Percorrer por autor "Bonnet, Richard"
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- Biochemical characterization of CTX-M-166, a new CTX-M β-lactamase produced by a commensal Escherichia coli isolatePublication . Manageiro, Vera; Graça, Rafael; Ferreira, Eugénia; Clemente, Lurdes; Bonnet, Richard; Caniça, ManuelaIn this study, we biochemically characterized the new CTX-M-166 β-lactamase detected in a ceftiofur-resistant Escherichia coli recovered in May 2014 from a six-week-old Gallus gallus broiler flock in an industrial poultry unit in the central region of Portugal.
- Biochemical study of a new inhibitor-resistant beta-lactamase, SHV-84, produced by a clinical Escherichia coli strainPublication . Manageiro, Vera; Ferreira, Eugénia; Albuquerque, Luís; Bonnet, Richard; Caniça, Manuela
- BlaGES-6 producing Pseudomonas aeruginosa ST235 is involved in resistance to different β-lactamsPublication . de Sousa, Telma; Machado, Sandro; Carvalho, Márcia; Caniça, Manuela; Ramos, Miguel J.N.; Santos, Daniela; Beyrouthy, Racha; Bonnet, Richard; Hébraud, Michel; Gomes, João Paulo; Igrejas, Gilberto; Poeta, PatríciaMultidrug resistance in Pseudomonas aeruginosa, particularly resistance to carbapenem, represents a major challenge for public health. This study investigated resistance mechanisms in three P. aeruginosa isolates: HU63 (blaGES-6 carbapenemase-positive), HU141 (carbapenem-resistant without carbapenemase), and PAO1 (control). Genomic analysis revealed distinct sequence types (ST235 for HU63, ST253 for HU141) and chromosomal integration of resistance genes. HU63 harbored diverse resistance mechanisms, including β-lactamases (bla, bla, bla) and efflux pumps. Minimum inhibitory concentration assays demonstrated HU63's resistance to all β-lactams tested (meropenem, imipenem-cilastatin, ceftazidime, piperacillin-tazobactam), while HU141 remained susceptible except to cefoxitin and cloxacillin. Time-kill assays revealed tolerance phenotypes, with HU63 showing regrowth after 8-24 h despite initial reductions in bacterial density. Gene expression varied significantlydepending on the antibiotic and the isolate. The HU63 isolate (GES-6 positive) stands out for its marked induction of bla in all the antibiotics tested, contributing to its resistance to carbapenems and broad-spectrum cephalosporins. These expression profiles corroborate the classic molecular mechanisms of resistance: regulation of entry pores (oprD), activation of efflux pumps (mexA) and production of β-lactamases (bla, ampC) adapted to each situation. These findings underscore the multifactorial nature of resistance in Carbapenem-resistant Pseudomonas aeruginosa (CRPA), combining enzymatic inactivation, efflux, and genetic adaptability. The study emphasizes the urgent need for genomic surveillance to track high-risk clones and develop therapies targeting tolerance mechanisms alongside traditional resistance.
- Characterization of the inhibitor-resistant SHV β-lactamase SHV-107 in a clinical Klebsiella pneumoniae strain co-producing GES-7 enzymePublication . Manageiro, Vera; Ferreira, Eugénia; Cougnoux, Antony; Albuquerque, Luís; Caniça, Manuela; Bonnet, RichardThe clinical Klebsiella pneumoniae INSRA6884 strain exhibited nonsusceptibility to all penicillins tested (MICs of 64 to>2,048 g/ml). The MICs of penicillins were weakly reduced by clavulanate (from 2,048 to 512 g/ml), and tazobactam restored piperacillin susceptibility. Molecular characterization identified the genes blaGES-7 and a new -lactamase gene, blaSHV-107, which encoded an enzyme that differed from SHV-1 by the amino acid substitutions Leu35Gln and Thr235Ala. The SHV-107-producing Escherichia coli strain exhibited only a -lactam resistance phenotype with respect to amoxicillin, ticarcillin, and amoxicillinclavulanate combination. The kinetic parameters of the purified SHV-107 enzyme revealed a high affinity for penicillins. However, catalytic efficiency for these antibiotics was lower for SHV-107 than for SHV-1. No hydrolysis was detected against oxyimino- -lactams. The 50% inhibitory concentration (IC50) for clavulanic acid was 9-fold higher for SHV-107 than for SHV-1, but the inhibitory effects of tazobactam were unchanged. Molecular dynamics simulation suggested that the Thr235Ala substitution affects the accommodation of clavulanate in the binding site and therefore its inhibitory activity.
- Mutational Analysis of Colistin-Resistant Pseudomonas aeruginosa Isolates: From Genomic Background to Antibiotic ResistancePublication . De Sousa, Telma; Wang, Hsin-Yao; Lin, Ting-Wei; Caniça, Manuela; Ramos, Miguel J.N.; Santos, Daniela; Silva, Catarina; Saraiva, Sónia; Beyrouthy, Racha; Bonnet, Richard; Hébraud, Michel; Igrejas, Gilberto; Poeta, PatríciaThis study analyzed eleven isolates of colistin-resistant Pseudomonas aeruginosa, originating from Portugal and Taiwan, which are associated with various pathologies. The results revealed significant genetic diversity among the isolates, with each exhibiting a distinct genetic profile. A prevalence of sequence type ST235 was observed, characterizing it as a high-risk clone, and serotyping indicated a predominance of type O11, associated with chronic respiratory infections in cystic fibrosis (CF) patients. The phylogenetic analysis demonstrated genetic diversity among the isolates, with distinct clades and complex evolutionary relationships. Additionally, transposable elements such as Tn3 and IS6 were identified in all isolates, highlighting their importance in the mobility of antibiotic resistance genes. An analysis of antimicrobial resistance profiles revealed pan-drug resistance in all isolates, with a high prevalence of genes conferring resistance to β-lactams and aminoglycosides. Furthermore, additional analyses revealed mutations in regulatory networks and specific loci previously implicated in colistin resistance, such as pmrA, cprS, phoO, and others, suggesting a possible contribution to the observed resistant phenotype. This study has a strong impact because it not only reveals the genetic diversity and resistance mechanisms in P. aeruginosa but also identifies mutations in regulatory genes associated with colistin resistance.
- Next-Generation Sequencing and MALDI Mass Spectrometry in the Study of Multiresistant Processed Meat Vancomycin-Resistant Enterococci (VRE)Publication . Sabença, Carolina; de Sousa, Telma; Oliveira, Soraia; Viala, Didier; Théron, Laetitia; Chambon, Christophe; Hébraud, Michel; Beyrouthy, Racha; Bonnet, Richard; Caniça, Manuela; Poeta, Patrícia; Igrejas, GilbertoVancomycin-resistant enterococci (VRE), due to their intrinsic resistance to various commonly used antibiotics and their malleable genome, make the treatment of infections caused by these bacteria less effective. The aims of this work were to characterize isolates of Enterococcus spp. that originated from processed meat, through phenotypic and genotypic techniques, as well as to detect putative antibiotic resistance biomarkers. The 19 VRE identified had high resistance to teicoplanin (89%), tetracycline (94%), and erythromycin (84%) and a low resistance to kanamycin (11%), gentamicin (11%), and streptomycin (5%). Based on a Next-Generation Sequencing NGS technique, most isolates were vanA-positive. The most prevalent resistance genes detected were erm(B) and aac(6')-Ii, conferring resistance to the classes of macrolides and aminoglycosides, respectively. MALDI-TOF mass spectrometry (MS) analysis detected an exclusive peak of the Enterococcus genus at m/z (mass-to-charge-ratio) 4428 ± 3, and a peak at m/z 6048 ± 1 allowed us to distinguish Enterococcusfaecium from the other species. Several statistically significant protein masses associated with resistance were detected, such as peaks at m/z 6358.27 and m/z 13237.3 in ciprofloxacin resistance isolates. These results reinforce the relevance of the combined and complementary NGS and MALDI-TOF MS techniques for bacterial characterization.
- Two novel CMY-2-type β-lactamases encountered in clinical Escherichia coli isolatesPublication . Manageiro, Vera; Ferreira, Eugénia; Pinto, Margarida; Fonseca, Fernando; Ferreira, Mónica; Bonnet, Richard; Caniça, ManuelaBACKGROUND: Chromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.