Rates of resistance to ceftazidime-avibactam and ceftolozane-tazobactam among patients treated for multidrug-resistant Pseudomonas aeruginosa bacteremia or pneumonia.

Among consecutive patients with multidrug-resistant Pseudomonas aeruginosa bacteremia or pneumonia we found those treated with ceftazidime-avibactam were more likely to develop resistance (defined as ≥4-fold increased MIC) than those treated with ceftolozane-tazobactam (40% vs. 10%; P=0.002). Ceftazidime-avibactam resistance was associated with new mutations in ampC and efflux regulatory pathways.

The growing threat of NDM-producing E. coli with penicillin-binding protein 3 mutations in the United States - is there a potential role for durlobactam?

We report identification of 5 patients with infections caused by NDM-5-producing E. coli harboring PBP3 mutations that showed reduced susceptibility to aztreonam-avibactam and cefiderocol. Durlobactam, a novel diazabicyclooctane ß-lactamase inhibitor, demonstrated minimum inhibitory concentrations ranging from 0.5 to 2 µg/mL supporting future investigations into a potential role in clinical management.

Differential in vitro susceptibility to ampicillin/ceftriaxone combination therapy among Enterococcus faecalis infective endocarditis clinical isolates.

OBJECTIVES: To investigate the genomic diversity and ß-lactam susceptibilities of Enterococcus faecalis collected from patients with infective endocarditis (IE). METHODS: We collected 60 contemporary E. faecalis isolates from definite or probable IE cases identified between 2018 and 2021 at the University of Pittsburgh Medical Center. We used whole-genome sequencing to study bacterial genomic diversity and employed antibiotic checkerboard assays and a one-compartment pharmacokinetic-pharmacodynamic (PK/PD) model to investigate bacterial susceptibility to ampicillin and ceftriaxone both alone and in combination. RESULTS: Genetically diverse E. faecalis were collected, however, isolates belonging to two STs, ST6 and ST179, were collected from 21/60 (35%) IE patients. All ST6 isolates encoded a previously described mutation upstream of penicillin-binding protein 4 (pbp4) that is associated with pbp4 overexpression. ST6 isolates had higher ceftriaxone MICs and higher fractional inhibitory concentration index values for ampicillin and ceftriaxone (AC) compared to other isolates, suggesting diminished in vitro AC synergy against this lineage. Introduction of the pbp4 upstream mutation found among ST6 isolates caused increased ceftriaxone resistance in a laboratory E. faecalis isolate. PK/PD testing showed that a representative ST6 isolate exhibited attenuated efficacy of AC combination therapy at humanized antibiotic exposures. CONCLUSIONS: We find evidence for diminished in vitro AC activity among a subset of E. faecalis IE isolates with increased pbp4 expression. These findings suggest that alternate antibiotic combinations against diverse contemporary E. faecalis IE isolates should be evaluated.

In vitro activity of clindamycin, doxycycline, and trimethoprim/sulfamethoxazole against clinical isolates of ß-hemolytic Streptococcus spp. via BD Phoenix and broth microdilution.

We tested 85 isolates of ß-hemolytic Streptococcus spp. against trimethoprim/sulfamethoxazole (TMP/SMX), clindamycin, and doxycycline by broth microdilution (BMD) and BD Phoenix. Susceptibility rates via BMD for TMP/SMX, clindamycin, and doxycycline were 100%, 85.5%, and 56.6%, respectively. TMP/SMX is a potential monotherapy agent for ß-hemolytic Streptococcus skin and soft tissue infections.

Mutations in ompK36 differentially impact in vitro synergy of meropenem/vaborbactam and ceftazidime/avibactam in combination with other antibiotics against KPC-producing Klebsiella pneumoniae.

Objectives: Ceftazidime/avibactam and meropenem/vaborbactam are preferred agents for Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) infections and are often used in combination with other agents. We aimed to characterize the synergy of combinations against KPC-Kp with varying ompK36 genotypes. Methods: KPC-Kp that harboured ompK36 WT, IS5 or glycine-aspartic acid duplication (GD) genotypes were selected. MICs were determined in triplicate. Synergy was assessed by time-kill assays for ceftazidime/avibactam and meropenem/vaborbactam in combination with colistin, gentamicin, tigecycline, meropenem or fosfomycin against 1 × 108 cfu/mL KPC-Kp. Results: KPC-Kp harboured ompK36 WT (n = 5), IS5 (n = 5) or GD (n = 5); 11 were KPC-2 and 4 were KPC-3. All were susceptible to ceftazidime/avibactam and meropenem/vaborbactam. In time-kill analysis, ceftazidime/avibactam and meropenem/vaborbactam 1 × MIC exhibited mean 24 h log-kills of -2.01 and -0.84, respectively. Ceftazidime/avibactam was synergistic in combination with colistin independent of ompK36 genotype. Ceftazidime/avibactam combinations impacted by porin mutations (compared to WT) were meropenem (-5.18 versus -6.62 mean log-kill, P < 0.001) and fosfomycin (-3.98 versus -6.58, P = 0.058). Mean log-kills with meropenem/vaborbactam were greatest in combination with gentamicin (-5.36). In the presence of porin mutations, meropenem/vaborbactam killing activity was potentiated by the addition of colistin (-6.65 versus -0.70, P = 0.03) and fosfomycin (-3.12 versus 1.54, P = 0.003). Conclusions: Our results shed new light on the synergy of ceftazidime/avibactam and meropenem/vaborbactam combinations against KPC-Kp with or without porin mutations. Killing activity of ceftazidime/avibactam with other cell wall active agents was decreased against isolates with porin mutations. On the other hand, some meropenem/vaborbactam combinations demonstrated enhanced killing in the presence of porin mutations.

In vitro activity of cefiderocol against Pseudomonas aeruginosa demonstrating evolved resistance to novel ß-lactam/ß-lactamase inhibitors.

Background: Cefiderocol demonstrates excellent activity against MDR Pseudomonas aeruginosa; however, the activity against isolates from patients previously treated with ß-lactam agents is unknown. We aimed to determine the activity of cefiderocol against P. aeruginosa collected before and after treatment with traditional ß-lactams and new ß-lactam/ß-lactamase inhibitors. Methods: Cefiderocol MICs were determined in triplicate in iron-depleted cation-adjusted Mueller-Hinton broth and compared with ß-lactam MICs tested by standard methods. All isolates underwent WGS analysis to identify mutations associated with resistance. Results: One hundred and seventy-eight P. aeruginosa isolates were evaluated; 48% (86/178) were non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam. The cefiderocol MIC50 and MIC90 were 0.12 and 1 mg/L, respectively. Median cefiderocol MICs did not vary against isolates classified as MDR, XDR, or those non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam when compared with non-MDR isolates. Against isolates collected from patients previously treated with ceftolozane/tazobactam, cefiderocol MICs were increased 4-fold compared with baseline. Cross-resistance to cefiderocol was identified in 21% (3/14) of patients who developed treatment-emergent resistance to ceftolozane/tazobactam. Overall, 6% (11/178) of isolates demonstrated cefiderocol MICs ≥2 mg/L, which were disproportionately collected from patients previously treated with ceftolozane/tazobactam (73%; 8/11). Isolates with reduced cefiderocol susceptibility harboured mutations in ampC, tonB-dependent receptors, the response regulator pirR and ftsI. Conclusions: Cefiderocol demonstrates excellent in vitro activity against P. aeruginosa isolates exposed to other novel ß-lactam agents; however, some exceptions were identified. Cross-resistance between cefiderocol and ceftolozane/tazobactam was evident, but not with ceftazidime/avibactam or imipenem/relebactam. Reduced cefiderocol susceptibility was mediated by mutations in ampC and tonB-dependent receptors.

Phage therapy in a lung transplant recipient with cystic fibrosis infected with multidrug-resistant Burkholderia multivorans.

BACKGROUND: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died. METHODS: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis. RESULTS: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate. CONCLUSIONS: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections.

Early initiation of three-drug combinations for the treatment of carbapenem-resistant A. baumannii among COVID-19 patients.

OBJECTIVES: We evaluated the clinical characteristics and outcomes of patients with COVID-19 who received three-drug combination regimens for treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) infections during a single-centre outbreak. Our objective was to describe the clinical outcomes and molecular characteristics and in vitro synergy of antibiotics against CRAB isolates. MATERIALS AND METHODS: Patients with severe COVID-19 admitted between April and July 2020 with CRAB infections were retrospectively evaluated. Clinical success was defined as resolution of signs/symptoms of infection without need for additional antibiotics. Representative isolates underwent whole-genome sequencing (WGS) and in vitro synergy of two- or three-drug combinations was assessed by checkerboard and time-kill assays, respectively. RESULTS: Eighteen patients with CRAB pneumonia or bacteraemia were included. Treatment regimens included high-dose ampicillin-sulbactam, meropenem, plus polymyxin B (SUL/MEM/PMB; 72%), SUL/PMB plus minocycline (MIN; 17%) or other combinations (12%). Clinical resolution was achieved in 50% of patients and 30-day mortality was 22% (4/18). Seven patients had recurrent infections, during which further antimicrobial resistance to SUL or PMB was not evident. PMB/SUL was the most active two-drug combination by checkerboard. Paired isolates collected before and after treatment with SUL/MEM/PMB did not demonstrate new gene mutations or differences in the activity of two- or three-drug combinations. CONCLUSIONS: Use of three-drug regimens for severe CRAB infections among COVID-19 resulted in high rates of clinical response and low mortality relative to previous studies. The emergence of further antibiotic resistance was not detected phenotypically or through WGS analysis. Additional studies are needed to elucidate preferred antibiotic combinations linked to the molecular characteristics of infecting strains.

Impact of ompk36 genotype and KPC subtype on the in vitro activity of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam against KPC-producing K. pneumoniae clinical isolates.

Objectives: The availability of new ß-lactam/ß-lactamase inhibitors ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam have redefined contemporary treatment of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) infections. We aimed to characterize and contrast the in vitro activity of these agents against genetically diverse KPC-Kp clinical isolates. Methods: We analysed genomes of 104 non-consecutive KPC-Kp isolates and compared the in vitro antibiotic activity by KPC subtype and ompK36 genotype. MICs were determined in triplicate by CLSI methods. Twenty representative isolates were selected for time-kill analyses against physiological steady-state and trough concentrations, as well as 4× MIC for each agent. Results: Fifty-eight percent and 42% of isolates harboured KPC-2 and KPC-3, respectively. OmpK36 mutations were more common among KPC-2- compared with KPC-3-producing Kp (P < 0.0001); mutations were classified as IS5 insertion, glycine-aspartic acid insertion at position 134 (GD duplication) and other mutations. Compared to isolates with WT ompK36, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam MICs were elevated for isolates with IS5 by 2-, 4- and 16-fold, respectively (P < 0.05 for each). Against isolates with GD duplication, imipenem/relebactam and meropenem/vaborbactam MICs were increased, but ceftazidime/avibactam MICs were not. In time-kill studies, ceftazidime/avibactam-mediated killing correlated with ceftazidime/avibactam MICs, and did not vary across ompK36 genotypes. Imipenem/relebactam was not bactericidal against any isolate at trough concentrations. At steady-state imipenem/relebactam concentrations, regrowth occurred more commonly for isolates with IS5 mutations. Log-kills were lower in the presence of meropenem/vaborbactam for isolates with GD duplication compared with IS5 mutations. Conclusions: Our investigation identified key genotypes that attenuate, to varying degrees, the in vitro activity for each of the new ß-lactam/ß-lactamase inhibitors. Additional studies are needed to translate the importance of these observations into clinical practice.

Evolution and Transmission of Cefiderocol-Resistant Acinetobacter baumannii During an Outbreak in the Burn Intensive Care Unit.

We report on 11 critically ill burn patients treated with cefiderocol for carbapenem-resistant Acinetobacter baumannii infections. Clinical success was achieved in 36% and complicated by treatment-emergent resistance and interpatient transmission of cefiderocol-resistant A. baumannii. Resistant isolates harbored disrupted pirA and piuA genes that were not disrupted among susceptible isolates.

Optimal ceftazidime/avibactam dosing exposure against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Infections due to carbapenem-resistant Enterobacterales are considered urgent public health threats and often treated with a ß-lactam/ß-lactamase inhibitor combination. However, clinical treatment failure and resistance emergence have been attributed to inadequate dosing. We used a novel framework to provide insights of optimal dosing exposure of ceftazidime/avibactam. METHODS: Seven clinical isolates of Klebsiella pneumoniae producing different KPC variants were examined. Ceftazidime susceptibility (MIC) was determined by broth dilution using escalating concentrations of avibactam. The observed MICs were characterized as response to avibactam concentrations using an inhibitory sigmoid Emax model. Using the best-fit parameter values, %fT>MICi was estimated for various dosing regimens of ceftazidime/avibactam. A hollow-fibre infection model (HFIM) was subsequently used to ascertain the effectiveness of selected regimens over 120 h. The drug exposure threshold associated with bacterial suppression was identified by recursive partitioning. RESULTS: In all scenarios, ceftazidime MIC reductions were well characterized with increasing avibactam concentrations. In HFIM, bacterial regrowth over time correlated with emergence of resistance. Overall, suppression of bacterial regrowth was associated with %fT>MICi ≥ 76.1% (100% versus 18.2%; P < 0.001). Using our framework, the optimal drug exposure could be achieved with ceftazidime/avibactam 2.5 g every 12 h in 5 out of 7 isolates. Furthermore, ceftazidime/avibactam 2.5 g every 8 h can suppress an isolate deemed resistant based on conventional susceptibility testing method. CONCLUSIONS: An optimal drug exposure to suppress KPC-producing bacteria was identified. The novel framework is informative and may be used to guide optimal dosing of other ß-lactam/ß-lactamase inhibitor combinations. Further in vivo investigations are warranted.

Evolution of Imipenem-Relebactam Resistance Following Treatment of Multidrug-Resistant Pseudomonas aeruginosa Pneumonia.

We report the emergence of imipenem-relebactam nonsusceptible Pseudomonas aeruginosa in 5 patients treated for nosocomial pneumonia for 10-28 days. Genome sequence analysis identified treatment-emergent mutations in MexAB-OprM and/or MexEF-OprN efflux operons that arose independently in each patient across distinct P. aeruginosa sequence types. Testing with efflux-inhibitor PAßN restored imipenem-relebactam susceptibility.

Ceftolozane/tazobactam for refractory P. aeruginosa endocarditis: A case report and pharmacokinetic analysis.

We describe a case of a 48 years old male with left sided endocarditis and septic emboli secondary to a Pseudomonas aeruginosa strain that developed resistance to other ß-lactam antibiotics during therapy resulting in prolonged bacteremia. Blood cultures sterilized within 1 day of initiating ceftolozane/tazobactam 3 g every 8 hours in combination with ciprofloxacin. Steady state free ceftolozane plasma Cmax and Cmin concentrations were calculated to be 122.2µg/mL and 24.3µg/mL, respectively. The multidrug-resistant strain harbored chromosomal ß-lactamases OXA-486 and PDC-3, mutations in ampD and dacB predicted to lead to ampC over-expression, and mutations in OprD predicted to decrease outer membrane permeability. Following completion of a 42 day course and aortic valve replacement, the patient was deemed clinically cured without recurrence of infection at follow up 2 years later. To our knowledge, this is the first reported case to measure ceftolozane concentrations during the treatment of endocarditis which supports dose optimization approaches of severe endovascular disease due to multidrug resistant pathogens.

Molecular Epidemiology, Natural History, and Long-Term Outcomes of Multidrug-Resistant Enterobacterales Colonization and Infections Among Solid Organ Transplant Recipients.

BACKGROUND: Multidrug-resistant Enterobacterales (MDR-E), including carbapenem-resistant and third-generation cephalosporin-resistant Enterobacterales (CRE, CefR-E), are major pathogens following solid organ transplantation (SOT). METHODS: We prospectively studied patients who underwent lung, liver, and small bowel transplant from February 2015 through March 2017. Weekly perirectal swabs (up to 100 days post-transplant) were cultured for MDR-E. Whole-genome sequencing (WGS) was performed on gastrointestinal (GI) tract-colonizing and disease-causing isolates. RESULTS: Twenty-five percent (40 of 162) of patients were MDR-E GI-colonized. Klebsiella pneumoniae was the most common CRE and CefR-E. Klebsiella pneumoniae carbapenemases and CTX-M were leading causes of CR and CefR, respectively. Thirty-five percent of GI colonizers developed MDR-E infection vs 2% of noncolonizers (P < .0001). The attack rate was higher among CRE colonizers than CefR-E colonizers (53% vs 21%, P = .049). GI colonization and high body mass index were independent risk factors for MDR-E infection (P ≤ .004). Thirty-day mortality among infected patients was 6%. However, 44% of survivors developed recurrent infections; 43% of recurrences were late (285 days to 3.9 years after the initial infection). Long-term survival (median, 4.3 years post-transplant) did not differ significantly between MDR-E-infected and MDR-E-noninfected patients (71% vs 77%, P = .56). WGS phylogenetic analyses revealed that infections were caused by GI-colonizing strains and suggested unrecognized transmission of novel clonal group-258 sublineage CR-K. pneumoniae and horizontal transfer of resistance genes. CONCLUSIONS: MDR-E GI colonization was common following SOT and predisposed patients to infections by colonizing strains. MDR-E infections were associated with low short- and long-term mortality, but recurrences were frequent and often occurred years after initial infections. Findings provide support for MDR-E surveillance in our SOT program.

Oxacillin plus ertapenem combination therapy leads to rapid blood culture clearance and positive outcomes among patients with persistent MSSA bacteraemia: a case series.

BACKGROUND: Bloodstream infections caused by MSSA are associated with significant morbidity and mortality. Traditional treatment of MSSA bacteraemia includes an IV antistaphylococcal ß-lactam and surgical source control when indicated. OBJECTIVES: To evaluate the time to blood culture clearance as well as in-hospital and 90 day mortality in patients with persistent MSSA bacteraemia treated with combination antistaphylococcal penicillin plus carbapenem therapy. METHODS: Consecutive patients with persistent MSSA bacteraemia treated with combination therapy were identified by study investigators and reviewed by independent clinicians. The decision to initiate combination therapy was made by the consulting clinician or by the institution's multidisciplinary endocarditis team. RESULTS: Among 10 patients with a median of 5 days of persistent MSSA bacteraemia, treatment with an antistaphylococcal penicillin plus carbapenem led to sterilization of blood cultures in all patients. Blood culture clearance occurred in a median of 1 day and patients received a median of 6 days of combination treatment. Four of seven patients who underwent source control of their primary site of infection cleared their bacteraemia on combination therapy prior to the surgical intervention. All patients survived to hospital discharge and 90 days post-discharge. CONCLUSIONS: These data extend prior findings and provide further evidence that suggests the potential benefits of combination therapy among patients with persistent MSSA bacteraemia.

In Vitro Susceptibility of Multidrug-Resistant Pseudomonas aeruginosa following Treatment-Emergent Resistance to Ceftolozane-Tazobactam.

We compared the in vitro susceptibility of multidrug-resistant Pseudomonas aeruginosa isolates collected before and after treatment-emergent resistance to ceftolozane-tazobactam. Median baseline and postexposure ceftolozane-tazobactam MICs were 2 and 64 µg/ml, respectively. Whole-genome sequencing identified treatment-emergent mutations in ampC among 79% (11/14) of paired isolates. AmpC mutations were associated with cross-resistance to ceftazidime-avibactam but increased susceptibility to piperacillin-tazobactam and imipenem. A total of 81% (12/16) of ceftolozane-tazobactam-resistant isolates with ampC mutations were susceptible to imipenem-relebactam.

Clinical Evolution of AmpC-Mediated Ceftazidime-Avibactam and Cefiderocol Resistance in Enterobacter cloacae Complex Following Exposure to Cefepime.

We report 2 independent patients from whom carbapenem and ceftazidime-avibactam-resistant Enterobacter cloacae complex strains were identified. The ceftazidime-avibactam resistance was attributed to a 2-amino acid deletion in the R2 loop of AmpC ß-lactamase, which concurrently caused resistance to cefepime and reduced susceptibility to cefiderocol, a novel siderophore cephalosporin.

Structural Basis of Reduced Susceptibility to Ceftazidime-Avibactam and Cefiderocol in Enterobacter cloacae Due to AmpC R2 Loop Deletion.

Ceftazidime-avibactam and cefiderocol are two of the latest generation ß-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales Here, we show that structural changes in AmpC ß-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant Enterobacter cloacae clinical strain (Ent385) was found to be resistant to ceftazidime-avibactam and cefiderocol without prior exposure to either agent. The AmpC ß-lactamase of Ent385 (AmpCEnt385) contained an alanine-proline deletion at positions 294 and 295 (A294_P295del) in the R2 loop. AmpCEnt385 conferred reduced susceptibility to ceftazidime-avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpCEnt385 showed increased hydrolysis of ceftazidime and cefiderocol compared to AmpCEnt385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpCEnt385 and AmpCP99, the canonical AmpC of E. cloacae complex, revealed that the two-residue deletion in AmpCEnt385 induced drastic structural changes of the H-9 and H-10 helices and the R2 loop, which accounted for the increased hydrolysis of ceftazidime and cefiderocol. The potential for a single mutation in ampC to confer reduced susceptibility to both ceftazidime-avibactam and cefiderocol requires close monitoring.

Plasma and Cerebrospinal Fluid Therapeutic Drug Monitoring of Ceftolozane and Tazobactam During Treatment of Multidrug-Resistant Pseudomonas aeruginosa Meningitis.

We report a case of multidrug-resistant Pseudomonas aeruginosa meningitis treated with ceftolozane-tazobactam with concomitant therapeutic drug monitoring of plasma and cerebral spinal fluid. The data suggest that ceftolozane-tazobactam may be an option for select central nervous system infections; however, treatment decisions should be interpreted on a case-by-case basis.

Early Experience With Meropenem-Vaborbactam for Treatment of Carbapenem-resistant Enterobacteriaceae Infections.

Twenty patients with carbapenem-resistant Enterobacteriaceae infections were treated with meropenem-vaborbactam. Thirty-day clinical success and survival rates were 65% (13/20) and 90% (18/20), respectively. Thirty-five percent of patients had microbiologic failures within 90 days. One patient developed a recurrent infection due to meropenem-vaborbactam-nonsusceptible, ompK36 porin mutant Klebsiella pneumoniae.