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Background: Multidrug-resistant (MDR) Pseudomonas aeruginosa is a major public health threat. Treatment with ceftolozane-tazobactam improves patient outcomes compared to salvage therapy; however, resistance has emerged in ~15% of patients following courses ranging from 7 to 53 days. Understanding the development and mechanisms of resistance in these difficult to treat MDR P. aeruginosa has public health importance. Our objective was to study the in vitro activity of alternative β-lactams in the setting of ceftolozane-tazobactam resistance. Methods: Isolates from 23 patients in whom ceftolozane-tazobactam resistance emerged were selected for analysis. Minimum inhibitory concentrations (MICs) were determined by standard broth microdilution in triplicate and interpreted by CLSI breakpoints. Mechanisms of resistance and relatedness of isolates were explored through whole-genome sequence (WGS) analysis in 15 patients from whom baseline and post-treatment isolates were available. Results: 23 baseline and 32 post-treatment isolates were included. The median baseline ceftolozane-tazobactam MIC was 2 µg/mL (range: 0.5 – 8 µg/mL). 75%, 25%, 82.6%, and 83.3% of baseline isolates were non-susceptible to ceftazidime, ceftazidime-avibactam, imipenem, and piperacillin-tazobactam respectively. Following a median 16 (range: 3- 60) days of therapy, the median post-exposure ceftolozane-tazobactam MIC was 64 µg/mL (range: 8 – >256 µg/mL). 100%, 72.7%, 69.6%, and 79.2% of post-treatment isolates were resistant to ceftazidime, ceftazidime-avibactam, imipenem, and piperacillin-tazobactam. The corresponding MIC foldchanges were 4, 8, -2, and 0, respectively. Median imipenem-relebactam MICs did not change before or after treatment with ceftolozane-tazobactam (median= 2 µg/mL for both) and 16.7% were classified as resistant. WGS data revealed several mutations in ampC and ampR sequences. Discussion: Our findings show that resistance to ceftolozane-tazobactam impacts the susceptibility of alternative β-lactams. Cross resistance occurs with ceftazidime and ceftazidimeavibactam (median 4 and 8 fold MIC increase, respectively). Imipenem MICs are decreased 2fold potentially demonstrating collateral sensitivity. Piperacillin-tazobactam MICs were unchanged and isolates remained resistant. Importantly, imipenem-relebactam MICs were unchanged suggesting the mechanism of ceftolozane-tazobactam resistance may be due to structural changes in ampC. WGS data showed a number of different mutations in both ampC and ampR. Certain mutations, such as F147L and mutations found in positions 234-244, were found to promote resistance to ceftolozane-tazobactam.

We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum β-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL−, 6 KPC−/ESBL+, and 38 KPC−/ESBL− isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL− (25% and 38%, respectively), KPC−/ESBL+ (50% and 17%, respectively), and KPC−/ESBL− (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and P < 0.01 for all). KPC+/ESBL+ isolates also had a greater number of AMEs (mean of 4.6 versus 1.5, 0.9, and 0.05, respectively; P < 0.01 for all). GEN and TOB MICs were higher against isolates with >1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 μg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter . PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority.

We identified four bla KPC-3 mutations in ceftazidime-avibactam-resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli , we conclusively demonstrated that mutant bla KPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop.

We determined imipenem, imipenem-relebactam, ceftazidime, and ceftazidime-avibactam MICs against 100 CRE isolates that underwent whole-genome sequencing. Klebsiella pneumoniae carbapenemases (KPCs) were the most common carbapenemases. Forty-six isolates carried extended-spectrum β-lactamases (ESBLs). With the addition of relebactam, imipenem susceptibility increased from 8% to 88%. With the addition of avibactam, ceftazidime susceptibility increased from 0% to 85%. Neither imipenem-relebactam nor ceftazidime-avibactam was active against metallo-β-lactamase (MBL) producers. Ceftazidime-avibactam (but not imipenem-relebactam) was active against OXA-48-like producers, including a strain not harboring any ESBL. Major OmpK36 porin mutations were independently associated with higher imipenem-relebactam MICs ( P < 0.0001) and showed a trend toward independent association with higher ceftazidime-avibactam MICs ( P = 0.07). The presence of variant KPC-3 was associated with ceftazidime-avibactam resistance ( P < 0.0001). In conclusion, imipenem-relebactam and ceftazidime-avibactam had overlapping spectra of activity and niches in which each was superior. Major OmpK36 mutations in KPC- K. pneumoniae may provide a foundation for stepwise emergence of imipenem-relebactam and ceftazidime-avibactam resistance.

We identified four blaKPC-3 mutations in ceftazidime-avibactam-resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli, we conclusively demonstrated that mutant blaKPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop.

We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum β-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL−, 6 KPC−/ESBL+, and 38 KPC−/ESBL− isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL− (25% and 38%, respectively), KPC−/ESBL+ (50% and 17%, respectively), and KPC−/ESBL− (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and 1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 μg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter. PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority.