report such a large group of OXA-48 carbapenemases with clonal dissemination among carbapenem-resistant K. pneumoniae in Valencia. This is also the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures.

OBJECTIVE: The aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. MATERIAL AND METHODS: Gram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. RESULTS: MALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n=68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5h. CONCLUSIONS: A simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients' clinical outcomes.

WGS characterization of MDR Enterobacterales with different ceftolozane/tazobactam susceptibility profiles during the SUPERIOR surveillance study in Spain.

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.

Activity of ceftolozane/tazobactam against Pseudomonas aeruginosa and Enterobacterales isolates recovered from intensive care unit patients in Spain: The SUPERIOR multicentre study.

Patients in intensive care units (ICUs) present a high risk of developing an infection caused by multidrug-resistant bacteria. Consequently, new antimicrobials and combinations are required. In this study, the activity of ceftolozane/tazobactam (C/T) was evaluated against Enterobacterales (n = 400) and Pseudomonas aeruginosa (n = 80) clinical isolates collected from patients in Spanish ICUs with complicated urinary tract infections (cUTI) and complicated intra-abdominal infections (cIAI). Overall susceptibility to C/T in P. aeruginosa isolates by infection type was 95.7% in cUTI (MIC50/90, 1/4 mg/L) and 85.3% in cIAI (MIC50/90, 1/64 mg/L). Activity against P. aeruginosa was maintained regardless of its resistance pattern, confirming that C/T is one of the best antipseudomonal agents along with colistin and amikacin. Susceptibility to C/T in Enterobacterales by infection type was 79.5/81.9% and 89.3/92.3% (EUCAST/CLSI) in cIAI and cUTI isolates, respectively. Activity was excellent against wild-type organisms, with 100% susceptible and inhibited at MIC ≤1 mg/L. Nevertheless, C/T susceptibility decreased against extended-spectrum ß-lactamase (ESBL)-producing isolates: Escherichia coli (80.4/84.8% susceptible by EUCAST/CLSI) and Klebsiella pneumoniae (59.1/77.3% susceptible by EUCAST/CLSI). No activity of C/T was observed in carbapenemase-producing isolates. The in vitro activity of C/T observed in this surveillance study suggests that this agent can be considered as a therapeutic option for cUTI and cIAI due to Enterobacterales and P. aeruginosa in ICU patients, particularly when carbapenemase-producing isolates are not involved.

Reporting identification of Acinetobacter spp genomic species: A nationwide proficiency study in Spain / Capacidad de los laboratorios españoles para identificar las genoespecies de Acinetobacter spp: estudio nacional multicéntrico español

Acinetobacter baumannii is the most important genomic species of Acinetobacter from a clinical and epidemiological point of view. Nevertheless, genomic species other than A. baumannii are increasingly recognized as nosocomial pathogens. Molecular methods of identification (genotypic and proteomic assays) are more accurate and reliable and have greater discriminatory power than phenotypic methods. Eleven genomic species of Acinetobacter spp. (8 A. baumannii, 1 A. pittii, 1 A. nosocomialis and 1 A. lwoffii) with different antimicrobial resistance phenotypes and mechanisms of resistance to antimicrobial agents were sent to 48 participating Spanish centers to evaluate their ability for correct identification at the genomic species level. Identification of the genomic species was performed at the two Clinical Microbiology reference laboratories (Hospital Universitario Virgen Macarena, Seville, Spain; and Complejo Hospitalario Universitario de A Coruña, A Coruña, Spain) by partial DNA sequencing of the rpoB gene and MALDI-TOF. The mean percentage of agreement was 76.1%. Fifty percent of CC-01 (A. pittii) and 50% of CC-02 (A. nosocomialis) identification results were reported as A. baumannii. Discrepancies by type of systems used for identification were: MicroScan WA (51.1%), Vitek 2 (19.5%), MALDI-TOF (18.0%), Phoenix (4.5%), Wider (3.8%) and API 20 NE (3.0%). In conclusion, clinical microbiology laboratories must improve their ability to correctly identify the most prevalent non A. baumannii genomic species

Acinetobacter baumannii es la genoespecie de Acinetobacter más importante desde el punto de vista clínico y epidemiológico. No obstante, otras genoespecies distintas de A. baumannii están adquiriendo cada vez mayor importancia como patógeno nosocomial. Los métodos moleculares (genotípicos y proteómicos) usados para la identificación de genoespecies de Acinetobacter son más precisos, fiables, y tienen mayor poder de discriminación que los métodos fenotípicos. En este estudio se incluyen 11 genoespecies de Acinetobacter spp. (8 A. baumannii, 1 A. pittii, 1 A. nosocomialis y 1 A. lwoffii) con diferentes fenotipos de resistencia y mecanismos de resistencia antimicrobiana. Los aislados se enviaron a 48 centros españoles participantes para evaluar su capacidad para identificar correctamente la genoespecie de Acinetobacter. Las identificaciones de referencia se realizaron en 2 Laboratorios de Microbiología Clínica (Hospital Universitario Virgen Macarena, Sevilla, España; Complejo Hospitalario Universitario de A Coruña, A Coruña, España) mediante secuenciación parcial del gen rpoB y MALDI-TOF. El porcentaje medio de concordancia fue del 76,1%. El 50% de los resultados de identificación del control CC-01 (A. pittii) y el 50% de los resultados de identificación del control CC-02 (A. nosocomialis) se informaron como A. baumannii. Considerando el tipo de sistema de identificación usado las discrepancias fueron: MicroScan/WA (51,1%), Vitek 2 (19,5%), MALDI-TOF (18,0%), Phoenix (4,5%), Wider (3,8%) y API 20 NE (3,0%). En conclusión, los laboratorios españoles de microbiología clínica deben mejorar su capacidad para identificar correctamente las genoespecies de Acinetobacter distintas de A. baumannii que son más prevalentes

Reporting identification of Acinetobacter spp genomic species: A nationwide proficiency study in Spain.

Acinetobacter baumannii is the most important genomic species of Acinetobacter from a clinical and epidemiological point of view. Nevertheless, genomic species other than A. baumannii are increasingly recognized as nosocomial pathogens. Molecular methods of identification (genotypic and proteomic assays) are more accurate and reliable and have greater discriminatory power than phenotypic methods. Eleven genomic species of Acinetobacter spp. (8 A. baumannii, 1 A. pittii, 1 A. nosocomialis and 1 A. lwoffii) with different antimicrobial resistance phenotypes and mechanisms of resistance to antimicrobial agents were sent to 48 participating Spanish centers to evaluate their ability for correct identification at the genomic species level. Identification of the genomic species was performed at the two Clinical Microbiology reference laboratories (Hospital Universitario Virgen Macarena, Seville, Spain; and Complejo Hospitalario Universitario de A Coruña, A Coruña, Spain) by partial DNA sequencing of the rpoB gene and MALDI-TOF. The mean percentage of agreement was 76.1%. Fifty percent of CC-01 (A. pittii) and 50% of CC-02 (A. nosocomialis) identification results were reported as A. baumannii. Discrepancies by type of systems used for identification were: MicroScan WA (51.1%), Vitek 2 (19.5%), MALDI-TOF (18.0%), Phoenix (4.5%), Wider (3.8%) and API 20 NE (3.0%). In conclusion, clinical microbiology laboratories must improve their ability to correctly identify the most prevalent non A. baumannii genomic species.

A survey on practices for active surveillance of carriage of multidrug-resistant bacteria in hospitals in the Autonomous Community of Valencia, Spain.

A questionnaire-based cross-sectional study was conducted to gather information on current microbiological practices for active surveillance of carriage of multidrug-resistant (MDR) bacteria in hospitals from 14 health departments of the Autonomous Community of Valencia (ACV), Spain, which together provided medical attention to 3,271,077 inhabitants in 2017, approximately 70% of the population of the ACV. The survey consisted of 35 questions on MDR bacteria screening policies, surveillance approach chosen (universal vs. targeted), and microbiological methods and processes in use for routine detection and reporting of colonization by MDR bacteria, including the anatomical sites scheduled to be sampled for each MDR bacterial species, and the methodology employed (culture-based, molecular-based, or both). Our study revealed striking differences across centers, likely attributable to the lack of consensus on optimal protocols for sampling, body sites for screening, and microbiological testing, thus underscoring the need for consensus guidelines on these issues.