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To ensure the cleaning and sanitization of the root canal system during mechanical and chemical preparation, potentiation by physical agents is important. The literature shows that an average of 35% to 53% of the canal walls remain untouched, exposing the limitations of mechanical instruments and emphasizing the importance of chemical substances for chemical-surgical preparation and tubular decontamination. The aim of the present study was to analyze the disinfecting capacity of three chemical substances used in endodontic treatment on an aggressive species of microorganism. The methodology applied was an experimental laboratory study to compare the antimicrobial potential of 1% Sodium Hypochlorite, 2% Chlorhexidine and 17% Silver Nanoparticle, used in endodontics against the pathogen E. Faecalis, selected from the microorganism bank of the laboratory of Molecular and Clinical Microbiology of the Graduate Program in Health Sciences at and whose storage and use was previously authorized by the Research Ethics Committee of the São Francisco University. After collecting data, it was possible to check the results and conclude that the 2% chlorhexidine solution presented the best results in terms of antimicrobial efficacy compared to the 1% sodium hypochlorite solution, followed by the silver nanoparticles at 17%. The latter was not able to form a growth inhibition halo against E.faecalis in vitro.

Klebsiella pneumoniae is a bacterium capable of colonizing mucous membranes, causing serious infections. Widespread antibiotic resistance in K. pneumoniae-either through intrinsic mechanisms or via acquisition from different species, especially in hospital environments-limits the therapeutic options against this pathogen, further aggravating the disease burden. To date, there are no vaccines available against K. pneumoniae infection. Although formulations based on capsular polysaccharides have been proposed, the high variability in capsular serotypes limits vaccine coverage. Recombinant vaccines based on surface exposed bacterial antigens are a promising alternative owing to their conservation among different serotypes and accessibility to the immune system. Many vaccine candidates have been proposed, some of which have reached clinical trials. The present review summarizes the current status of K. pneumoniae vaccine development. Different strategies including whole cell vaccines, outer membrane vesicles (OMVs), ribosome, polysaccharide, lipopolysaccharide (LPS), and protein-based formulations are discussed. The contribution of antibody and cell-mediated responses is also presented. In summary, K. pneumoniae vaccines are feasible and a promising strategy to prevent infections and to reduce the antimicrobial resistance burden worldwide.

In the last decades, the ortho-aesthetic-functional rehabilitation had significant advances with the advent of implantology. Despite the success in implantology surgeries, there is a percentage of failures mainly due to in loco infections, through bacterial proliferation, presence of fungi and biofilm formation, originating peri-implantitis. In this sense, several studies have been conducted since then, seeking answers to numerous questions that remain unknown. Thus, the present work aims to discuss the interaction between host-oral microbiome and the development of peri-implantitis. Peri-implantitis was associated with a diversity of bacterial species, being Porphiromonas gingivalis, Treponema denticola and Tannerella forsythia described in higher proportion of peri-implantitis samples. In a parallel role, the injury of peri-implant tissue causes an inflammatory response mediated by activation of innate immune cells such as macrophages, dendritic cells, mast cells, and neutrophils. In summary, the host immune system activation may lead to imbalance of oral microbiota, and, in turn, the oral microbiota dysbiosis is reported leading to cytokines, chemokines, prostaglandins, and proteolytic enzymes production. These biological processes may be responsible for implant loss.

We characterized by whole-genome sequencing (WGS) six carbapenem-resistant Acinetobacter baumannii strains isolated from a Brazilian tertiary hospital during a 14-day period. The ISAba1-blaOXA-23 s...

Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide’s bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.

Salmonella Heidelberg is a clinically-important serovar linked to food-borne illness, and commonly isolated from poultry products. Since 1962, Salmonella Heidelberg has been widely reported from poultry production systems in several countries, including Brazil. The emergence of multidrug-resistant (MDR) Salmonella Heidelberg strains in food animals underscores a significant food safety hazard. In our study, we performed antimicrobial susceptibility testing (AST) and Whole-genome sequencing (WGS) to identify the antimicrobial resistance (AMR) genes, pathogenicity mechanisms and virulence factors (VF) in Salmonella Heidelberg E2 strain recovered from a chicken carcass in Southern Brazil. Salmonella Heidelberg strain belonged to ST15 and showed to be susceptible to colistin (MIC ≤2 μg/mL) and multidrug-resistant to amoxicillin-clavulanic acid, gentamicin, ampicillin, cefaclor, cefazolin, ceftiofur, nalidixic acid, azithromycin, erythromycin, doxycycline, tetracycline and sulfonamide. We identified AMR genes mediating resistance to aminoglycosides (aac(6')-Iaa, aac(3)-VIa, aph(3')-Ia, aadA, 16S rrsD), β-lactams (blaCTX-M-2), quinolones (parC), macrolides (acrB), tetracyclines (tet(A)), fosfomycin (fosA7) and sulfonamide (sul1). Interestingly, the mutation in parC T255S has never been reported among Salmonella Heidelberg strains. To our knowledge, this is the first report of a Salmonella enterica strain harbouring 16S rrsD 471G A, acrB F28L and acrB L40P chromosomal point mutations. Three plasmid replicon types, ST2-IncHI2, ST2-IncHI2A and IncX1 were identified. Nine Salmonella Pathogenicity Islands and 98 virulence genes encoding virulence factors were identified associated with cell adhesion, invasion, intracellular survival and resistance to antimicrobial peptides. Although Salmonella Heidelberg E2 strain likely originated from poultry, cross-contamination during meat processing cannot be excluded. This study adds to our understanding of Salmonella Heidelberg transmission along the food-chain and informs ongoing regulatory discussions on Salmonella Heidelberg in poultry.

Childhood respiratory diseases, such as asthma, are important public health problems worldwide and could be associated with tooth enamel defects. This study aimed to verify the relationship between asthma and enamel defects in teeth, to answer the following question: ''Could asthma in children be significantly associated with enamel defects in deciduous dentition and young permanent teeth?''. PUBMED-MEDLINE, EMBASE, LILACS, and COCHRANE databases were systematically searched and assessed articles (2000-2021) were cautiously scored according to a predetermined criterion. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses were considered. Twenty-two articles were critically appraised and used as a basis for conclusions. The relationship between asthma and enamel defects was confirmed in the majority of appraised papers, one with a high level of evidence, nine with a moderate level, and four with a low level. Out of the 8 manuscripts investigating the influence of asthma medication on enamel defects, only three (one with high, one moderate, and another with a low level of evidence) suggested an association. It can be concluded that asthma is closely connected with enamel defects in young permanent teeth. However, as most of the papers appraised were of cross-sectional or case-control design, further well-designed clinical investigations with a prospective design are welcome to reinforce our findings. This article is protected by copyright. All rights reserved.

Introdução: Microrganismos endofíticos são bactérias ou fungos que vivem dentro das plantas e geram vantagens de sobrevivência para seu hospedeiro, e assim, esses microrganismos podem proporcionar vantagens seletivas contra estresses ambientais, pragas e doenças, contribuindo na maior eficiência em filtração de nutrientes e elevação do crescimento. Dentre os produtos desses microrganismos, estão substâncias com atividade antimicrobiana, que protegem a planta contra infecções por microrganismos fitopatogênicos. O papel de muitos microrganismos endofíticos nas suas plantas hospedeiras ainda não está completamente elucidado, mas diversas espécies já foram caracterizadas apresentando, entre outras características, a capacidade de produzir moléculas antimicrobianas. A descoberta e utilização de moléculas naturais, com atividade antimicrobiana, podem trazer avanços significativos para o controle de infecções graves e redução das taxas de mortalidade. Objetivo: Dessa forma, este estudo tem por objetivo descrever as principais espécies do bioma brasileiro que apresentam microrganismos endofíticos que produzem substâncias com atividade antimicrobiana. Material e Métodos: Este estudo tem caráter descritivo e foi realizado por meio de uma revisão bibliográfica, a partir da coleta de informações de fontes secundárias: Google Acadêmico, National Library of Medicine (PUBMED) e o Scientific Electronic Library Online (SCIELO). Resultados: Foram identificados na literatura 15 citações de bactérias e 129 de fungos endofíticos, como produtores de substâncias antimicrobianas. Nossos resultados mostraram uma frequência maior dos gêneros Bacillus spp. e Diaporthe spp. como bactérias e fungos endofíticos, respectivamente, isolados de 41 espécies de plantas brasileiras. Conclusão: A busca de substâncias produzidas por microrganismos, com atividade antimicrobiana, pode reduzir significativamente o problema da resistência aos antibióticos. A diversidade do bioma brasileiro pode ser uma grande fonte de estudos para busca e caracterização dessas substâncias.

Nine carbapenem-resistant Acinetobacter baumannii isolates carrying bla OXA-231 and an IS Aba1 upstream occAB1 were evaluated. They were clonally related and belonged to ST107. An OXA-143-producing A. baumannii ST107 strain (Ac-148) that did not possess IS Aba1 upstream occAB1 was included in the analysis. Reduction in the expression of occAB1 and a 4-fold increase of carbapenem MICs were observed for all isolates, except for the Ac-148 strain, probably due to the presence of IS Aba1 upstream occAB1 but in the same transcriptional orientation. We reported an A. baumannii ST107 clone carrying bla OXA-143 that acquired a mutation resulting into bla OXA-231 and mobilized IS Aba1 upstream occAB1 .

We characterized by whole-genome sequencing (WGS) six carbapenem-resistant

The emergence of resistance to polymyxins in KPC-producing Klebsiella pneumoniae isolates has been a major clinical problem. This study evaluated the molecular mechanisms associated with polymyxin B (PMB) resistance that emerged in a previously PMB-susceptible KPC-2-producing K. pneumoniae during PMB therapy for a bloodstream infection in a neutropenic patient. The first isolate (PMB-susceptible) was obtained while the patient was receiving meropenem and other isolates were recovered from 2 sets of blood cultures in different dates while the patient was receiving PMB therapy (4 of 6 blood cultures bottles yielded isolates with full PMB resistance). The population analysis profile of the first isolate revealed the growth of resistant subpopulations with PFGE profile distinct from the parental isolate but undistinguishable from those obtained in subsequent days under PMB exposure. Resistant subpopulations were obtained from all parental PMB-susceptible and in one PMB-resistant isolate recovered from the patient. The molecular mechanism observed in the hetero-resistant subpopulations (IS1-like in mgrB-promoter region, increased rstB transcription with no mutation and non-identified mechanism) differed from those found in the PMB-resistant isolates, in which no mutation or transcriptional alterations were detected. This study showed that the mechanism of resistance to PMB that emerged during PMB therapy was not related to those observed in subpopulations selected in vitro from PMB-susceptible isolates recovered from the patient. The absence of mutations in the former isolates may be due to adaptive resistance occurred because of sub-optimal PMB levels as well as amikacin and meropenem used in combination.

Colistin resistance involving Gram-negative bacilli infections is a challenge for health institutions around of the world. Carbapenem-resistance among these isolates makes colistin the last therapeutic option for this treatment. Colistin resistance among Enterobacteriaceae, Acinetobacter spp., and Pseudomonas spp. was evaluated between 2010 and 2014 years, at Hospital das Clínicas, São Paulo, Brazil. Over five years 1346 (4.0%) colistin resistant Gram-negative bacilli were evaluated. Enterobacteriaceae was the most frequent (86.1%) pathogen isolated, followed by Acinetobacter spp. (7.6%), and Pseudomonas spp. (6.3%). By temporal analysis there was a trend for an increase of colistin resistance among Enterobacteriaceae, but not among non-fermentative isolates. Among 1346 colistin resistant isolates, carbapenem susceptibility was observed in 21.5%. Colistin resistance in our hospital has been alarmingly increased among Klebsiella pneumoniae isolates in both KPC positive and negative, thus becoming a therapeutic problem. Keywords: Colistin-resistant, Polymyxin B-resistant, Enterobacteriaceae, Brazil

Broth microdilution, agar dilution, Etest® and disk diffusion techniques were compared to evaluate the susceptibility profile of 82 Bcc clinical isolates against six antimicrobials as recommended by CLSI. Broth microdilution was considered the "gold standard" method. The regression analysis was applied to determine the essential (EA) and categorical (CA) agreement rates. STX (MIC50, 1 mg/L) was the most potent antimicrobial tested against Bcc isolates. The worst in vitro activity was observed for chloramphenicol (MIC50, 16 mg/L) and ticarcillin-clavulanic acid (MIC50, >256 mg/L). The EA among broth microdilution and agar dilution results was good for the majority of antimicrobial tested. When comparing broth microdilution and Etest®, ceftazidime, SXT and chloramphenicol exhibited EA rates below 90%. SXT showed an excellent CA (100%) when dilution methodologies were compared. However, a low CA rate was found for this agent between dilution and disk diffusion methodologies resulting in unacceptable very major and minor error rates.

Nine carbapenem-resistant Acinetobacter baumannii isolates carrying bla

Polymyxins have become drugs of last resort for treatment of multi-drug resistant (MDR) Gram-negative infections. However, the mechanisms of resistance to this compound have not been completely elucidated. In this study, we evaluated the mechanisms of resistance to this antimicrobial in two A. baumannii clinical isolates, respectively, susceptible (A027) and resistant (A009) to polymyxin B before and after polymyxin B exposure (A027ind and A009ind). The pmrAB and lpxACD were sequenced and their transcriptional levels were analyzed by qRT-PCR. The bacterial cell morphology was evaluated by transmission electronic microscopy (TEM) and the membrane potential was measured using Zeta-potential analyzer. The virulence of strains was studied using a Caenorhabditis elegans model. Both clinical isolates exhibited an elevation of the polymyxin B MIC after exposure to this compound. On the other hand, A027ind showed decreased values of MIC for β-lactams, aminoglycosides, vancomycin, teicoplanin, oxacillin and erythromycin. A027ind harbored two mutations in pmrB and the ISAba125 disrupting the lpxA. In contrast, A009ind strain exhibited increase of pmrB transcriptional level, after polymyxin B exposure, despite the absence of mutations in the pmrAB genes. The TEM images revealed a thicker and more electron-dense peptidoglycan layer for A009 than that of A027. The exposure to polymyxin B induced a strong condensation and darkening of intracellular material, mainly in A009ind. In addition, the surface charge of A009 was significantly less negative than the one of A027. Using the C. elegans model, only A027ind strain showed a reduction on virulence. The diversity of polymyxin B resistance mechanisms among A. baumannii strains evaluated in this study confirms the complexity of these mechanisms, which may vary depending of the background of each strain.

Polymyxins have been the only alternative therapeutic option for the treatment of serious infections caused by multidrug-resistant Acinetobacter baumannii or Pseudomonas aeruginosa isolates. For this reason, it is of crucial importance that susceptibility tests provide accurate results when testing these drug-pathogen combinations. In this study, the effect of cation concentration variability found on different commercial brands of Mueller-Hinton agar (MHA) for testing polymyxin B susceptibility was evaluated. The polymyxin B susceptibilities determined using Etest and disk diffusion were compared to those determined by the CLSI reference broth microdilution method. In general, the polymyxin B MIC values were higher when determined by Etest than when determined by broth microdilution against both A. baumannii and P. aeruginosa isolates. A high very major error rate (10%) was observed, as well as a trend toward lower MICs, compared to those determined by broth microdilution when the Merck MHA was tested by Etest. Poor essential agreement rates (10 to 70%) were observed for P. aeruginosa when all MHA brands were tested by Etest. Although an excellent categorical agreement rate (100%) was seen between the disk diffusion and broth microdilution methods for P. aeruginosa , larger zones of inhibition were shown obtained using the Merck MHA. The high cation concentration variability found for the MHA brands tested correlated to the low accuracy, and discrepancies in the polymyxin B MICs were determined by Etest method, particularly for P. aeruginosa isolates.

A total of 31 unrelated OXA-23-producing Acinetobacter baumannii strains isolated from 14 hospitals located in distinct Brazilian regions were evaluated in this study. These isolates were grouped into 12 different sequence types (STs), of which 7 had unique allelic sequences (ST188, ST189, ST190, ST191, ST192, ST228, and ST299). Most isolates belonged to the clonal complex CC79 followed by CC15 and CC1. Only polymyxin B and minocycline showed good activity against the OXA-23-producing A. baumannii clones. The ISAba1 upstream blaOXA-23, blaOXA-51-like, or ampC was found in 100%, 54.8%, and 77.4% of the isolates, respectively. High resistance rates to ceftazidime and cefotaxime were observed among those isolates possessing ISAba1 upstream ampC, in contrast to those isolates that did not carry this configuration. Moreover, a ≥2 Log2 decrease in the MICs of meropenem and ceftazidime was observed in the presence of phenyl-arginine-β-naphthylamide for 80.6% and 54.8% of isolates, respectively. Overexpression of the adeB was observed in 61.3% of isolates, particularly among those isolates belonging to the ST1 (CC1). It was also verified that ompW was down-regulated in all isolates belonging to the ST15 (CC15). On the other hand, carO and omp33-36 genes were overexpressed in 48.4% and 58.1% of the isolates, respectively. In this study, we show that overexpression of AdeABC system could significantly contribute for resistance to meropenem and ceftazidime among OXA-23-producing A. baumannii clones in Brazil, demonstrating the complexity involved in the β-lactam resistance in such isolates.

Ceftobiprole is a broad-spectrum cephalosporin with potent activity against staphylococci, including those resistant to oxacillin, as well as against most gram-negative bacilli including Pseudomonas aeruginosa. In this study, the in vitro activity of ceftobiprole and comparator agents was tested against bacterial isolates recently collected from Brazilian private hospitals. A total of 336 unique bacterial isolates were collected from hospitalized patients between February 2008 and August 2009. Each hospital was asked to submit 100 single bacterial isolates responsible for causing blood, lower respiratory tract or skin and soft tissue infections. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using CLSI microdilution method at a central laboratory. The CLSI M100-S21 (2011) was used for interpretation of the antimicrobial susceptibility results. Among the 336 pathogens collected, 255 (75.9%) were gram-negative bacilli and 81 (24.1%) were gram-positive cocci. Although ceftobiprole MIC50 values for oxacillin resistant strains were two-fold higher than for methicillin susceptible S. aureus, ceftobiprole inhibited 100% of tested S. aureus at MICs4 µg/mL. Polymyxin B was the only agent to show potent activity against Acinetobacter spp. (MIC50/90, 0.5/1 µg/mL), and P. aeruginosa (MIC50/90, 1/2 µg/mL). Resistance to broad-spectrum cephalosporins varied from 55.3-68.5% and 14.3-28.5% among E. coli and Klebsiella spp. isolates, respectively; with ceftobiprole MIC506 µg/mL for both species. Our results showed that ceftobiprole has potent activity against staphylococci and E. faecalis, which was superior to that of vancomycin. Our data also indicates that ceftobiprole demonstrated potency comparable to that of cefepime and ceftazidime against key gram-negative species.

Cateteres venosos centrais (CVC) sao utilizados na terapia intravenosa com a finalidade de facilitar o diagnostico e o tratamento do paciente, pois permitem a administracao de medicamentos, nutricao parenteral, alem de serem usados como acesso vascular para hemodialise. Entretanto, o uso desses cateteres oferece riscos de infeccao local e sistemica, incluindo endocardite e bacteremia. O presente estudo teve por objetivo isolar microrganismos de CVC, utilizando a tecnica de cultura semi-quantitativa, e identificar os mesmos, mediante provas bioquimicas convencionais e por sistema automatizado. Neste estudo, 198 pontas de CVC foram avaliadas e 105 (53%) foram consideradas positivas, ou seja, apresentaram crescimento microbiano ³ 15 UFC. Os microrganismos encontrados foram os seguintes: 63,8% de bacterias Gram-positivas; 30,5% de bacterias Gram-negativas e 5,7% de leveduras. Os microrganismos predominantes foram: Staphylococci coagulase-negativa (35,2%), Staphylococcus aureus (25,7%), Klebsiella pneumoniae (8,5%), Pseudomonas aeruginosa (7,6%), Acinetobacter baumannii (7,6%) e Candida albicans (4,7%). O aumento da incidencia de infeccao relacionada a cateter tem sido observado mundialmente, e decorre do aumento de procedimentos medicos invasivos, utilizados para o tratamento de pacientes. Este estudo possibilitou identificar os microrganismos predominantes em pontas de CVC, em um hospital escola da regiao de Londrina-PR. A identificacao destes microrganismos e de extrema importância para otimizar o tratamento da infeccao e estabelecer medidas preventivas.

Infecções Relacionadas à Assistência à Saúde (IRAS) representam uma preocupação no mundo. Muitas destas infecções são ocasionadas por microorganismos resistentes aos antimicrobianos, dentre os quais se destaca Acinetobacter baumannii. As infecções causadas por este patógeno eram tratadas com carbapenêmicos, porém diante da emergência e disseminação de cepas resistentes a estes antimicrobianos, outras opções terapêuticas tornam-se necessárias. Atualmente a disseminação de A. baumannii resistentes aos carbapenêmicos é relacionada a determinados clones de circulação mundial, como os Complexos Clonais CC1, CC2 e CC3. No Brasil, entretanto, há a prevalência do CC1, CC15 e CC79, denominados clones de alto risco. Desta maneira, o objetivo deste trabalho foi avaliar a suscetibilidade antimicrobiana e os determinantes genéticos de resistência em isolados clínicos de A. baumannii pertencentes ao CC1, CC15 e CC79, utilizando microdiluição em caldo e sequenciamento de genoma como principais metodologias. Foram estudados 63 isolados clínicos de A. baumannii de pulsotipos diferentes (ApaI-PFGE), provenientes de 23 instituições brasileiras entre 2016 e 2017. Destes isolados, 21 (33,3%) pertenciam ao CC1, 20 (31,8%) ao CC15 e 22 (35%) ao CC79. A maior parte dos isolados foi classificada em XDR (extensivamente resistente) (CC1= 90,5%; CC15= 65%; CC79= 95,5%), sendo que o fenótipo PDR (pan-resistente) foi detectado em um isolado do CC15. Por meio do sequenciamento de genoma completo foi possível analisar a estrutura clonal dos isolados, categorizando-os nas sequências-tipo (ST) ST1, ST15, ST79, ST20, ST730 e ST1447 (ST novo). Além do MLST, os genes de resistência, sistemas de efluxo e mutações presentes nas cepas dos Complexos Clonais estudados foram caracterizados. Isolados pertencentes aos CC1 (100%) e CC15 (95%) apresentaram o gene blaOXA-23. Por outro lado, nos isolados do CC79, o gene blaOXA-23 foi detectado em 54,5% e blaOXA-72 em 50% dos isolados. Observou-se a presença de genes em comum aos isolados do CC1, CC15 e CC79, tais como blaOXA-23 e aph(3)-VIa. Entretanto, a comparação entre o perfil de suscetibilidade e a análise do genoma demonstrou dados relacionados aos diferentes Complexos Clonais, como aac(3)-Ia, aac(6)-Ib, aac(6)-Ib-cr, sul1 para CC1 e blaOXA-72, blaTEM-1A, dfrA1,sat2, sul2, strA, strB, aadA1, msrE, mphE e floR para CC79 (p < 0,05). Dessa forma, nossa análise sugere que a seleção dos clones de alto risco seja impulsionada pelos determinantes de resistência sob pressão seletiva do uso de antimicrobianos Healthcare associated infections (HAI) are a worldwide concern, many of these infections are caused by bacterial agents resistant to antimicrobials, among which the Acinetobacter baumannii stands out. Infections caused by this pathogen were treated with carbapenems, however, in view of the emergence and spread of strains resistant to these antimicrobials, other therapeutic options are necessary. Currently the dissemination of carbapenem-resistant A. baumannii is related to known clones of worldwide circulation, such as Clonal Complexes CC1, CC2 and CC3. In Brazil, however, there is a prevalence of CC1, CC15 and CC79, called high-risk clones. Thus, the present study aims to evaluate the antimicrobial susceptibility and the genetic determinants of resistance in clinical isolates of A. baumannii belonging to high risk-clones (CC1, CC15 and CC79), using broth microdilution and genome sequencing as main methodologies. 63 clinical A. baumannii isolates from different pulsotypes (ApaI-PFGE), from 23 Brazilian institutions, between 2016 and 2017, were studied. Of these, 21 (33.3%) belonged to CC1, 20 (31.8%) to CC15 and 22 (35%) to CC79. The isolates were classified as XDR (extensively resistant) (CC1 = 90.5%; CC15 = 65%; CC79 = 95.5%), and the PDR (pan-resistant) phenotype was detected in an isolate from CC15. Through the complete genome sequencing, it was possible to analyze the clonal structure of the isolates, categorizing them in the sequencing type (ST) ST1, ST15, ST79, ST20, ST730 and ST1447 (new ST). In addition to MLST, resistance genes, efflux systems and mutations present in the strains of the studied Clonal Complexes were characterized. Isolates belonging to CC1 (100%) and CC15 (95%) presented the blaOXA-23 gene. On the other hand, in the CC79 isolates, the blaOXA-23 gene was detected in 54.5% and blaOXA-72 in 50% of the isolates. The presence of genes in common to isolates from CC1, CC15 and CC79 was observed, such as blaOXA-23 and aph(3\')-Via. However, the comparison between the susceptibility profile and the genome analysis showed data related to the different Clonal Complexes, such as aac(3)-Ia,aac(6)-Ib, aac(6)-Ib-cr, sul1 for CC1 and blaOXA-72, blaTEM-1A, dfrA1,sat2, sul2, strA, strB, aadA1, msrE, mphE and floR for CC79 (p < 0,05). Thus, our analysis suggests that the selection of high- risk clones is driven by the determinants of resistance under selective pressure of the use of antimicrobials

A especiação de isolados clínicos de Aspergillus ganhou relevância nos últimos anos devido à descrição de espécies crípticas resistentes aos derivados azólicos. A identificação morfológica convencional não é capaz de discriminar as espécies de Aspergillus e o sequenciamento de DNA é uma técnica pouco adaptada a laboratórios clínicos. A espectrometria de massas por ionização/dessorção a laser auxiliada por matriz tempo-devoo (EM MALDI-TOF) é uma metodologia emergente que vem sendo explorada com intuito de fornecer identificação rápida e acurada de microrganismos, incluindo os fungos filamentosos de importância clínica. Entretanto, há poucos estudos avaliando a plataforma VITEK MS® para a identificação de espécies de Aspergillus. O presente estudo teve como objetivo fornecer dados adicionais sobre a performance dos sistemas do VITEK MS® e suas bibliotecas de espectros de referências (ER) In Vitro Diagnostics (IVD) e Research Use Only (RUO) para identificar as espécies de Aspergillus de relevância clínica. Uma biblioteca de ER in-house também foi construída e avaliada. Um total de 106 organismos foram avaliados por EM, incluindo 47 cepas provenientes das coleções de fungos do Westerdijk Fungal Biodiversity Institute (Holanda) e do LEMIUNIFESP, seis isolados ambientais (IMT-USP) e 53 isolados cínicos do HC-FMUSP. Foram utilizados dois protocolos de extração proteica, um recomendado pelo fabricante e outro empregando meio de cultura líquido para os isolados/cepas com baixa esporulação. Trinta e cinco organismos foram selecionadas para construir os ERs, e os 71 restantes foram usados para avaliação de desempenho das bibliotecas IVD, RUO e RUO+in-house. Entre os 71 organismos analisados, 91,5%, 84,5% e 100% tiveram identificação correta de gênero pelos bibliotecas IVD, RUO e RUO+in-house, respectivamente. Enquanto para identificação de espécie, as bibliotecas IVD, RUO e RUO+in-house mostraram 83,1%, 77,4% e 90,1% de identificações de espécie, respectivamente. Para as 16 espécies crípticas de Aspergillus analisadas, a identificação foi correta em 31,2%, 18,7% e 62,5%, pelos sistemas IVD, RUO e RUO+in-house, respectivamente. Entre as espécies crípticas resistentes aos derivados azólicos, o sistema IVD forneceu identificação correta para as espécies Aspergillus lentulus, Aspergillus calidoustus e Aspergillus sydowii. Entretanto, Aspergillus fumigatiaffinis e Neosartorya pseudofischeri foram erroneamente identificadas como Aspergillus fumigatus pelo sistema IVD. A biblioteca in-house demonstrou melhor performance, mas espécies filogeneticamente próximas como A. fumigatiaffinis e A. lentulus tiveram identificações cruzadas. Concluímos que o VITEK® MS demonstrou boa performance para a identificação das espécies de Aspergillus, porém para algumas espécies crípticas, há necessidade de melhoria das bibliotecas de espectro de referência comercializadas. Algumas espécies crípticas filogeneticamente relacionadas apresentaram espectros similares e são de difícil diferenciação por EM MALDI, mesmo com a construção de uma biblioteca de ER in-house com vários representantes de cada espécie Aspergillus spp. identification has become more relevant in clinical practice since azoleresistant cryptic species have emerged in the last years. Conventional morphologic identification is not able to discriminate Aspergillus species and DNA sequencing is not feasible for clinical laboratories. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is an emergent technology that has been explored to provide fast and accurate identification of microorganisms, including clinically relevant molds. However, only a few studies have explored the platform VITEK MS® for the identification of Aspergillus species. The present study aimed to provide additional data regarding the performance of the In Vitro Diagnostics (IVD) and Research Use Only (RUO) systems for the identification of Aspergillus species, including azoleresistant ones, and also to construct and to evaluate an in-house reference spectrum library. A total of 106 organisms were evaluated by MS, including 47 Aspergillus strains (Westerdijk Fungal Biodiversity Institute and LEMI-UNIFESP collections), six environmental (IMT-USP) and 53 clinical isolates (HC-FMUSP). Two protein extraction protocols were used, one recommended by the manufacturer and another with liquid broth for the organisms with poor sporulation. Thirty-five organisms were selected to construct the in-house reference spectrum library and the remaining 71 were used for the performance evaluation. Correct genus identification was provided in 91.5%, 84.5% and 100% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Correct species identification was provided in 83.1%, 77.4% and 90.1% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Among the 16 Aspergillus cryptic species, correct identification was provided in 31.5%, 18.7% and 62.5% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Among the azole-resistant cryptic species, the IVD system provided correct identification for Aspergillus lentulus, Aspergillus calidoustus and Aspergillus sydowii. However, Aspergillus fumigatiaffinis and Neosartorya pseudofischeri were misidentified as Aspergillus fumigatus by the IVD system. The in-house library had better performance for the identification of Aspergillus cryptic species, but closely related taxa may be difficult to have correct differentiation by MALDI-TOF MS. In conclusion, VITEK® MS showed good performance for the identification of Aspergillus species and some azoleresistant species. However, a more robust reference spectrum library including more representatives of azole-resistant cryptic species may be necessary to achieve better identification performance of closely related taxa