Browsing by Author "Alcalde-Rico, Manuel"

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    Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in Chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa
    (2024) Soto, Katherine D.; Alcalde-Rico, Manuel; Ugalde, Juan A.; Olivares-Pacheco, Jorge; Quiroz, Valeria; Brito, Barbara; Rivas, Lina M.; Munita, Jose M.; Garcia, Patricia C.; Wozniak, Aniela
    Introduction Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum beta-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Methods Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla(PER) gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla(PER-3) gene was induced in a PER-positive isolate by successive passages at 43 degrees C without antibiotics. Results Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla(PER). One isolate carried bla(PER) but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla(PER-3) gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum beta-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla(PER-3) gene restored susceptibility to CZA, ceftolozane/tazobactam and other beta-lactamsin the in vitro evolved isolate. Discussion PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with bla(PER-3) gene implicated in resistance to CZA and other beta-lactams.
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    Dynamics of the MRSA Population in a Chilean Hospital: a Phylogenomic Analysis (2000-2016)
    (2023) Martinez, Jose R. W.; Planet, Paul J.; Spencer-Sandino, Maria; Rivas, Lina; Diaz, Lorena; Moustafa, Ahmed M.; Quesille-Villalobos, Ana; Riquelme-Neira, Roberto; Alcalde-Rico, Manuel; Hanson, Blake; Carvajal, Lina P.; Rincon, Sandra; Reyes, Jinnethe; Lam, Marusella; Calderon, Juan F.; Araos, Rafael; Garcia, Patricia; Arias, Cesar A.; Munita, Jose M.
    Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or more limited typing techniques that lack the resolution to represent an accurate description of the genomic landscape.
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    Real-World Performance of Susceptibility Testing for Ceftolozane/Tazobactam against Non-Carbapenemase- Producing Carbapenem-Resistant Pseudomonas aeruginosa
    (2022) Rivas, Lina; Martinez, José R.W.; Munita, José M.; Alcalde-Rico, Manuel; Olivares Pacheco, Jorge; García Cañete, Patricia; Olivares-Pacheco J.; Moreno, María Victoria; Rojas, Pamela; Wozniak Banchero, Aniela; Miller, William; Arias, Cesar A.; Khan, Ayesha
    Ceftolozane/tazbactam (C/T) is a potent anti-pseudomonal agent that has clinical utility against infections caused by non-carbapenemase, producing-carbapenemresistant Pseudomonas aeruginosa (non-CP-CR-PA). Accurate, precise, and reliable antimicrobial susceptibility testing (AST) is crucial to guide clinical decisions. However, studies assessing the performance of different AST methods against non-CP-CR-PA (the main clinical niche for C/T), are lacking. Here, we evaluated performance of gradient strips (Etest and MIC test strip [MTS], and disk diffusion [DD]) using CLSI breakpoints. Additionally, we assessed the performance of DD using EUCAST breakpoints. For all susceptibility tests, we used a collection of 97 non-CP-CR-PA clinical isolates recovered from 11 Chilean hospitals. Both gradient strips and DD had acceptable performance when using CLSI breakpoints, yielding a categorical agreement (CA) of .90% and 92%, respectively. In contrast, DD using EUCAST breakpoints performed suboptimally (CA 81%). MTS yielded a higher essential agreement (EA, .90%) than Etest (84%). Importantly, the performance of all methods varied significantly when the isolates were stratified by their degree of susceptibility to other anti-pseudomonal b-lactams. All methods had 100% CA when testing isolates that were pan-susceptible to all b-lactams (Pan-β-S). However, the CA markedly decreased when testing isolates resistant to all b-lactams (Pan-β-R). Indeed, the CA was 81% for Etest (six errors), 78% for MTS (seven errors), and 78% and 56% for DD when using CLSI (seven errors) or EUCAST breakpoints (14 errors), respectively. Our results suggest that all manual AST methods have strikingly decreased performance in the context of Pan-β-R P. aeruginosa with potentially major clinical implications.
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    Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa
    (2022) Jose Contreras-Gomez, Maria; Martinez, Jose R. W.; Rivas, Lina; Riquelme-Neira, Roberto; Ugalde, Juan A.; Wozniak, Aniela; Garcia, Patricia; Munita, Jose M.; Olivares-Pacheco, Jorge; Alcalde-Rico, Manuel
    Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine beta-naphthylamide (PA beta N). In the absence of PA beta N, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PA beta N showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PA beta N [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.