ß-lactamase expression induces collateral sensitivity in Escherichia coli.

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, ß-lactam resistance genes -encoding ß-lactamases- stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to eliminate AMR selectively. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissect the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the ß-lactamase gene blaOXA-48 induces CS to colistin and azithromycin. We next show that other clinically relevant mobile ß-lactamases produce similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we show that ß-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of ß-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.

Evolution of ceftazidime-avibactam and cefiderocol resistance in ST131-H30R1-Escherichia coli isolates with KPC-3 mutants and application of FTIR biotyping.

Ceftazidime-avibactam and cefiderocol represent two of the few alternatives for infections by KPC-producing Enterobacterales. We reported the emergence of resistance to both ceftazidime-avibactam and cefiderocol in a KPC-producing ST131-Escherichia coli (KPC-ST131-Ec) clinical isolate. Antimicrobial susceptibility testing, Fourier-transform infrared (FTIR) spectroscopy, whole-genome sequencing, and cloning experiments were performed. A KPC-49-Ec isolate resistant to ceftazidime-avibactam (MICCZA > 16/4 mg/L) and susceptible to cefiderocol (MICFDC: 2 mg/L) was recovered in a blood sample from an oncologic patient hospitalized in the medical ICU (June 2019) during ceftazidime-avibactam treatment. After 44 days, a KPC-31-Ec resistant to both ceftazidime-avibactam and cefiderocol (MICCZA > 16/4 mg/L, MICFDC: 8 mg/L) was found in a rectal sample during a second cycle of ceftazidime-avibactam treatment. Both KPC-49 (R163S) and KPC-31 (D179Y) were detected in the epidemic ST131-H30R1-Ec high-risk clone and showed a phenotype resembling that of ESBL producers. FTIR spectroscopy managed to differentiate cefiderocol-susceptible and resistant ST131-Ec isolates, and these from others belonging to different clones. After cloning and transformation experiments, KPC-49 and KPC-31 were responsible for ceftazidime-avibactam resistance (MICCZA > 16/4 mg/L) and decreased carbapenem MICs (MICMER ≤ 0.12 mg/L, MICIMI ≤ 1 mg/L). KPC-31 was also shown to be associated with increased MICs of cefiderocol (twofold and threefold dilutions over KPC-3 and KPC-49, respectively). However, mutations in proteins participating in outer membrane stability and integrity, such as TolR, could have a more relevant role in cefiderocol resistance. The effects of ceftazidime-avibactam and cefiderocol co-resistance in clinical isolates of Enterobacterales producing KPC mutants make their identification challenging for clinical laboratories.IMPORTANCEThroughout four admissions in our hospital of a single patient, different KPC-3 variants (KPC-3, KPC-49, and KPC-31) were found in surveillance and clinical ST131-Escherichia coli isolates, after prolonged therapies with meropenem and ceftazidime-avibactam. Different patterns of resistance to cefiderocol and ceftazidime-avibactam emerged, accompanied by restored carbapenem susceptibility. The inability to detect these variants with some phenotypic methods, especially KPC-31 by immunochromatography, and the expression of a phenotype similar to that of ESBL producers, posed challenge to identify these variants in the clinical microbiology laboratory. Molecular methods and whole-genome sequencing are necessary and new techniques able to cluster or differentiate related isolates could also be helpful; this is the case of Fourier-transform infrared spectroscopy, which managed in our study to discriminate isolates by cefiderocol susceptibility within ST131, and those from the non-ST131 ones.

Outbreak by KPC-62-producing ST307 Klebsiella pneumoniae isolates resistant to ceftazidime/avibactam and cefiderocol in a university hospital in Madrid, Spain.

OBJECTIVES: Ceftazidime/avibactam and cefiderocol are two of the latest antibiotics with activity against a wide variety of Gram-negatives, including carbapenem-resistant Enterobacterales. We sought to describe the phenotypic and genotypic characteristics of ceftazidime/avibactam- and cefiderocol-resistant KPC-Klebsiella pneumoniae (KPC-Kp) detected during an outbreak in 2020 in the medical ICU of our hospital. METHODS: We collected 11 KPC-Kp isolates (6 clinical; 5 surveillance samples) resistant to ceftazidime/avibactam and cefiderocol from four ICU patients (November 2020 to January 2021), without prior exposure to these agents. All patients had a decontamination regimen as part of the standard ICU infection prevention protocol. Additionally, one ceftazidime/avibactam- and cefiderocol-resistant KPC-Kp (June 2019) was retrospectively recovered. Antibiotic susceptibility was determined by broth microdilution. ß-Lactamases were characterized and confirmed. WGS was also performed. RESULTS: All KPC-Kp isolates (ceftazidime/avibactam MIC  ≥16/4 mg/L; cefiderocol MIC ≥4 mg/L) were KPC + CTX-M-15 producers and belonged to the ST307 high-risk-clone (ST307-HRC). KPC-62 (L168Q) was detected in all isolates involved in the 2020 outbreak, contained in January 2021. KPC-31 (D179Y) was identified in the KPC-Kp from 2019. Cloning experiments demonstrated that both blaKPC-62 and blaKPC-31 were responsible for ceftazidime/avibactam resistance (MIC >16 mg/L) and an increased cefiderocol MIC. Additionally, mutations in OmpA and EnvZ/OmpR porin proteins (in KPC-62-Kp) and in PBP2 (in KPC-31-Kp) were found and may be involved in cefiderocol resistance. CONCLUSIONS: The emergence of resistance to both ceftazidime/avibactam and cefiderocol in KPC-Kp-HRCs, together with the diversification of novel KPC enzymes displaying different antibiotic resistance phenotypes, is an epidemiological and clinical risk.

Impact of Automated Blood Culture Systems on the Management of Bloodstream Infections: Results from a Crossover Diagnostic Clinical Trial.

Bloodstream infections are associated with high rates of morbidity and mortality. Blood culture remains the gold standard for the diagnosis of BSIs. We report a prospective crossover diagnostic clinical trial comparing the performances of two blood culture incubation systems: Virtuo and Bactec FX. The primary outcome was the time to detection (TTD) (from the loading of the sample into the incubator to the positivity signal). Patients over 16 years old suspected of having bacteremia/fungemia were included. They were divided into two strata with a total of 9,957 blood extractions. Initially, each stratum was randomly assigned to one of the incubators and then alternated every 2 weeks for 6 months. Each sample was inoculated into an aerobic bottle and an anaerobic bottle. All bottles were processed equally according to the laboratory's standard procedures after they were flagged positive. We analyzed 4,797 samples in the Virtuo system and 5,160 in the Bactec FX system. The median TTD was significantly lower for the Virtuo group (Virtuo, 15.2 h; Bactec FX, 16.3 h [P < 0.0001]). The turnaround time (TAT) (from sample loading to the Gram stain report) was also reduced with Virtuo (Virtuo, 26.2 h; Bactec FX, 28.3 h [P < 0.004]). When considering only samples from patients with antimicrobial treatment prior to blood culture extraction, the TTD was shorter for Virtuo (median differences in the TTD of 4.5 h for all bottles and 8.7 h for aerobic bottles only [P = 0.0001]). In conclusion, virtuo provided shorter TTD and TAT than Bactec FX. The difference in the median TTD was increased when considering samples incubated in aerobic bottles from patients with antimicrobial treatment. This could have an important effect on the faster diagnosis of BSIs. IMPORTANCE Bloodstream infections are associated with high rates of morbidity and mortality. Blood culture remains the gold standard for its diagnosis. While the identification of the pathogen and its antibiotic susceptibility is required to confirm the optimal antimicrobial regimen, reductions in the times to the detection of positivity and reporting of Gram stain results may be important and time-saving to reduce inappropriate antimicrobial use, improve patient outcomes, and decrease health care costs. We report the first clinical diagnostic study of this scale in a "real-world" setting with a crossover design, comparing two automatic blood culture incubators using samples from patients with a suspected diagnosis of bacteremia/sepsis, as opposed to spiked vials. Our study design mimics that of clinical trials performed for drug marketing authorization, but patient randomization was replaced with the crossover design. A shorter time to detection could have an important effect on the faster identification of causative microorganisms of BSIs and antimicrobial stewardship.

Antibacterial spectrum of cefiderocol

Cefiderocol, a siderophore catechol cephalosporin, recently introduced in the market has been developed to enhance the in vitro activity of extended spectrum cephalosporins and to avoid resistance mechanisms affecting cephalosporins and carbapenems. The in vitro study of cefiderocol in the laboratory requires iron depleted media when MIC values are determined by broth microdilution. Disk diffusion presents good correlation with MIC values. In surveillance studies and in clinical trials it has been demonstrated excellent activity against Gram-negatives, including carbapenemase producers and non-fermenters such as Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia. Few cefiderocol resistant isolates have been found in surveillance studies. Resistance mechanisms are not directly associated with porin deficiency and or efflux pumps. On the contrary, they are related with gene mutations affecting iron transporters, AmpC mutations in the omega loop and with certain beta-lactamases such us KPC-variants determining also ceftazidime-avibactam resistance, certain infrequent extended-spectrum betalactamases (PER, BEL) and metallo-beta-lactamases (certain NDM variants and SPM enzyme). (AU)

Impact of Ceftazidime-Avibactam Treatment in the Emergence of Novel KPC Variants in the ST307-Klebsiella pneumoniae High-Risk Clone and Consequences for Their Routine Detection.

The emergence of Klebsiella pneumoniae isolates carrying novel blaKPC variants conferring ceftazidime-avibactam (CAZ/AVI) resistance is being increasingly reported. We evaluated the accuracy of phenotypic methods commonly used in routine clinical laboratories in the detection of novel K. pneumoniae carbapenemase (KPC) enzymes. Additionally, we characterized by whole-genome sequencing (WGS) the KPC-ST307-K. pneumoniae isolates recovered in our hospital before and after CAZ/AVI therapy. Rectal colonization or infection by carbapenem-resistant KPC-3 K. pneumoniae isolates (imipenem MIC, 16 mg/L; meropenem MIC, 8 to >16 mg/L) and CAZ/AVI-susceptible isolates (CAZ/AVI MIC, 1 to 2 mg/L) were first detected in three intensive care unit (ICU) patients admitted between March 2020 and July 2020. KPC K. pneumoniae isolates with increased CAZ/AVI MICs (8 to 32 mg/L) and carbapenem susceptibility (imipenem and meropenem MIC, <1 mg/L) were recovered within 6 to 24 days after CAZ/AVI treatment. WGS confirmed that all KPC K. pneumoniae isolates belonged to the sequence type 307 (ST307) high-risk clone and carried identical antimicrobial resistance genes and virulence factors. The presence of the novel blaKPC-46, blaKPC-66, and blaKPC-92 genes was confirmed in the K. pneumoniae isolates with increased CAZ/AVI MICs and restored carbapenem activity. KPC production was confirmed by immunochromatography, the eazyplex Superbug CRE system, and the Xpert Carba-R assay in all KPC K. pneumoniae isolates, but not in any isolate using chromogenic agar plates for carbapenemase producers (ChromID-CARBA), the KPC/MBL/OXA-48 Confirm kit, and the ß-CARBA test. Nevertheless, all grew in chromogenic agar plates for extended-spectrum ß-lactamase (ESBL) producers (ChromID-ESBL). We report the failure of the most common phenotypic methods used for the detection of novel KPC carbapenemases but not of rapid molecular or immunochromatography assays, thus highlighting their relevance in microbiology laboratories.