Successful phage-antibiotic therapy of P. aeruginosa implant-associated infection in a Siamese cat.

Antibiotic-resistant pathogens are a growing global issue, leading to untreatable infectious diseases in both humans and animals. Personalized bacteriophage (phage) therapy, the use of specific anti-bacterial viruses, is currently a leading approach to combat antibiotic-resistant infections. The implementation of phage therapy has primarily been focused on humans, almost neglecting the impact of such infections on the health and welfare of companion animals. Pets also have the potential to spread resistant infections to their owners or the veterinary staff through zoonotic transmission. Here, we showcase personalized phage-antibiotic treatment of a cat with a multidrug-resistant Pseudomonas aeruginosa implant-associated infection post-arthrodesis surgery. The treatment encompassed a tailored combination of an anti-P. aeruginosa phage and ceftazidime, precisely matched to the pathogen. The phage was topically applied to the surgical wound while the antibiotic was administered intramuscularly. After two treatment courses spanning 7 and 3 weeks, the surgical wound, which had previously remained open for five months, fully closed. To the best of our knowledge, this is the first case of personalized phage therapy application in felines, which provides further evidence of the effectiveness of this approach. The successful outcome paves the way for personalized phage-antibiotic treatments against persistent infections therapy in veterinary practice.

Evaluation of a simple method for testing aztreonam and ceftazidime-avibactam synergy in New Delhi metallo-beta-lactamase producing Enterobacterales.

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4µg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 µg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae.

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

Post-Transplant Fecal Carriage of Antibiotic Resistant and B-Lactamases-Producing Enterobacteriales among Renal Transplant Recipients.

BACKGROUND: The intestinal colonization and transmission of antibiotic-resistant Enterobacteriales to renal transplant recipients may pose a threat to them because they are profoundly immunocompromised and vulnerable to infection. Hence, it is crucial to identify these antibiotic-resistant fecal Enterobacteriales harboring high-risk populations. The objective of this study was to determine antibiotic resistance as well as ß-lactamases production in fecal Enterobacteriales among renal transplant recipients. METHODS: The stool samples, one collected from each transplant recipient, were processed for isolation and identification of Enterobacteriales and were tested for their antibiotic susceptibility, extended-spectrum ß-lactamase, and metallo-ß-lactamase production by standard methods. RESULTS: A total of 103 Enterobacteriales comprising of Escherichia coli (86.4%), Klebsiella species (11.7%), and Citrobacter species (1.9%) were isolated and more than 60% of the E. coli were found resistant to ceftazidime and ciprofloxacin and around half of the Klebsiella species were resistant to ceftazidime and fluroquinolones. The extended-spectrum ß-lactamase production was seen in 3.4% and 8.3% and metallo-ß-lactamase production in 24.7% and 33.3% of E. coli and Klebsiella species, respectively. The high proportion of ß-lactamase-producers were resistant to piperacillin-tazobactam, meropenem, gentamicin, and amikacin than ß-lactamases non-producers. CONCLUSION: Since the antibiotic resistance is higher in fecal Enterobacteriales, each renal transplant recipient should be screened for these highly resistant intestinal colonizers after transplantation in order to prevent infections and to reduce the rate of transplant failure due to infections.

Evaluation of the synergistic effect of eravacycline and tigecycline against carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a substantial healthcare challenge. This study assessed the in vitro efficacy of selected antibiotic combinations against CRKP infections. METHODS: Our research involved the evaluation of 40 clinical isolates of CRKP, with half expressing Klebsiella pneumoniae carbapenemase (KPC) and half producing Metallo-ß-lactamase (MBL), two key enzymes contributing to carbapenem resistance. We determined the minimum inhibitory concentrations (MICs) of four antibiotics: eravacycline, tigecycline, polymyxin-B, and ceftazidime/avibactam. Synergistic interactions between these antibiotic combinations were examined using checkerboard and time-kill analyses. RESULTS: We noted significant differences in the MICs of ceftazidime/avibactam between KPC and MBL isolates. Checkerboard analysis revealed appreciable synergy between combinations of tigecycline (35%) or eravacycline (40%) with polymyxin-B. The synergy rates for the combination of tigecycline or eravacycline with polymyxin-B were similar among the KPC and MBL isolates. These combinations maintained a synergy rate of 70.6% even against polymyxin-B resistant isolates. In contrast, combinations of tigecycline (5%) or eravacycline (10%) with ceftazidime/avibactam showed significantly lower synergy than combinations with polymyxin-B (P < 0.001 and P = 0.002, respectively). Among the MBL CRKP isolates, only one exhibited synergy with eravacycline or tigecycline and ceftazidime/avibactam combinations, and no synergistic activity was identified in the time-kill analysis for these combinations. The combination of eravacycline and polymyxin-B demonstrated the most promising synergy in the time-kill analysis. CONCLUSION: This study provides substantial evidence of a significant synergy when combining tigecycline or eravacycline with polymyxin-B against CRKP strains, including those producing MBL. These results highlight potential therapeutic strategies against CRKP infections.

A Multidrug-Resistant Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacter hormaechei Strain from Mixed Sprouts.

Fresh vegetables can harbor antibiotic-resistant bacteria, including extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales. Enterobacter hormaechei is a bacterium belonging to the Enterobacterales order and the most commonly identified nosocomial pathogen of Enterobacter cloacae complex. The purpose of this study was to characterize a multi-drug resistant ESBL-producing E. hormaechei strain isolated from a sample of mixed sprouts. Vegetable samples were pre-enriched in buffered peptone water, followed by enrichment in Enterobacteria Enrichment Broth, and isolation on Chromagar™ ESBL plates. One isolate from a sprout sample was confirmed to produce both ESBL and AmpC ß-lactamases through the combination disk diffusion assay using antibiotic disks containing cefotaxime and ceftazidime with or without clavulanate, and with or without cloxacillin, respectively. The isolate was also resistant to multiple antibiotics, including cefotaxime, ceftazidime, chloramphenicol, trimethoprim-sulfamethoxazole, tetracycline, gentamicin, ampicillin, and amoxicillin-clavulanate, as determined by antimicrobial susceptibility testing. Through whole genome sequencing, the isolate was identified as E. hormaechei 057-E1, which carried multiple antibiotic resistance (AR) genes and a sul2-aph(3″)-Ib-aph(6)-Id-blaTEM-1-ISEcp1 -blaCTX-M-15 gene cluster. Our results further demonstrate the important role of fresh vegetables in AR and highlight the need to develop strategies for AR mitigation in fresh vegetables.

Assessing Clinician Utilization of Next-Generation Antibiotics Against Resistant Gram-Negative Infections in U.S. Hospitals : A Retrospective Cohort Study.

BACKGROUND: The U.S. antibiotic market failure has threatened future innovation and supply. Understanding when and why clinicians underutilize recently approved gram-negative antibiotics might help prioritize the patient in future antibiotic development and potential market entry rewards. OBJECTIVE: To determine use patterns of recently U.S. Food and Drug Administration (FDA)-approved gram-negative antibiotics (ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, plazomicin, eravacycline, imipenem-relebactam-cilastatin, and cefiderocol) and identify factors associated with their preferential use (over traditional generic agents) in patients with gram-negative infections due to pathogens displaying difficult-to-treat resistance (DTR; that is, resistance to all first-line antibiotics). DESIGN: Retrospective cohort. SETTING: 619 U.S. hospitals. PARTICIPANTS: Adult inpatients. MEASUREMENTS: Quarterly percentage change in antibiotic use was calculated using weighted linear regression. Machine learning selected candidate variables, and mixed models identified factors associated with new (vs. traditional) antibiotic use in DTR infections. RESULTS: Between quarter 1 of 2016 and quarter 2 of 2021, ceftolozane-tazobactam (approved 2014) and ceftazidime-avibactam (2015) predominated new antibiotic usage whereas subsequently approved gram-negative antibiotics saw relatively sluggish uptake. Among gram-negative infection hospitalizations, 0.7% (2551 [2631 episodes] of 362 142) displayed DTR pathogens. Patients were treated exclusively using traditional agents in 1091 of 2631 DTR episodes (41.5%), including "reserve" antibiotics such as polymyxins, aminoglycosides, and tigecycline in 865 of 1091 episodes (79.3%). Patients with bacteremia and chronic diseases had greater adjusted probabilities and those with do-not-resuscitate status, acute liver failure, and Acinetobacter baumannii complex and other nonpseudomonal nonfermenter pathogens had lower adjusted probabilities of receiving newer (vs. traditional) antibiotics for DTR infections, respectively. Availability of susceptibility testing for new antibiotics increased probability of usage. LIMITATION: Residual confounding. CONCLUSION: Despite FDA approval of 7 next-generation gram-negative antibiotics between 2014 and 2019, clinicians still frequently treat resistant gram-negative infections with older, generic antibiotics with suboptimal safety-efficacy profiles. Future antibiotics with innovative mechanisms targeting untapped pathogen niches, widely available susceptibility testing, and evidence demonstrating improved outcomes in resistant infections might enhance utilization. PRIMARY FUNDING SOURCE: U.S. Food and Drug Administration; NIH Intramural Research Program.

In vitro activity of ceftazidime/avibactam, cefiderocol, meropenem/vaborbactam and imipenem/relebactam against clinical strains of the Stenotrophomonas maltophilia complex.

BACKGROUND: Infections caused by Stenotrophomonas maltophilia and related species are increasing worldwide. Unfortunately, treatment options are limited, whereas the antimicrobial resistance is increasing. METHODS: We included clinical isolates identified as S. maltophilia by VITEK 2 Compact. Ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, quinolones, and tetracycline family members were evaluated by broth microdilution method and compared with first-line treatment drugs. Minimum inhibitory concentrations (MICs) were reported for all antibiotics. We sequenced the Whole Genome of cefiderocol resistant strains (CRSs) and annotated their genes associated with cefiderocol resistance (GACR). Presumptive phylogenetic identification employing the 16S marker was performed. RESULTS: One hundred and one clinical strains were evaluated, sulfamethoxazole and trimethoprim, levofloxacin and minocycline showed susceptibilities of 99.01%, 95.04% and 100% respectively. Ceftazidime was the antibiotic with the highest percentage of resistance in all samples (77.22%). Five strains were resistant to cefiderocol exhibiting MIC values ≥ 2 µg/mL (4.95%). The ß-lactamase inhibitors meropenem/vaborbactam and imipenem/relebactam, failed to inhibit S. maltophilia, preserving both MIC50 and MIC90 ≥64 µg/mL. Ceftazidime/avibactam restored the activity of ceftazidime decreasing the MIC range. Tigecycline had the lowest MIC range, MIC50 and MIC90. Phylogeny based on 16S rRNA allowed to identify to cefiderocol resistant strains as putative species clustered into Stenotrophomonas maltophilia complex (Smc). In these strains, we detected GARCs such as Mutiple Drug Resistance (MDR) efflux pumps, L1-type ß-lactamases, iron transporters and type-1 fimbriae. CONCLUSION: Antimicrobial resistance to first-line treatment is low. The in vitro activity of new ß-lactamase inhibitors against S. maltophilia is poor, but avibactam may be a potential option. Cefiderocol could be considered as a potential new option for multidrug resistant infections. Tetracyclines had the best in vitro activity of all antibiotics evaluated.

Difficult-to-treat (DTR) Pseudomonas aeruginosa harboring Verona-Integron metallo-ß-lactamase (blaVIM): infection management and molecular analysis.

Pseudomonas aeruginosa harboring Verona Integron-encoded metallo-ß-lactamase enzymes (VIM-CRPA) have been associated with infection outbreaks in several parts of the world. In the US, however, VIM-CRPA remain rare. Starting in December 2018, we identified a cluster of cases in our institution. Herein, we present our epidemiological investigation and strategies to control/manage these challenging infections. This study was conducted in a large academic healthcare system in Miami, FL, between December 2018 and January 2022. Patients were prospectively identified via rapid molecular diagnostics when cultures revealed carbapenem-resistant P. aeruginosa. Alerts were received in real time by the antimicrobial stewardship program and infection prevention teams. Upon alert recognition, a series of interventions were performed as a coordinated effort. A retrospective chart review was conducted to collect patient demographics, antimicrobial therapy, and clinical outcomes. Thirty-nine VIM-CRPA isolates led to infection in 21 patients. The majority were male (76.2%); the median age was 52 years. The majority were mechanically ventilated (n = 15/21; 71.4%); 47.6% (n = 10/21) received renal replacement therapy at the time of index culture. Respiratory (n = 20/39; 51.3%) or bloodstream (n = 13/39; 33.3%) were the most common sources. Most infections (n = 23/37; 62.2%) were treated with an aztreonam-avibactam regimen. Six patients (28.6%) expired within 30 days of index VIM-CRPA infection. Fourteen isolates were selected for whole genome sequencing. Most of them belonged to ST111 (12/14), and they all carried blaVIM-2 chromosomally. This report describes the clinical experience treating serious VIM-CRPA infections with either aztreonam-ceftazidime/avibactam or cefiderocol in combination with other agents. The importance of implementing infection prevention strategies to curb VIM-CRPA outbreaks is also demonstrated.

Novel spore-forming species exhibiting intrinsic resistance to third- and fourth-generation cephalosporins and description of Tigheibacillus jepli gen. nov., sp. nov.

A comprehensive microbial surveillance was conducted at NASA's Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HST, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HST is most closely related to Virgibacillus halophilus 5B73CT, sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus Virgibacillus is polyphyletic, strain 179-BFC-A-HST is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HST = DSM 115946T = NRRL B-65666T), and its closest neighbor, V. halophilus, is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73CT = DSM 21623T = JCM 21758T = KCTC 13935T). It was also necessary to reclassify its second closest neighbor Virgibacillus soli, as a member of a novel genus Paracerasibacillus, reflecting its phylogenetic position relative to the genus Cerasibacillus, for which we propose Paracerasibacillus soli comb. nov. (type strain CC-YMP-6T = DSM 22952T = CCM 7714T). Within Amphibacillaceae (n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known ß-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy. P. soli was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins. IMPORTANCE: The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.

Ceftazidime/avibactam serum concentration in patients on ECMO.

OBJECTIVES: The use of extracorporeal membrane oxygenation (ECMO) may alter blood levels of several drugs, including antibiotics, leading to under dosing of these drugs and thus to potential treatment failure. No data exist on pharmacokinetics of new antimicrobial, in particular ceftazidime/avibactam. We therefore perform this study to evaluate ceftazidime/avibactam blood levels in ECMO patients and find factors associated with underdosing. METHODS: Retrospective observational study of patients on ECMO having received ceftazidime/avibactam and in whom trough blood levels of ceftazidime and avibactam were available. Main outcome measurement was the number of patients with ceftazidime and avibactam blood levels above predefined cut-off values, derived from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for Enterobacteriaceae and Pseudomonas aeruginosa, namely 8 mg/L for ceftazidime and 4 mg/L for avibactam, and explored factors associated with underdosing. RESULTS: Twenty-three ceftazidime/avibactam trough levels were available in 14 ECMO patients, all of them having received veno-venous ECMO for SARS-CoV-2-associated pneumonia. Although ceftazidime levels were above 8 mg/L in all except one patient, nine (39%) of the avibactam dosages were below 4 mg/L. Increased renal clearance (creatinine clearance > 130 mL/min) was the main factor associated with under dosing, since 7 out of the 10 dosages below the predefined cut-offs were measured in patients with this condition. CONCLUSIONS: In ECMO patients receiving ceftazidime/avibactam, ceftazidime and avibactam serum levels are above EUCAST breakpoints in most cases, justifying the use of normal dosing in ECMO patients. Increased renal clearance may lead to ceftazidime and avibactam under dosing.

Zein nanoparticles containing ceftazidime and tobramycin: antibacterial activity against Gram-negative bacteria.

Aims: This work describes the encapsulation of ceftazidime and tobramycin in zein nanoparticles (ZNPs) and the characterization of their antibacterial and antibiofilm activities against Gram-negative bacteria. Materials & methods: ZNPs were synthesized by nanoprecipitation. Cytotoxicity was assessed by MTT assay and antibacterial and antibiofilm assays were performed by broth microdilution and violet crystal techniques. Results: ZNPs containing ceftazidime (CAZ-ZNPs) and tobramycin (TOB-ZNPs) showed drug encapsulation and thermal stability. Encapsulation of the drugs reduced their cytotoxicity 9-25-fold. Antibacterial activity, inhibition and eradication of biofilm by CAZ-ZNPs and TOB-ZNPs were observed. There was potentiation when CAZ-ZNPs and TOB-ZNPs were combined. Conclusion: CAZ-ZNPs and TOB-ZNPs present ideal physical characteristics for in vivo studies of antibacterial and antibiofilm activities.

A nanotechnology product was developed to treat diseases caused by bacteria. This prototype showed the ideal characteristics and could be administered by ingestion through the mouth, aspiration through the nose or injection into the veins. The prototype did not harm or kill human cells. It killed the bacteria and prevented the formation of a type of protection against antibiotics that bacteria can produce, called a biofilm. Nanotechnology products are a promising alternative for the treatment of bacterial infections.

In vitro development of resistance against antipseudomonal agents: comparison of novel ß-lactam/ß-lactamase inhibitor combinations and other ß-lactam agents.

We subjected seven P. aeruginosa isolates to a 10-day serial passaging against five antipseudomonal agents to evaluate resistance levels post-exposure and putative resistance mechanisms in terminal mutants were analyzed by whole-genome sequencing analysis. Meropenem (mean, 38-fold increase), cefepime (14.4-fold), and piperacillin-tazobactam (52.9-fold) terminal mutants displayed high minimum inhibitory concentration (MIC) values compared to those obtained after exposure to ceftolozane-tazobactam (11.4-fold) and ceftazidime-avibactam (5.7-fold). Fewer isolates developed elevated MIC values for other ß-lactams and agents belonging to other classes when exposed to meropenem in comparison to other agents. Alterations in nalC and nalD, involved in the upregulation of the efflux pump system MexAB-OprM, were common and observed more frequently in isolates exposed to ceftazidime-avibactam and meropenem. These alterations, along with ones in mexR and amrR, provided resistance to most ß-lactams and levofloxacin but not imipenem. The second most common gene altered was mpl, which is involved in the recycling of the cell wall peptidoglycan. These alterations were mainly noted in isolates exposed to ceftolozane-tazobactam and piperacillin-tazobactam but also in one cefepime-exposed isolate. Alterations in other genes known to be involved in ß-lactam resistance (ftsI, oprD, phoP, pepA, and cplA) and multiple genes involved in lipopolysaccharide biosynthesis were also present. The data generated here suggest that there is a difference in the mechanisms selected for high-level resistance between newer ß-lactam/ß-lactamase inhibitor combinations and older agents. Nevertheless, the isolates exposed to all agents displayed elevated MIC values for other ß-lactams (except imipenem) and quinolones tested mainly due to alterations in the MexAB-OprM regulators that extrude these agents.

Spread and evolution of blaKPC-plasmid between Serratia marcescens and Klebsiella pneumoniae.

OBJECTIVES: blaKPC-carrying Enterobacterales have post great challenges to global healthcare systems. In this study, we reported the evolution and spread of blaKPC between Serratia marcescens and Klebsiella pneumoniae. METHODS: Four S. marcescens and one K. pneumoniae strains were isolated from the sputum samples of the patient. Antimicrobial susceptibility tests and whole genome sequencing were performed to investigate the phenotype & genotype of strains. Conjugation assays, cloning experiment and kinetic parameters measuring were performed to explore the spread and antimicrobial resistance mechanisms. RESULTS: The evolution and transmission of blaKPC-2 occurred during the treatment of ceftazidime-avibactam and trimethoprim-sulfamethoxazole. Analysis of the antimicrobial susceptibility and genetic profiles of the clinical strains showed that blaKPC-2 evolved into blaKPC-71 and blaKPC-44, together with resistance to ceftazidime-avibactam and carbapenems susceptibility recovery under antimicrobial pressure. Cloning and expression of blaKPC-44 & blaKPC-71 in E. coli DH5α showed that KPC-44 and KPC-71 resulted in a 64∼128-fold increase in the MIC value for ceftazidime-avibactam. Meanwhile, the kinetic assays also showed that the enzyme activity of KPC-44 and KPC-71 towards carbapenems was destroyed and couldn't be inhibited by avibactam. Based on the conjugation assay and whole genome sequence analyses, we provided evolutionary insights into the transmission pathway trace of blaKPC-bearing plasmids between S. marcescens and K. pneumoniae. CONCLUSIONS: Mixed-species co-infection is one of the risk factors leading to the spread of plasmids carrying carbapenem-resistant genes, and increased surveillance of multidrug-resistant Enterobacterales is urgently needed.

Efficacy of ceftazidime-avibactam in the treatment of carbapenem-resistant Klebsiella pneumoniae infections: Focus on solid organ transplantation recipients.

INTRODUCTION: Ceftazidime-avibactam (CAZ-AVI) is a new option to treat KPC- and OXA-48 carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. However, clinical evidence is limited regarding its use in treating CRKP infections, especially in solid organ transplantation (SOT) recipients. In this study, we assessed the efficacy of CAZ-AVI in treating CRKP infections in both the general population and the SOT recipients in comparison with other antibiotic regimens. METHODS: This is a single-centre retrospective cohort study of patients admitted between January 1, 2018 and June 30, 2021 with the diagnosis of CRKP infections receiving either CAZ-AVI or other regimens ≥ 72 hours and clinical outcomes were analysed. RESULTS: Of 200 patients with CRKP infections, 67 received CAZ-AVI, 133 received other regimens, and 50 were SOT recipients. In the SOT cohort, 30 patients received CAZ-AVI, and 20 received other regimens. The overall 30-day mortality was 38% in the SOT cohort. Compared with patients receiving other regimens, CAZ-AVI therapy resulted in lower 30-day mortality (23.3% vs. 60%, P = 0.014) and 90-day mortality (35.7% vs. 86.7%, P = 0.003), higher clinical cure (93.3% vs. 40%, P < 0.001) and microbiological clearance. Similar promising results of CAZ-AVI were also shown in the whole population cohort. Moreover, clinical outcomes of SOT recipients receiving CAZ-AVI were not inferior to those without SOT. CONCLUSIONS: CAZ-AVI therapy was associated with better clinical outcomes in CRKP infections in both the general population and SOT recipients. Considering the limitations of the present study, well-conducted RCTs are still warranted to confirm these findings.

Activity of cefiderocol and innovative ß-lactam/ß-lactamase inhibitor combinations against isogenic strains of Escherichia coli expressing single and double ß-lactamases under high and low permeability conditions.

OBJECTIVES: To analyse the impact of the most clinically relevant ß-lactamases and their interplay with low outer membrane permeability on the activity of cefiderocol, ceftazidime/avibactam, aztreonam/avibactam, cefepime/enmetazobactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/xeruborbactam and meropenem/nacubactam against recombinant Escherichia coli strains. METHODS: We constructed 82 E. coli laboratory transformants expressing the main ß-lactamases circulating in Enterobacterales (70 expressing single ß-lactamase and 12 producing double carbapenemase) under high (E. coli TG1) and low (E. coli HB4) permeability conditions. Antimicrobial susceptibility testing was determined by reference broth microdilution. RESULTS: Aztreonam/avibactam, cefepime/zidebactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam were active against all E. coli TG1 transformants. Imipenem/relebactam, meropenem/vaborbactam, cefepime/taniborbactam and cefepime/enmetazobactam were also highly active, but unstable against most of MBL-producing transformants. Combination of ß-lactamases with porin deficiency (E. coli HB4) did not significantly affect the activity of aztreonam/avibactam, cefepime/zidebactam, cefiderocol or meropenem/nacubactam, but limited the effectiveness of the rest of carbapenem- and cefepime-based combinations. Double-carbapenemase production resulted in the loss of activity of most of the compounds tested, an effect particularly evident for those E. coli HB4 transformants in which MBLs were present. CONCLUSIONS: Our findings highlight the promising activity that cefiderocol and new ß-lactam/ß-lactamase inhibitors have against recombinant E. coli strains expressing widespread ß-lactamases, including when these are combined with low permeability or other enzymes. Aztreonam/avibactam, cefiderocol, cefepime/zidebactam and meropenem/nacubactam will help to mitigate to some extent the urgency of new compounds able to resist MBL action, although NDM enzymes represent a growing challenge against which drug development efforts are still needed.

In vitro activity of ceftazidime-avibactam against gram-negative bacteria in patients with bacteremia and skin and soft-tissue infections in Colombia 2019-2021.

OBJECTIVES: Ceftazidime-avibactam (CAZ-AVI) is an option for infections caused by MDR gram-negative bacilli. In this study, we aimed to analyze the in vitro antimicrobial activity of CAZ-AVI and other antimicrobial agents against gram-negative bacilli that were collected in Colombia between 2019 and 2021 from patients with bacteremia and skin and soft-tissue infections (SSTIs). METHODS: A total of 600 Enterobacterales and 259 P. aeruginosa strains were analyzed. The phenotypic resistance of isolates, particularly non-susceptibility to meropenem, multidrug-resistant (MDR) isolates, and difficult-to-treat (DTR) P. aeruginosa, was evaluated according to CLSI breakpoints. RESULTS: Enterobacterales had the most susceptibility to CAZ-AVI (96.5 %) and tigecycline (95 %). Tigecycline and CAZ-AVI were the antimicrobial agents with the most in vitro activity against carbapenem-resistant Enterobacterales (CRE). CAZ-AVI was the antimicrobial treatment with the most activity against P. aeruginosa. CONCLUSIONS: Tigecycline and CAZ-AVI were the antimicrobial agents with the most activity against CRE and MDR Enterobacterales. For P. aeruginosa, CAZ-AVI was the antimicrobial treatment with the most in vitro activity.

Determination of aztreonam/ceftazidime-avibactam synergism and proposal of a new methodology for the evaluation of susceptibility in vitro.

We proposed a new methodology, the microelution ATM/CZA (mATM/CZA), based on the antibiotic disc elution and the use of resazurin, for rapid (<4h) determination of in vitro susceptibility to aztreonam combined with ceftazidime-avibactam among Enterobacterales. The mATM/CZA presented excellent accuracy with 1.9 %, 98.1 % and 100 % of major error, specificity and sensitivity, respectively. Furthermore, we assessed synergism between aztreonam and ceftazidime-avibactam in Enterobacterales and Pseudomonas aeruginosa, which was observed in 37/55 Enterobacterales and 31/56 P. aeruginosa. As reference methodologies (checkerboard, time-kill curve) are not compatible with the routine of the clinical microbiology laboratories, mATM/CZA is an important alternative to evaluate susceptibility of the combination in a scenario where its clinical use is increasingly important.