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ABSTRACTWe compared ceftazidime-avibactam, ceftolozane-tazobactam, ceftazidime, cefepime, and piperacillin-tazobactam MICs for 38 meropenem-resistant Pseudomonas aeruginosaisolates. No isolates harbored carbapenemases; 74% were oprDmutants. Ceftazidime-avibactam and ceftolozane-tazobactam were active against 92% of the isolates, including 80% that were resistant to all three β-lactams. Forty-three percent of ceftazidime-avibactam-susceptible isolates and 6% of ceftolozane-tazobactam-susceptible isolates exhibited MICs at the respective breakpoints. Ceftolozane-tazobactam and ceftazidime-avibactam are therapeutic options for meropenem-resistant P. aeruginosainfections that should be used judiciously to preserve activity.

ABSTRACTPrecise FKSmutation rates among Candidaspecies are undefined because studies have not systematically screened consecutive, disease-causing isolates. The Sensititre YeastOne (SYO) assay measures echinocandin MICs against Candidawith less variability than reference broth microdilution methods. However, clinical breakpoint MICs may overstate caspofungin nonsusceptibility compared to other agents. Our objectives were to determine CandidaFKSmutation rates by studying consecutive bloodstream isolates and to determine if discrepant susceptibility results were associated with FKSmutations. FKShot spots were sequenced in echinocandin-intermediate and -resistant isolates and those from patients with breakthrough candidemia or ≥3 days of prior echinocandin exposure. Overall, 453 isolates from 384 patients underwent susceptibility testing; 16% were echinocandin intermediate or resistant. Intermediate susceptibility rates were higher for Candida glabratathan for other species (P< 0.0001) and higher for caspofungin than for other agents (P< 0.0001). Resistance rates were similar between agents. FKSmutations were detected in 5% of sequenced isolates and 2% of isolates overall. Corresponding rates among C. glabrataisolates were 8% and 4%, respectively. Among Candida albicansisolates, rates were 5% and <1%, respectively. Mutations occurred exclusively with prior echinocandin exposure and were not detected in other species. Isolates with discrepant susceptibility results did not harbor FKSmutations. Mutation rates among isolates resistant to ≥2, 1, and 0 agents were 75%, 13%, and 0%, respectively. In conclusion, FKSmutations were uncommon among non-C. glabrataspecies, even with prior echinocandin exposure. Discrepancies in echinocandin susceptibility by SYO testing were not driven by mutations and likely reflect imprecise caspofungin clinical breakpoints.

ABSTRACTAvibactam is a novel β-lactamase inhibitor with affinity for Klebsiella pneumoniaecarbapenemases (KPCs). In combination with ceftazidime, the agent demonstrates activity against KPC-producing K. pneumoniae(KPC-Kp). KPC-Kp strains are genetically diverse and harbor multiple resistance determinants, including defects in outer membrane proteins and extended-spectrum β-lactamases (ESBLs). Mutations in porin gene ompK36confer high-level carbapenem resistance to KPC-Kp strains. Whether specific mechanisms of antimicrobial resistance also influence the activity of ceftazidime-avibactam is unknown. We defined the effects of ceftazidime-avibactam against 72 KPC-Kp strains with diverse mechanisms of resistance, including various combinations of KPC subtypes and ESBL and ompK36mutations. Ceftazidime MICs ranged from 64 to 4,096 μg/ml and were lowered by a median of 512-fold with the addition of avibactam. All strains exhibited ceftazidime-avibactam MICs at or below the CLSI breakpoint for ceftazidime (≤4 μg/ml; range, 0.25 to 4). However, the MICs were within two 2-fold dilutions of the CLSI breakpoint against 24% of the strains, and those strains would be classified as nonsusceptible to ceftazidime by EUCAST criteria (MIC > 1 μg/ml). Median ceftazidime-avibactam MICs were higher against KPC-3 than KPC-2 variants (P= 0.02). Among KPC-2-Kp strains, the presence of both ESBL and porin mutations was associated with higher drug MICs compared to those seen with either factor alone (P= 0.003 and P= 0.02, respectively). In conclusion, ceftazidime-avibactam displays activity against genetically diverse KPC-Kp strains. Strains with higher-level drug MICs provide a reason for caution. Judicious use of ceftazidime-avibactam alone or in combination with other agents will be important to prevent the emergence of resistance.

ABSTRACTTreatment failures of a carbapenem-colistin regimen among patients with bacteremia due to sequence type 258 (ST258), KPC-2-producing Klebsiella pneumoniaewere significantly more likely if both agents were inactive in vitro, as defined by a colistin MIC of >2 μg/ml and the presence of either a major ompK36porin mutation (guanine and alanine insertions at amino acids 134 and 135 [ins aa 134–135 GD], IS5promoter insertion [P= 0.007]) or a doripenem MIC of >8 μg/ml (P= 0.01). Major ompK36mutations among KPC-K. pneumoniaestrains are important determinants of carbapenem-colistin responses in vitroand in vivo.

ABSTRACTThe aminoglycoside-modifying enzyme AAC(6′)-Ib is common among carbapenem-resistant Klebsiella pneumoniae(CR-Kp) strains. We investigated amikacin (AMK) activity against 20 AAC(6′)-Ib-producing CR-Kp strains. MICs clustered at 16 to 32 μg/ml. By the time-kill study, AMK (1× and 4× the MIC) was bactericidal against 30% and 85% of the strains, respectively. At achievable human serum concentrations, however, the majority of strains showed regrowth, suggesting that AAC(6′)-Ib confers intermediate AMK resistance. AMK and trimethoprim-sulfamethoxazole (TMP-SMX) were synergistic against 90% of the strains, indicating that the combination may overcome resistance.

ABSTRACTWe measured in vitroactivity of plazomicin, a next-generation aminoglycoside, and other aminoglycosides against 50 carbapenem-resistant Klebsiella pneumoniaestrains from two centers and correlated the results with the presence of various aminoglycoside-modifying enzymes (AMEs). Ninety-four percent of strains were sequence type 258 (ST258) clones, which exhibited 5 ompK36genotypes; 80% and 10% of strains produced Klebsiella pneumoniaecarbapenemase 2 (KPC-2) and KPC-3, respectively. Ninety-eight percent of strains possessed AMEs, including AAC(6′)-Ib (98%), APH(3′)-Ia (56%), AAC(3)-IV (38%), and ANT(2″)-Ia (2%). Gentamicin, tobramycin, and amikacin nonsusceptibility rates were 40, 98, and 16%, respectively. Plazomicin MICs ranged from 0.25 to 1 μg/ml. Tobramycin and plazomicin MICs correlated with gentamicin MICs (r= 0.75 and 0.57, respectively). Plazomicin exerted bactericidal activity against 17% (1× MIC) and 94% (4× MIC) of strains. All strains with AAC(6′)-Ib were tobramycin-resistant; 16% were nonsusceptible to amikacin. AAC(6′)-Ib combined with another AME was associated with higher gentamicin, tobramycin, and plazomicin MICs than AAC(6′)-Ib alone (P= 0.01, 0.0008, and 0.046, respectively). The presence of AAC(3)-IV in a strain was also associated with higher gentamicin, tobramycin, and plazomicin MICs (P= 0.0006, P< 0.0001, and P= 0.01, respectively). The combination of AAC(6′)-Ib and another AME, the presence of AAC(3)-IV, and the presence of APH(3′)-Ia were each associated with gentamicin resistance (P= 0.0002, 0.003, and 0.01, respectively). In conclusion, carbapenem-resistant K. pneumoniaestrains (including ST258 clones) exhibit highly diverse antimicrobial resistance genotypes and phenotypes. Plazomicin may offer a treatment option against strains resistant to other aminoglycosides. The development of molecular assays that predict antimicrobial responses among carbapenem-resistant K. pneumoniaestrains should be a research priority.

ABSTRACTWe compared in vitrokilling of colistin, doripenem, and sulbactam by time-kill methods against Acinetobacter baumanniiisolates collected from patients before and after colistin-doripenem treatment (initial and recurrent isolates, respectively). Colistin-doripenem bactericidal activity against recurrent isolates was attenuated (mean log10kill, −5.74 versus −2.88; P= 0.01) but was restored by adding sulbactam. Doripenem MICs rather than colistin MICs correlated with the activity of colistin-doripenem. Among colistin-resistant isolates, colistin-doripenem-sulbactam combinations achieved greater killing than colistin-doripenem alone (−5.65 versus −2.43; P= 0.04).

ABSTRACTBy CLSI interpretive criteria, anidulafungin and micafungin MICs determined by various methods were sensitive (60 to 70%) and highly specific (94 to 100%) for identifying FKSmutations among 120 Candida glabrataisolates. Anidulafungin and micafungin breakpoints were more specific than CLSI's caspofungin breakpoint in identifying FKSmutant strains and patients with invasive candidiasis who were likely to fail echinocandin treatment (P≤ 0.0001 for both). Echinocandin MICs were most useful clinically when interpreted in the context of prior echinocandin exposure.

ABSTRACTMutations in Candida glabrata FKSgenes, which encode the echinocandin target enzyme, are independent risk factors for treatment failures during invasive candidiasis. We retrospectively compared the ability of caspofungin susceptibility testing methods to identify C. glabrataFKSmutant isolates and predict outcomes among patients at our center. Eight percent (10/120) of sterile-site C. glabrataisolates harbored FKS1(n= 3) or FKS2(n= 7) mutations, including 32% (10/32) recovered from patients with prior echinocandin exposure. Median echinocandin exposures for mutant and nonmutant isolates were 55 (range, 7 to 188) and 13 (3 to 84) days, respectively (P= 0.004). Sensitivity and specificity of the CLSI caspofungin resistance breakpoint MIC (>0.12 μg/ml by broth microdilution using RPMI medium [BMD-RPMI]) were 90% (9/10) and 3% (3/110), respectively, for identifying FKSmutants. Sensitivity and specificity of receiver-operator characteristic (ROC) curve-derived breakpoints by BMD-RPMI, BMD-antibiotic medium 3, Etest, and YeastOne ranged from 70 to 100% and 89 to 95%, respectively; susceptibility rates varied from 83 to 90%. The 14-day echinocandin treatment success rate was 67% (44/66); failure was more likely with prior echinocandin exposure (P= 0.002) or infection with an FKSmutant (P= 0.0001) or echinocandin-resistant isolates by BMD-AM3, Etest, and YeastOne (P≤ 0.03). The failure rate among patients with prior exposure and infection with a resistant isolate was 91% (10/11); it was 22% (12/55) among others (P< 0.0001). In conclusion, ROC-derived caspofungin MIC breakpoints by several methods were sensitive and specific for identifying C. glabrataFKSmutant isolates. Mutations were seen exclusively among patients with prior echinocandin exposure. A paradigm that considers prior echinocandin exposure and caspofungin MICs accurately classified treatment outcomes for C. glabratainvasive candidiasis.

ABSTRACTEchinocandins are frontline agents against invasive candidiasis (IC), but predictors for echinocandin therapeutic failure have not been well defined. Mutations in Candida FKSgenes, which encode the enzyme targeted by echinocandins, result in elevated MICs and have been linked to therapeutic failures. In this study, echinocandin MICs by broth microdilution and FKS1and FKS2mutations among C. glabrataisolates recovered from patients with IC at our center were correlated retrospectively with echinocandin therapeutic responses. Thirty-five patients with candidemia and 4 with intra-abdominal abscesses were included, 92% (36/39) of whom received caspofungin. Twenty-six percent (10) and 74% (29) failed and responded to echinocandin therapy, respectively. Caspofungin, anidulafungin, and micafungin MICs ranged from 0.5 to 8, 0.03 to 1, and 0.015 to 0.5 μg/ml, respectively. FKSmutations were detected in 18% (7/39) of C. glabrataisolates (FKS1, n= 2; FKS2, n= 5). Median caspofungin and anidulafungin MICs were higher for patients who failed therapy (P= 0.04 and 0.006, respectively). By receiver operating characteristic (ROC) analyses, MIC cutoffs that best predicted failure were >0.5 (caspofungin), >0.06 (anidulafungin), and >0.03 μg/ml (micafungin), for which sensitivity/specificity were 60%/86%, 50%/97%, and 40%/90%, respectively. Sensitivity/specificity of an FKSmutation in predicting failure were 60%/97%. By univariate analysis, recent gastrointestinal surgery, prior echinocandin exposure, anidulafungin MIC of >0.06 μg/ml, caspofungin MIC of >0.5 μg/ml, and an FKSmutation were significantly associated with failure. The presence of an FKSmutation was the only independent risk factor by multivariate analysis (P= 0.002). In conclusion, detection of C. glabrata FKSmutations was superior to MICs in predicting echinocandin therapeutic responses among patients with IC.

ABSTRACTCeftazidime-avibactam and ceftolozane-tazobactam are newly approved agents for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. Resistance to both agents has been described clinically. Susceptibility testing on automated systems is unavailable for either agent. Our objective was to compare the disk diffusion and Etest methods to standard broth microdilution (BMD) methods for testing ceftazidime-avibactam and ceftolozane-tazobactam against a diverse collection of carbapenem-resistant Enterobacteriaceae(CRE) and carbapenem-resistant Pseudomonas aeruginosa(CRP) isolates, respectively. Among 74 ceftazidime-avibactam-susceptible and -resistant CRE isolates, BMD categorical agreement was higher with Etest (96%) than with disk diffusion (72%; P= 0.0003). Twenty-eight percent of ceftazidime-avibactam-susceptible CRE isolates were classified as resistant by disk diffusion. Results were comparable to those obtained with resistance defined genotypically. Among 72 ceftolozane-tazobactam-susceptible and -resistant CRP isolates, the levels of BMD categorical agreement with disk diffusion and Etest were 94% and 96%, respectively; the only errors identified were minor. Our findings demonstrate that Etest measurements of ceftazidime-avibactam and ceftolozane-tazobactam susceptibility correlate closely with standard BMD methods, suggesting a useful role clinically. On the other hand, disk diffusion measurements overcalled CRE resistance to ceftazidime-avibactam. A better understanding of ceftazidime-avibactam interpretive breakpoints is needed before disk diffusion is used routinely in the clinic. Until clinicians and microbiologists understand Etest and disk diffusion performance at their centers, test results should be interpreted cautiously.

ABSTRACTCeftazidime-avibactam resistance is mediated by blaKPC-3mutations, which restore carbapenem susceptibility. We subjected Klebsiella pneumoniaeisolates with different blaKPC-3mutations (n= 5) or wild-type blaKPC-3(n= 2) to serial passages with meropenem. The meropenem MIC against each isolate increased. Mutations in the ompK36porin gene evolved in 5 isolates. Among isolates with D179Y substitutions in KPC-3, blaKPC-3mutations reverted to wild type, were replaced by new mutations, or were retained. Carbapenem treatment of ceftazidime-avibactam-resistant K. pneumoniaeinfections may select for carbapenem resistance.

ABSTRACTCeftazidime-avibactam is a novel β-lactam/β-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae(CRE) that produce Klebsiella pneumoniaecarbapenemase (KPC). We report the first cases of ceftazidime-avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniaeemerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniaeisolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne blaKPC-3, which were not present in baseline isolates. blaKPC-3mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and blaKPCcloning into competent Escherichia coli. In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ≥4-fold from baseline, restoring susceptibility in K. pneumoniaefrom two patients. Cefepime and ceftriaxone MICs were also reduced ≥4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of blaKPC-3encoding D179Y/T243M and D179Y variants was diminished compared to blaKPC-3expression in baseline isolates. In conclusion, the development of resistance-conferring blaKPC-3mutations in K. pneumoniaewithin 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.