Journal of Antimicrobial Chemotherapy最新文献

Objectives: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is compromising gonorrhoea treatment, and enhanced N. gonorrhoeae AMR and genome-based epidemiological surveillance is imperative. Molecular tests are replacing N. gonorrhoeae culture internationally, excluding possibilities to perform WGS. We describe and evaluate a novel approach using a custom SureSelectXTHS Target-Enrichment probe panel automated on the Magnis NGS Prep System and Illumina sequencing to generate accurate N. gonorrhoeae genomes directly from clinical urogenital and extragenital specimens.

Methods: One hundred thirteen clinical N. gonorrhoeae-positive APTIMA Combo 2 (AC2) specimens (with 89 linked N. gonorrhoeae isolates) were included. DNA was extracted using QIAsymphony DSP Virus/Pathogen kit. Amplisens multiplex RT-PCR assay (AM-PCR) identified 105 (92.9%) of the AC2 specimens as N. gonorrhoeae positive, which were further examined. Sequence libraries for AC2 specimens were prepared on the Magnis NGS Prep System using the Magnis SureSelectXTHS Reagent kit for Illumina paired-end platforms. Paired-end sequencing was performed on Illumina platforms.

Results: Seventy-four of the 105 (70.5%) AC2 samples remained N. gonorrhoeae positive with a cycle threshold <20 in the AM-PCR and subjected to SureSelectXTHS target enrichment and subsequently Illumina WGS. Seventy-two (97.3%) of all target-enriched specimens were successfully genome-sequenced. All linked AC2 specimens and N. gonorrhoeae isolates from the same anatomical site had identical AMR determinants and molecular epidemiological sequence types.

Conclusions: We show that custom SureSelectXTHS target enrichment automated on the Magnis NGS Prep System, followed by Illumina sequencing, enables culture-independent genome-based surveillance of N. gonorrhoeae AMR and molecular epidemiology. This novel methodological advancement provides an efficient and accurate WGS of N. gonorrhoeae directly from clinical urogenital and extragenital NAAT specimens.

Objectives: To evaluate clinical impact of ceftazidime/avibactam on treating infections due to MDR Gram-negative bacteria in patients with haematological malignancies (HMs).

Methods: We conducted a retrospective, observational study at 17 Italian haematological wards that included patients with HMs receiving ceftazidime/avibactam for the treatment of suspected or proven infections. The primary endpoint was all-cause mortality 30 days after infection onset. Secondary endpoints included the development of in vitro ceftazidime/avibactam resistance, adverse reactions and infection relapse.

Results: Of 198 patients enrolled, 66 had fever of unknown origin and 132 had microbiologically proven infections (MPIs). Enterobacterales were responsible for 98 MPIs, with KPC producers accounting for 75% of these, and carbapenem-resistant Pseudomonas aeruginosa caused 25% of MPIs. The overall 30-day mortality rate was 17.7%. Infection relapse occurred in four patients with MPI. Patients who died within 30 days of infection onset tended to have pre-existing cerebrovascular diseases, a Charlson Comorbidity Index > 4 and septic shock at infection onset and had received inadequate initial antibiotic therapy. Thirty-day mortality was independently associated with septic shock at infection onset and inappropriate initial antibiotic therapy.

Conclusions: Our study provides further evidence about the effectiveness of ceftazidime/avibactam in treating infections in patients with HMs.

Objectives: To investigate the carbapenem resistance mechanism of a carbapenem-resistant clinical Pseudomonas aeruginosa isolate.

Methods: A clinical isolate of P. aeruginosa was sent to the German National Reference Centre for multidrug-resistant Gram-negative bacteria for carbapenemase detection. Phenotypic tests for carbapenemase detection and an EDTA-combined disc test were positive, therefore PCR-screenings were done for the most prevalent metallo-β-lactamase (MBL) encoding genes. As no MBL gene could be found, whole-genome sequencing was performed. For characterization, heterologous expression in a E. coli strain with subsequent MIC testing and purification of the new MBL to determine enzyme kinetics with in vitro hydrolysis assays was performed.

Results: WGS revealed the putative gene for a B3 MBL located on the chromosome between several disrupted IS elements with 67% identity to EVM-1, which was named NWM-1. MIC studies and enzyme kinetics confirmed MBL activity. No activity against ceftazidime was observed.

Conclusions: The identification of NWM-1 shows the importance of WGS to identify yet unknown carbapenemases and underlines the diversity of subclass B3 β-lactamases. It also shows that although several carbapenemase variants have already been identified and characterized, there are always new variants to be found in clinical isolates.

Background: The lack of research on the dose-response relationship of adjuvants in reversing colistin resistance will lead to a lack of scientific theoretical basis for determining the dosage of adjuvants in clinical combination therapy plans or their compound formulations.

Objectives: This study investigates the dose-response relationship of the deworming drug closantel (CST) on the reversal of colistin resistance in mcr-1-positive Escherichia coli (E. coli).

Methods: Firstly, the reversal effect of different concentrations of CST on colistin resistance in mcr-1-positive E. coli was analysed using broth microdilution method, checkerboard method and time-killing curves. Then, the inhibitory effect of CST on the development of colistin resistance, as well as the haemolytic and cytotoxic properties of CST, was analysed. Finally, the in vivo efficacy of the combination of CST and colistin was evaluated.

Results: Both the checkerboard assays and the time-killing curves indicate that there is a special dose-response relationship between CST and its reversal effect on colistin resistance, which is not concentration-dependent. High reversal efficiency can be achieved within a low concentration range. However, as the CST concentration increases, the ability to reverse colistin resistance remains unchanged or decreases, which resulted in a gradual decrease in reversal efficiency. Additionally, CST can inhibit the development of colistin resistance and reduce the cytotoxicity of colistin. Importantly, in a mouse model of E. coli infection, the combination of CST and colistin showed a significant therapeutic effect.

Conclusions: This study indicates a special dose-response relationship between CST and its reversal effect on colistin resistance, which was not concentration-dependent.

Background: Heteroresistance, frequently observed in diverse bacterial species, imposes clinical challenges. For this study, we investigated the stability and resilience of tigecycline heteroresistance in Acinetobacter baumannii.

Methods: Four tigecycline-heteroresistant (HR) A. baumannii strains and resistant populations (RPs) obtained from them were subjected to laboratory evolution assays for 30 days in antibiotic-free media. The heteroresistance phenotype was determined using a population analysis. Bacterial growth curves and in vitro competitiveness were determined to investigate the fitness cost of heteroresistance. Tigecycline efficacy was evaluated using an in vitro time-killing assay. Genetic mutations were identified using whole genome sequencing, and expression of genes in the two-component systems was also evaluated.

Results: Tigecycline heteroresistance was preserved even in antibiotic-free media, and tigecycline-RPs reverted to heteroresistance during serial culture without tigecycline pressure. The tigecycline-RPs showed a higher fitness cost than their respective HR strains, and the HR strains exhibited a survival advantage upon tigecycline treatment. Although the AdeABC efflux pump was overexpressed in the tigecycline-RPs, it was down-regulated in the HR strains.

Conclusions: Our data indicate that tigecycline heteroresistance is a highly resilient phenotype in A. baumannii that gives a high fitness advantage to bacteria in terms of competitiveness and response to antibiotic pressure.

Background: Nuclear import, dependent on the transporter importin α (IMPα), is a drug target for apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Indeed, a panel of small molecule inhibit interactions between IMPα and nuclear localization signals (NLSs) in vitro and the growth of rapidly dividing stages (P. falciparum blood stages and T. gondii tachyzoites) in culture.

Objectives: As new drugs targeting multiple life cycle stages of both parasites are required, the panel of IMPα inhibitors was tested for their ability to inhibit nuclear transport in the rapidly dividing stages and the maturation of differentiated stages (P. falciparum gametocytes and T. gondii bradyzoites).

Methods: Using biophysical assays, Bay 11-7082, a Bay 11-7085 structural analogue, was tested for inhibition of IMPα:NLS interactions. The effect of the panel of inhibitors on the nuclear localization of reporter proteins was analysed in both parasites using transfections and microscopy. Also, using microscopy, the effect of inhibitors on differentiated stages of both parasites was tested.

Results: Bay 11-7085 can inhibit nuclear transport in tachyzoites, while GW5074 and Caffeic Acid Phenethyl Ester (CAPE) can inhibit nuclear transport in the blood stages. Interestingly, CAPE can strongly inhibit gametocyte maturation, and Bay 11-7082 and Bay 11-7085 weakly inhibit bradyzoite differentiation.

Conclusions: As differentiation of gametocytes and bradyzoites is dependent on the activation of gene expression triggered by the nuclear translocation of transcription factors, our work provides a 'proof of concept' that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of which are recalcitrant to existing drugs.

Background: Nacubactam (NAC), a new diazabicyclooctane β-lactamase inhibitor, is being developed for use together with aztreonam (AZT) and cefepime (CFPM). However, the effective clinical dosages of AZT/NAC and CFPM/NAC have not yet been established. We have previously shown that free time above instantaneous MIC (fT > MICi) is a valuable pharmacokinetic (PK)/pharmacodynamic parameter for β-lactam (BL)/NAC in a mouse thigh infection model.

Objectives: This study simulated the fT > MICi (%) for AZT/NAC and CFPM/NAC against carbapenemase-producing Enterobacterales (CPE) with different MIC in humans to estimate the clinical efficacy at practically achievable combination doses of AZT/NAC and CFPM/NAC.

Methods: Using previously reported PK parameters of each drug in humans and chequerboard MIC data, we calculated the fT > MICi (%) for AZT/NAC and CFPM/NAC in 10 000 simulated patients to predict the percentages of target attainment of bacteriostatic and bactericidal efficacies at various combined doses.

Results: The results predicted that both BL/NAC combinations could achieve 100% 2 log10-kill against CPE strains at the lowest combination dose (0.5 g/0.5 g q8h). Additionally, in MIC studies examining BLs/NAC at a 1:1 ratio, the dosage regimen for strains with MICcomb ≤ 1 mg/L was expected to offer 100% bactericidal efficacy (2 log10-kill) at 0.5 g/0.5 g q8h or higher doses. For strains with 1 mg/L < MICcomb ≤ 16 mg/L, BLs/NAC at a 2 g/2 g q8h was predicted to produce bactericidal efficacy (1 log10-kill). At MICcomb = 32 mg/L, some bacteriostatic effect was expected at high BL doses.

Conclusions: AZT/NAC and CFPM/NAC are bactericidal against CPE at practically achievable dosages.

Background: Pseudomonas aeruginosa biofilms limit the efficacy of currently available antibacterial therapies and pose significant clinical challenges. Pseudomonal biofilms are complicated further when other markers of persistence such as mucoid and hypermutable phenotypes are present. There is currently a paucity of data regarding the activity of the newer β-lactam/β-lactamase inhibitor combination ceftolozane/tazobactam against P. aeruginosa biofilms.

Methods: We evaluated the efficacy of ceftolozane/tazobactam against clinical P. aeruginosa isolates, the laboratory isolate PAO1 and its isogenic mutS-deficient hypermutator derivative (PAOMS) grown under static and dynamic biofilm conditions. The clinical isolate collection included strains with mucoid and hypermutable phenotypes.

Results: Ceftolozane/tazobactam exposure led to a bactericidal (≥3 log cfu/cm2) biofilm reduction in 15/18 (83%) clinical isolates grown under static conditions, irrespective of carbapenem susceptibility or mucoid phenotype, with greater activity compared with colistin (P < 0.05). Dynamically grown biofilms were less susceptible to ceftolozane/tazobactam with active biofilm reduction (≥1 log cfu/cm2) observed in 2/3 isolates. Hypermutability did not affect the antibiofilm efficacy of ceftolozane/tazobactam in either static or dynamic conditions when comparing PAO1 and PAOMS. Consistent with the activity of ceftolozane/tazobactam as a potent inhibitor of PBP3, dramatic impacts on P. aeruginosa morphology were observed.

Conclusions: Our data demonstrate that ceftolozane/tazobactam has encouraging properties in the treatment of P. aeruginosa biofilm infections, and its activity is not diminished against mucoid or hypermutable variants at the timepoints examined.

Background: Catheter-associated urinary tract infections (CA-UTIs) are a common hospital-acquired infection. We examined ciprofloxacin activity in a novel CA-UTI in vitro model.

Methods: Three ATCC strains [Escherichia coli (ECO)-25922, Klebsiella pneumoniae (KPN)-700721, Pseudomonas aeruginosa (PAE)-27853] and 45 clinical urinary isolates were assessed. Biofilm mass and planktonic bacterial density were quantified during drug-free incubation (72 h) and following ciprofloxacin exposure (equivalent 750 mg orally q12h, 3 days).

Results: ECO produced smaller biofilms (6.3 ± 1.1 log10 cfu/cm2) compared with KPN (7.1 ± 0.7 log10 cfu/cm2) and PAE (7.0 ± 1.2 log10 cfu/cm2), which extended along the entire catheter length. Following ciprofloxacin, all isolates with MIC > 4 mg/L had minimal biofilm disruption or planktonic kill. Ciprofloxacin resistance was most common in PAE isolates (10/16 isolates), compared with ECO (3/16 isolates) and KPN (6/16 isolates). Greater ciprofloxacin exposure (AUC0-24/MIC) was required for a 3 log10 biofilm kill for KPN (5858; R2 = 0.7774) compared with ECO (2117; R2 = 0.7907) and PAE (2485; R2 = 0.8260). Due to persistent growth in the bladder, ECO required greater ciprofloxacin exposure for a 3 log10 planktonic kill (5920; R2 = 0.8440) compared with KPN (2825; R2 = 0.9121) and PAE (1760; R2 = 0.8781). Monte Carlo simulation supported a 95% PTA for both a 3 log10 biofilm and planktonic kill for ECO and KPN isolates with MIC ≤ 0.5 mg/L and PAE isolates with MIC ≤ 1 mg/L.

Conclusions: In a novel CA-UTI model, following simulated ciprofloxacin therapy, KPN biofilms were comparatively more difficult to disrupt, ECO planktonic growth frequently persisted in the bladder, and PAE had greater propensity for emergence of ciprofloxacin resistance.