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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.

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...

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.

KPC-producing K. pneumoniae is an endemic challenge. Seven KPC-producing Enterobacterales showed unusual carbapenems susceptibility profile. These strains were resistance at least one carbapenem and the ertapenem MIC was lower than imipenem and/or meropenem MICs using Vitek 2™ system (bioMerieux). When E-test™ and disk diffusion methods were performed the carbapenems showed susceptible results. Keywords: Enterobacterales, Vitek 2™ system, KPC, Carbapenems, Carbapenemases

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.

Background The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. Results We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13pH), magnesium deprivation (Kp13Mg), high concentrations of calcium (Kp13Ca) and iron (Kp13Fe), and a control condition with PB (Kp13PolB). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. Conclusions We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3070-y) contains supplementary material, which is available to authorized users.

Nine carbapenem-resistant Acinetobacter baumannii isolates carrying bla

OBJECTIVE: We describe an IncX4 pHC891/16mcr plasmid carrying mcr-1 in a colistin-resistant and carbapenem-susceptible E. coli isolate (HC891/16), ST156, which caused a blood infection in a Brazilian patient with gallbladder adenocarcinoma. METHODS: Strain HC891/16 was subjected to whole genome sequencing using the MiSeq Platform (Illumina, Inc., USA). Assembly was performed using Mira and ABACAS. RESULTS: The isolates showed resistance only to ciprofloxacin, ampicillin and cefoxitin, and whole-genome sequencing revealed the presence of aac(6’)Ib-cr and blaTEM1. CONCLUSION: Our findings warn of the possible silent dissemination of colistin resistance by carbapenem-susceptible mcr-1 producers, as colistin susceptibility is commonly tested only among carbapenem-resistant isolates.

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.

We read with great interest C. S. Antonio et al.'s letter describing the high prevalence of Acinetobacter baumannii carrying bla OXA-143 in Brazilian hospitals ([1][1]). Recently, we carried out a similar study, and although the bla OXA-143 gene was identified, its frequency was lower than that

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 MICs < 4 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 MIC50 > 6 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. Janssen-Cilag Universidade Federal de São Paulo (UNIFESP) Post-graduation Course in Sciences UNIFESP UNIFESP, Post-graduation Course in Sciences UNIFESP SciELO

In 2008 isolates of KPC-producing Klebsiella pneumoniae (KPC-KPN) were detected for the first time at Hospital Heliópolis, São Paulo, Brazil. The aim of this study was to characterize the clinical and microbiological outcomes of infections caused by KPC-KPN. A historical cohort of patients from whom KPC-KPN strains were isolated was performed. Isolates were identified as resistant to ertapenem by automated broth microdilution system and screened as carbapenemase producers by the modified Hodge test. The beta-lactamase resistance gene blaKPC was detected by PCR. The genetic relatedness of isolates was determined by PFGE. The study provides early clinical experience in treating KPC-KPN infections in a Brazilian tertiary center. Hospital Heliópolis Hospital Heliópolis Department of Infectious Diseases UNIFESP-EPM Hospital Heliópolis Microbiology Laboratory UNIFESP-EPM Infectious Diseases Division UNIFESP, EPM, Infectious Diseases Division SciELO

A infecção do trato urinário é uma das infecções hospitalares mais freqüentes. A formação de biofilme é um grande problema em pacientes que necessitam a utilização de cateter vesical. Pseudomonas aeruginosa é um microrganismo que consegue aderir ao cateter após poucas horas de inserção e formar um biofilme. Este, por sua vez, promove vantagens de sobrevivência aos microrganismos que o compõe como resistência a antibióticos, a biocidas, proteção contra o sistema imunológico do hospedeiro, entre outros. Sendo assim, o microrganismo permanece no paciente causando a infecção. O objetivo deste trabalho foi estudar a formação de biofilme em 111 cepas de P. aeruginosa isoladas de pacientes com infecção urinária, internados ou atendidos no Hospital Universitário e no Ambulatório do Hospital das Clínicas de Londrina no período de 2003 a 2005. Através da PCR foi possível verificar a presença do gene algC, um dos genes responsável pela síntese do alginato, em todas as 111 cepas testadas. Noventa e um porcento (91%) das cepas aderiram na microplaca de poliestireno. Em células Vero, as amostras apresentaram um modelo de adesão agregativo semelhante ao de Escherichia coli enteroagregativa. As cepas testadas apresentaram uma superfície celular altamente hidrofílica. Este fato pode ser explicado pela produção do alginato que é um polissacarídeo e conseqüentemente deixa a superfície da bactéria hidrofílica. P. aeruginosa demonstrou, através da Microscopia Eletrônica de Varredura, grande capacidade de formar biofilme na superfície de cateter vesical. Urinary tract infections are one of the most frequent hospital infections. The development of biofilm is a serious problem in patients who require vesical catheter use. Pseudomonas aeruginosa is a microorganism that can adhere to the catheter a few hours after its insertion and form a biofilm. The biofilm provides survival advantages to the microorganisms that it is consisted of, such as antibiotics and biocides resistance, protection against the host?s immune system, amongst others. That way, the microorganism stays in the patient causing the infection. The objective of this work was to study the biofilm?s formation in 111 strains of P. aeruginosa isolated in patients with urinary tract infections, who were either hospitalized or received care at Hospital Universitário and at Ambulatório do Hospital das Clínicas of Londrina from 2003 to 2005. It was possible to verify the presence of the algC gene through the PCR, one of the genes responsible for alginate synthesis, in all 111 tested strains. Ninety one percent (91%) of strains adhered to the polyethylene micro plaque. In Vero cells, the samples presented a collective adhesion model similar to the Enteroaggregative Escherichia coli. The tested strains presented highly hydrophilic cell surface. This fact can be explained by the production of alginate, which is a polysaccharide and consequentially leaves the cell surface hydrophilic. P. aeruginosa demonstrated, through a Scanning Electron Microscope, great capacity of developing biofilm in vesical catheter surfaces.

Background Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial and community-acquired infections. A wide repertoire of virulence and antimicrobial resistance genes is present in K. pneumoniae genomes, which can constitute extra challenges in the treatment of infections caused by some strains. K. pneumoniae Kp13 is a multidrug-resistant strain responsible for causing a large nosocomial outbreak in a teaching hospital located in Southern Brazil. Kp13 produces K. pneumoniae carbapenemase (KPC-2) but is unrelated to isolates belonging to ST 258 and ST 11, the main clusters associated with the worldwide dissemination of KPC-producing K. pneumoniae. In this report, we perform a genomic comparison between Kp13 and each of the following three K. pneumoniae genomes: MGH 78578, NTUH-K2044 and 342. Results We have completely determined the genome of K. pneumoniae Kp13, which comprises one chromosome (5.3 Mbp) and six plasmids (0.43 Mbp). Several virulence and resistance determinants were identified in strain Kp13. Specifically, we detected genes coding for six beta-lactamases (SHV-12, OXA-9, TEM-1, CTX-M-2, SHV-110 and KPC-2), eight adhesin-related gene clusters, including regions coding for types 1 (fim) and 3 (mrk) fimbrial adhesins. The rmtG plasmidial 16S rRNA methyltransferase gene was also detected, as well as efflux pumps belonging to five different families. Mutations upstream the OmpK35 porin-encoding gene were evidenced, possibly affecting its expression. SNPs analysis relative to the compared strains revealed 141 mutations falling within CDSs related to drug resistance which could also influence the Kp13 lifestyle. Finally, the genetic apparatus for synthesis of the yersiniabactin siderophore was identified within a plasticity region. Chromosomal architectural analysis allowed for the detection of 13 regions of difference in Kp13 relative to the compared strains. Conclusions Our results indicate that the plasticity occurring at many hierarchical levels (from whole genomic segments to individual nucleotide bases) may play a role on the lifestyle of K. pneumoniae Kp13 and underlie the importance of whole-genome sequencing to study bacterial pathogens. The general chromosomal structure was somewhat conserved among the compared bacteria, and recombination events with consequent gain/loss of genomic segments appears to be driving the evolution of these strains.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.Results: Aztreonam exhibited the highest in vitro activity against the P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. the MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC beta-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. in addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. the MBL-encoding genes bla(SPM-1) and bla(IMP-1) were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. the bla(GES-1) was detected in 5.1% of the isolates, while bla(GES-5) and bla(CTX-M-2) were observed in 1.7% of the isolates evaluated. in the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance.Conclusions: Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary beta-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates. Universidade Federal de São Paulo, Div Infect Dis, BR-04039032 São Paulo, Brazil Universidade Federal de São Paulo, Div Infect Dis, BR-04039032 São Paulo, Brazil FAPESP: FAPESP - 2006/01716-8 CNPq: 307714/2006-3 Web of Science

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.

In vitro activity of doripenem and comparator antimicrobial agents was evaluated against Gram-negative bacilli recently isolated from Brazilian private hospitals that were enrolled in the INVITA-A-DORI Brazilian Study. A total of 805 unique Gram-negative bacilli were collected from patients hospitalized at 18 medical centers between May/08 and March/09. Each hospital was asked to submit 50 single Gram-negative bacilli isolated from blood, lower respiratory tract or intraabdominal secretions. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using Clinical Laboratory Standards Institute (CLSI) microdilution method at a central laboratory. CLSI M100-S21 (2011) or US-FDA package insert criteria (tigecycline) was used for interpretation of the antimicrobial susceptibility results. Doripenem was as active as meropenem and more active than imipenem against E. coli and K. pneumoniae isolates. A total of 50.0% of Enterobacter spp. isolates were resistant to ceftazidime but 85.7% of them were inhibited at doripenem MICs ≤ 1 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). Although high rates of imipenem (53.1%) and meropenem (44.5%) resistance were detected among P. aeruginosa, doripenem showed MIC50 of 16 μg/mL against imipenem-resistant P. aeruginosa and inhibited a greater number of imipenem-resistant P. aeruginosa (10.5%) at MIC values of ≤ 4 μg/mL than did meropenem (0.0%). In this study, doripenem showed similar in vitro activity to that of meropenem and retained some activity against imipenem-resistant P. aeruginosa isolated from Brazilian medical centers. Keywords: carbapenems, Gram-negative bacteria, Brazil