Drug resistance in Mycobacterium tuberculosis clinical isolates from Brazil: Phenotypic and genotypic methods

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antimicrobial use and incidence of multidrug-resistant Pseudomonas aeruginosa in a teaching hospital: an ecological approach

Introduction: Multidrug-resistant Pseudomonas aeruginosa is a major threat in healthcare settings. The use of antimicrobials can influence the incidence of resistant strains by direct and indirect mechanisms. The latter can be addressed by ecological studies. Methods: Our group attempted to analyze the relation between the use of antipseudomonal drugs and the incidence of MDR-PA among 18 units from a 400-bed teaching hospital. The study had a retrospective, ecological design, comprising data from 2004 and 2005. Data on the use of four antimicrobials (amikacin, ciprofloxacin, ceftazidime and imipenem) were tested for correlation with the incidence of MDR-PA (defined as isolates resistant to the four antimicrobials of interest) in clinical cultures. Univariate and multivariate linear regression analyses were performed. Results: Significant correlations were determined between use and resistance for all antimicrobials in the univariate analysis: amikacin (standardized correlation coefficient = 0.73, p = 0.001); ciprofloxacin (0.71, p = 0.001); ceftazidime (0.61, p = 0.007) and imipenem (0.87, p < 0.001). In multivariate analysis, only imipenem (0.67, p = 0.01) was independently related to the incidence of multidrug-resistant strains. Conclusions: These findings share similarities with those reported in individual-based observational studies, with possible implications for infection control.

Trends in multidrug-resistant tuberculosis

Multidrug-resistant tuberculosis (MDR-TB) is an increasing global problem. The extent and burden of MDR-TB varies significantly from country to country and region to region. Globally, about three per cent of all newly diagnosed patients have MDR-TB and the proportion is higher in patients who had previously received anti-tuberculosis (anti-TB) treatment reflecting the failure of programs designed to ensure complete cure of patients with tuberculosis. The management of MDR-TB is a challenge that should be undertaken by experienced clinicians at centers equipped with reliable laboratory services and implementation of DOTS-Plus strategy.

Preditores espaço-temporais do andar para testes de capacidade funcional em pacientes com doença de Parkinson

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High Prevalence of bla(CTX-M) Extended Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae Isolates from a Tertiary Care Hospital: First report of bla(SHV-12), bla(SHV-31), bla(SHV-38), and bla(CTX-M-15) in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural studies of shikimate 5-dehydrogenase from Mycobacterium tuberculosis

Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50 degrees C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of similar to 63-64 degrees C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents.

Alkyl Hydroxybenzoic Acid Derivatives that Inhibit HIV-1 Protease Dimerization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Shikimate kinase: A potential target for development of novel antitubercular agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

Investigation of the cytocidal potential of Rhinella jimi skin methanol extracts

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Implication of the RDRio Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal

Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis control programs. Genotyping methods, such as spoligotyping and MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Units), are useful in monitoring potentially epidemic strains and estimating strain phylogenetic lineages and/or genotypic families. M. tuberculosis Latin American Mediterranean (LAM) family is a major worldwide contributor to tuberculosis (TB). LAM specific molecular markers, Ag85C(103) single nucleotide polymorphism (SNP) and RDRio long-sequence polymorphism (LSP), were used to characterize spoligotype signatures from 859 patient isolates from Portugal. LAM strains were found responsible for 57.7% of all tuberculosis cases. Strains with the RDRio deletion (referred to as RDRio) were estimated to represent 1/3 of all the strains and over 60% of the multidrug resistant (MDR) strains. The major spoligotype signature SIT20 belonging to the LAM1 RDRio sublineage, represented close to 1/5th of all the strains, over 20% of which were MDR. Analysis of published datasets according to stipulated 12 loci MIRU-VNTR RDRio signatures revealed that 96.3% (129/134) of MDR and extensively drug resistant (XDR) clusters were RDRio. This is the first report associating the LAM RDRio sublineage with MDR. These results are an important contribution to the monitoring of these strains with heightened transmission for future endeavors to arrest MDR-TB and XDR-TB. (c) 2012 Elsevier B.V. All rights reserved.

Beef Carcass Contamination by Shiga Toxin-Producing Escherichia coli Strains in an Abattoir in Brazil: Characterization and Resistance to Antimicrobial Drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

DAHP synthase from Mycobacterium tuberculosis H37Rv: Cloning, expression, and purification of functional enzyme

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now super strains, resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47 U mg-1 under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development. © 2004 Elsevier Inc. All rights reserved.

Multidrug-resistant urinary tract isolates of Escherichia coli from Ribeirão Preto, São Paulo, Brazil

Multiple resistances to antimicrobial drugs arising in Escherichia coli isolates may complicate therapeutic management of urinary tract infection (UTI) by this organism. In order to assess the multidrug resistance (MDR) among urinary E. coli isolates, we have tested 11 antimicrobial drugs against 67 isolates from outpatients attended in a tertiary-care teaching hospital and of 78 isolates from a municipal health unit, respectively in Ribeirão Preto, State of São Paulo, Brazil. Seventy-six percent and 22% of the isolates from the tertiary-care hospital and the municipal unit, respectively, were resistant to three or more different classes of agents, and were considered to present MDR. Among the isolates from the hospital patients, 73.0%, 65.0%, 58.0%, 58.0% and 31.0% were resistant to tetracycline, ampicillin, cephalothin, trimethoprim-sulfamethoxazole (TMP/SMX) and norfloxacin, respectively; resistance from the municipal unit patients were 31.0%, 37.0%, 8.0%, 29.0% and 12.0% respectively, to the same drugs. The predominant phenotype among the MDR isolates presented is ampicillin, TMP/SMX and tetracycline resistance. The high prevalence of drug resistance among UTI patients calls for continuous surveillance to assure effective control of this infection. © 2007 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

Contamination of cattle carcasses by Escherichia coli shiga like toxin with high antimicrobials resistence

During processing of cattle carcasses, contamination may occurs with the transfer of microbiota of animals feaces to carcasses. This contamination many times may be by Escherichia coli carriers of virulence factor as stx and eae genes being classified as Shiga like toxin. Shiga toxin-producing Escherichia coli (STEC) is recognized wordwide as human pathogen. A survey was performed to determine the sensibility profile to several antimicrobial drugs of STEC in carcasses obtained from an abattoir in Brazil between March 2008 and August at 2009. A total of 120 STEC were isolated. All isolates were confirmed as being E. coli by their biochemical analysis and submitted to polymerase chain reaction (PCR) for detection of stx, eae and ehly genes. No strains was isolated being carriers of ehly gene. The number of isolates carriers of eae gene were 48/120. The most frequent resistance was seen against cephalothin (84.0%), streptomycin (45.0%), nalidixic acid (42.0%) and tetracycline (20.0%). Multidrug resistance (MDR) to three or more antimicrobial agents was observed in 46 (38.3%) E. coli isolates. The findings of STEC and MRD show that cattle carcasses may be a reservoir of pathogenic bacterial for the consumer public. © 2011 Academic Journals.

In Vitro and In Vivo Activities of Ruthenium(II) Phosphine/Diimine/Picolinate Complexes (SCAR) against Mycobacterium tuberculosis

Rifampicin, discovered more than 50 years ago, represents the last novel class of antibiotics introduced for the first-line treatment of tuberculosis. Drugs in this class form part of a 6-month regimen that is ineffective against MDR and XDR TB, and incompatible with many antiretroviral drugs. Investments in R&D strategies have increased substantially in the last decades. However, the number of new drugs approved by drug regulatory agencies worldwide does not increase correspondingly. Ruthenium complexes (SCAR) have been tested in our laboratory and showed promising activity against Mycobacterium tuberculosis. These complexes showed up to 150 times higher activity against MTB than its organic molecule without the metal (free ligand), with low cytotoxicity and high selectivity. In this study, promising results inspired us to seek a better understanding of the biological activity of these complexes. The in vitro biological results obtained with the SCAR compounds were extremely promising, comparable to or better than those for first-line drugs and drugs in development. Moreover, SCAR 1 and 4, which presented low acute toxicity, were assessed by Ames test, and results demonstrated absence of mutagenicity. © 2013 Pavan et al.