CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases.

Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction.

Activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against a clinical and laboratory collection of ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa ß-lactamase mutants. METHODS: The activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam, cefepime/zidebactam and comparators was evaluated against a collection of 30 molecularly characterized ceftolozane/tazobactam- and/or ceftazidime/avibactam-resistant P. aeruginosa isolates from patients previously treated with cephalosporins. To evaluate how the different ß-lactamases in the clinical isolates affected the resistance to these agents, a copy of each blaPDC, blaOXA-2 and blaOXA-10 ancestral and mutant allele from the clinical isolates was cloned in pUCp24 and expressed in dual blaPDC-oprD (for blaPDC-like genes) or single oprD (for blaOXA-2-like and blaOXA-10-like genes) PAO1 knockout mutants. MICs were determined using reference methodologies. RESULTS: For all isolates, MICs were higher than 4 and/or 8 mg/L for ceftolozane/tazobactam and ceftazidime/avibactam, respectively. Cefiderocol was the most active agent, showing activity against all isolates, except one clinical isolate that carried an R504C substitution in PBP3 (MIC = 16 mg/L). Imipenem/relebactam was highly active against all isolates, except two clinical isolates that carried the VIM-20 carbapenemase. Cefepime/zidebactam and cefepime/taniborbactam displayed activity against most of the isolates, but resistance was observed in some strains with PBP3 amino acid substitutions or that overexpressed mexAB-oprM or mexXY efflux pumps. Evaluation of transformants revealed that OXA-2 and OXA-10 extended-spectrum variants cause a 2-fold increase in the MIC of cefiderocol relative to parental enzymes. CONCLUSIONS: Cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam show promising and complementary in vitro activity against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa. These agents may represent potential therapeutic options for ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa infections.

In vivo translational assessment of the GES genotype on the killing profile of ceftazidime, ceftazidime/avibactam and meropenem against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the in vivo killing profile of human-simulated exposures of ceftazidime, ceftazidime/avibactam and meropenem against GES-harbouring Pseudomonas aeruginosa in the murine thigh infection model. METHODS: Five P. aeruginosa isolates [three isogenic (GES-1, GES-5 and GES-15) and two clinical (GES-5 and GES-15)] were evaluated. MICs were determined using broth microdilution. Human-simulated regimens (HSRs) of ceftazidime 2 g IV q8h as a 2 h infusion, ceftazidime/avibactam 2.5 g IV q8h as a 2 h infusion and meropenem 2 g IV q8h as a 3 h infusion were administered. Change in bacterial burden relative to baseline was assessed. RESULTS: Modal MICs ranged from 8 to >64 mg/L for ceftazidime, from 1 to 16 mg/L for ceftazidime/avibactam and from 1 to >64 mg/L for meropenem. In vivo, for the isogenic strains, avibactam augmented ceftazidime activity against the GES-1- and GES-15-harbouring isolates. Both ceftazidime and ceftazidime/avibactam resulted in significant kill against the GES-5 isogenic isolate. The meropenem HSR produced >1 log10 kill against each isogenic isolate (MICs of 1-4 mg/L). Against the GES-5 clinical isolate, ceftazidime and ceftazidime/avibactam resulted in >1 log10 kill compared with bacterial growth with the meropenem HSR. In the clinical isolate harbouring GES-15, the elevated MICs of ceftazidime and ceftazidime/avibactam reduced the effectiveness of both compounds, while the observed reduction in meropenem MIC translated into in vivo efficacy of the HSR regimen, predictive of clinical efficacy. CONCLUSIONS: In GES-harbouring P. aeruginosa, quantitative reductions in bacterial density observed with the translational murine model suggest that the phenotypic profile of ceftazidime, ceftazidime/avibactam and meropenem is predictive of clinical efficacy when using the evaluated dosing regimens.

Selection of AmpC ß-Lactamase Variants and Metallo-ß-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections.

Infections caused by ceftolozane-tazobactam and ceftazidime-avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane-tazobactam and ceftazidime-avibactam resistance mechanisms in all multidrug-resistant or extensively drug-resistant (MDR/XDR) P. aeruginosa isolates recovered during 1 year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane-tazobactam and ceftazidime-avibactam resistance were evaluated. Clinical conditions, previous positive cultures, and ß-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. Multilocus sequence types (MLSTs) and resistance mechanisms were determined using short- and long-read whole-genome sequencing (WGS). The impact of Pseudomonas-derived cephalosporinases (PDCs) on ß-lactam resistance was demonstrated by cloning into an ampC-deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired ß-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane-tazobactam or ceftazidime-avibactam. Seven isolates from different sequence types (STs) owed their ß-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219, and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. The 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13) and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlighted that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection.

BACKGROUND: Pseudomonas aeruginosa may develop resistance to novel cephalosporin/ß-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. OBJECTIVES: To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. METHODS: Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. RESULTS: WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. CONCLUSIONS: We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/ß-lactamase inhibitors in P. aeruginosa.

Monitoring the antimicrobial susceptibility of Gram-negative organisms involved in intraabdominal and urinary tract infections recovered during the SMART study (Spain, 2016 and 2017) / Seguimiento de la sensibilidad antimicrobiana de microorganismos gramnegativos procedentes de infecciones intraabdominales y urinarias del estudio SMART (España, 2016 y 2017)

Introduction: Continuous antimicrobial resistance surveillance is recommended by Public Health authorities. We up-dated data from the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. Material and methods: The antimicrobial susceptibility data and extended-spectrum beta-lactamase (ESBL) production in isolates recovered from intra-abdominal (IAI) (n=1,429) and urinary tract (UTI) (n=937) infections during the 2016- 2017 SMART study in 10 Spanish hospitals were analysed. Results: Escherichia coli was the most frequently microorganism isolated (48.3% and 53.7%) followed by Klebsiella spp. (11.5% and 21.9%) in IAIs and UTIs, respectively. Figures for Pseudomonas aeruginosa were 9.0% and 6.1%, being more frequently recovered from patients with nosocomial infections. Overall, 9.9% (IAI) and 14.0% (UTI) of E. coli, Klebsiella spp. and Proteus mirabilis isolates were ESBL-producers, being Klebsiella pneumoniae (34.5%) from UTI of nosocomial origin the most frequent. ESBL-producers were higher in patients >60 years in both IAIs and UTIs. As in previous years, amikacin (96.3%-100% susceptibility), ertapenem (84.2%-100%) and imipenem (70.3%- 100%) were the most active antimicrobials tested among Enterobacterales species. The activity of amoxicillin-clavulanic, piperacillin-tazobactam, and ciprofloxacin susceptibility was lower, particularly among ESBL-producers. Ertapenem susceptibility (88.9%-100%) was retained in ESBL-E. coli isolates that were resistant to these antimicrobials but decreased (28.6%-100%) in similar isolates of K. pneumoniae. Conclusions: Continuous antimicrobial resistance surveillance from the SMART study reveals overall maintenance of ESBL-producers in Spain, although with higher presence in isolates from UTIs than from IAIs. Moreover, ertapenem activity was high in E. coli irrespective of ESBL production but decreased in K. pneumoniae, particularly among ESBL-producers

Introducción: Las autoridades de Salud Pública recomiendan la vigilancia continua de la resistencia a los antimicrobianos. Se actualizan los datos del estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) en España. Material y métodos: Se analizaron los datos de sensibilidad antimicrobiana y la producción de betalactamasas de espectro extendido (BLEE) en aislamientos obtenidos en el estudio SMART de infecciones intraabdominales (IIA) (n=1.429) y del tracto urinario (ITU) (n=937) durante 2016-2017 en 10 hospitales españoles. Resultados: Escherichia coli fue el microorganismo más frecuente (54,5% y 57,5%, respectivamente), seguido de Klebsiella spp. (18,4% y 25,4%) en IIA y en ITU. En Pseudomonas aeruginosa estas cifras fueron 9% y 6%, siendo más frecuente en la infección nosocomial. El 9,9% (IIA) y el 14% (ITU) del to-tal de los aislados de E. coli, Klebsiella spp. y Proteus mirabilis producían BLEE, obteniéndose la tasa más alta en Klebsiella pneumoniae (34.5%) en ITU nosocomial. El mayor porcentaje de aislados con BLEE se observó en pacientes >60 años, tanto en IIA como en ITU. Como en años anteriores, amikacina (sensibilidad 96,3%-100%), ertapenem (84,2%-100%) e imipenem (70,3%-100%) fueron los antimicrobianos más activos en Enterobacterales. La sensibilidad a amoxicilina-ácido clavulánico, piperacilina-tazobactam y ciprofloxacino fue menor, en particular en los productores de BLEE. La sensibilidad a ertapenem (88,9%-100%) se mantuvo en E. coli con BLEE resistente a estos antimicrobianos, pero disminuyó (28,6%-100%) en aislados similares de K. pneumoniae. Conclusiones: La vigilancia continua de la resistencia a los antimicrobianos en el estudio SMART revela el mantenimiento de la frecuencia de aislados productores de BLEE en España, pero con mayor presencia en las ITUs que en las IIAs. Además, la sensibilidad a ertapenem fue alta en E. coli con independencia de la producción de BLEE, pero disminuyó en K. pneumoniae, sobre todo en los productores de BLEE