The need for synergy testing is driven by the necessity to extend the antimicrobial spectrum, reducing drug dosage/toxicity and the development of resistance. Despite the abundance of synergy testing methods, there is the absence of a gold standard and a lack of synergy correlation among methods. The most popular method (checkerboard) is labor-intensive and is not practical for clinical use. Most clinical laboratories use several gradient synergy methods which are quicker/easier to use. This study sought to evaluate three gradient synergy methods (direct overlay, cross, MIC:MIC ratio) with the checkerboard, and compare two interpretative criteria (the fractional inhibitory concentration index (FICI) and susceptibility breakpoint index (SBPI)) regarding these methods. We tested 70 multidrug-resistant Pseudomonas aeruginosa, using a tobramycin and ceftazidime combination. The agreement between the checkerboard and gradient methods was 60 to 77% for FICI, while agreements for SBPI that ranged between 67 and 82.86% were statistically significant (p ≤ 0.001). High kappa agreements were observed using SBPI (Ƙ > 0.356) compared to FICI (Ƙ < 0.291) criteria, and the MIC:MIC method demonstrated the highest, albeit moderate, intraclass correlation coefficient (ICC = 0.542) estimate. Isolate resistance profiles suggest method-dependent synergism for isolates, with ceftazidime susceptibility after increased exposure. The results show that when interpretative criteria are considered, gradient diffusion (especially MIC:MIC) is a valuable and practical method that can inform the treatment of cystic fibrosis patients who are chronically infected with P. aeruginosa.

Background: Antimicrobial combination therapy is a time/resource- intensive procedure commonly employed in the treatment of cystic fibrosis (CF) pulmonary exacerbations caused by P. aeruginosa. Ten years ago the most promising antimicrobial combinations were proposed, but there has since been the introduction of new β-lactam+β-lactamase inhibitor antimicrobial combinations. The aims of this study were i) to compare in vitro activity of these new antimicrobials with other anti-pseudomonals agents and suggest their most synergistic antimicrobial combinations. ii) to determine antimicrobial resistance rates and study inherent trends of antimicrobials over ten years. Methods: A total of 721 multidrug-resistant P. aeruginosa isolates from 183 patients were collated over the study period. Antimicrobial susceptibility and combination testing were carried out using the Etest method. The results were further assessed using the fractional inhibitory concentration index (FICI) and the susceptible breakpoint index (SBPI). Results: Resistance to almost all antimicrobial agents maintained a similar level during the studied period. Colistin (p<0.001) and tobramycin (p=0.001) were the only antimicrobials with significant increasing isolate susceptibility while an increasing resistance trend was observed for levofloxacin. The most active antimicrobials were colistin, ceftolozane/tazobactam, ceftazidime/avibactam, and gentamicin. All combinations with β-lactam+β-lactamase inhibitors produced some synergistic results. Ciprofloxacin+ceftolozane/tazobactam (40%) and amikacin+ceftazidime (36.7%) were the most synergistic combinations while colistin combinations gave the best median SPBI (50.11). Conclusions: This study suggests that effective fluoroquinolone stewardship should be employed for CF patients. It also presents in vitro data to support the efficacy of novel combinations for use in the treatment of chronic P. aeruginosa infections.

Background: Achromobacter spp. are recognized as an emerging pathogen in patients with Cystic Fibrosis (CF). Though recent works have established species-level identification using nrdA sequencing, there is a dearth in knowledge relating to species-level antimicrobial susceptibility patterns and antimicrobial combinations which hampers the use of optimal antimicrobial combinations for the treatment of chronic infections. The aims of this study were i) to identify at species-level referred Achromobacter isolates ii) to describe species-level antimicrobial susceptibility profiles iii) to determine the most promising antimicrobial combination for chronic Achromobacter infections. Methods: A total of 112 multidrug-resistant (MDR) Achromobacter spp. isolates from 39 patients were identified using nrdA sequencing. Antimicrobial susceptibility and combination testing were carried out using the Etest method. Results: We detected six species of Achromobacter and found that A. xylosoxidans was the most prevalent species. Interestingly, sequence analysis showed it was responsible for persistent infection (18/28 patients) followed by A. ruhlandii (2/3 patients). Piperacillin-tazobactam (70.27%) and cotrimoxazole (69.72%) were the most active antimicrobials. Differences were observed in species-level susceptibility to ceftazidime, carbapenems, ticarcillin-clavulanate, and tetracycline. Antimicrobial combinations with cotrimoxazole or tobramycin demonstrate the best synergy while cotrimoxazole gave the best susceptibility breakpoint Index values. Conclusions: This study enriches the understanding of MDR Achromobacter spp. epidemiology, confirms prevalence and chronic colonization of A. xylosoxidans in CF lungs. It presents in vitro data to support the efficacy of new combinations for use in the treatment of chronic Achromobacter infections.

Pseudomonas aeruginosa is a major pathogen in humans and other animals, frequently harboring mechanisms of resistance to commonly used antimicrobials. Here, we describe the isolation of P. aeruginosa bacteriophage Zikora. The full 65,837-bp genome was annotated and demonstrates similarity to Pbunavirus phages, making Zikora a new member of this genus of the Myoviridae family.