Real-World Impact of the Accelerate PhenoTest BC Kit on Patients With Bloodstream Infections in the Improving Outcomes and Antimicrobial Stewardship Study: A Quasiexperimental Multicenter Study.

BACKGROUND: Bloodstream infections (BSIs) are a leading cause of morbidity and mortality. The Improving Outcomes and Antimicrobial Stewardship study seeks to evaluate the impact of the Accelerate PhenoTest BC Kit (AXDX) on antimicrobial use and clinical outcomes in BSIs. METHODS: This multicenter, quasiexperimental study compared clinical and antimicrobial stewardship metrics, prior to and after implementation of AXDX, to evaluate the impact this technology has on patients with BSIs. Laboratory and clinical data from hospitalized patients with BSIs (excluding contaminants) were compared between 2 arms, 1 that underwent testing on AXDX (post-AXDX) and 1 that underwent alternative organism identification and susceptibility testing (pre-AXDX). The primary outcomes were time to optimal therapy (TTOT) and 30-day mortality. RESULTS: A total of 854 patients with BSIs (435 pre-AXDX, 419 post-AXDX) were included. Median TTOT was 17.2 hours shorter in the post-AXDX arm (23.7 hours) compared with the pre-AXDX arm (40.9 hours; P<.0001). Compared with pre-AXDX, median time to first antimicrobial modification (24.2 vs 13.9 hours; P<.0001) and first antimicrobial deescalation (36.0 vs 27.2 hours; P=.0004) were shorter in the post-AXDX arm. Mortality (8.7% pre-AXDX vs 6.0% post-AXDX), length of stay (7.0 pre-AXDX vs 6.5 days post-AXDX), and adverse drug events were not significantly different between arms. Length of stay was shorter in the post-AXDX arm (5.4 vs 6.4 days; P=.03) among patients with gram-negative bacteremia. CONCLUSIONS: For BSIs, use of AXDX was associated with significant decreases in TTOT, first antimicrobial modification, and time to antimicrobial deescalation.

Improving outcomes and antibiotic stewardship (IOAS) for patients with Gram-positive bloodstream infections through use of rapid testing: a quasi-experimental multicentre study of the Accelerate PhenoTest™ BC Kit.

BACKGROUND: Data from the Improving Outcomes and Antibiotic Stewardship for Patients with Bloodstream Infections: Accelerate PhenoTest™ BC Kit (AXDX) Registry Study were analysed to determine the impact of rapid organism identification and antimicrobial susceptibility testing (AST) for Gram-positive bacteraemia. PATIENTS AND METHODS: This multicentre, quasi-experimental study evaluated clinical and antimicrobial stewardship metrics following the implementation of AXDX. Data from hospitalized patients with bacteraemia were compared between groups, one that underwent testing on AXDX (post-AXDX) and one that underwent traditional identification and AST (pre-AXDX). An analysis of patients with Gram-positive bacteraemia was performed. The primary outcome was time to optimal therapy (TTOT). Secondary outcomes included time to first antibiotic modification (overall and Gram-positive), duration of unnecessary MRSA coverage, incidence of adverse events, length of stay and mortality. RESULTS: A total of 219 (109 pre-AXDX, 110 post-AXDX) patients with Gram-positive bacteraemia were included. Median TTOT was 36.3 h (IQR, 16.9-56.7) in the pre-AXDX group and 20.4 h (IQR, 7.5-36.7) in the post-AXDX group (P = 0.01). Compared with pre-AXDX, median time to first antibiotic modification (29.1 versus 15.9 h; P = 0.002), time to first Gram-positive antibiotic modification (33.2 versus 17.2 h; P = 0.003) and median duration of unnecessary MRSA coverage (58.4 versus 29.7 h; P = 0.04) were reduced post-AXDX. A trend towards decreased acute kidney injury (24% versus 13%; P = 0.06) was observed in the post-AXDX group. Groups did not differ in other secondary outcomes. CONCLUSIONS: Implementation of AXDX testing for patients with Gram-positive bacteraemia shortened the TTOT and reduced unnecessary antibiotic exposure due to faster antibiotic modifications.

Time to Result for Pathogen Identification and Antimicrobial Susceptibility Testing of Bronchoalveolar Lavage and Endotracheal Aspirate Specimens in U.S. Acute Care Hospitals.

Identification (ID) and antimicrobial susceptibility testing (AST) of respiratory pathogens are critical to the management of patients with pneumonia to facilitate optimal antibiotic therapy selection. Few studies have examined the time to results (TTR) for this critical specimen, and such data can be valuable for benchmarking the current paradigm of diagnostic approaches. TTR for bronchoalveolar lavage (BAL) and endotracheal aspirate (ETA) specimens from hospitalized patients was evaluated using the Premier Healthcare Database, a comprehensive database of 194 U.S. hospitals. Times from specimen collection to reporting of organism ID/AST were evaluated and compared by specimen types and characteristics. A total of 79,662 (43,129 BAL; 36,533 ETA) specimens were included, of which 19.3% harbored no growth, 47.1% contained normal respiratory flora alone (including yeast), and 0.6% contained mycobacteria/molds. Potential bacterial pathogens (PBP) were recovered from 33.0%. ETA specimens had a higher proportion of specimens with isolation of PBP (39.2% versus 27.7%) and with normal respiratory flora (52.0% versus 43.0%) and were less likely to be negative (8.2% versus 28.6%) than BAL specimens (all P < 0.0001). Staphylococcus aureus and Pseudomonas aeruginosa were isolated in 10.5 and 6.4% of the specimens, respectively, and were the most common organisms identified. Median (interquartile range) TTR were 37.0 h (21.8 to 51.7 h) and 60.5 h (46.6 to 72.4 h) for ID and AST, respectively. Median TTR for major respiratory pathogens by organism ranged from 29.2 to 43.9 h for ID and from 47.9 to 73.9 h for AST. Organism type, specimen collection time, and hospital teaching status influenced TTR. Mechanically vented patients and ETA specimens were more likely to recover PBP.

Evaluation of rapid polymerase chain reaction-based organism identification of gram-positive cocci for patients with a single positive blood culture.

For patients with a single-positive blood culture growing gram-positive cocci, organism identification can provide supportive information for differentiating contamination from infection. We investigated the effect of a rapid blood culture identification panel (BCID) on vancomycin-prescribing patterns and patient outcomes for single positive blood culture (PBC) growing gram-positive cocci. Adult patients with single-positive blood culture growing gram-positive cocci with conventional organism identification (pre-BCID) were compared with organism identification by BCID (post-BCID). Antimicrobial Stewardship Program (ASP) review of PBC was performed in both study groups. Vancomycin prescribing patterns were studied. Secondary endpoints were the incidence of nephrotoxicity, length of stay (LOS), readmission rate, mortality, and hospital costs. A total of 188 patients (86 pre-BCID, 102 post-BCID) were included. Organism identification was known 21 h sooner in the post-BCID group (P < 0.001). Coagulase-negative staphylococci were the most commonly isolated organisms (73%). In patients where vancomycin was deemed unnecessary (n = 133), vancomycin use (51% pre-BCID vs 36% post-BCID; P = 0.09) and time from culture positivity to vancomycin discontinuation (1.5 vs. 1.7 days; P = 0.92) did not differ between groups. We found no differences in the development of nephrotoxicity, LOS, readmission, mortality, or hospital costs. Earlier identification of single positive blood culture growing gram-positive cocci did not significantly influence prescribing patterns of vancomycin. However, baseline antimicrobial stewardship review of single positive blood culture growing gram-positive cocci may have lessened the opportunity for detectable differences. Larger studies, accounting for the impact of ASP intervention, should be performed to determine the value of each individual component.

Isavuconazole pharmacokinetics in a patient with cystic fibrosis following bilateral orthotopic lung transplantation.

Previous studies of patients with cystic fibrosis (CF) treated with azole antifungals have shown altered pharmacokinetics relative to healthy patients. Data regarding the pharmacokinetic profile of isavuconazole in patients with CF undergoing lung transplantation are currently not available. Serum trough concentrations assessed in a single CF patient following transplant revealed significantly lower values relative to available literature. Larger studies are required to validate CF population pharmacokinetics of isavuconazole.

Emergence of Ceftolozane-Tazobactam-Resistant Pseudomonas aeruginosa during Treatment Is Mediated by a Single AmpC Structural Mutation.

Ceftolozane-tazobactam is a cephalosporin-ß-lactamase inhibitor combination that exhibits potent in vitro activity against Pseudomonas aeruginosa, including strains that are resistant to other ß-lactams. The emergence of ceftolozane-tazobactam resistance among clinical isolates of P. aeruginosa has rarely been described. Here we characterized ceftolozane-tazobactam-resistant P. aeruginosa strains recovered from a patient who was treated with this agent for 6 weeks for a recurrent wound infection. The results showed that the resistance was mediated by a single AmpC structural mutation.

Antimicrobial Resistance in the Intensive Care Unit: A Focus on Gram-Negative Bacterial Infections.

Bacterial infections are a frequent cause of hospitalization, and nosocomial infections are an increasingly common condition, particularly within the acute/critical care setting. Infection control practices and new antimicrobial development have primarily focused on gram-positive bacteria; however, in recent years, the incidence of infections caused by gram-negative bacteria has risen considerably in intensive care units. Infections caused by multidrug-resistant (MDR) gram-negative organisms are associated with high morbidity and mortality, with significant direct and indirect costs resulting from prolonged hospitalizations due to antibiotic treatment failures. Of particular concern is the increasing prevalence of antimicrobial resistance to ß-lactam antibiotics (including carbapenems) among Pseudomonas aeruginosa and Acinetobacter baumannii and, recently, among pathogens of the Enterobacteriaceae family. Treatment options for infections caused by these pathogens are limited. Antimicrobial stewardship programs focus on optimizing the appropriate use of currently available antimicrobial agents with the goals of improving outcomes for patients with infections caused by MDR gram-negative organisms, slowing the progression of antimicrobial resistance, and reducing hospital costs. Newly approved treatment options are available, such as ß-lactam/ß-lactamase inhibitor combinations, which significantly extend the armamentarium against MDR gram-negative bacteria.

Benefits of Adding a Rapid PCR-Based Blood Culture Identification Panel to an Established Antimicrobial Stewardship Program.

Studies have demonstrated that the combination of antimicrobial stewardship programs (ASP) and rapid organism identification improves outcomes in bloodstream infections (BSI) but have not controlled for the incremental contribution of the individual components. Hospitalized adult patients with blood culture pathogens on a rapid, multiplex PCR-based blood culture identification panel (BCID) that included 19 bacterial species, 5 Candida spp., and 4 antimicrobial resistance genes were studied over sequential time periods in a pre-post quasiexperimental study in 3 groups in the following categories: conventional organism identification (controls), conventional organism identification with ASP (AS), and BCID with ASP (BCID). Clinical and economic outcomes were compared between groups. There were 783 patients with positive blood cultures; of those patients, 364 (115 control, 104 AS, and 145 BCID) met inclusion criteria. The time from blood culture collection to organism identification was shorter in the BCID group (17 h; P < 0.001) than in the control group (57 h) or the AS group (54 h). The BCID group had a shorter time to effective therapy (5 h; P < 0.001) than the control group (15 h) or AS group (13 h). The AS (57%) and BCID (52%) groups had higher rates of antimicrobial de-escalation than the control group (34%), with de-escalation occurring sooner in the BCID group (48 h; P = 0.034) than in the AS group (61 h) or the control group (63 h). No difference between the control group, AS group, and BCID group was seen with respect to mortality, 30-day readmission, intensive care unit length of stay (LOS), postculture LOS, or costs. In patients with BSI, ASP alone improved antimicrobial utilization. Addition of BCID to an established ASP shortened the time to effective therapy and further improved antimicrobial use compared to ASP alone, even in a setting of low antimicrobial resistance rates.

Characterizing in vivo pharmacodynamics of carbapenems against Acinetobacter baumannii in a murine thigh infection model to support breakpoint determinations.

Pharmacodynamic profiling data of carbapenems for Acinetobacter spp. are sparse. This study aimed to determine the pharmacodynamic targets of carbapenems for Acinetobacter baumannii based on a range of percentages of the dosing interval in which free drug concentrations remained above the MIC (fT>MIC) in the neutropenic murine thigh infection model. fT>MIC values of 23.7%, 32.8%, and 47.5% resulted in stasis, 1-log reductions, and 2-log reductions in bacterial density after 24 h, respectively. The pharmacodynamic targets of carbapenems for A. baumannii demonstrated in vivo are similar to those of other Gram-negative bacteria.

Clinical pharmacodynamics of antipseudomonal cephalosporins in patients with ventilator-associated pneumonia.

Advanced-generation cephalosporins are frequently used for empirical coverage of ventilator-associated pneumonia (VAP) due to their activity against a broad spectrum of Gram-positive and Gram-negative aerobic bacteria, including Pseudomonas aeruginosa and Enterobacteriaceae. Providing optimal antibiotic exposure is essential to achieving successful response in patients with VAP. We evaluated exposures of two antipseudomonal cephalosporins, ceftazidime and cefepime, in patients with VAP due to Gram-negative bacilli to identify the pharmacodynamic parameter predictive of microbiological success. Population pharmacokinetic models were used to estimate individual free drug exposures. Pharmacodynamic indices were determined for each patient using the baseline Gram-negative bacilli with the highest drug MIC. Classification and regression tree analysis was utilized to partition exposure breakpoints, and multivariate logistic regression was conducted to identify predictors of microbiological success. A total of 73 patients (18 receiving ceftazidime therapy and 55 receiving cefepime therapy) were included. MICs ranged widely from 0.047 to 96 µg/ml. The microbiological success rate was 58.9%. Predictive breakpoints were identified for all pharmacodynamic parameters, including a serum fT>MIC greater than 53% (P=0.02). When controlling for APACHE II (odds ratio [OR], 1.01; 95% confidence interval, 0.93 to 1.09; P=0.85) and combination therapy (OR, 0.74; 95% confidence interval, 0.25 to 2.19; P=0.59), achieving a greater than 53% fT>MIC remained a significant predictor of success (OR, 10.3; 95% confidence interval, 1.1 to 92.3; P=0.04). In patients with VAP due to Gram-negative bacilli, serum exposure of greater than 53% fT>MIC was found to be a significant predictor of favorable microbiological response for antipseudomonal cephalosporins. These data are useful when determining dosing regimens for cephalosporin agents under development for pneumonia.

In vitro activity of human-simulated epithelial lining fluid exposures of ceftaroline, ceftriaxone, and vancomycin against methicillin-susceptible and -resistant Staphylococcus aureus.

Staphylococcus aureus, including methicillin-susceptible (MSSA) and -resistant (MRSA) strains, is an important pathogen of bacterial pneumonia. As antibiotic concentrations at the site of infection are responsible for killing, we investigated the activity of human-simulated epithelial lining fluid (ELF) exposures of three antibiotics (ceftaroline, ceftriaxone, and vancomycin) commonly used for treatment of S. aureus pneumonia. An in vitro pharmacodynamic model was used to simulate ELF exposures of vancomycin (1 g every 12 h [q12h]), ceftaroline (600 mg q12h and q8h), and ceftriaxone (2 g q24h and q12h). Four S. aureus isolates (2 MSSA and 2 MRSA) were evaluated over 72 h with a starting inoculum of ∼ 10(6) CFU/ml. Time-kill curves were constructed, and microbiological response (change in log10 CFU/ml from 0 h and the area under the bacterial killing and regrowth curve [AUBC]) was assessed in duplicate. The change in 72-h log10 CFU/ml was largest for ceftaroline q8h (reductions of >3 log10 CFU/ml against all strains). This regimen also achieved the lowest AUBC against all organisms (P < 0.05). Vancomycin produced reliable bacterial reductions of 0.9 to 3.3 log10 CFU/ml, while the activity of ceftaroline q12h was more variable (reductions of 0.2 to 2.3 log10 CFU/ml against 3 of 4 strains). Both regimens of ceftriaxone were poorly active against MSSA tested (0.1 reduction to a 1.8-log10 CFU/ml increase). Against these S. aureus isolates, ELF exposures of ceftaroline 600 mg q8h exhibited improved antibacterial activity compared with ceftaroline 600 mg q12h and vancomycin, and therefore, this q8h regimen deserves further evaluation for the treatment of bacterial pneumonia. These data also suggest that ceftriaxone should be avoided for S. aureus pneumonia.

Unexpected in vivo activity of ceftazidime alone and in combination with avibactam against New Delhi metallo-ß-lactamase-producing Enterobacteriaceae in a murine thigh infection model.

The emergence of the New Delhi metallo-ß-lactamase (NDM) among Enterobacteriaceae has become a global concern because of its high levels of in vitro resistance to nearly all available antibiotics. However, recent in vivo studies demonstrated the efficacies of carbapenems against NDM-1-producing isolates despite high MICs. Herein, we report in vivo findings with ceftazidime and ceftazidime-avibactam against an isogenic pair (wild type and NDM-1) and four clinical NDM-producing isolates that demonstrate discordance between MICs measured in vitro and the in vivo activity of ceftazidime-avibactam against this resistant genotype.

In vivo efficacy of humanized exposures of Ceftazidime-Avibactam in comparison with Ceftazidime against contemporary Enterobacteriaceae isolates.

Ceftazidime-avibactam is a ß-lactam ß-lactamase inhibitor combination under investigation for the treatment of serious Gram-negative infections. When combined with avibactam, a novel non-ß-lactam ß-lactamase inhibitor, ceftazidime has activity against isolates that produce Ambler class A, class C, and some class D ß-lactamases. However, little is known of the in vivo efficacy of the combination against these targeted ceftazidime- and carbapenem-resistant Enterobacteriaceae. Using humanized exposures in the murine thigh model, we evaluated the efficacy of ceftazidime-avibactam against Enterobacteriaceae exhibiting MICs of ≥8 µg/ml to aid in the assignment of interpretive susceptibility criteria. Eighteen clinical Enterobacteriaceae isolates, including nine carbapenem-resistant strains, were evaluated against ceftazidime-avibactam (2,000 mg/500 mg) as a 2-h infusion every 8 h. To highlight the impact of avibactam, 13 select isolates were tested in the neutropenic model against a humanized regimen of 2,000 mg ceftazidime every 8 h (2-h infusion). Additionally, nine isolates were evaluated in immunocompetent animals. The efficacy was evaluated as the change in log10 CFU compared with that of 0-h controls after 24 h. The vast majority (17/18, 94%) of the isolates were resistant to ceftazidime alone. The ceftazidime monotherapy failed to have activity against 10 of 13 isolates, while ceftazidime-avibactam produced reductions in bacterial density against 16 of 18 isolates. Ceftazidime-avibactam (2,000 mg/500 mg) every 8 h (2-h infusion) displayed dependable activity against the Enterobacteriaceae isolates, exhibiting MICs of ≤16 µg/ml (free drug concentration above the MIC [fT>MIC] of ≥62%) and variable activity was noted at an MIC of 32 µg/ml (fT>MIC of 34%). The presence of a functioning immune system enhanced the efficacy for both regimens against all tested isolates. These data support further examination of the use of ceftazidime-avibactam as an effective therapy against infections due to Gram-negative infections, including carbapenem-resistant Enterobacteriaceae.

Novel method of calculating adjusted antibiotic use by microbiological burden.

OBJECTIVE: To determine the usefulness of adjusting antibiotic use (AU) by prevalence of bacterial isolates as an alternative method for risk adjustment beyond hospital characteristics. DESIGN: Retrospective, observational, cross-sectional study. SETTING: Hospitals in the southeastern United States. METHODS: AU in days of therapy per 1,000 patient days and microbiologic data from 2015 and 2016 were collected from 26 hospitals. The prevalences of Pseudomonas aeruginosa, extended-spectrum ß-lactamase (ESBL)-producing bacteria, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) were calculated and compared to the average prevalence of all hospitals in the network. This proportion was used to calculate the adjusted AU (a-AU) for various categories of antimicrobials. For example, a-AU of antipseudomonal ß-lactams (APBL) was the AU of APBL divided by (prevalence of P. aeruginosa at that hospital divided by the average prevalence of P. aeruginosa). Hospitals were categorized by bed size and ranked by AU and a-AU, and the rankings were compared. RESULTS: Most hospitals in 2015 and 2016, respectively, moved ≥2 positions in the ranking using a-AU of APBL (15 of 24, 63%; 22 of 26, 85%), carbapenems (14 of 23, 61%; 22 of 25; 88%), anti-MRSA agents (13 of 23, 57%; 18 of 26, 69%), and anti-VRE agents (18 of 24, 75%; 15 of 26, 58%). Use of a-AU resulted in a shift in quartile of hospital ranking for 50% of APBL agents, 57% of carbapenems, 35% of anti-MRSA agents, and 75% of anti-VRE agents in 2015 and 50% of APBL agents, 28% of carbapenems, 50% of anti-MRSA agents, and 58% of anti-VRE agents in 2016. CONCLUSIONS: The a-AU considerably changes how hospitals compare among each other within a network. Adjusting AU by microbiological burden allows for a more balanced comparison among hospitals with variable baseline rates of resistant bacteria.

Multicenter, Observational Cohort Study Evaluating Third-Generation Cephalosporin Therapy for Bloodstream Infections Secondary to Enterobacter, Serratia, and Citrobacter Species.

OBJECTIVES: There is debate on whether the use of third-generation cephalosporins (3GC) increases the risk of clinical failure in bloodstream infections (BSIs) caused by chromosomally-mediated AmpC-producing Enterobacterales (CAE). This study evaluates the impact of definitive 3GC therapy versus other antibiotics on clinical outcomes in BSIs due to Enterobacter, Serratia, or Citrobacter species. METHODS: This multicenter, retrospective cohort study evaluated adult hospitalized patients with BSIs secondary to Enterobacter, Serratia, or Citrobacter species from 1 January 2006 to 1 September 2014. Definitive 3GC therapy was compared to definitive therapy with other non-3GC antibiotics. Multivariable Cox proportional hazards regression evaluated the impact of definitive 3GC on overall treatment failure (OTF) as a composite of in-hospital mortality, 30-day hospital readmission, or 90-day reinfection. RESULTS: A total of 381 patients from 18 institutions in the southeastern United States were enrolled. Common sources of BSIs were the urinary tract and central venous catheters (78 (20.5%) patients each). Definitive 3GC therapy was utilized in 65 (17.1%) patients. OTF occurred in 22/65 patients (33.9%) in the definitive 3GC group vs. 94/316 (29.8%) in the non-3GC group (p = 0.51). Individual components of OTF were comparable between groups. Risk of OTF was comparable with definitive 3GC therapy vs. definitive non-3GC therapy (aHR 0.93, 95% CI 0.51-1.72) in multivariable Cox proportional hazards regression analysis. CONCLUSIONS: These outcomes suggest definitive 3GC therapy does not significantly alter the risk of poor clinical outcomes in the treatment of BSIs secondary to Enterobacter, Serratia, or Citrobacter species compared to other antimicrobial agents.

Clinical experience with ceftazidime/avibactam for treatment of antibiotic-resistant organisms other than Klebsiella pneumoniae.

BACKGROUND: Ceftazidime/avibactam is a newly approved ß-lactam/ß-lactamase inhibitor combination with activity against antibiotic-resistant Gram-negative organisms, including many carbapenem-resistant strains. Although this agent may offer a promising treatment option for serious infections with limited alternatives available, clinical experience with ceftazidime/avibactam in treatment of infections caused by multidrug-resistant Gram-negative organisms other than Klebsiella pneumoniae is limited. METHODS: A retrospective case series was performed to evaluate patients treated with ceftazidime/avibactam for infections caused by organisms other than K. pneumoniae at our institution over a 1-year period. Patients aged at least 18 years who received at least one dose of ceftazidime/avibactam were eligible for inclusion. Clinical and microbiological data were collected, and investigators assessed adverse effects, microbiological cure, clinical success, and 30-day in-hospital mortality following completion of ceftazidime/avibactam therapy. RESULTS: Ten patients were included. The most common index infection was pneumonia (n = 6/13, 46%) and the most frequently isolated organism was Pseudomonas aeruginosa (n = 8/21, 38%). Fifty percent of patients received ceftazidime/avibactam as monotherapy. Microbiological cure was achieved in 67% (n = 6/9) of patients and 70% (n = 7/10) of patients met criteria for clinical success. The 30-day in-hospital mortality rate was 30%. No patients experienced adverse events because of ceftazidime/avibactam therapy. CONCLUSIONS: For infections caused by antibiotic-resistant Gram-negative organisms other than K. pneumoniae, clinical and microbiological success rates for patients treated with ceftazidime/avibactam were similar to those that have been reported for K. pneumoniae. Ceftazidime/avibactam appears to be a promising treatment option for infections caused by a variety of resistant Gram-negative organisms when limited alternatives exist.

Analyses of a Ceftazidime-Avibactam-Resistant Citrobacter freundii Isolate Carrying blaKPC-2 Reveals a Heterogenous Population and Reversible Genotype.

A blaKPC-2-carrying Citrobacter freundii isolate developed ceftazidime-avibactam resistance during treatment with this agent. The initial and follow-up isolates exhibited ceftazidime-avibactam MICs of 4 and 64 µg/ml, respectively. Overexpression of AcrAB-TolC and porin alterations were detected in both isolates, but no other resistance mechanism was observed. After passaging the initial clinical isolate in ceftazidime-avibactam at a fixed concentration of 4 µg/ml and a 4:1 ratio, resistance to all ß-lactams was noted, and a percentage of the blaKPC-2 sequencing reads had mutations leading to the alterations D176Y (blaKPC-2-D176Y [78%]) or R164S plus P147L (blaKPC-2-R164S + P147L [82%]). Further investigation of the follow-up isolate showed that 11% of the blaKPC-2 reads had mutations leading to D179Y substitution (blaKPC-2-D179Y). In the absence of selective pressure, ceftazidime-avibactam MICs of the passaged and follow-up isolates revealed that 7 or 8 out of 20 screened colonies reverted to susceptible and possessed blaKPC-2 wild-type sequences. Recombinant plasmids carrying the blaKPC-2 alterations observed were transformed in Escherichia coli, and MIC values for ceftazidime ± avibactam were elevated. Lower MICs for ceftriaxone, cefepime, aztreonam, meropenem, and imipenem for the mutated KPC-2-producing isolates were observed compared to those of the isolates producing a wild-type KPC-2. Avibactam at a fixed concentration of 4 µg/ml restored the activity of all ß-lactams tested for the recombinant strains. The heterogenous population of wild-type and mutated blaKPC-2 and the reversibility of the genotypes observed suggest a significant challenge for managing KPC-producing isolates that develop ceftazidime-avibactam resistance during therapy.IMPORTANCE The development of ceftazidime-avibactam resistance among KPC-producing isolates during treatment with this agent has been reported. Usually isolates that become resistant have a mutated blaKPC gene that confers resistance to ceftazidime-avibactam and susceptibility to meropenem. We report a Citrobacter freundii isolate that developed ceftazidime-avibactam resistance due to mutations within the coding region of the blaKPC-2 Ω-loop previously reported; however, in this case, only 11% of the whole-genome sequencing reads had mutations, making this alteration difficult to detect and the treatment of these isolates more challenging. In addition to blaKPC, the initial and the follow-up patient isolates displayed hyperexpression of the AcrAB-TolC efflux system and disruption of the outer membrane protein (OMP) OmpF, which contribute to carbapenem resistance. Experiments performed to confirm our findings included generating mutant isolates from the initial patient isolate, passaging the isolates for purity in drug-free medium, resulting in a reversible phenotype, and cloning the mutations to demonstrate the resistance conferred.