| 1. |
- Barceló, Isabel M., et al.
(author)
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Filling knowledge gaps related to AmpC-dependent β-lactam resistance in Enterobacter cloacae
- 2024
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 14:1
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Journal article (peer-reviewed)abstract
- Enterobacter cloacae starred different pioneer studies that enabled the development of a widely accepted model for the peptidoglycan metabolism-linked regulation of intrinsic class C cephalosporinases, highly conserved in different Gram-negatives. However, some mechanistic and fitness/virulence-related aspects of E. cloacae choromosomal AmpC-dependent resistance are not completely understood. The present study including knockout mutants, β-lactamase cloning, gene expression analysis, characterization of resistance phenotypes, and the Galleria mellonella infection model fills these gaps demonstrating that: (i) AmpC enzyme does not show any collateral activity impacting fitness/virulence; (ii) AmpC hyperproduction mediated by ampD inactivation does not entail any biological cost; (iii) alteration of peptidoglycan recycling alone or combined with AmpC hyperproduction causes no attenuation of E. cloacae virulence in contrast to other species; (iv) derepression of E. cloacae AmpC does not follow a stepwise dynamics linked to the sequential inactivation of AmpD amidase homologues as happens in Pseudomonas aeruginosa; (v) the enigmatic additional putative AmpC-type β-lactamase generally present in E. cloacae does not contribute to the classical cephalosporinase hyperproduction-based resistance, having a negligible impact on phenotypes even when hyperproduced from multicopy vector. This study reveals interesting particularities in the chromosomal AmpC-related behavior of E. cloacae that complete the knowledge on this top resistance mechanism.
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| 2. |
- Barceló, Isabel M., et al.
(author)
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Role of enzymatic activity in the biological cost associated with the production of ampC b-lactamases in pseudomonas aeruginosa
- 2022
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In: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 10:5
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Journal article (peer-reviewed)abstract
- In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC β-lactamase hyperproduction is probably themost important resistancemechanismof Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new β-lactam-β-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that β-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type β-lactamases such as FOX enzymes, some of which also provide resistance to new β-lactam-β-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type β-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations.
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| 3. |
- Barceló, Isabel M., et al.
(author)
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Transferable AmpCs in Klebsiella pneumoniae : interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications
- 2024
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In: Antimicrobial Agents and Chemotherapy. - : American Society for Microbiology. - 0066-4804 .- 1098-6596. ; 68:5
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Journal article (peer-reviewed)abstract
- Chromosomal and transferable AmpC β-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to β-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most β-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable β-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.
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| 4. |
- Escobar-Salom, María, et al.
(author)
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Bacterial virulence regulation through soluble peptidoglycan fragments sensing and response : knowledge gaps and therapeutic potential
- 2023
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In: FEMS Microbiology Reviews. - : Oxford University Press. - 0168-6445 .- 1574-6976. ; 47:2
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Research review (peer-reviewed)abstract
- Given the growing clinical-epidemiological threat posed by the phenomenon of antibiotic resistance, new therapeutic options are urgently needed, especially against top nosocomial pathogens such as those within the ESKAPE group. In this scenario, research is pushed to explore therapeutic alternatives and, among these, those oriented toward reducing bacterial pathogenic power could pose encouraging options. However, the first step in developing these antivirulence weapons is to find weak points in the bacterial biology to be attacked with the goal of dampening pathogenesis. In this regard, during the last decades some studies have directly/indirectly suggested that certain soluble peptidoglycan-derived fragments display virulence-regulatory capacities, likely through similar mechanisms to those followed to regulate the production of several β-lactamases: binding to specific transcriptional regulators and/or sensing/activation of two-component systems. These data suggest the existence of intra- and also intercellular peptidoglycan-derived signaling capable of impacting bacterial behavior, and hence likely exploitable from the therapeutic perspective. Using the well-known phenomenon of peptidoglycan metabolism-linked β-lactamase regulation as a starting point, we gather and integrate the studies connecting soluble peptidoglycan sensing with fitness/virulence regulation in Gram-negatives, dissecting the gaps in current knowledge that need filling to enable potential therapeutic strategy development, a topic which is also finally discussed.
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