Ceftazidime-avibactam Combination Research Articles

Practical Application of Aztreonam-Avibactam as a Treatment Strategy for Ambler Class B Metallo-β-Lactamase Producing Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae infections are a considerable challenge for clinicians. In recent years, novel antibiotic options have resulted in a tremendous advance in medical therapy; however, current treatment options are primarily effective for resistance derived from serine-based carbapenemases. The Ambler class B metallo-β-lactamases (MBLs) remain a critical challenge with decidedly fewer effective options. One intriguing option for these MBL pathogens is the combination of ceftazidime-avibactam with aztreonam. While clinical experience with this regimen is limited, in vitro studies are promising, and limited case reports describe success with this regimen; however, significant challenges preclude widespread adoption of this novel treatment regimen. A systemic literature review was performed to offer recommendations based on current evidence for a practical strategy on how to best integrate the use of aztreonam with avibactam combination therapy.

Drug utilization evaluation of ceftazidime-avibactam in a tertiary care hospital

Background: The World Health Organization considers antimicrobial resistance a major global threat, and ceftazidime-avibactam (CAZ-AVI) is a treatment for multidrug-resistant Gram-negative infections. The FDA approved its use in 2015 for infections with limited treatment options. Methods: A retrospective study was conducted in a hospital in India to analyse the use of CAZ-AVI formulation in patients. Data was collected from electronic records and included information on patient demographics, clinical and microbiological profiles, antibiotic treatment, and patient outcomes for those in the ICU. Results: A total of 110 patients were included in the study. On assessing the results of this study, it was found that, the median age of population who received CAZ-AVI was 63 years and hypertension, diabetes mellitus were the common comorbidities present in the patients. Out of 110 subjects, 66 (60%) patients were started with a high-end antibiotic as an empirical therapy. While analysing the resistance pattern, it was found that 22 (20%) were resistant and 84 (76%) of the study subjects were sensitive to ceftazidime-avibactam. There was higher clinical success for BSI, CUTI, VAP amongst the indications of CAZ-AVI and combination of CAZ-AVI with aztreonam has given the most successful treatment outcome. The combination of CAZ-AVI with one or two high end antibiotics was observed as a common clinical practice. Conclusions: The analysis of results showed encouraging clinical cure rates. Our study results suggest that ceftazidime-avibactam could be an effective standard therapy for managing MDR Gram -negative organisms.

Ceftazidime-avibactam combination therapy versus monotherapy for treating carbapenem-resistant gram-negative infection: a systemic review and meta-analysis.

This meta-analysis was conducted to compare the efficacy of ceftazidime-avibactam combination therapy with that of monotherapy in the treatment of carbapenem-resistant Gram-negative bacterial (CR-GNB). A literature search of PubMed, Embase, the Cochrane Library, and ClinicalTrials.gov was conducted until September 1, 2023. Only studies that compared CZA combination therapy with monotherapy for CR-GNB infections were included. A total of 25 studies (23 retrospective observational studies and 2 prospective studies) involving 2676 patients were included. There was no significant difference in 30-day mortality between the study group receiving combination therapy and the control group receiving monotherapy (risk ratio [RR] 0.91; 95% confidence interval [CI] 0.71-1.18). In addition, no significant differences were observed between the study and the control group in terms of in-hospital mortality (RR 1.00; 95% CI 0.79-1.27), 14-day mortality (RR 1.54; 95% CI 0.24-9.91), 90-day mortality (RR 1.18; 95% CI 0.62-2.22), and clinical cure rate (RR 0.95; 95% CI 0.84-1.08). However, the combination group had a borderline higher microbiological eradication rate than the control group (RR 1.15; 95% CI 1.00-1.32). Compared to monotherapy, CZA combination therapy did not yield additional clinical benefits. However, combination therapy may be associated with favorable microbiological outcomes.

PAM-1: an antimicrobial peptide with promise against ceftazidime-avibactam resistant Escherichia coli infection.

Antibiotic misuse and overuse have led to the emergence of carbapenem-resistant bacteria. The global spread of resistance to the novel antibiotic combination ceftazidime-avibactam (CZA) is becoming a severe problem. Antimicrobial peptide PAM-1 offers a novel approach for treating infections caused by antibiotic-resistant bacteria. This study explores its antibacterial and anti-biofilm activities and mechanisms against CZA-resistant Escherichia. Coli (E. coli), evaluating its stability and biosafety as well. The broth microdilution method, growth curve analysis, crystal violet staining, scanning electron microscopy, and propidium iodide staining/N-phenyl-1-naphthylamine uptake experiments were performed to explore the antibacterial action and potential mechanism of PAM-1 against CZA-resistant E. coli. The biosafety in diverse environments of PAM-1 was evaluated by red blood cell hemolysis, and cytotoxicity tests. Its stability was further assessed under different temperatures, serum concentrations, and ionic conditions using the broth microdilution method to determine its minimum inhibitory concentration (MIC). Galleria mellonella infection model and RT-qPCR were used to investigate the in vivo antibacterial and anti-inflammatory effects. In vitro antibacterial experiments demonstrated that the MICs of PAM-1 ranged from 2 to 8 μg/mL, with its effectiveness sustained for a duration of 24 h. PAM-1 exhibited significant antibiofilm activities against CZA-resistant E. coli (p < 0.05). Furthermore, Membrane permeability test revealed that PAM-1 may exert its antibacterial effect by disrupting membrane integrity by forming transmembrane pores (p < 0.05). Red blood cell hemolysis and cytotoxicity tests revealed that PAM-1 exerts no adverse effects at experimental concentrations (p < 0.05). Moreover, stability tests revealed its effectiveness in serum and at room temperature. The Galleria mellonella infection model revealed that PAM-1 can significantly improve the survival rate of Galleria mellonella (>50%)for in vivo treatment. Lastly, RT-qPCR revealed that PAM-1 downregulates the expression of inflammatory cytokines (p < 0.05). Overall, our study findings highlight the potential of PAM-1 as a therapeutic agent for CZA-resistant E. coli infections, offering new avenues for research and alternative antimicrobial therapy strategies.

Activities of aztreonam in combination with several novel β-lactam-β-lactamase inhibitor combinations against carbapenem-resistant Klebsiella pneumoniae strains coproducing KPC and NDM.

Isolates coproducing serine/metallo-carbapenems are a serious emerging public health threat, given their rapid dissemination and the limited number of treatment options. The purposes of this study were to evaluate the in vitro antibacterial activity of novel β-lactam-β-lactamase inhibitor combinations (BLBLIs) against carbapenem-resistant Klebsiella pneumoniae (CRKP) coproducing metallo-β-lactamase and serine-β-lactamase, and to explore their effects in combination with aztreonam, meropenem, or polymyxin in order to identify the best therapeutic options. Four CRKP isolates coproducing K. pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) were selected, and a microdilution broth method was used to determine their susceptibility to antibiotics. Time-kill assay was used to detect the bactericidal effects of the combinations of antibiotics. The minimum inhibitory concentration (MIC) values for imipenem and meropenem in three isolates did not decrease after the addition of relebactam or varbobactam, but the addition of avibactam to aztreonam reduced the MIC by more than 64-fold. Time-kill assay demonstrated that imipenem-cilastatin/relebactam (ICR) alone exerted a bacteriostatic effect against three isolates (average reduction: 1.88 log10 CFU/mL) and ICR combined with aztreonam exerted an additive effect. Aztreonam combined with meropenem/varbobactam (MEV) or ceftazidime/avibactam (CZA) showed synergistic effects, while the effect of aztreonam combined with CZA was inferior to that of MEV. Compared with the same concentration of aztreonam plus CZA combination, aztreonam/avibactam had a better bactericidal effect (24 h bacterial count reduction >3 log10CFU/mL). These data indicate that the combination of ATM with several new BLBLIs exerts powerful bactericidal activity, which suggests that these double β-lactam combinations might provide potential alternative treatments for infections caused by pathogens coproducing-serine/metallo-carbapenems.

A multi-species outbreak of VIM-producing carbapenem-resistant bacteria in a burn unit and subsequent investigation of rapid development of cefiderocol resistance.

Carbapenem resistance due to metallo-β-lactamases (MBLs) such as the Verona integron-encoded metallo-β-lactamase (VIM) is particularly problematic due to the limited treatment options. We describe a case series of bacterial infections in a tertiary care hospital due to multi-species acquisition of a VIM gene along with our experience using novel β-lactam antibiotics and antibiotic combinations to treat these infections. Four patients were treated with the combination of ceftazidime-avibactam and aztreonam, with no resistance to the combination detected. However, cefiderocol-resistant Klebsiella pneumoniae isolates were detected in two out of the five patients who received cefiderocol within 3 weeks of having started the antibiotic. Strain pairs of sequential susceptible and resistant isolates from both patients were analyzed using whole-genome sequencing. This analysis revealed that the pairs of isolates independently acquired point mutations in both the cirA and fiu genes, which encode siderophore receptors. These point mutations were remade in a laboratory strain of K. pneumoniae and resulted in a significant increase in the MIC of cefiderocol, even in the absence of a beta-lactamase enzyme or a penicillin-binding protein 3 (PBP3) mutation. While newer β-lactam antibiotics remain an exciting addition to the antibiotic armamentarium, their use must be accompanied by diligent monitoring for the rapid development of resistance.

Evaluation of the MTS™ aztreonam-avibactam strip (Liofilchem) on New Delhi metallo-β-lactamase-producing Enterobacterales.

The combination of ceftazidime-avibactam (CAZ-AVI) and aztreonam (ATM) is used to treat MBL-producing Enterobacterales-related infections. The new combination aztreonam-avibactam (AZA) is currently in development. We compared results obtained with the new MIC test strip (MTS) AZA (Liofilchem) with broth microdilution method (BMD) on 41 MBL-producing Enterobacterales from 41 clinical samples. The MTS AZA was also compared to combination testing method using CAZ-AVI and ATM strips. Compared to BMD, categorical agreement (CA) was 100%. Compared with combination testing method, CA was 97.6%. The MTS AZA can be used to determine MICs levels of AZA or CAZ-AVI/ATM combinations.

Ceftazidime-avibactam/aztreonam combination synergy against carbapenem-resistant Gram-negative isolates: In vitro study

ABSTRACT Background: The ceftazidime-avibactam combination is able to inhibit ESBLs, AmpCs and Class A carbapenemases and has been recommended for the treatment of complicated UTIs and ventilator-associated bacterial pneumonia. Aims: The present study was undertaken to evaluate the in vitro activity of ceftazidime-avibactam and to determine the synergistic activity of aztreonam/ceftazidime-avibactam combination against carbapenem-resistant Gram-negative bacilli. Materials and Methods: Gram-negative isolates that exhibited resistance to at least one of the carbapenems (imipenem or meropenem) by the Kirby–Bauer disc diffusion method were subjected to phenotypic characterisation by the Vitek-2 automated method. Phenotypically confirmed isolates were subjected to ceftazidime/avibactam-aztreonam synergy testing by disc diffusion method. Results: Twenty-two carbapenem-resistant isolates showed a minimum inhibitory concentration of 4–64 μg/mL for imipenem and 8–64 μg/mL for meropenem. Out of 22 carbapenem-resistant isolates, 18 (81.82%) isolates showed resistance to ceftazidime-avibactam and aztreonam, and 2 (9.09%) isolates showed intermediate resistance to aztreonam. Nine (40.91%) isolates showed synergy to ceftazidime-avibactam/aztreonam combination by disc diffusion method. An increase in zone diameter of 5–23 mm and 5–16 mm was observed with the ceftazidime-avibactam/aztreonam combination for Klebsiella pneumoniae and Escherichia coli, respectively, when compared to ceftazidime-avibactam and aztreonam disc tested alone. Out of 21 Enterobacterales studied, 21 (100%) isolates showed resistance to amoxicillin-clavulanate (≥32 μg/mL) and piperacillin-tazobactam (128 μg/mL), 12 (57.14%) isolates showed resistance to gentamicin (≥16 μg/mL), 5 (23.81%) isolates were resistant to amikacin (≥64 μg/mL), 21 (100%) isolates were resistant to ciprofloxacin (≥4 μg/mL), 19 (90.48%) isolates were resistant to cotrimoxazole (≥320 μg/mL), 21 (100%) isolates were resistant to cefepime (≥16 μg/mL) and 6 (28.57%) isolates were resistant to tigecycline (2 μg/mL). Conclusion: In the present study, 42.86% of Enterobacterales isolates showed synergism to the ceftazidime-avibactam/aztreonam combination. The optimal dosing strategy and in vivo efficacy of this combination need to be evaluated.

In vitro effectiveness of ceftazidime-avibactam in combination with aztreonam on carbapenemase-producing Enterobacterales

ObjectiveThis work aimed to describe the in vitro performance of the combined activity of ceftazidime-avibactam (CZA) plus aztreonam (ATM) against carbapenemase-producing Enterobacterales (CPE). MethodsWe studied 44 CPE clinical isolates: NDM-1 (31), KPC-2 (5), KPC-3 (3), VIM-2 (2), NDM-1+KPC-2 (2), and OXA-48 (1). The efficacy of CZA in combination with were determined by two methods: (i) Kirby-Bauer's double disk synergy test and; (ii) Determination of the minimum inhibitory concentration to CZA by E-test, in either Mueller-Hinton agar alone or, supplemented with ATM 4 mg/L. Additionally, the Fractional inhibitory concentration index (FICI) was determined; values of ≤ 0.5 were interpreted as synergistic, while FICI > 0.5 were considered indifferent. ResultsAll isolates were carbapenem-resistant, 14 were resistant to CZA and ATM, 15 were only CZA resistant, 12 were only ATM resistant, and three were susceptible to both. 34/44 isolates presented positive double disk synergy tests between CZA and ATM regardless of their susceptibility profile, the isolates with negative synergy tests were susceptible to at least one of the agents. On the other hand, the 21 isolates selected to compare the MIC to CZA alone and CZA plus 4 mg/L ATM of exhibited FICI values between 0.016 and 0.125, indicating a synergistic effect. ConclusionsThis method is available to clinical laboratories and would provide valuable information to guide the treatment of infections with CZA and ATM. In this sense, the use of CZA together with ATM is a potentially suitable combination for the treatment of carbapenemase-producing microorganisms.

Looking for Stenotrophomonas maltophilia treatment: in vitro activity of ceftazidime/avibactam alone and in combination with aztreonam

During the Sars-Cov-2 pandemic, Stenotrophomonas maltophilia (S.maltophilia) secondary pulmonary infections have increased, especially in critically ill patients, highlighting the need for new therapeutic options. Trimethoprim-sulfamethoxazole (SXT) is the treatment of choice but the increase of resistant strains or adverse drug reactions limited its clinical use. Recently ceftazidime/avibactam (CZA) has been approved for the treatment of multi drug resistant (MDR) Gram-negative bacteria infections, including hospital acquired pneumonia. The aim of this study was to evaluate the in vitro activity of ceftazidime/avibactam (CZA) alone and in combination with aztreonam (ATM) against S. maltophilia clinical isolates by E-test method. Susceptibility of SXT and levofloxacin (LEV) was also investigated. Our results showed 22% of resistance to CZA, 2% to SXT and 26% to LEV. CZA in combination with ATM demonstrated synergistic activity against 86% of the strains, including all those resistant to CZA. The combination of CZA with ATM provides a new therapeutic option for the treatment of severe respiratory infections in critically ill patients.

Evaluation of Aztreonam and Ceftazidime/Avibactam Synergism against Klebsiella pneumoniae by MALDI-TOF MS.

Resistance to carbapenems due to the co-production of NDM and ESBL or NDM and KPC is increasing. Therefore, combined therapy with aztreonam (ATM) plus ceftazidime/avibactam (CZA) has been recommended. Then, it is necessary to develop and evaluate fast and simple methods to determine synergism in vitro in microbiology laboratories. To develop a method to determine the synergism of ATM and CZA by MALDI-TOF MS (SynMALDI). Klebsiella pneumoniae (n = 22) isolates with blaNDM and/or blaKPC genes were tested. The time-kill curve assay was performed for four isolates (three positives for blaNDM and blaKPC and one positive for blaNDM only). For SynMALDI, each isolate was incubated for 3 h in 4 tubes containing brain-heart infusion broth with the following: (1) no antibiotic; (2) ATM at 64 mg/L; (3) CZA at 10/4 mg/L; and (4) ATM at 64 mg/L plus CZA at 10/4 mg/L. After incubation, the bacterial protein extract was analyzed by MALDI-TOF MS, and the relative growth (RG) was determined for each isolate, considering intensities of the peaks of the bacterium incubated with antibiotic (tubes 2, 3, and 4) to the same bacterium incubated without antibiotic (tube 1), as follows: RG = IntensityWith antibiotic/IntensityWithout antibiotic. The combination was determined as synergistic when there was an RG decrease of 0.3 in the antibiotic combination in relation to the RG of the most active antibiotic alone. The combination of ATM plus CZA proved to be synergic by time-kill curve assay. All isolates tested with the SynMALDI method also presented synergism. Detection of synergism for ATM plus CZA combination can be determined by MALDI-TOF MS, providing fast results in order to improve patient treatment.

Aktifitas Antibiotik Ceftazidime-Avibactam Dan Ceftaroline Secara In Vitro Terhadap Isolat Klinis Di Pekanbaru

Ceftaroline and Ceftazidime–avibactam are relatively new antibiotics that show good effectiveness against antibiotic-resistant pathogens. Both of these antibiotics are now available worldwide, but reports of resistance to them are also starting to emerge. This study aimed to evaluate the in vitro activity of the antibiotic Ceftazidime-avibactam combination of 100 clinical isolates consisting of Enterobacteriaceae and Pseudomonas aeruginosa bacteria and to evaluate the antibiotic effect of Ceftaroline against 100 clinical isolates of Enterobacteriaceae and Staphylococcus sp. This study is observational with cross-sectional study design. The study was conducted at the microbiology laboratory of a private hospital at Pekanbaru Riau in Januari-June 2021. There were 100 clinical isolates tested with Ceftaroline and Ceftazidime-avibactam. The sensitivity to Ceftaroline and Ceftazidime-avibactam were reported as a number isolate and percentage. The results showed the sensitivity of bacteria K. pneumoniae, E. coli, Enterobacter sp, and P. aeruginosa, to Ceftazidime-Avibactam were 87%, 90%, 66%, and 82%, respectively. The sensitivity of the antibiotic Ceftaroline to the bacteria K. pneumoniae, E. coli, Enterobacter sp and Staphylococcus sp. were 39%, 54%, 36.4%, 67% and 75%, respectively. Based on these results, the antibiotic ceftazidime avibactam can be used for the treatment of suitable pathogenic infections because of its fairly good sensitivity (&gt;80%) to Enterobacteriaceae and P. aeruginosa bacteria. Ceftaroline antibiotics are not recommended to be used as a therapeutic option, because of the high resistance to Enterobacteriaceae and Staphylococcus sp. Further research is needed to determine the molecular mechanism of resistance to Ceftaroline antibiotics

Enhanced bacterial killing with a combination of sulbactam/minocycline against dual carbapenemase-producing Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAB) is often difficult to treat. Considering the current circumstances, there is an unquestionable need for new therapeutic options to treat CRAB infections. In the present study, the synergistic activity of sulbactam-based combination was determined against genetically characterized CRAB isolates. Non-duplicate CRAB isolates (n = 150) recovered from blood culture and endotracheal aspirates were included in this study. The minimum inhibitory concentrations (MICs) of tetracyclines (minocycline, tigecycline, eravacycline) and their comparators (meropenem, sulbactam, cefoperazone/sulbactam, ceftazidime/avibactam, and colistin) were determined using the microbroth dilution method. Six isolates were tested for the synergistic activity of various sulbactam-based combinations using time-kill experiments. Tigecycline and minocycline showed a wide spread of MICs with most isolates in the range of1 to 16mg/L. The MIC90 of eravacycline (0.5mg/L) was four dilutions lower than that of tigecycline (8mg/L). Minocycline with sulbactam was the most active dual combination against OXA-23 like (n = 2) and NDM with OXA-23 like producers (n = 1), which resulted in ≥ 2 log10 kill. The combination of ceftazidime-avibactam with sulbactam showed ≥ 3 log10 kill against all the three tested OXA-23 like producing CRAB isolates, but showed no activity against dual carbapenemase producers. Sulbactam with meropenem showed ≥ 2 log10 kill against one OXA-23 like producing CRAB isolate. The findings suggest that sulbactam-based combination may confer therapeutic benefits against CRAB infections.

In vitro and in vivo Antimicrobial Activities of Ceftazidime/Avibactam Alone or in Combination with Aztreonam Against Carbapenem-Resistant Enterobacterales.

To examine the in vitro and in vivo antimicrobial activities of ceftazidime/avibactam (CZA) alone or in combination with aztreonam (ATM) against KPC-, NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains. A total of 67 clinical non-repetitive carbapenem-resistant Enterobacterales (CRE) strains were selected for the microdilution broth method that was performed to analyze the minimal inhibitory concentration (MIC) and the combination antimicrobial susceptibility test using checkerboard titration method. The fractional inhibitory concentration (FIC) was calculated to determine the antimicrobial effect. The time-kill assays and the mouse infection model were used to study the bactericidal effect and therapeutic effect of CZA alone or in combination with ATM. The CZA minimal inhibitory concentration (MIC) values of CZA revealed that 29 KPC-producing strains and 1 OXA-producing strain were ≤4µg/mL. The CZA MIC values of 37 metal-β-lactamase (MBLs)-producing strains such as NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains were ≥128µg/mL, after combining with ATM, the FIC values were all below 0.51. The time-kill assays revealed that CZA at various concentrations of 2, 4 and 8 MIC showed significant bactericidal efficiency to the KPC-producing strains. For NDM-, IMP-producing strains, no colony growth was detected after 8 hours of incubation with CZA in combination with ATM. Six percent of the mice in the treatment group and 58% of the mice in the infection group died within 3 days. Our in vitro results showed that CZA had a good antimicrobial effect on the KPC-producing and OXA-producing strains. CZA combined with ATM showed synergistic bacteriostatic or bactericidal activity against NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains. The combination of CZA and ATM reduced mortality and prolonged lifespan of mice infected with NDM-, IMP-, KPC+IMP-, and KPC+NDM-producing strains, which provides fundamental knowledge for improving treatment strategies and initializing clinical trials.

Outbreak of Klebsiella pneumoniae ST11 Resistant To Ceftazidime-Avibactam Producing KPC-31 and the Novel Variant KPC-115 during COVID-19 Pandemic in Argentina.

We describe an outbreak of Klebsiella pneumoniae sequence type 11 (ST11) producing KPC variants resistant to ceftazidime-avibactam. Six patients hospitalized in the intensive care unit (mostly due to critical COVID pneumonia) presented infection or colonization by this bacterium. They had several comorbidities and required mechanical ventilation, central venous catheters, and urinary catheters. All 6 patients had a history of fecal colonization with KPC-producing Enterobacterales (KPC-E). Three of them had previous episodes of infection with ceftazidime-avibactam-susceptible KPC-producing K. pneumoniae, which were treated with ceftazidime-avibactam. Several phenotypic methods failed to detect carbapenemase production in these 6 ceftazidime-avibactam-resistant isolates, and they showed in vitro susceptibility to imipenem and meropenem. All of them rendered positive results for blaKPC by PCR, and amplicon sequencing identified blaKPC-31 variant in 5 isolates and a novel variant, named blaKPC-115, in the other. Moreover, matrix-assisted laser desorption ionization-time of flight mass spectrometry was able to detect KPC in all isolates. Ceftazidime-avibactam-resistant isolates, as well as those recovered from previous infection episodes (KPC-3-producing K. pneumoniae, ceftazidime-avibactam susceptible), displayed a unique pulse type and belonged to ST11. Based on whole-genome sequencing results of selected isolates, less than 7 single-nucleotide polymorphisms were identified among them, which was indicative of the presence of a unique clone. Both in vivo selection and horizontal transmission seemed to have occurred in our hospital. Detection of these strains is challenging for the laboratory. History of previous KPC-E infections or colonization and systematic testing for resistance to ceftazidime-avibactam might help raise awareness of this possibility. IMPORTANCE Klebsiella pneumoniae is one of the main bacteria that cause infections in health care settings. This pathogen has developed a high level of resistance to many antibiotics. Some K. pneumoniae isolates can produce an enzyme known as carbapenemase KPC, making carbapenems (considered the last line for therapy) not effective to treat their infections. The combination ceftazidime-avibactam, approved by FDA in 2015, is useful to treat infections caused by KPC-producing K. pneumoniae. This study describes the emergence, in one hospital in Argentina, of K. pneumoniae isolates that produce KPC variants (KPC-31 and KPC-115) resistant to ceftazidime-avibactam. The ceftazidime-avibactam-resistant bacteria were isolated in inpatients, including some that previously received this combination as treatment. Transmission of this strain to other patients also occurred in the studied period. Detection of these bacteria is challenging for the laboratory. The knowledge and awareness of the emergence of this pathogen in our region are highly valuable.

Emergence of KPC-31, a KPC-3 Variant Associated with Ceftazidime-Avibactam Resistance, in an Extensively Drug-Resistant ST235 Pseudomonas aeruginosa Clinical Isolate.

A ceftazidime-avibactam-resistant KPC-producing Pseudomonas aeruginosa strain was isolated in Argentina from a tracheal aspirate. The patient was treated with ceftazidime-avibactam in combination with other agents for 130 days. Whole-genome sequencing of P. aeruginosa identified a D179Y substitution in the Ω loop of KPC-3, corresponding to KPC-31, integrated at the chromosome. The strain belonged to the sequence type 235/O11 (ST235/O11) high-risk clone. Evaluation of carbapenemase detection assays most used by clinical laboratories failed to identify the isolate as a KPC producer.

In-vitro susceptibility testing methods for the combination of ceftazidime-avibactam with aztreonam in metallobeta-lactamase producing organisms: Role of combination drugs in antibiotic resistance era.

Resistance in Gram-negative organisms has become one of the leading threats in recent years. Of the different mechanisms described in the literature, resistance due to beta-lactamases genes have been overcomed by the use of a beta-lactamase inhibitor in combination with a beta-lactam antibiotic. When this combination is insufficient to counter metallo-beta-lactamases, a third antibiotic, has been added to restore susceptibility. One such recent combination is ceftazidime-avibactam with aztreonam. In this study, 60 isolates of multidrug-resistant organisms producing metallo-beta-lactamases were included to perform in-vitro antibiotic susceptibility testing against ceftazidime-avibactam and aztreonam alone and in combination using three different methods. Individual testing revealed 100% (60/60) resistance to both ceftazidime-avibactam and aztreonam in all the isolates. The disk diffusion method showed an inhibition zone size of 21 mm in all the isolates, with 16 isolates showing an increase in inhibition zone size of >16 mm. In the E-test fixed ratio method, MICs of ceftazidime-avibactam and aztreonam when used alone ranged from 8/4 µg l−1 to ≥256/4 µg l−1 and 16 µg l−1 to 256 µg l−1, respectively, but in combination, these MICs were reduced to 0.016/4 µg l−1 to 2/4 µg l−1 with FIC < 0.5 in all the isolates. Similar results were obtained with the E-test agar dilution method with more than a 16-fold reduction in MIC in all the isolates when avibactam concentration was fixed at 4 µg l−1. All three methods showed a 100% correlation with each other. The current study depicted the usefulness of combining ceftazidime-avibactam with aztreonam against organisms producing metallo-beta-lactamases and that disk diffusion methods can be used as a method for performing in-vitro antibiotic susceptibility testing of this combination.

Extensively Drug-Resistant Klebsiellapneumoniae Counteracts Fitness and Virulence Costs That Accompanied Ceftazidime-Avibactam Resistance Acquisition.

ABSTRACTThe ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages.IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

Structural Characterization of the D179N and D179Y Variants of KPC-2 β-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) present a global clinical threat, as these β-lactamases confer resistance to carbapenems and oxyimino-cephalosporins. Recent clinically identified KPC variants with substitutions at Ambler position D179, located in the Ω loop, are resistant to the β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam, but susceptible to meropenem-vaborbactam. To gain insights into ceftazidime-avibactam resistance conferred by D179N/Y variants of KPC-2, crystal structures of these variants were determined. The D179N KPC-2 structure revealed that the change of the carboxyl to an amide moiety at position 179 disrupted the salt bridge with R164 present in wild-type KPC-2. Additional interactions were disrupted in the Ω loop, causing a decrease in the melting temperature. Shifts originating from N179 were also transmitted toward the active site, including ∼1-Å shifts of the deacylation water and interacting residue N170. The structure of the D179Y KPC-2 β-lactamase revealed more drastic changes, as this variant exhibited disorder of the Ω loop, with other flanking regions also being disordered. We postulate that the KPC-2 variants can accommodate ceftazidime because the Ω loop is displaced in D179Y or can be more readily displaced in D179N KPC-2. To understand why the β-lactamase inhibitor vaborbactam is less affected by the D179 variants than avibactam, we determined the crystal structure of D179N KPC-2 in complex with vaborbactam, which revealed wild-type KPC-2-like vaborbactam-active site interactions. Overall, the structural results regarding KPC-2 D179 variants revealed various degrees of destabilization of the Ω loop that contribute to ceftazidime-avibactam resistance, possible substrate-assisted catalysis of ceftazidime, and meropenem and meropenem-vaborbactam susceptibility.

Molecular Characterization of WCK 5222 (Cefepime/Zidebactam)-Resistant Mutants Developed from a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate.

ABSTRACTWCK 5222 (cefepime/zidebactam) is a β-lactam/β-lactamase inhibitor combination that is effective against a broad range of highly drug-resistant bacterial pathogens, including those producing metallo-β-lactamase. In this study, we isolated a multidrug-resistant Pseudomonas aeruginosa clinical strain that is resistant to a variety of β-lactam antibiotics and the ceftazidime-avibactam combination. A metallo-β-lactamase gene blaDIM-2 was identified on a self-transmissible megaplasmid in the strain, which confers the resistance to β-lactam antibiotics, leaving WCK 5222 potentially one of the last treatment resorts. In vitro passaging assay combined with whole-genome sequencing revealed mutations in the pbpA gene (encoding the zidebactam target protein PBP2) in the evolved resistant mutants. Among the mutations, a V516M mutation increased the bacterial virulence in a murine acute pneumonia model. Reconstitution of the mutations in the reference strain PAO1 verified their roles in the resistance to zidebactam and revealed their influences on cell morphology in the absence and presence of zidebactam. Microscale thermophoresis (MST) assays demonstrated that the mutations reduced the affinity between PBP2 and zidebactam to various extents. Overall, our results revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings.IMPORTANCE Antibiotic resistance imposes a severe threat on human health. WCK 5222 is a β-lactam/β-lactamase inhibitor combination that is composed of cefepime and zidebactam. It is one of the few antibiotics in clinical trials that are effective against multidrug-resistant Pseudomonas aeruginosa, including those producing metallo-β-lactamases. Understanding the mechanisms and development of bacterial resistance to WCK 5222 may provide clues for the development of strategies to suppress resistant evolvement. In this study, we performed an in vitro passaging assay by using a multidrug-resistant P. aeruginosa clinical isolate. Our results revealed that mutations in the zidebactam target protein PBP2 play a major role in the bacterial resistance to WCK 5222. We further demonstrated that the mutations reduced the affinities between PBP2 and zidebactam and resulted in functional resistance of PBP2 to zidebactam.