Novel phenotypic and molecular approaches for tackling the problem of microbial drug resistance

Over the past decade, infections caused by multi-resistant bacteria have undergone a rapid worldwide spread, which represents a public health problem of major concern, with a significative impact on morbidity, mortality, and healthcare-associated costs. Among Gram-negative bacteria, carbapenemase-producing Enterobacterales along with carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa are considered high priority pathogens for which the development of novel antimicrobials is crucial. On the other hand, vancomycin-resistant Enterococcus faecium and methicillin and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) represent the most critical pathogens among Gram-positives. An effective approach against antimicrobial resistance would require multiple strategies, including the optimization of antimicrobial drug use and the development of new antimicrobial agents, and the improvement of current diagnostic systems. In this scenario, the general aim of this project was to characterize local and nationwide collections of both Gram-negative and Gram-positive clinical isolates at a phenotypic and genotypic level, evaluating the activity of novel antimicrobial agents and novel and commercially available molecular and phenotypic methods. The first part of this project has included the development of a new phenotypic method (the colistin-MAC test) for phenotypic screening of acquired colistin resistance mediated by transferable mcr-1 resistance determinants, based on colistin MIC reduction in the presence of dipicolinic acid. The advantages of this test include the simple and inexpensive execution for each clinical laboratory, especially in low-income settings, and the possibility to screen large collections of isolates to detect new mcr-like genes not yet targeted by the current molecular assays. Furthermore, the comparison of reference methods (broth microdilution and agar dilution) and different commercial diagnostic systems was performed for susceptibility testing of carbapenems and fosfomycin, which are considered as last-resort antibiotics. This evaluation showed that most commercial methods can be unreliable for testing susceptibility to carbapenems and fosfomycin in KPC-producing Escherichia coli and Klebsiella pneumoniae, respectively. The second line of research has explored the in vitro activity of novel antibiotics (ceftobiprole and cefiderocol) and of novel combinations of β-lactam/β-lactamase inhibitors (cefepime/VNRX-5133, meropenem/vaborbactam, ceftazidime/avibactam) against bacterial isolates of clinical origin from large multicentric collections. All tested agents exhibited a potent activity against at least 90% of the isolates tested and most of them have been evaluated prior to their introduction in the market. In particular, ceftobiprole was able to inhibit the growth of 95.5% of MRSA isolates which were also characterized by an NGS approach, providing an updated image of the Italian epidemiology of S. aureus from hospital-acquired pneumonia. Among antibiotics against Gram-negative pathogens, cefiderocol exhibited the highest activity against both carbapenem-resistant Enterobacterales and non-fermentative Gram-negatives including metallo-β-lactamase-producers and carbapenemase-producing A. baumannii for which no β-lactam/ β-lactamase inhibitor combinations are currently effective. Among anti-pseudomonas agents, cefepime/VNRX-5133 exhibited activity comparable to ceftolozane/tazobactam (91,5% vs 90.0%) against a multicentric collection of 934 P. aeruginosa. Furthermore, VNRX-5133 restored cefepime susceptibility in 43.8% of VIM-producers. Moreover, both ceftazidime/avibactam and meropenem/vaborbactam resulted active (93.9% vs 96.1%) against a multicentric collection of 407 carbapenem-resistant Enterobacterales (mainly KPC-producers) from bloodstream infections. In particular, meropenem/vaborbactam showed efficacy also in some cases of ceftazidime/avibactam resistant KPC-producing isolates. Finally, the ceftazidime/avibactam resistance mechanism was characterized in two Klebsiella pneumoniae isolates from urine and bloodstream infections, respectively, using a whole-genome sequencing approach (Illumina MiSeq and Oxford Nanopore MinION) and gene transfer experiments. The resistance was associated with increased blaKPC-3 gene dosage, mediated by a pKpQIL plasmid derivative carrying two copies of Tn4401a, and can be considered as a novel type of potentially transferable ceftazidime/avibactam resistance mechanism among Enterobacterales.