RESUMO

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 ß-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°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 ß-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 ß-lactamsin the in vitro evolved isolate. Discussion: PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other ß-lactams.

Assuntos

Antibacterianos , Compostos Azabicíclicos , Proteínas de Bactérias , Ceftazidima , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas , Pseudomonas aeruginosa , beta-Lactamases , Ceftazidima/farmacologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Compostos Azabicíclicos/farmacologia , Humanos , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Infecções por Pseudomonas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Chile , Sequenciamento Completo do Genoma , Mutação

RESUMO

OBJECTIVES: Extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) are a main cause of human deaths associated with antimicrobial resistance (AMR). Despite hundreds of reports of the faecal carriage of ESBL-E. coli in domestic and wild animals, the dynamics of its circulation remains poorly understood. METHODS: We used whole genome sequencing of 19 ESBL-E. coli previously isolated in the same local setting from dogs, livestock, and a wild rodent in Central Chile to assess potential cross-species transmission of ESBL-E. coli. RESULTS: Isolates harboured a large number of AMR (n = 95) and virulence (n = 45) genes, plasmids replicons (n = 24), and E. coli sequence types including top extraintestinal pathogenic E. coli ST410, ST58, ST88, and ST617. Almost identical clones (<50 single nucleotide polymorphisms difference, same antibiotic and heavy metal resistance genes, virulence genes, and plasmids) were found in faeces of dogs, cattle, or sheep from the same farm, and in a dog and a wild rodent living in proximity. CONCLUSIONS: To our knowledge, this is the first report of multiple clonal cross-species transmission of ESBL-E. coli in domestic and potentially wild animals of Latin America. Our results suggest that relatively rare spread of AMR across animal species can still occur by both clonal and plasmid dissemination. Our study highlights the need for establishing preventive measures to limit the circulation of these bacteria among animals in agricultural settings, particularly given the highly pathogenic profile of several E. coli strains detected in these animals.

Assuntos

Infecções por Escherichia coli , Escherichia coli , Humanos , Animais , Cães , Bovinos , Ovinos , Escherichia coli/genética , Animais Selvagens , Gado/microbiologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Infecções por Escherichia coli/microbiologia , Chile/epidemiologia , beta-Lactamases/genética

RESUMO

BACKGROUND: High rates of antibiotic use (AU) among inpatients with coronavirus disease 2019 (COVID-19) despite low rates of bacterial coinfection and secondary infection have been reported. We evaluated the impact of the COVID-19 pandemic on AU in healthcare facilities (HCFs) in South America. METHODS: We conducted an ecologic evaluation of AU in inpatient adult acute care wards in 2 HCFs each in Argentina, Brazil, and Chile. The AU rates for intravenous antibiotics were calculated as the defined daily dose per 1000 patient-days, using pharmacy dispensing records and hospitalization data from March 2018-February 2020 (prepandemic) and March 2020-February 2021 (pandemic). Differences in median AU were compared between the prepandemic and pandemic periods, using the Wilcoxon rank sum test to determine significance. Interrupted time series analysis was used to analyze changes in AU during the COVID-19 pandemic. RESULTS: Compared with the prepandemic period, the median difference in AU rates for all antibiotics combined increased in 4 of 6 HCFs (percentage change, 6.7%-35.1%; P < .05). In the interrupted time series models, 5 of 6 HCFs had significant increases in use of all antibiotics combined immediately at the onset of the pandemic (immediate effect estimate range, 15.4-268), but only 1 of these 5 HCFs experienced a sustained increase over time (change in slope, +8.13; P < .01). The effect of the pandemic onset varied by antibiotic group and HCF. CONCLUSIONS: Substantial increases in AU were observed at the beginning of the COVID-19 pandemic, suggesting the need to maintain or strengthen antibiotic stewardship activities as part of pandemic or emergency HCF responses.

Assuntos

Antibacterianos , COVID-19 , Humanos , Adulto , Antibacterianos/uso terapêutico , COVID-19/epidemiologia , Pacientes Internados , Pandemias , Chile/epidemiologia , Argentina/epidemiologia , Brasil

RESUMO

BACKGROUND: Antimicrobial resistance is a global threat, heavily impacting low- and middle-income countries. This study estimated antimicrobial-resistant gram-negative bacteria (GNB) fecal colonization prevalence in hospitalized and community-dwelling adults in Chile before the coronavirus disease 2019 pandemic. METHODS: From December 2018 to May 2019, we enrolled hospitalized adults in 4 public hospitals and community dwellers from central Chile, who provided fecal specimens and epidemiological information. Samples were plated onto MacConkey agar with ciprofloxacin or ceftazidime added. All recovered morphotypes were identified and characterized according to the following phenotypes: fluoroquinolone-resistant (FQR), extended-spectrum cephalosporin-resistant (ESCR), carbapenem-resistant (CR), or multidrug-resistant (MDR; as per Centers for Disease Control and Prevention criteria) GNB. Categories were not mutually exclusive. RESULTS: A total of 775 hospitalized adults and 357 community dwellers were enrolled. Among hospitalized subjects, the prevalence of colonization with FQR, ESCR, CR, or MDR-GNB was 46.4% (95% confidence interval [CI], 42.9-50.0), 41.2% (95% CI, 37.7-44.6), 14.5% (95% CI, 12.0-16.9), and 26.3% (95% CI, 23.2-29.4). In the community, the prevalence of FQR, ESCR, CR, and MDR-GNB colonization was 39.5% (95% CI, 34.4-44.6), 28.9% (95% CI, 24.2-33.6), 5.6% (95% CI, 3.2-8.0), and 4.8% (95% CI, 2.6-7.0), respectively. CONCLUSIONS: A high burden of antimicrobial-resistant GNB colonization was observed in this sample of hospitalized and community-dwelling adults, suggesting that the community is a relevant source of antibiotic resistance. Efforts are needed to understand the relatedness between resistant strains circulating in the community and hospitals.

Assuntos

Anti-Infecciosos , COVID-19 , Infecções por Bactérias Gram-Negativas , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias , Carbapenêmicos , Cefalosporinas , Chile/epidemiologia , Resistência Microbiana a Medicamentos , Farmacorresistência Bacteriana Múltipla , Fluoroquinolonas , Bactérias Gram-Negativas , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Hospitais , Fatores de Risco , Adulto

RESUMO

BACKGROUND: The impact of coronavirus disease 2019 (COVID-19) on antimicrobial use (AU) and resistance has not been well evaluated in South America. These data are critical to inform national policies and clinical care. METHODS: At a tertiary hospital in Santiago, Chile, between 2018 and 2022, subdivided into pre- (3/2018-2/2020) and post-COVID-19 onset (3/2020-2/2022), we evaluated intravenous AU and frequency of carbapenem-resistant Enterobacterales (CRE). We grouped monthly AU (defined daily doses [DDD]/1000 patient-days) into broad-spectrum ß-lactams, carbapenems, and colistin and used interrupted time-series analysis to compare AU during pre- and post-pandemic onset. We studied the frequency of carbapenemase-producing (CP) CRE and performed whole-genome sequencing analyses of all carbapenem-resistant (CR) Klebsiella pneumoniae (CRKpn) isolates collected during the study period. RESULTS: Compared with pre-pandemic, AU (DDD/1000 patient-days) significantly increased after the pandemic onset, from 78.1 to 142.5 (P < .001), 50.9 to 110.1 (P < .001), and 4.1 to 13.3 (P < .001) for broad-spectrum ß-lactams, carbapenems, and colistin, respectively. The frequency of CP-CRE increased from 12.8% pre-COVID-19 to 51.9% after pandemic onset (P < .001). The most frequent CRE species in both periods was CRKpn (79.5% and 76.5%, respectively). The expansion of CP-CRE harboring blaNDM was particularly noticeable, increasing from 40% (n = 4/10) before to 73.6% (n = 39/53) after pandemic onset (P < .001). Our phylogenomic analyses revealed the emergence of two distinct genomic lineages of CP-CRKpn: ST45, harboring blaNDM, and ST1161, which carried blaKPC. CONCLUSIONS: AU and the frequency of CP-CRE increased after COVID-19 onset. The increase in CP-CRKpn was driven by the emergence of novel genomic lineages. Our observations highlight the need to strengthen infection prevention and control and antimicrobial stewardship efforts.

Assuntos

Anti-Infecciosos , COVID-19 , Enterobacteriáceas Resistentes a Carbapenêmicos , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Klebsiella pneumoniae/genética , Chile/epidemiologia , Colistina , Pacientes Internados , Filogenia , Pandemias , COVID-19/epidemiologia , Proteínas de Bactérias/genética , beta-Lactamases/genética , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Hospitais , beta-Lactamas , Testes de Sensibilidade Microbiana

RESUMO

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P < 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. IMPORTANCE 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. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000 and 2016 in Chile providing the largest and most detailed study of clonal dynamics of MRSA in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing in frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

Assuntos

Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/epidemiologia , Chile/epidemiologia , Filogenia , Centros de Atenção Terciária , Antibacterianos

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

RESUMO

Background: Antimicrobial resistance (AMR) is among the most critical global health threats of the 21st century. AMR is primarily driven by the use and misuse of antibiotics but can be affected by socioeconomic and environmental factors. Reliable and comparable estimates of AMR over time are essential to making public health decisions, defining research priorities, and evaluating interventions. However, estimates for developing regions are scant. We describe the evolution of AMR for critical priority antibiotic-bacterium pairs in Chile and examine their association with hospital and community-level characteristics using multivariate rate-adjusted regressions. Methods: Drawing on multiple data sources, we assembled a longitudinal national dataset to analyse AMR levels for critical priority antibiotic-bacterium combinations in 39 private and public hospitals (2008-2017) throughout the country and characterize the population at the municipality level. We first described trends of AMR in Chile. Second, we used multivariate regressions to examine the association of AMR with hospital characteristics and community-level socioeconomic, demographic, and environmental factors. Last, we estimated the expected distribution of AMR by region in Chile. Findings: Our results show that AMR for priority antibiotic-bacterium pairs steadily increased between 2008 and 2017 in Chile, driven primarily by Klebsiella pneumoniae resistant to third-generation cephalosporins and carbapenems, and vancomycin-resistant Enterococcus faecium. Higher hospital complexity, a proxy for antibiotic use, and poorer local community infrastructure were significantly associated with greater AMR. Interpretation: Consistent with research in other countries in the region, our results show a worrisome increase in clinically relevant AMR in Chile and suggest that hospital complexity and living conditions in the community may affect the emergence and spread of AMR. Our results highlight the importance of understanding AMR in hospitals and their interaction with the community and the environment to curtail this ongoing public health crisis. Funding: This research was supported by the Agencia Nacional de Investigación y Desarrollo (ANID), Fondo Nacional de Desarrollo Científico y Tecnológico FONDECYT, The Canadian Institute for Advanced Research (CIFAR), and Centro UC de Políticas Públicas, Pontificia Universidad Católica de Chile.

RESUMO

Ceftazidime-avibactam (CZA) is the combination of a third-generation cephalosporin and a new non-ß-lactam ß-lactamase inhibitor capable of inactivating class A, C, and some D ß-lactamases. From a collection of 2,727 clinical isolates of Enterobacterales (n = 2,235) and P. aeruginosa (n = 492) that were collected between 2016 and 2017 from five Latin American countries, we investigated the molecular resistance mechanisms to CZA of 127 (18/2,235 [0.8%] Enterobacterales and 109/492 [22.1%] P. aeruginosa). First, by qPCR for the presence of genes encoding KPC, NDM, VIM, IMP, OXA-48-like, and SPM-1 carbapenemases, and second, by whole-genome sequencing (WGS). From the CZA-resistant isolates, MBL-encoding genes were detected in all 18 Enterobacterales and 42/109 P. aeruginosa isolates, explaining their resistant phenotype. Resistant isolates that yielded a negative qPCR result for any of the MBL encoding genes were subjected to WGS. The WGS analysis of the 67 remaining P. aeruginosa isolates showed mutations in genes previously associated with reduced susceptibility to CZA, such as those involved in the MexAB-OprM efflux pump and AmpC (PDC) hyperproduction, PoxB (blaOXA-50-like), FtsI (PBP3), DacB (PBP4), and OprD. The results presented here offer a snapshot of the molecular epidemiological landscape for CZA resistance before the introduction of this antibiotic into the Latin American market. Therefore, these results serve as a valuable comparison tool to trace the evolution of the resistance to CZA in this carbapenemase-endemic geographical region. IMPORTANCE In this manuscript, we determine the molecular mechanisms of ceftazidime-avibactam resistance in Enterobacterales and P. aeruginosa isolates from five Latin American countries. Our results reveal a low rate of resistance to ceftazidime-avibactam among Enterobacterales; in contrast, resistance in P. aeruginosa has proven to be more complex, as it might involve multiple known and possibly unknown resistance mechanisms.

Assuntos

Ceftazidima , Infecções por Pseudomonas , Humanos , Ceftazidima/farmacologia , Pseudomonas aeruginosa , América Latina , Antibacterianos/farmacologia , Hospitais

RESUMO

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCC mec I) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in the last years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary healthcare center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000-2016 in a tertiary healthcare center in Chile. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r=0.8748, p<0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016. The temporal trend analysis revealed that in the period 2000-2003 most of the isolates (94.2%; n=98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013-2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCC mec II and ST72-SCC mec VI. In conclusion, the ChC clone remains the most frequent MRSA lineage in Chile. However, this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCC mec II. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. Importance: 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 classical typing techniques with several limitations to depict an accurate description of their genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000-2016 in Chile to provide the largest and most detailed study of clonal dynamics of MRSA carried out in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing their frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

RESUMO

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the distribution and clinical significance of carbapenemases are unclear. The aim of this study was to define characteristics and outcomes of CRPA infections and the global frequency and clinical impact of carbapenemases harboured by CRPA. METHODS: We conducted an observational, prospective cohort study of CRPA isolated from bloodstream, respiratory, urine, or wound cultures of patients at 44 hospitals (10 countries) between Dec 1, 2018, and Nov 30, 2019. Clinical data were abstracted from health records and CRPA isolates were whole-genome sequenced. The primary outcome was 30-day mortality from the day the index culture was collected. We compared outcomes of patients with CRPA infections by infection type and across geographic regions and performed an inverse probability weighted analysis to assess the association between carbapenemase production and 30-day mortality. FINDINGS: We enrolled 972 patients (USA n=527, China n=171, south and central America n=127, Middle East n=91, Australia and Singapore n=56), of whom 581 (60%) had CRPA infections. 30-day mortality differed by infection type (bloodstream 21 [30%] of 69, respiratory 69 [19%] of 358, wound nine [14%] of 66, urine six [7%] of 88; p=0·0012) and geographical region (Middle East 15 [29%] of 52, south and central America 20 [27%] of 73, USA 60 [19%] of 308, Australia and Singapore three [11%] of 28, China seven [6%] of 120; p=0·0002). Prevalence of carbapenemase genes among CRPA isolates also varied by region (south and central America 88 [69%] of 127, Australia and Singapore 32 [57%] of 56, China 54 [32%] of 171, Middle East 27 [30%] of 91, USA ten [2%] of 527; p<0·0001). KPC-2 (n=103 [49%]) and VIM-2 (n=75 [36%]) were the most common carbapenemases in 211 carbapenemase-producing isolates. After excluding USA patients, because few US isolates had carbapenemases, patients with carbapenemase-producing CRPA infections had higher 30-day mortality than those with non-carbapenemase-producing CRPA infections in both unadjusted (26 [22%] of 120 vs 19 [12%] of 153; difference 9%, 95% CI 3-16) and adjusted (difference 7%, 95% CI 1-14) analyses. INTERPRETATION: The emergence of different carbapenemases among CRPA isolates in different geographical regions and the increased mortality associated with carbapenemase-producing CRPA infections highlight the therapeutic challenges posed by these organisms. FUNDING: National Institutes of Health.

Assuntos

Antibacterianos , Infecções por Pseudomonas , Estados Unidos , Humanos , Antibacterianos/uso terapêutico , Pseudomonas aeruginosa/genética , Estudos Prospectivos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/epidemiologia , Carbapenêmicos/uso terapêutico

RESUMO

BACKGROUND: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) compromise the clinical efficacy of vancomycin. The hVISA isolates spontaneously produce vancomycin-intermediate Staphylococcus aureus (VISA) cells generated by diverse and intriguing mechanisms. OBJECTIVE: To characterize the biomolecular profile of clinical hVISA applying genomic, transcriptomic and metabolomic approaches. METHODS: 39 hVISA and 305 VSSA and their genomes were included. Core genome-based Bayesian phylogenetic reconstructions were built and alterations in predicted proteins in VISA/hVISA were interrogated. Linear discriminant analysis and a Genome-Wide Association Study were performed. Differentially expressed genes were identified in hVISA-VSSA by RNA-sequencing. The undirected profiles of metabolites were determined by liquid chromatography and hydrophilic interaction in six CC5-MRSA. RESULTS: Genomic relatedness of MRSA associated to hVISA phenotype was not detected. The change Try38 → His in Atl (autolysin) was identified in 92% of the hVISA. We identified SNPs and k-mers associated to hVISA in 11 coding regions with predicted functions in virulence, transport systems, carbohydrate metabolism and tRNA synthesis. Further, capABCDE, sdrD, esaA, esaD, essA and ssaA genes were overexpressed in hVISA, while lacABCDEFG genes were downregulated. Additionally, valine, threonine, leucine tyrosine, FAD and NADH were more abundant in VSSA, while arginine, glycine and betaine were more abundant in hVISA. Finally, we observed altered metabolic pathways in hVISA, including purine and pyrimidine pathway, CoA biosynthesis, amino acid metabolism and aminoacyl tRNA biosynthesis. CONCLUSIONS: Our results show that the mechanism of hVISA involves major changes in regulatory systems, expression of virulence factors and reduction in glycolysis via TCA cycle. This work contributes to the understanding of the development of this complex resistance mechanism in regional strains.

Assuntos

Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Vancomicina/farmacologia , Staphylococcus aureus/genética , Staphylococcus aureus Resistente à Vancomicina/genética , Estudo de Associação Genômica Ampla , América Latina , Teorema de Bayes , Multiômica , Filogenia , Resistência a Vancomicina/genética , RNA de Transferência , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia

RESUMO

Objectives: Identify molecular mechanisms responsible for the in vitro non-susceptibility to ceftolozane/tazobactam (TOL) in a group of 158 clinical isolates of Pseudomonas aeruginosa from five Latin American countries collected before the introduction of TOL into the clinical practice. Methods: Clinical isolates of P. aeruginosa (n = 504) were collected between January 2016 and October 2017 from 20 hospitals located in Argentina, Brazil, Chile, Colombia, and Mexico. Minimum inhibitory concentrations (MICs) to TOL were determined by standard broth microdilution and interpreted according to CLSI breakpoints. Initially, production of carbapenemases in TOL non-susceptible isolates was assessed by Rapidec® followed by qPCR to detect bla KPC, bla NDM-1, bla VIM, and bla IMP. Illumina® WGS was performed for isolates in which non-susceptibility to TOL was not mediated by carbapenemases. Results: A total of 158 (31.3%) isolates were non-susceptible to TOL. In 74 (46.8%) of these isolates, non-susceptibility to TOL was explained by the production of at least one carbapenemase. WGS revealed that some isolates carried ESBLs, mutated bla PDC and ampD, associated with decreased susceptibility to TOL. Conclusion: Substitutions found in PDC and carbapenemase production were the most common presumed mechanisms of resistance to TOL detected in this study. This study shows that epidemiological surveillance is warranted to monitor the emergence of novel mechanisms of resistance to TOL that might compromise its clinical utility.

RESUMO

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 ß-naphthylamide (PAßN). In the absence of PAßN, 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 PAßN 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 PAßN [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.

RESUMO

La disponibilidad de cepas bacteriana para el estudio de la resistencia bacteriana es clave para los avances en la investigación básica y clínica respecto del tema. Existen pocos biorrepositorios o bancos de bacterias con mecanismos de resistencia conocidos, aisladas de infecciones clínicamente significativas. Una revisión de la literatura revela que sólo en los Estados Unidos de América existe un biobanco de aislados resistentes disponibles para estudios. En esta publicación se cuenta cómo se creó el primer biorrepositorio de bacterias resistentes en Chile asociados a la Red de Laboratorios MICROB-R, con la participación de 11 centros distribuidos a lo largo del país, que a la fecha cuenta con más de 3.500 aislados bacterianos estudiados fenotípica y genotípicamente, disponibles para la comunidad científica chilena.

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The availability of bacterial strains for the study of bacterial resistance is key to advances in basic and clinical research. There are few biobanks of bacteria with known resistance mechanisms, isolated from clinically significant infections. A review of the literature reveals that only in the United States of America is there a biobank of resistant isolates. This publication shows the creation of the first biorepository of resistant bacteria Chile associated with the MICROB-R Laboratory Network, with the participation of 11 centers distributed throughout the country, which to date has more than 3,500 bacterial isolates studied phenotypically and genotypically, available to the Chilean scientific community.

Assuntos

Bactérias/isolamento & purificação , Bancos de Espécimes Biológicos , Farmacorresistência Bacteriana , Chile , Laboratórios

RESUMO

Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing Pseudomonas aeruginosa (KPC-PA) and Enterobacterales. CAZ/AVI resistance is driven by several mechanisms. In P. aeruginosa this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in Enterobacterales it frequently occurs through D179Y substitution in the active site of KPC enzyme. This aminoacid change abolishes AVI binding to the KPC active site, hence inhibition is impaired. However, this substitution also decreases KPC-mediated resistance to carbapenems ("see-saw" effect). The goal of this work was to characterize the in vivo acquisition of CAZ/AVI resistance through D179Y substitution in a KPC-PA isolated from a hospitalized patient after CAZ/AVI treatment. Two KPC-PA isolates were obtained. The first isolate, PA-1, was obtained before CAZ/AVI treatment and was susceptible to CAZ/AVI. The second isolate, PA-2, was obtained after CAZ/AVI treatment and exhibited high-level CAZ/AVI resistance. Characterization of isolates PA-1 and PA-2 was performed through short and long-read whole genome sequencing analysis. The hybrid assembly showed that PA-1 and PA-2A had a single plasmid of 54,030 bp, named pPA-1 and pPA-2 respectively. Each plasmid harbored two copies of the bla KPC-containing Tn4401b transposon. However, while pPA-1 carried two copies of bla KPC-2, pPA-2 had one copy of bla KPC-2 and one copy of bla KPC-33, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the "see-saw" effect. The bla KPC-33 allele was detected only through hybrid assembly using a long-read-first approach. The present work describes a KPC-PA isolate harboring a plasmid-borne CAZ/AVI resistance mechanism based on two copies of bla KPC-2-Tn4401b and D179Y mutation in one of them, that is not associated with loss of resistance to carbapenems. These findings highlight the usefulness of a fine-tuned combined analysis of short and long-read data to detect similar emerging resistance mechanisms.

Assuntos

Ceftazidima , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos , Carbapenêmicos/farmacologia , Ceftazidima/farmacologia , Combinação de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Mutação , Porinas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo

RESUMO

BACKGROUND: Ceftolozane/tazobactam (C/T) is a combination of an antipseudomonal oxyiminoaminothiazolyl cephalosporin with potent in vitro activity against Pseudomonas aeruginosa and tazobactam, a known ß-lactamase inhibitor. The aim of this study was to evaluate the activity of C/T against clinical isolates of P. aeruginosa and Enterobacterales collected from five Latin American countries between 2016 and 2017, before its clinical use in Latin America, and to compare it with the activity of other available broad-spectrum antimicrobial agents. METHODS: a total of 2760 clinical isolates (508 P. aeruginosa and 2252 Enterobacterales) were consecutively collected from 20 hospitals and susceptibility to C/T and comparator agents was tested and interpreted following the current guidelines. RESULTS: according to the CLSI breakpoints, 68.1% (346/508) of P. aeruginosa and 83.9% (1889/2252) of Enterobacterales isolates were susceptible to C/T. Overall, C/T demonstrated higher in vitro activity than currently available cephalosporins, piperacillin/tazobactam and carbapenems when tested against P. aeruginosa, and its performance in vitro was comparable to fosfomycin. When tested against Enterobacterales, it showed higher activity than cephalosporins and piperacillin/tazobactam, and similar activity to ertapenem. CONCLUSIONS: these results show that C/T is an active ß-lactam agent against clinical isolates of P. aeruginosa and Enterobacterales.

RESUMO

Macrolides, lincosamides, and type B streptogramins (MLSB) are important therapeutic options to treat methicillin-resistant Staphylococcus aureus (MRSA) infections; however, resistance to these antibiotics has been emerging. In Chile, data on the MLSB resistance phenotypes are scarce in both community-(CA) and hospital-acquired (HA) MRSA isolates. Antimicrobial susceptibility to MLSB was determined for sixty-eight non-repetitive isolates of each HA-(32) and CA-MRSA (36). Detection of SCCmec elements, ermA, ermB, ermC, and msrA genes was performed by PCR. The predominant clones were SCCmec I-ST5 (HA-MRSA) and type IVc-ST8 (CA-MRSA). Most of the HA-MRSA isolates (97%) showed resistance to clindamycin, erythromycin, azithromycin, and clarithromycin. Among CA-MRSA isolates, 28% were resistant to erythromycin, azithromycin, and 25% to clarithromycin. All isolates were susceptible to linezolid, vancomycin, daptomycin and trimethoprim/sulfamethoxazole, and over 97% to rifampicin. The ermA gene was amplified in 88% of HA-MRSA and 17% of CA-MRSA isolates (p < 0.001). The ermC gene was detected in 6% of HA-SARM and none of CA-SARM isolates, whereas the msrA gene was only amplified in 22% of CA-MRSA (p < 0.005). Our results demonstrate the prevalence of the cMLSB resistance phenotype in all HA-MRSA isolates in Chile, with the ermA being the predominant gene identified among these isolates.