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Bacterial antibiotic resistance is a public health problem affecting humans and animals. This study focuses on identifying Gram-negative bacilli (GNB) (MALDI-TOF MS and Klebsiella MALDI TypeR) resistant to antimicrobials in freshly emitted feces of healthy captive and rescued wild birds from a zoo in Brazil. Birds from the zoo and rescued from sixteen different orders were investigated. Resistant bacteria from feces were selected (MacConkey agar with 2 µg/mL cefotaxime). Genomic similarity and plasmid were investigated by Pulsed-Field Gel Electrophoresis of XbaI fragments (XbaI-PFGE) and S1-PFGE. Polymerase Chain Reaction (PCR) was performed to search for beta-lactamase genes. From 80 birds included, 26 from the zoo (50 %) and 18 rescued wild birds (64 %) presented cefotaxime-resistant GNB. E. coli and Klebsiella spp were the most prevalent species. Among 65 isolates from the zoo and rescued wild birds, 75 % were considered multidrug-resistant (MDR). The majority of the isolates were extended-spectrum beta-lactamases (ESBL) producing and resistant to enrofloxacin. blaCTX-M-GROUP-1, blaTEM, and blaSHV were the most detected genes, and blaKPC was detected in K. pneumoniae complex. According to genomic similarity results, some identical profiles were found in birds with no known contact among the zoo or rescued birds. Several isolates carried one to three plasmids (15-350 kb). The presence of multidrug-resistant (MDR) isolates from healthy captive and wild birds brings novel data on the dissemination of these elements to the environment.

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Pseudomonas aeruginosa belong to the special pathogen group capable of causing serious infections, with high mortality rates. The aim of this study was to describe the antibiotic resistance and genomic characteristics of Pseudomonas aeruginosa belonging to international high-risk clone ST235 (GPAE0131 isolate), obtained from hospital wastewater. P. aeruginosa GPAE0131 was isolated from ward tertiary hospital in Brazil and the antibiotic resistance profile was determined by the disc-diffusion method. Genomic characteristics related to antibiotic resistance and virulence factors were evaluated by genomic DNA sequencing on the Illumina MiSeq platform and bioinformatic analysis. GPAE0131 isolate showed resistance to piperacillin-tazobactam, cefepime, ceftazidime, imipenem, meropenem, ciprofloxacin, levofloxacin and tobramycin. Resistome comprehend of resistance genes to ß-lactams (blaVIM-2, blaOXA-4, blaOXA-488, blaPDC-35), aminoglycosides (aph(3')-IIb, aac(6')-IIc, aac(6')-Ib9, aadA1), fosfomycin (fosA), chloramphenicol (catB7) and sulfonamides (sul1). Genome comparisons evidence insertion of blaVIM-2 and blaOXA-4 genes. GPAE0131 isolate was predicted to be pathogenic to humans and several virulence factors were found, including encoding gene for ExoU and exotoxin A. All of these features into a pathogenic international high-risk clone (ST235), classified as critical priority, stands out as public health concern due to the widespread dispersal of human pathogens through wastewater. It is suggested that mitigating measures be implemented, such as the treatment of hospital sewage and the addition of tertiary treatment, to prevent the escape of pathogens at this level into the environment.

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Salmonella spp. is an important global issue in food-producing animals. The present study evaluated antimicrobial resistance and virulence profiles in Salmonella spp. isolates from chickens in Brazil. Identification of serotypes, virulence and antimicrobial resistance genes, and plasmid profiles were performed. Three different serovars were found, S. Schwarzengrund, S. Newport and S. Kentucky. All isolates were considered Multidrug- resistance (MDR). Among the 32 Salmonella spp. isolates analysed, 29 isolates carried blaCTX-M-2 gene and showed the insertion sequence ISCR1 and a class 1 integron structure upstream from blaCTX-M-2. This gene was harboured in large IncHI2A plasmids with approximately 280kb. Furthermore, 30 isolates harboured tetA and tetB genes and 25 also harboured qnrB. The virulence genes invA, misL, orfL, spiC and pipD were detected in all isolates. The study shows a high prevalence of MDR Salmonella isolates disseminated in poultry farms. The association of the replicon IncHI2A with the resistance genes found, elevate the risk of foodborne disease outbreaks.

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Klebsiella quasipneumoniae subsp. similipneumoniae has emerged as a human pathogen and sporadic isolates from non-clinical sources were reported. Here, we described the phenotypic- and genomic-characteristics of a multidrug-resistant (MDR) and potentially hypervirulent (MDR-hv) Klebsiella quasipneumoniae subsp. similipneumoniae (KqA1) isolated from hospital wastewater. The antibiotic susceptibility profile of KqA1 was investigated using disk-diffusion method, broth microdilution method, and agar dilution method, and the genetic characteristics of antimicrobial resistance, mobile genetics elements, and virulence were evaluated by genomic DNA sequencing on the Illumina® NovaSeq6000 platform as well as by bioinformatic analysis. Resistome analyses revealed the presence of genes related to resistance to ß-lactams, aminoglycosides, quinolones, tetracyclines, sulfonamides, trimethoprim, chloramphenicol, macrolides, and fosfomycin. New genetic contexts to blaGES-16 (carbapenemase gene) and to fosA (fosfomycin resistance gene) were described. A set of mechanisms that can contribute to antibiotic resistance, commonly detected in Klebsiella spp., was also found including chromosomal mutations, efflux systems, proteins, and regulators. Moreover, KqA1 presented genes related to tolerance to metals (arsenic, copper, nickel, cobalt, magnesium, cadmium, zinc, tellurium, selenium) and to biocides (quaternary-ammonium compounds). The isolate was classified as potentially hypervirulent due to a wide range of virulence factors found associated to regulation, motility, biofilm, effector delivery systems, immune modulation, nutritional/metabolic factors, adherence, invasion, and competitive advantage. The occurrence of MDR-hv KqA1 in hospital wastewater points out how this environment matrix plays a crucial role in the maintenance and selection of critical bacterial pathogens. Regarding One Health perspective, it is evident the need for multidisciplinary implementation of control measures for antibiotic-resistant bacteria, not only in hospital settings but also in a general environmental context to mitigate the dissemination of MDR and hv bacteria.

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OBJECTIVE AND DESIGN: Our research aimed to investigate the role of CD14 in pulmonary infection by Achromobacter xylosoxidans in an experimental murine model. METHODS: C57Bl/6 or CD14-deficient mice were infected intratracheally with non-lethal inoculum of A. xylosoxidans. At times 1, 3 and 7 days after infection, lungs, bronchoalveolar lavage and blood were collected. CD14 gene expression was determined by RT-PCR. The bacterial load in the lungs was assessed by counting colony forming units (CFU). Cytokines, chemokines, lipocalin-2 and sCD14 were quantified by the ELISA method. Inflammatory infiltrate was observed on histological sections stained with HE, and leukocyte subtypes were assessed by flow cytometry. In another set of experiments, C57Bl/6 or CD14-deficient mice were inoculated with lethal inoculum and the survival rate determined. RESULTS: CD14-deficient mice are protected from A. xylosoxidans-induced death, which is unrelated to bacterial load. The lungs of CD14-deficient mice presented a smaller area of tissue damage, less neutrophil and macrophage infiltration, less pulmonary edema, and a lower concentration of IL-6, TNF-α, CXCL1, CCL2 and CCL3 when compared with lungs of C57Bl/6 mice. We also observed that A. xylosoxidans infection increases the number of leukocytes expressing mCD14 and the levels of sCD14 in BALF and serum of C57Bl/6-infected mice. CONCLUSIONS: In summary, our data show that in A. xylosoxidans infection, the activation of CD14 induces intense pulmonary inflammatory response resulting in mice death.

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Salmonella enterica serovar Choleraesuis (S. Choleraesuis) is a swine-adapted serovar associated to invasive infections in humans. In Brazil, data of strains of this serovar are scarce. In the present study, six S. Choleraesuis strains of animal (n = 5) and human (n = 1) origin from Brazil were screened for phenotypic antimicrobial resistance using disk-diffusion assay and using whole-genome sequencing data to search for antimicrobial resistance genes, plasmids, prophages, and Salmonella pathogenicity islands (SPIs). Its genetic relatedness was evaluated by MLST and SNP analysis. A single isolate from swine gallbladder harbored the colistin resistance gene mcr-1.1 into a IncX4 plasmid. In the six strains analyzed, resistance was found to tetracycline, nalidixic acid, ciprofloxacin, ampicillin, piperacillin, streptomycin, cefazoline, gentamycin, sulfamethoxazole-trimethoprim, and choloramphenicol, along with resistance genes aac(6')-Iaa, aac(3)-IV, aph(3'')-Ib, aph(6)-Id, aph(4)-Ia, aadA1, aph(3')-IIa, blaTEM-1A, floR, sul1, sul2, tet(B), drfA1, erm(B), mph(B), lnu(G), qacE, and gyrA point mutation Serine83 → Tyrosine and parC Threonine57 → Serine. Furthermore, IncF and IncH plasmids, ten SPIs, and seven prophage types were detected. All strains were assigned to ST145 and five belonged to a common SNP cluster of S. Choleraesuis strains from Brazil. The presence of S. Choleraesuis isolated from animals harboring relevant antimicrobial resistance profiles and virulence determinants reinforced the urge for enhanced surveillance to avoid its transmission to humans through food items.

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Information regarding resistance and virulence traits of meningitis-causing enterobacteria in hospital environment remains scarce. The aim of this study was to characterize virulence and acquired resistance genes of carbapenem-resistant and/or 3rd to 4th generation cephalosporin-resistant Klebsiella pneumoniae isolated from the cerebrospinal fluid of inpatients. Antimicrobial susceptibility testing was performed by disk diffusion. The string test was performed to identify hypermucoviscous phenotype. Galleria mellonella infection model was used to evaluate the virulence profile of the isolates. Screening for virulence determinants and acquired resistance genes were performed by PCR. The blaCTX-M and/or blaKPC and/or rmtG were detected in all the isolates. Genetic virulence determinants, including mrkD, entB, iroD, fecIRA, uge, wabG, fimH, ureA, ybtS, and clb were detected in the majority of multidrug-resistant K. pneumoniae isolates. One isolate presented hypermucoviscous phenotype, and several isolates showed enhanced virulence in G. mellonella infection model. The combination of the virulence genes found here seems to support not only the known virulence genetic context among nosocomial infections-causing K. pneumoniae but also the role that clb and ybtS may play in K. pneumoniae virulence.

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This study aimed to assess the ultrapure cannabidiol (CBD) antibacterial activity and to investigate the antibacterial activity of the combination CBD + polymyxin B (PB) against Gram-negative (GN) bacteria, including PB-resistant Gram-negative bacilli (GNB). We used the standard broth microdilution method, checkerboard assay, and time-kill assay. CBD exhibited antibacterial activity against Gram-positive bacteria, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis), and Mycobacterium tuberculosis, but not against GNB. For most of the GNB studied, our results showed that low concentrations of PB (≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii), including PB-resistant GNB. CBD + PB also showed additive and/or synergistic effect against LOS-expressing GND. Time-kill assays results showed that the combination CBD + PB leads to a greater reduction in the number of colony forming units per milliliter compared to CBD and PB alone, at the same concentration used in combination, and the combination CBD + PB was synergistic for all four PB-resistant K. pneumoniae isolates evaluated. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K. pneumoniae, is particularly promising.

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Background and Objectives: carbapenem resistance in Acinetobacter baumannii has reached extremely high levels worldwide, and class D OXA-type carbapenemases are the main associated mechanism. This study aimed to assess the phenotypic and molecular profile of clinical carbapenem-resistant A. baumannii (CRAb) isolates from a southern Brazilian border region. Methods: A. baumannii species was identified by the presence of the blaOXA-51 gene, and the susceptibility profile was determined by broth microdilution. The main carbapenemases were investigated by PCR and the molecular typing was performed by PFGE. Results: during the study, a total of 36 CRAb were recovered, of which 85.7% were from respiratory tract samples from ICU patients. High level resistance to were found in contrast to 100% of susceptibility for polymyxin B. The blaOXA-23 gene was present in 34 isolates and was the only one detected other than blaOXA-51. Molecular typing revealed the presence of four clonal strains, two of them endemic during the period of the study. Conclusion: to the best of our knowledge, our study brings the first data about resistance profile in Acinetobacter in the western border of southern Brazil and make aware of endemic clones of CRAb-producing-OXA-23 in this region of state, contributing for the construction of the national epidemiologic scenario of CRAb.(AU)

Justificativa e Objetivos: a resistência aos carbapenêmicos em Acinetobacter baumannii atingiu níveis extremamente altos em todo o mundo, e as carbapenemases do tipo OXA classe D são o principal mecanismo associado. O objetivo deste estudo foi avaliar o perfil fenotípico e molecular de isolados clínicos de A. baumannii resistentes aos carbapenêmicos (CRAb) de uma região de fronteira do sul do Brasil. Métodos: a espécie A. baumannii foi identificada através da presença do gene blaOXA-51, e o perfil de sensibilidade foi determinado por microdiluição em caldo. As principais carbapenemases foram investigadas por PCR, e a tipagem dos isolados de CRAb foi realizada por PFGE. Resultados: durante o período do estudo, 36 CRAb foram recuperados, dos quais 85,7% foram provenientes de amostras do trato respiratório de pacientes de UTI. Uma elevada resistência a aminoglicosídeos e fluoroquinolonas foi encontrada em contraste com 100% de sensibilidade a polimixina B. O gene blaOXA-23 foi encontrado em 34 isolados e foi o único detectado além do blaOXA-51. A tipagem molecular revelou a presença de quatro linhagens clonais, duas delas endêmicas ao longo do período do estudo. Conclusão: nosso estudo traz os primeiros dados sobre o perfil de resistência em Acinetobacter na fronteira oeste do sul do Brasil e alerta para a presença de clones endêmicos de CRAb produtores de OXA-23 nessa região, contribuindo para a construção do cenário epidemiológico nacional de CRAb.(AU)

Justificación y Objetivos: la resistencia a carbapenémicos en Acinetobacter baumannii ha alcanzado niveles extremadamente altos en todo el mundo y las carbapenemases OXA de clase D son el principal mecanismo asociado. El objetivo de este estudio fue evaluar el perfil fenotípico y molecular de los aislados clínicos de A. baumannii resistentes a carbapenémicos (CRAb) de una región fronteriza en el sur de Brasil. Métodos: la especie A. baumannii se identificó a través de la presencia del gen blaOXA-51 y el perfil de sensibilidad se determinó por microdilución en caldo. Las principales carbapenemasas fueron investigadas por PCR y la tipificación se hizo con PFGE. Resultados: durante el período de estudio, se recuperaron 36 CRAb, 85,7% de muestras del tracto respiratorio de pacientes de la UCI. Se encontró una alta resistencia a los aminoglucósidos y las fluoroquinolonas en contraste con 100% de sensibilidad a polimixina B. El gen blaOXA-23 se encontró en 34 aislamientos y fue el único detectado además de blaOXA-51. La tipificación molecular reveló la presencia de cuatro cepas clonales, dos de ellas endémicas durante el período de estudio. Conclusiones: hasta donde sabemos, nuestro estudio trae los primeros datos sobre el perfil de resistencia en Acinetobacter en la frontera oeste del sur de Brasil y reconoce los clones endémicos de CRAb productores de OXA.(AU)

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Some heavy metals have antimicrobial activity and are considered as potential alternatives to traditional antibiotic therapy. However, heavy metal tolerance genes (HMTG) have been already detected and coding different tolerance mechanisms. Considering that certain metals are promising for antimicrobial therapy, evaluation of HMTG dissemination in bacteria from sewage is essential to understand the evolution of these bacteria and to predict antimicrobial use and control. The present study aimed to evaluate the occurrence of bacteria carrying HMTG in samples of hospital wastewater and from urban wastewater treatment plant (WWTP). The acquired HMTG were investigated by PCR in bacterial collection previously characterized for antibiotic resistant genes (ARGs). HMTG searched include arsB (arsenic efflux pump), czcA (cadmium, zinc and cobalt efflux pump), merA (mercuric reductase), pcoD (copper efflux pump), silA (silver efflux pump) and terF (tellurite resistance protein). Among 45 isolates, 82% of them carried at last one HMTG, in which the silA and pcoD tolerance genes were the most prevalent. A very strong positive correlation was found between these genes (r = 0.91, p < 0.0001). Tolerance genes merA, arsB, czcA and terF were detected in 47%, 13%, 13% and 7% of the isolates, respectively. It was found that 15 isolates co-harbored ARGs (ß-lactamase encoding genes). HMTG are probably more dispersed than ARGs in bacteria, representing a new concern for heavy metals use as effective antimicrobials. To the best of our knowledge, this is the first study on the HMTG searched in Hafnia alvei, Serratia fonticola and Serratia liquefaciens. Hospital wastewater treatment implementation and additional technologies for treatment in WWTP can reduce the impacts on water resources and HMTG spread, ensureing the environmental and human health safety.

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IMP-1-producing Pseudomonas aeruginosa was first reported in Japan and since then, bacteria with this metallo-ß-lactamase have been detected worldwide. Pseudomonas monteilii (part of P. putida group) were considered an environmental pathogen with low virulence potential; however, multidrug-resistant and carbapenem-resistant P. monteilii have emerged. The present study reports the draft sequence of an extensively drug-resistant IMP-16-producing P. monteilii 597/14 isolated from cerebrospinal fluid in 2014. The sequencing data revealed blaIMP-16 as a gene cassette on class 1 integron, In1738 characterized in this study. Furthermore, the resistome of Pm597/14 consisted of 7 resistance genes (aadA1b, strA, strB, aacA4, blaIMP-16, blaOXA-2, sul1) and diverse virulence determinants involved in the adherence, LPS, antiphagocytosis, iron uptake and mercuric resistance. Although different virulence determinants were found in this study, using Galleria mellonella infection model, Pm597/14 did not kill any larvae between 7 days post-infection. P. monteilii isolates have been reported from clinical and environmental sources, carrying different MBL genes showing its potential role as their reservoir.

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Multidrug resistance mediated by ß-lactamase in Gram-negative bacilli is a serious public health problem. Sewers are considered reservoirs of multiresistant bacteria due to presence of antibiotics that select them and favor their dissemination. The present study evaluated the antibiotic resistance profile and ß-lactamases production in Gram-negative bacilli isolates from hospital sewage and urban wastewater treatment plants (UWWTP) in Brazil. Bacteria were isolated and identified with biochemical tests. Antibiotic susceptibility testing was performed by the disk-diffusion method and detection of extended-spectrum ß-lactamase and carbapenemases by enzymatic inhibitor and conventional PCR. Differences in resistance to amoxicillin clavulanic, aztreonam, cefepime, and cefotaxime were observed in hospital sewage compared with urban sewage (p < 0.05). The multidrug-resistant phenotype was observed in 33.3% of hospital sewage isolates (p = 0.0025). ß-lactamases genes were found in 35.6% of isolates, with the most frequent being blaKPC and blaTEM (17.8%), and blaSHV and blaCTX-M (13.3% and 8.9%, respectively). The data obtained are relevant, since the bacteria detected are on the priority pathogens list from the World Health Organization and hospital sewage could be released untreated into the municipal collection system, which may favor the spread of resistance. Changes in hospital sewage discharge practices, as well as additional technologies regarding effluent disinfection in the UWWTP, can prevent the spread of these bacteria into the environment and negative impact on water resources.

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The number of foodborne gastroenteritis caused by nontyphoidal Salmonella (NTS) worldwide is estimated to be 80.3 million each year. Currently, antimicrobial-resistant NTS disseminated in the animal environment increases the risk of aggravated foodborne outbreaks. Poultry are important source of foodborne NTS infections. This study was conducted to evaluate the phenotypic and genotypic characteristics of 83 NTS isolates from poultry, classified within 36 different serovars. The most prevalent serovar was S. Schwarzengrund (10/83), from which 8/10 were multidrug resistant (MDR). The antimicrobial susceptibility testing showed a total of 18 MDR isolates, from which 8/18 coharbored blaCTX-M-2 and qnrB5. The genes qnrB5, blaCTX-M-2, qnrB2, or blaCMY-2 were also found alone in other MDR isolates. All resistance genes were harbored in large plasmids, ranging from 30 to 270 kb. The pColE replicon was present in 8 MDR isolates; however it was not associated with resistance. ISCR1 and class I integron structures were always associated with blaCTX-M-2.

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We describe the mobilome of Escherichia fergusonii 40A isolated from poultry, consisting of four different plasmids, p46_40A (IncX1, 45,869 bp), p80_40A (non-typable, 79,635 bp), p150_40A (IncI1-ST1, 148,340 bp) and p280_40A (IncHI2A-ST2, 279,537 bp). The mobilome-40A carries a blend of several different resistance and virulence genes, heavy metal tolerance operons and conjugation system. This mobilome 40A is a perfect tool to preserve and disseminate antimicrobial resistance and makes the bacterial isolate incredibly adapted to survive under constant antimicrobial pressure.

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Background: Lateral gene transfer plays a central role in the dissemination of carbapenem resistance in bacterial pathogens associated with nosocomial infections, mainly Enterobacteriaceae and Pseudomonas aeruginosa. Despite their clinical significance, there is little information regarding the mobile genetic elements and mechanism of acquisition and propagation of lateral genes in P. aeruginosa, and they remain largely unknown. Objectives: The present study characterized the genetic context of bla KPC-2 in carbapenem-resistant P. aeruginosa strain BH9. Methods: Pseudomonas aeruginosa BH9 sequencing was performed using the long-read PacBio SMRT platform and the Ion Proton System. De novo assembly was carried out using the SMRT pipeline and Canu, and gene prediction and annotation were performed using Prokka and RAST. Results: Pseudomonas aeruginosa BH9 exhibited a 7.1 Mb circular chromosome. However, the bla KPC-2 gene is located in an additional contig composed by a small plasmid pBH6 from P. aeruginosa strain BH6 and several phage-related genes. Further analysis revealed that the beginning and end of the contig contain identical sequences, supporting a circular plasmid structure. This structure spans 41,087 bp, exhibiting all the Mu-like phage landmarks. In addition, 5-bp direct repeats (GGATG) flanking the pBH6 ends were found, strongly indicating integration of the Mu-like phage into the pBH6 plasmid. Mu phages are commonly found in P. aeruginosa. However, for the first time showing a potential impact in shaping the vehicles of the dissemination of antimicrobial (e.g., plasmid pBH6) resistance genes in the Pseudomonas genus. Conclusion: pBH6 captured the Mu-like Phage BH9, creating a co-integrate pBH6::Phage BH9, and this phage-plasmid complex may represent novel case of a phage-like plasmid.

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The emergence and spread of bacteria tolerant to heavy metal ions used in disinfectants have become a new threat to antimicrobial therapies. In this study, metal tolerance genes were analyzed in multidrug-resistant (MDR) Enterobacteriaceae isolated from chickens in Brazil. Different tolerance genes were found disseminated among the MDR isolates.

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The dissemination of multiresistant Klebsiella pneumoniae carbapenemase (KPC)-2-producing Klebsiella pneumoniae isolates belonging to international high-risk clones poses a major health care threat. In this study, 48 nonduplicated, carbapenem-resistant K. pneumoniae isolated from 2011 to 2014 in a tertiary hospital were investigated. The blaKPC-2 gene was the only determinant for carbapenem resistance. The blaCTX-M-15 gene was the main determinant for the production of extended-spectrum beta-lactamase (ESBL), whereas aph(3')-Ia and qnrB were the most common genes associated with resistance to aminoglycosides and quinolones, respectively. Nine different sequence types (STs) were identified. The most common was ST340. Molecular typing by enterobacterial repetitive intergenic consensus-PCR placed 48 strains among 10 different clusters. In the studied hospital, the high-risk clone of KPC-2-producing K. pneumoniae ST340, harboring genes that codify aminoglycoside modifying enzymes, QnrB and CTX-M-15 plus CTXM-2-type ESBLs, is disseminated and acts as a major agent of infections in critically ill patients.

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