Browsing by Author "Olivares-Pacheco, Jorge"

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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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    Genomic characterisation of the population structure and antibiotic resistance of Salmonella enterica serovar Infantis in Chile, 2009-2022
    (2024) Pina-Iturbe, Alejandro; Diaz-Gavidia, Constanza; Alvarez, Francisca P.; Barron-Montenegro, Rocio; Alvarez-Espejo, Diana M.; Garcia, Patricia; Solis, Doina; Constenla-Albornoz, Rodrigo; Toro, Magaly; Olivares-Pacheco, Jorge; Reyes-Jara, Angelica; Meng, Jianghong; Bell, Rebecca L.; Moreno-Switt, Andrea I.
    Background Multidrug-resistant (MDR) Salmonella Infantis has disseminated worldwide, mainly linked to the consumption of poultry products. Evidence shows dissemination of this pathogen in Chile; however, studies are primarily limited to phenotypic data or involve few isolates. As human cases of Salmonella Infantis infections have substantially increased in recent years, this study aimed to characterise the genomic epidemiology and antimicrobialresistance profiles of isolates obtained from different sources, aiming to inform effective surveillance and control measures. Methods We sequenced 396 Salmonella Infantis genomes and analysed them with all publicly available genomes of this pathogen from Chile (440 genomes in total), representing isolates from environmental, food, animal, and human sources obtained from 2009 to 2022. Based on bioinformatic and phenotypic methods, we assessed the population structure, dissemination among different niches, and antimicrobial resistance (AMR) profiles of Salmonella Infantis in the country. Findings The genomic and phylogenetic analyses showed that Salmonella Infantis from Chile comprised several clusters of highly related isolates dominated by sequence type 32. The HC20_343 cluster grouped an important proportion of all isolates. This was the only cluster associated with pESI-like megaplasmids, and up to 12 acquired AMR genes/mutations predicted to result in an MDR phenotype. Accordingly, antimicrobial -susceptibility testing revealed a strong concordance between the AMR genetic determinants and their matching phenotypic expression, indicating that a significant proportion of HC20_343 isolates produce extended -spectrum beta-lactamases and have intermediate fluoroquinolone resistance. HC20_343 Salmonella Infantis were spread among environmental, animal, food, and human niches, showing a close relationship between isolates from different years and sources, and a low intra-source genomic diversity. Interpretation Our findings show a widespread dissemination of MDR Salmonella Infantis from the HC20_343 cluster in Chile. The high proportion of isolates with resistance to first -line antibiotics and the evidence of active transmission between the environment, animals, food, and humans highlight the urgency of improved surveillance and control measures in the country. As HC20_343 isolates predominate in the Americas, our results suggest a high prevalence of ESBL-producing Salmonella Infantis with intermediate fluoroquinolone resistance in the continent.
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    Humans and Hoofed Livestock Are the Main Sources of Fecal Contamination of Rivers Used for Crop Irrigation: A Microbial Source Tracking Approach
    (FRONTIERS MEDIA SA, 2022) Diaz-Gavidia, Constanza; Barria, Carla; Weller, Daniel L.; Salgado-Caxito, Marilia; Estrada, Erika M.; Araya, Anibal; Vera, Leonardo; Smith, Woutrina; Kim, Minji; Moreno-Switt, Andrea I.; Olivares-Pacheco, Jorge; Adell, Aiko D.
    Freshwater bodies receive waste, feces, and fecal microorganisms from agricultural, urban, and natural activities. In this study, the probable sources of fecal contamination were determined. Also, antibiotic resistant bacteria (ARB) were detected in the two main rivers of central Chile. Surface water samples were collected from 12 sampling sites in the Maipo (n = 8) and Maule Rivers (n = 4) every 3 months, from August 2017 until April 2019. To determine the fecal contamination level, fecal coliforms were quantified using the most probable number (MPN) method and the source of fecal contamination was determined by Microbial Source Tracking (MST) using the Cryptosporidium and Giardia genotyping method. Separately, to determine if antimicrobial resistance bacteria (AMB) were present in the rivers, Escherichia coli and environmental bacteria were isolated, and the antibiotic susceptibility profile was determined. Fecal coliform levels in the Maule and Maipo Rivers ranged between 1 and 130 MPN/100-ml, and 2 and 30,000 MPN/100-ml, respectively. Based on the MST results using Cryptosporidium and Giardia host-specific species, human, cattle, birds, and/or dogs hosts were the probable sources of fecal contamination in both rivers, with human and cattle host-specific species being more frequently detected. Conditional tree analysis indicated that coliform levels were significantly associated with the river system (Maipo versus Maule), land use, and season. Fecal coliform levels were significantly (p < 0.006) higher at urban and agricultural sites than at sites immediately downstream of treatment centers, livestock areas, or natural areas. Three out of eight (37.5%) E. coli isolates presented a multidrug-resistance (MDR) phenotype. Similarly, 6.6% (117/1768) and 5.1% (44/863) of environmental isolates, in Maipo and Maule River showed and MDR phenotype. Efforts to reduce fecal discharge into these rivers should thus focus on agriculture and urban land uses as these areas were contributing the most and more frequently to fecal contamination into the rivers, while human and cattle fecal discharges were identified as the most likely source of this fecal contamination by the MST approach. This information can be used to design better mitigation strategies, thereby reducing the burden of waterborne diseases and AMR in Central Chile.
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    Qualitative Risk Assessment for Antimicrobial Resistance among Humans from Salmon Fillet Consumption Due to the High Use of Antibiotics against Bacterial Infections in Farmed Salmon
    (MDPI, 2022) Salgado-Caxito, Marilia; Zimin-Veselkoff, Natalia; Adell, Aiko D.; Olivares-Pacheco, Jorge; Mardones, Fernando O.
    Background: Worldwide, aquaculture is considered as a hotspot environment for antimicrobial resistance (AMR) due to the intense use of antibiotics in its productive systems. Chile is the second largest producer of farmed salmon worldwide, and tons of antibiotics are used to control bacterial diseases, such as Salmon Rickettsial Syndrome (SRS) and Bacterial Kidney Disease (BKD). However, studies determining the risk of consuming salmon fillets that have been treated with antibiotics during the salmon production are limited. Consulting leading experts in the field could provide a knowledge base to identify and address this question and research gaps. Methods: Multisectoral risk perception of AMR through salmon fillet consumption was evaluated by eliciting expert data obtained through discussions during a workshop and from questionnaires given to experts from academia (n = 15, 63%), the public sector (n = 5, 21%), and the salmon industry (n = 4, 17%). Results: The qualitative risk analysis suggested an overall 'low' probability of AMR acquisition by consumption of salmon fillet that had been treated during the production cycle. The risk perception varied slightly between production stages in freshwater and seawater. In consensus with all sectors, this overall 'low', but existing, risk was probably associated with bacterial infections and the use of antibiotics. Conclusions: As it is essential to reduce the use of antibiotics in the Chilean salmon industry, this intersectoral approach and consensual results could favor effective implementation of targeted initiatives for the control and prevention of major bacterial diseases.
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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.
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    Widespread dissemination of ESBL-producing Salmonella enterica serovar Infantis exhibiting intermediate fluoroquinolone resistance and harboring blaCTX-M-65-positive pESI-like megaplasmids in Chile
    (bioRxiv, 2023) Piña Iturbe, Luis Alejandro; Díaz Gavidia, Constanza Paz; Álvarez Rojas, Francisca Pía; Barron Montenegro, Rocío Nicole; Álvarez Espejo, Diana Claudia Marcela; García Cañete, Patricia; Solís, Doina; Constenla-Albornoz, Rodrigo; Toro Parada, Magaly Rosana; Olivares-Pacheco, Jorge; Reyes-Jara, Angélica; Meng, Jianghong; Bell, Rebecca L.; Moreno Switt, Andrea Isabel