Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling.

A double ampC (AmpCG183D) and ampD (AmpDH157Y) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpCG183D, PAO1-AmpDH157Y, PAO1-AmpCG183D/AmpDH157Y) and in PaR (PaR-AmpCPaS/AmpDPaS). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC50 values increased by 1.4, 4.1, and 29-fold after AmpCG183D , AmpDH157Y and AmpCG183D/AmpDH157Y mutations, respectively. EC50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC50 of PAO1-AmpCG183D/AmpDH157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpCG183D, and AmpDH157Y mutations led to an important decrease of EC50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpCPaS/AmpDPaS, respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpCG183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpCG183D and AmpDH157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.

Simultaneous and divergent evolution of resistance to cephalosporin/ß-lactamase inhibitor combinations and imipenem/relebactam following ceftazidime/avibactam treatment of MDR Pseudomonas aeruginosa infections.

OBJECTIVES: To describe and characterize the emergence of resistance to ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam in a patient receiving ceftazidime/avibactam treatment for an MDR Pseudomonas aeruginosa CNS infection. METHODS: One baseline (PA1) and two post-exposure (PA2 and PA3) isolates obtained before and during treatment of a nosocomial P. aeruginosa meningoventriculitis were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. The impact on ß-lactam resistance of mutations in blaPDC and mexR was determined through cloning experiments and complementation assays. RESULTS: Isolate PA1 showed baseline resistance mutations in DacB (I354A) and OprD (N142fs) conferring resistance to conventional antipseudomonals but susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. Post-exposure isolates showed two divergent ceftazidime/avibactam-resistant phenotypes associated with distinctive mutations affecting the intrinsic P PDC ß-lactamase (S254Ins) (PA2: ceftolozane/tazobactam and ceftazidime/avibactam-resistant) or MexAB-OprM negative regulator MexR in combination with modification of PBP3 (PA3: ceftazidime/avibactam and imipenem/relebactam-relebactam-resistant). Cloning experiments demonstrated the role of PDC modification in resistance to ceftolozane/tazobactam and ceftazidime/avibactam. Complementation with a functional copy of the mexR gene in isolate PA3 restored imipenem/relebactam susceptibility. CONCLUSIONS: We demonstrated how P. aeruginosa may simultaneously develop resistance and compromise the activity of new ß-lactam/ß-lactamase inhibitor combinations when exposed to ceftazidime/avibactam through selection of mutations leading to PDC modification and up-regulation of MexAB-OprM-mediated efflux.

Activity of newest generation ß-lactam/ß-lactamase inhibitor combination therapies against multidrug resistant Pseudomonas aeruginosa.

Multidrug resistant (MDR) P. aeruginosa accounts for 35% of all P. aeruginosa isolated from respiratory samples of patients with cystic fibrosis (CF). The usefulness of ß-lactam antibiotics for treating CF, such as carbapenems and later generation cephalosporins, is limited by the development of antibacterial resistance. A proven treatment approach is the combination of a ß-lactam antibiotic with a ß-lactamase inhibitor. New ß-lactam/ß-lactamase inhibitor combinations are available, but data are lacking regarding the susceptibility of MDR CF-associated P. aeruginosa (CFPA) to these new combination therapies. In this study we determined MIC values for three new combinations; imipenem-relebactam (I-R), ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T) against MDR CFPA (n = 20). The MIC90 of I-R, CZA, and C/T was 64/4, 32/4, and 16/8 (all µg/mL), respectively. The susceptibility of isolates to imipenem was not significantly improved with the addition of relebactam (p = 0.68). However, susceptibility to ceftazidime was significantly improved with the addition of avibactam (p < 0.01), and the susceptibility to C/T was improved compared to piperacillin/tazobactam (p < 0.05) These data provide in vitro evidence that I-R may not be any more effective than imipenem monotherapy against MDR CFPA. The pattern of susceptibility observed for CZA and C/T in the current study was similar to data previously reported for non-CF-associated MDR P. aeruginosa.

Treatment of multidrug-resistant Pseudomonas aeruginosa bacteremia using ceftolozane-tazobactam-based or colistin-based antibiotic regimens: A multicenter retrospective study.

BACKGROUND: Ceftolozane-tazobactam is an emerging treatment for severe infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. However, limited data support its use in bacteremia treatment. This study aimed to assess the effectiveness of the treatment of MDR P. aeruginosa bacteremia using ceftolozane- tazobactam-based or colistin-based regimens. PATIENTS AND METHODS: This retrospective, cohort, multicentre study included adult patients with MDR P. aeruginosa bacteremia treated with either ceftolozane-tazobactam or colistin, between September 2018 and August 2021, at four hospitals in Saudi Arabia. The primary endpoint was the 30-day risk-adjusted mortality. Secondary endpoints included the 14-day risk of mortality, bacterial eradication, and clinical success. Cox proportional hazards regression and relative risk estimation were used for analysis, as appropriate. RESULTS: In total, 46 patients were included; 17 patients received ceftolozane- tazobactam-based regimen, and 29 received a colistin-based regimen. There was no association with the use of ceftolozane-tazobactam compared to colistin and the 30-day risk-adjusted mortality (hazard ratio [HR] 0.58, 95% confidence interval [CI] 0.16-2.13, P = 0.42). Also, the 14-day risk of mortality and bacterial eradication were not different between the ceftolozane-tazobactam and colistin regimens, HR 2.1, 95% CI 0.42-10.48; P = 0.36; and relative risk (RR) 0.65; 95% CI 0.28-1.52; P = 0.30; respectively. On the other hand, ceftolozane-tazobactam use was associated with higher clinical success than colistin (RR 1.84, 95% CI 1.11-3.06: P = 0.021). CONCLUSION: The risk of mortality of MDR P.aeruginosa bacteremia was similar when treated with ceftolozane-tazobactam-based or colistin-based antimicrobial regimens. A higher clinical success was observed with the ceftolozane- tazobactam-based regimen compared to the colistin-based regimen. .

Therapeutic Drug Monitoring and Prolonged Infusions of Ceftolozane/Tazobactam for MDR/XDR Pseudomonas aeruginosa Infections: An Observational Study.

BACKGROUND AND OBJECTIVE: Prolonged infusion of ceftolozane/tazobactam (C/T) is a strategy used to increase achievement of pharmacokinetic/pharmacodynamic targets for the treatment of multi- or extensively drug-resistant MDR/XDR Gram-negative microorganisms. The objective of this study was to describe our therapeutic drug monitoring (TDM) experience of C/T administered by prolonged infusion or intermittent infusion to patients with MDR/XDR Pseudomonas aeruginosa infections. Our outcomes of interest were pharmacokinetic/pharmacodynamic target achievement and clinical cure. METHODS: Patients with MDR/XDR P. aeruginosa infections treated with C/T were enrolled between February 2018 and February 2020. Blood samples were obtained as part of a TDM program. The pharmacokinetic/pharmacodynamic therapeutic target of C/T was defined as 100% of the duration of the dosing interval that free concentrations are above the minimum inhibitory concentration (MIC) (100 %ƒT ≥ MIC) of the causative pathogen. Dose changes were performed according to TDM results. RESULTS: Forty patients were included: 13 (32.5%) with a proven MDR and 27 (67.5%) with a XDR P. aeruginosa infection. C/T was administered by prolonged infusion in 32 (80%) patients and by intermittent infusion in 8 (20%) patients. Lower doses were administered in the prolonged infusion compared to the intermittent infusion group [3 (9.4%) vs. 5 (62.5%] patients received a dose of 9 g/day (ceftolozane 2 g + tazobactam 1 g, every 8 h; p = 0.004). All patients achieved the pharmacokinetic/pharmacodynamic target and C/T concentrations exceeded 10 × MIC in > 50% of patients in both groups. TDM-recommended dose reductions occurred in 19 (47.5%) patients, being 16 (84.2%) in the prolonged infusion group. A high proportion of patients achieved clinical cure (82.5%). CONCLUSIONS: The administration of C/T by prolonged infusion with TDM-guided dosing allowed the achievement of a pharmacokinetic/pharmacodynamic target even at lower doses. C/T showed a high efficacy for treating MDR/XDR P. aeruginosa infections.

Are there differences between ceftolozane/tazobactam and ceftazidime/avibactam in treating patients with complicated abdominal infections? Evidence from clinical trials.

Ceftolozane/tazobactam (C/T) and ceftazidime/avibactam (CZA) are new possibilities of antimicrobial treatment that combined a ß-lactam with a ß-lactamase inhibitor. The United States (US) and European regulatory agencies approved their clinical use in adults with complicated intra-abdominal infections. This study aims to know if one of the two antibiotics obtain better efficacy in adults with complicated intra-abdominal infections and by specific pathogens such as P. aeruginosa or E. coli. A search of all trials in MEDLINE, Scopus, and Web of Science comparing a C/T or CZA based antimicrobial regimen with other treatments in patients with intraabdominal infections until August 2021 was performed. To make indirect comparisons, we used a frequentist approach using the R package netmeta.The effects have been expressed through the relative risk (RR) with its confidence interval. Considering the clinical cure and failure rates between the different trial populations (mMITT, CE, ME) and the mortality at the end of the study, we have not found significant differences between CZA and C/T. In the case of Pseudomonas, the RR of treatment failure between these two antibiotics is 1 (95% CI 0.55-1.18). In the case of E. Coli, although it seems that CZA would have a worse result than C/T, differences did not reach statistical significance (RR1.06; 95% CI 0.9-1.14). In conclusion, we have not found statistically significant differences between ceftolozane-tazobactam and ceftazidime-avibactam in treating cIAI. In regards to E. Coli, our results do not reach significance, but it would be possible that C/T and meropenem had better results than CZA. Perhaps new trials would allow a better profile of the role in different types of patients or infections caused by specific microorganisms in the future.

Real-Life Use of Ceftolozane/Tazobactam for the Treatment of Bloodstream Infection Due to Pseudomonas aeruginosa in Neutropenic Hematologic Patients: a Matched Control Study (ZENITH Study).

We sought to assess the characteristics and outcomes of neutropenic hematologic patients with Pseudomonas aeruginosa (PA) bloodstream infection (BSI) treated with ceftolozane-tazobactam (C/T). We conducted a multicenter, international, matched-cohort study of PA BSI episodes in neutropenic hematologic patients who received C/T. Controls were patients with PA BSI treated with other antibiotics. Risk factors for overall 7-day and 30-day case fatality rates were analyzed. We compared 44 cases with 88 controls. Overall, 91% of episodes were caused by multidrug-resistant (MDR) strains. An endogenous source was the most frequent BSI origin (35.6%), followed by pneumonia (25.8%). There were no significant differences in patient characteristics between groups. C/T was given empirically in 11 patients and as definitive therapy in 41 patients. Treatment with C/T was associated with less need for mechanical ventilation (13.6% versus 33.3%; P = 0.021) and reduced 7-day (6.8% versus 34.1%; P = 0.001) and 30-day (22.7% versus 48.9%; P = 0.005) mortality. In the multivariate analysis, pneumonia, profound neutropenia, and persistent BSI were independent risk factors for 30-day mortality, whereas lower mortality was found among patients treated with C/T (adjusted OR [aOR] of 0.19; confidence interval [CI] 95% of 0.07 to 0.55; P = 0.002). Therapy with C/T was associated with less need for mechanical ventilation and reduced 7-day and 30-day case fatality rates compared to alternative agents in neutropenic hematologic patients with PA BSI. IMPORTANCE Ceftolozane-tazobactam (C/T) has been shown to be a safe and effective alternative for the treatment of difficult to treat infections due to Pseudomonas aeruginosa (PA) in the general nonimmunocompromised population. However, the experience of this agent in immunosuppressed neutropenic patients is very limited. Our study is unique because it is focused on extremely immunosuppressed hematological patients with neutropenia and bloodstream infection (BSI) due to PA (mainly multidrug resistant [MDR]), a scenario which is often associated with very high mortality rates. In our study, we found that the use of C/T for the treatment of MDR PA BSI in hematological neutropenic patients was significantly associated with improved outcomes, and, in addition, it was found to be an independent risk factor associated with increased survival. To date, this is the largest series involving neutropenic hematologic patients with PA BSI treated with C/T.

Ceftolozane/tazobactam versus colistin in the treatment of ventilator-associated pneumonia due to extensively drug-resistant Pseudomonas aeruginosa.

Resistant strains of Pseudomonas aeruginosa are common pathogens in the intensive care unit (ICU), limiting available therapeutic options. We aimed to compare ceftolozane/tazobactam (C/T) with colistimethate sodium (CMS) in the treatment of ventilator-associated pneumonia (VAP) due to extensively drug-resistant (XDR) Pseudomonas aeruginosa. A retrospective, observational study was performed at a tertiary care ICU. Clinical and microbiological success rate, 28-day all-cause mortality, and adverse events were compared in patients who received C/T with those treated with systemic CMS. A total of 51 patients were included (18 in the C/T and 33 in the CMS group). Clinical success rates in the C/T and CMS groups were 13 (72.2%) and 10 (30.3%), respectively. On multivariate regression analysis, treatment with C/T was independently associated with clinical success (odds ratio 4.47, 95% CI 1.17-17.08). There was no difference in 28-day all-cause mortality (27.8% and 33.3% in the C/T and CMS group, p = 0.76). Acute kidney injury was more common in patients who received CMS (48.5% vs 11.1%, p = 0.01). In our study, ceftolozane/tazobactam was more efficacious in the treatment of XDR Pseudomonas aeruginosa VAP and showed a better safety profile compared to CMS.

Ceftolozane-tazobactam vs. colistin for the treatment of infections due to multidrug-resistant Pseudomonas aeruginosa: a multicentre cohort study.

OBJECTIVES: The aim of this study was to compare the safety and effectiveness of ceftolozane-tazobactam (C-T) to colistin-based regimen for treating infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. METHODS: This was a retrospective, multicentre, observational cohort study of inpatients who received either C-T or intravenous colistin for treating infections caused by MDR P. aeruginosa. The study was conducted in five tertiary care hospitals in Saudi Arabia. The main study outcomes included clinical cure at end of treatment, in-hospital mortality, and acute kidney injury (AKI). Univariate analysis and multivariate logistic regression model were conducted to evaluate the independent effect of C-T on the clinical outcome. RESULTS: A total of 184 patients were included in the study: 82 patients received C-T, and 102 patients received colistin-based regimen. Clinical cure (77% vs. 57%; P = 0.005; OR, 2.52; 95% CI, 1.32-4.79) was significantly more common in patients who received C-T. After adjusting the difference between the two groups, treatment with C-T is independently associated with clinical cure (adjusted OR, 2.47; 95% CI, 1.16-5.27). In-hospital mortality (39% vs. 49%; P = 0.175; OR, 0.67; 95% CI, 0.37-1.20) was lower in patients who received C-T, but the difference was not significant. AKI (15% vs. 41%; P < 0.001; OR, 0.25; 95% CI, 0.12-0.51) was significantly less common in patients who received C-T. CONCLUSION: C-T is associated with a higher rate of clinical cure and lower rate of AKI compared to colistin. Our findings support the preferential use of C-T over colistin-based regimen for treating these infections.

Selection of AmpC ß-Lactamase Variants and Metallo-ß-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections.

Infections caused by ceftolozane-tazobactam and ceftazidime-avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane-tazobactam and ceftazidime-avibactam resistance mechanisms in all multidrug-resistant or extensively drug-resistant (MDR/XDR) P. aeruginosa isolates recovered during 1 year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane-tazobactam and ceftazidime-avibactam resistance were evaluated. Clinical conditions, previous positive cultures, and ß-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. Multilocus sequence types (MLSTs) and resistance mechanisms were determined using short- and long-read whole-genome sequencing (WGS). The impact of Pseudomonas-derived cephalosporinases (PDCs) on ß-lactam resistance was demonstrated by cloning into an ampC-deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired ß-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane-tazobactam or ceftazidime-avibactam. Seven isolates from different sequence types (STs) owed their ß-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219, and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. The 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13) and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlighted that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

[Utilization study in real clinical practice of ceftolozane/tazobactam vs aminoglycosides and/or colistin in the treatment of multirresistant or extremely resistant Pseudomonas aeruginosa]. / Estudio de utilización en práctica clínica real de ceftolozano/tazobactam frente a aminoglucósidos y/o colistina en el tratamiento de Pseudomonas aeruginosa.

OBJECTIVE: Comparative "real life" data on the effectiveness and safety of ceftolozane/tazobactam (C/T) versus other regimens (aminoglycosides/colistin/combination), in the treatment of multi-resistant (MDR) and extremely resistant (XDR) Pseudomonas aeruginosa (PA), are needed to establish positions. METHODS: Observational, retrospective study of patients with microbiological confirmation of MDR and XDR PA from July 2016 up to December 2018 in a tertiary hospital. Variables: age, sex, comorbidities, risk factors for multidrug resistance, variables related to infection, source of infection, microorganism and type of sample, antibiotic treatment, clinical cure, microbiological cure, recurrence, mortality on admission and 30 days post-discharge. Patients were classified according to received antibiotic treatment, C/T or aminoglycosides/colistin/combination. RESULTS: A total of 405 patients with PA MDR and XDR infection (73.1% men, mean age 63 ± 15 years) were studied. An 87.1% of PA XDR and a 12.9% MDR were observed. All patients received C/T as targeted therapy and in the aminoglycosides/colistin/combination group were 73.5%. Patients in the C/T group present worse prognostic factors: septic shock (30.0%) and catheterization (90.0%) (p<0.05). There were not statistically significant differences in microbiological cure (p=0.412), recurrence (p=0.880) and clinical cure (p=0.566). There were not statistically significant differences in mortality at admission (p=0.352) or at 30 days after discharge (p=0.231). A 17.2% of the patients with aminoglycosides/colistin/combination had acute kidney injury according to RIFLE criteria and 4.3% with C/T. CONCLUSIONS: The data obtained suggest that there have been no differences in effectiveness (clinical or microbiological cure) in favour of C/T, although, in the period studied, it was used in most cases in multitreated patients with a worse prognosis. Randomized and prospective studies would be needed to establish an adequate positioning.

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.

Systematic review and meta-analysis of in vitro efficacy of antibiotic combination therapy against carbapenem-resistant Gram-negative bacilli.

The superiority of combination therapy for carbapenem-resistant Gram-negative bacilli (CR-GNB) infections remains controversial. In vitro models may predict the efficacy of antibiotic regimens against CR-GNB. A systematic review and meta-analysis was performed including pharmacokinetic/pharmacodynamic (PK/PD) and time-kill (TK) studies examining the in vitro efficacy of antibiotic combinations against CR-GNB [PROSPERO registration no. CRD42019128104]. The primary outcome was in vitro synergy based on the effect size (ES): high, ES ≥ 0.75, moderate, 0.35 < ES < 0.75; low, ES ≤ 0.35; and absent, ES = 0). A network meta-analysis assessed the bactericidal effect and re-growth rate (secondary outcomes). An adapted version of the ToxRTool was used for risk-of-bias assessment. Over 180 combination regimens from 136 studies were included. The most frequently analysed classes were polymyxins and carbapenems. Limited data were available for ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. High or moderate synergism was shown for polymyxin/rifampicin against Acinetobacter baumannii [ES = 0.91, 95% confidence interval (CI) 0.44-1.00], polymyxin/fosfomycin against Klebsiella pneumoniae (ES = 1.00, 95% CI 0.66-1.00) and imipenem/amikacin against Pseudomonas aeruginosa (ES = 1.00, 95% CI 0.21-1.00). Compared with monotherapy, increased bactericidal activity and lower re-growth rates were reported for colistin/fosfomycin and polymyxin/rifampicin in K. pneumoniae and for imipenem/amikacin or imipenem/tobramycin against P. aeruginosa. High quality was documented for 65% and 53% of PK/PD and TK studies, respectively. Well-designed in vitro studies should be encouraged to guide the selection of combination therapies in clinical trials and to improve the armamentarium against carbapenem-resistant bacteria.

Molecular Characterization of ß-Lactam Resistance and Antimicrobial Susceptibility to Possible Therapeutic Options of AmpC-Producing Multidrug-Resistant Proteus mirabilis in a University Hospital of Split, Croatia.

This study was performed to elucidate genetic relatedness and molecular resistance mechanisms of AmpC-producing multidrug-resistant Proteus mirabilis isolates in University Hospital of Split (UHS), and define efficient antibiotics in vitro. A total of 100 nonrepeated, consecutive, amoxicillin/clavulanate- and cefoxitin-resistant P. mirabilis isolates were collected, mostly from urine (44%) and skin and soft-tissue samples (30%). They were all positive in cefoxitin Hodge test and negative for extended spectrum beta-lactamase production. Pulsed field gel electrophoresis identified four clusters and two singletons, with 79% of isolates in dominant cluster. Molecular characterization and I-CeuI analysis of representatives revealed blaCMY-16 gene located on chromosome, and insertion element ISEcp1 positioned 110 pb upstream of blaCMY-16 starting codon. They also harbored blaTEM-1, except one with blaTEM-2. They were all resistant to trimethoprim-sulfamethoxazole, all but one to quinolones, and 81% to all aminoglycosides, while 77% were susceptible (S) and 22% intermediate (I) to piperacillin/tazobactam, and 4% were S and 68% I to cefepime. Only 15% were S to ceftolozane/tazobactam. Meropenem, ertapenem, ceftazidime/avibactam, temocillin, and fosfomycin were 100% efficient in vitro. This is the first report of blaCMY-16 gene in P. mirabilis from hospital samples in Croatia. The findings are in accordance with Italian and Greek reports. The clonal nature of outbreak suggests the high potential of clonal spread. Alternative agents should be considered to spare carbapenem usage.

Short-Term Effects of Appropriate Empirical Antimicrobial Treatment with Ceftolozane/Tazobactam in a Swine Model of Nosocomial Pneumonia.

The rising frequency of multidrug-resistant and extensively drug-resistant (MDR/XDR) pathogens is making more frequent the inappropriate empirical antimicrobial therapy (IEAT) in nosocomial pneumonia, which is associated with increased mortality. We aim to determine the short-term benefits of appropriate empirical antimicrobial treatment (AEAT) with ceftolozane/tazobactam (C/T) compared with IEAT with piperacillin/tazobactam (TZP) in MDR Pseudomonas aeruginosa pneumonia. Twenty-one pigs with pneumonia caused by an XDR P. aeruginosa strain (susceptible to C/T but resistant to TZP) were ventilated for up to 72 h. Twenty-four hours after bacterial challenge, animals were randomized to receive 2-day treatment with either intravenous saline (untreated) or 25 to 50 mg of C/T per kg body weight (AEAT) or 200 to 225 mg of TZP per kg (IEAT) every 8 h. The primary outcome was the P. aeruginosa burden in lung tissue and the histopathology injury. P. aeruginosa burden in tracheal secretions and bronchoalveolar lavage (BAL) fluid, the development of antibiotic resistance, and inflammatory markers were secondary outcomes. Overall, P. aeruginosa lung burden was 5.30 (range, 4.00 to 6.30), 4.04 (3.64 to 4.51), and 4.04 (3.05 to 4.88) log10CFU/g in the untreated, AEAT, and IEAT groups, respectively (P = 0.299), without histopathological differences (P = 0.556). In contrast, in tracheal secretions (P < 0.001) and BAL fluid (P = 0.002), bactericidal efficacy was higher in the AEAT group. An increased MIC to TZP was found in 3 animals, while resistance to C/T did not develop. Interleukin-1ß (IL-1ß) was significantly downregulated by AEAT in comparison to other groups (P = 0.031). In a mechanically ventilated swine model of XDR P. aeruginosa pneumonia, appropriate initial treatment with C/T decreased respiratory secretions' bacterial burden, prevented development of resistance, achieved the pharmacodynamic target, and may have reduced systemic inflammation. However, after only 2 days of treatment, P. aeruginosa tissue concentrations were moderately affected.

Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections.

BACKGROUND: The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. OBJECTIVES: Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. METHODS: Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of ß-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. RESULTS: The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. CONCLUSIONS: Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/ß-lactamase inhibitor combinations in P. aeruginosa.

Approach to the Treatment of Patients with Serious Multidrug-Resistant Pseudomonas aeruginosa Infections.

Multidrug resistance(MDR) among Pseudomonas aeruginosa (PSA) isolates presents a significant clinical challenge and can substantially complicate the approach to selection of optimal antibiotic therapy. This review addresses major considerations in antibiotic selection for patients with suspected or documented serious MDR-PSA infections. Common mechanisms contributing to MDR among clinical PSA isolates are summarized. Empiric and definitive therapy considerations are addressed including the potential role of combination therapy. Newer agents with in vitro activity against MDR-PSA (e.g., ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, and cefiderocol) and their potential roles in clinical settings are discussed. Although these newer agents are promising options for the treatment of MDR-PSA, clinical data remain generally limited. Future studies are needed to determine optimal agents for the empiric and definitive treatment of MDR-PSA.

Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection.

BACKGROUND: Pseudomonas aeruginosa may develop resistance to novel cephalosporin/ß-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. OBJECTIVES: To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. METHODS: Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. RESULTS: WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. CONCLUSIONS: We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/ß-lactamase inhibitors in P. aeruginosa.

Characterization of AmpC ß-lactamase mutations of extensively drug-resistant Pseudomonas aeruginosa isolates that develop resistance to ceftolozane/tazobactam during therapy.

INTRODUCTION: We characterized AmpC ß-lactamase mutations that resulted in ceftolozane/tazobactam resistance in extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates recovered from patients treated with this agent from June 2016 to December 2018. METHODS: Five pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa XDR isolates were included among a total of 49 patients treated. Clonal relationship among isolates was first evaluated by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was further performed. AmpC mutations were investigated by PCR amplification of the blaPDC gene followed by sequencing. RESULTS: The ST175 high-risk clone was detected in four of the pairs of isolates and the ST1182 in the remaining one. All resistant isolates showed a mutation in AmpC: T96I in two of the isolates, and E247K, G183V, and a deletion of 19 amino acids (G229-E247) in the other three. The G183V mutation had not been described before. The five isolates resistant to ceftolozane/tazobactam showed cross-resistance to ceftazidime/avibactam and lower MICs of imipenem and piperacillin/tazobactam than the susceptible isolates. CONCLUSIONS: Ceftolozane/tazobactam resistance was associated in all of the cases with AmpC mutations, including a novel mutation (G183V) not previously described. There is a vital need for surveillance and characterization of emerging ceftolozane/tazobactam resistance, in order to preserve this valuable antipseudomonal agent.