A collection of 611
Pseudomonas
isolated from 14 sampling sites along the Danube River were identified previously by MALDI-TOF MS with the VITEK MS system and were grouped in 53 clusters by their main protein profiles. The strains were identified in the present study at the phylospecies level by
rpoD
gene sequencing. Partial sequences of the
rpoD
gene of 190 isolates representatives of all clusters were analyzed. Strains in the same MALDI-TOF cluster were grouped in the same phylospecies when they shared a minimum 95% similarity in their
rpoD
sequences. The sequenced strains were assigned to 34 known species (108 strains) and to 32 possible new species (82 strains). The 611 strains were identified at the phylospecies level combining both methods. Most strains were assigned to phylospecies in the
Pseudomonas putida
phylogenetic group of species. Special attention was given to 14 multidrug resistant strains that could not be assigned to any known
Pseudomonas
species and were considered environmental reservoir of antibiotic resistance genes. Coverage indices and rarefaction curves demonstrated that at least 50% of the
Pseudomonas
species in the Danube River able to grow in the isolation conditions have been identified at the species level. Main objectives were the confirmation of the correlation between the protein profile clusters detected by MALDI-TOF MS and the phylogeny of
Pseudomonas
strains based on the
rpoD
gene sequence, the assessment of the higher species discriminative power of the
rpoD
gene sequence, as well as the estimation of the high diversity of
Pseudomonas
ssp. along the Danube river. This study highlights the
Pseudomonas
species diversity in freshwater ecosystems and the usefulness of the combination of MALDI-TOF mass spectrometry for the dereplication of large sets of strains and the
rpoD
gene sequences for rapid and accurate identifications at the species level.
The growing problem of antibiotic resistance in Gram-negative pathogens is linked to three key aspects, (i) the progressive worldwide epidemic spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) Gram-negative strains, (ii) a decrease in the number of effective new antibiotics against multiresistant isolates, and (iii) the lack of mechanistically informed combinations and dosing strategies. Our combined efforts should focus not only on the development of new antimicrobial agents but the adequate administration of these in combination with other agents currently available in the clinic.
Avibactam belongs to the new class of diazabicyclooctane β-lactamase inhibitors. Its inhibitory spectrum includes class A, C and D enzymes, including P. aeruginosa AmpC. Nonetheless, recent reports have revealed strain-dependent avibactam AmpC induction. In the present work, we wanted to assess the mechanistic basis underlying AmpC induction and determine if derepressed PDC-X mutated enzymes from ceftazidime/avibactam-resistant clinical isolates were further inducible. We determined avibactam concentrations that half-maximally inhibited (IC50) bocillin FL binding. Inducer β-lactams were also studied as comparators. Live cells’ time-course penicillin-binding proteins (PBPs) occupancy of avibactam was studied. To assess the ampC induction capacity of avibactam and comparators, qRT-PCR was performed in wild-type PAO1, PBP4, triple PBP4, 5/6 and 7 knockout derivatives and two ceftazidime/avibactam-susceptible/resistant XDR clinical isolates belonging to the epidemic high-risk clone ST175. PBP4 inhibition was observed for avibactam and β-lactam comparators. Induction capacity was consistently correlated with PBP4 binding affinity. Outer membrane permeability-limited PBP4 binding was observed in the live cells’ assay. As expected, imipenem and cefoxitin showed strong induction in PAO1, especially for carbapenem; avibactam induction was conversely weaker. Overall, the inducer effect was less remarkable in ampC-derepressed mutants and nonetheless absent upon avibactam exposure in the clinical isolates harboring mutated AmpC variants and their parental strains.
The manuscript represents the first
N. gonorrhoeae
PBP-binding data set for 22 chemically different drugs in two type strains with different genetic background. We have identified three clusters of drugs according to their PBP binding IC
50
s and highlighted the binding differences across the two strains studied.
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