Selection of AmpC ß-Lactamase Variants and Metallo-ß-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections.

Infections caused by ceftolozane-tazobactam and ceftazidime-avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane-tazobactam and ceftazidime-avibactam resistance mechanisms in all multidrug-resistant or extensively drug-resistant (MDR/XDR) P. aeruginosa isolates recovered during 1 year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane-tazobactam and ceftazidime-avibactam resistance were evaluated. Clinical conditions, previous positive cultures, and ß-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. Multilocus sequence types (MLSTs) and resistance mechanisms were determined using short- and long-read whole-genome sequencing (WGS). The impact of Pseudomonas-derived cephalosporinases (PDCs) on ß-lactam resistance was demonstrated by cloning into an ampC-deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired ß-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane-tazobactam or ceftazidime-avibactam. Seven isolates from different sequence types (STs) owed their ß-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219, and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. The 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13) and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlighted that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

Anti-adhesive activity of a Vaccinium corymbosum polyphenolic extract targeting intestinal colonization by Klebsiella pneumoniae.

The therapeutic effect of Vaccinium polyphenols against uropathogens has been widely studied. Most attention has focused on the antimicrobial activity against P-fimbriated Escherichia coli strains. The present study investigated the anti-adhesive and anti-biofilm activity of a saline extract of blueberry (Vaccinium corymbosum) targeting intestinal colonization by a highly adherent Klebsiella pneumoniae strain. This strain, responsible for a large outbreak of infection in Spain, was selected on the basis of its remarkable capacity to colonize the gastrointestinal tract of patients. The blueberry extract was obtained using a medium scale ambient temperature system (MSAT) in a novel approach based on the use of an aqueous solvent and addition of mineral salts. The polyphenolic content was determined by liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS). The findings confirmed that the blueberry extract is a rich source of phenolic compounds, including the most polar polyphenols (mostly non-flavonoids), intermediate polarity compounds (flavan-3-ols and most procyanidins) and low polarity compounds (flavonols and anthocyanins). The extract significantly inhibited biofilm formation and bacterial adhesion to HT-29 colorectal cells by a highly adherent multidrug-resistant K. pneumoniae. Although some individual anthocyanidins (malvidin, delphinidin and cyanidin) and one hydroxycinnamic acid (caffeic acid) proved capable of reducing bacterial adhesion, the unfractionated extract was more active than any of the individual polyphenolic compounds. In addition, the extract displayed considerable potential as an intestinal decolonization treatment in a murine model. The study findings demonstrate the potential value of the V. corymbosum extract as an alternative treatment for K. pneumoniae infections.