Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

"Multiplex PCR" identification of the atypical and monophasic Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- in São Paulo State, Brazil: frequency and antibiotic resistance patterns

Salmonella spp. are the etiologic agents of salmonellosis, a worldwide spread zoonoses causing foodborne outbreaks and clinical diseases. By serological identification, Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- accounted for 8.8% of human and 1.6% of nonhuman Salmonella strains isolated in São Paulo State, during 1991-2000. A total of 28.6% of them amplified a fragment corresponding to H:1,2 (flagellar phase two) through PCR analysis and were further assigned as S. Typhimurium. Antimicrobial resistance was detected in 36.3% of the 369 PCR-negative strains tested, including the multiresistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin.

Genetic diversity and antimicrobial resistance of enterococcal isolates from Southern region of Brazil

In the present study, a total of 455 enterococcal isolates, recovered from patients living in the city of Porto Alegre, State of Rio Grande do Sul, Brazil, during the period from July 1996 to June 1997, were identified to the species level by conventional biochemical and microbiological tests, and assayed for their susceptibilities to antimicrobial agents. The genetic diversity of antimicrobial resistant strains was evaluated by pulsed-field gel electrophoresis (PFGE) analysis of SmaI restricted chromosomal DNA. The most frequent species was Enterococcus faecalis (92.8%). Other species identified were: E. faecium (2.9%), E. gallinarum (1.5%), E. avium (1.1%), E. hirae (0.7%), E. casseliflavus (0.4%), E. durans (0.4%) and E. raffinosus (0.2%). The overall prevalence of isolates with high-level resistance (HLR) to aminoglycosides was 37.8%. HLR to gentamicin was found in 24.8%. No strains with acquired resistance to vancomycin were found. PFGE analysis showed the predominance of clonal group A, comprising strains isolated from different clinical specimens obtained from patients in three hospitals. These results suggest intra and inter-hospital dissemination of one predominant clonal group of E. faecalis isolates with HLR to gentamicin in the hospitals included in this study.

Clonal types and antimicrobial resistance profiles of methicillin-resistant Staphylococcus aureus isolates from hospitals in south Brazil

In the present study were evaluated the DNA macrorestriction profile and SCCmec types for nine multi-resistant MRSA selected. Also antimicrobial susceptibility testing by disk diffusion method was evaluated for 68 MRSA isolates against 12 antimicrobial agents. The isolates were recovered from blood culture collected from hospitalized patients in three hospitals of Porto Alegre, Brazil. PFGE and PCR for mecA and SCCmec I, II, III, IV types genes were done on selected nine isolates with susceptibility only to vancomycin, teicoplanin and linezolid. Two clone profiles, with five subtypes, were demonstrated among multi-resistant MRSA analyzed. Eight isolates showed harbor SCCmec type III and one isolate was not typeable. The knowledge of SCCmec type, clone and antimicrobial profiles among S. aureus is essential mainly to prevention and control of dissemination of the antimicrobial resistance.

Epidemiological characterization, antimicrobial resistance and virulence mechanisms of human and animal streptococci

Dissertação para obtenção do Grau de Doutor em Biologia

Indications of carbapenem resistance evolution through heteroresistance as an intermediate stage in Acinetobacter baumannii after carbapenem administration

We describe an in vivo evolution of an antimicrobial profile from susceptibility to full-resistance to carbapenems, with heteroresistance as an intermediate stage, in an Acinetobacter baumannii strain. Heteroresistance was characterized by the growth of sub-populations within the susceptibility halo in both disk-diffusion and Etest. PCRs for the main A. baumannii carbapenemases were negative. The exact resistance mechanism, diagnostic methods and clinical relevance of heteroresistance in A. baumannii warrant further investigations. This is the first description of such phenomenon in vivo and the second report of heteroresistance to carbapenems in A. baumannii.

Risk factors for and mortality of extended-spectrum-β-lactamase-producing Klebsiella pneumoniae and Escherichia coli nosocomial bloodstream infections

A case-control study, involving patients with positive blood cultures for Klebsiella pneumoniae (KP) or Escherichia coli (EC) EC and controls with positive blood cultures for non-ESBL-KP or EC, was performed to assess risk factors for extended-spectrum-β-lactamase (ESBL) production from nosocomial bloodstream infections (BSIs). Mortality among patients with BSIs was also assessed. The study included 145 patients (81, 59.5% with K. pneumoniae and 64, 44.1% with E. coli BSI); 51 (35.2%) isolates were ESBL producers and 94 (64.8%) nonproducers. Forty-five (55.6%) K. pneumoniae isolates were ESBL producers, while only six (9.4%) E. coli isolates produced the enzyme. Multivariate analysis showed that recent exposure to piperacillin-tazobactam (adjusted Odds Ratio [aOR] 6.2; 95%CI 1.1-34.7) was a risk factor for ESBL BSI. K. pneumoniae was significantly more likely to be an ESBL-producing isolate than E. coli (aOR 6.7; 95%CI 2.3-20.2). No cephalosporin class was independently associated with ESBLs BSI; however, in a secondary model considering all oxymino-cephalosporins as a single variable, a significant association was demonstrated (aOR 3.7; 95%CI 1.3-10.8). Overall 60-day mortality was significantly higher among ESBL-producing organisms. The finding that piperacillin-tazobactam use is a risk factor for ESBL-production in KP or EC BSIs requires attention, since this drug can be recommended to limit the use of third-generation cephalosporins.

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36% of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

Estudio de la gestión de la variable ambiental en la generación termoeléctrica

En la actualidad, el cambio climático es uno de los temas de mayor preocupación para la población mundial y los científicos de todo el mundo. Debido al crecimiento de la población de forma exponencial, la demanda de energía aumenta acorde con ello, por lo que las actividades de producción energética aumentan consecuentemente, siendo éstas las principales causantes de la aceleración del cambio climático. Pese a que muchos países previamente habían apostado por la producción energética mediante tecnologías limpias a partir de energías renovables, hoy en día es imposible prescindir de los combustibles fósiles pues, junto a la energía nuclear, suponen el mayor porcentaje dentro del mix energético de los países más grandes del mundo, por lo que el cambio debe ser global y con todos los países implicados al unísono. Por ello, los países desarrollados decidieron acordar una serie de leyes y normas para la regulación y el control de la expansión energética en el mundo, mediante programas de incentivo a las empresas para la producción de energía limpia, libre de emisiones, sustituyendo y mejorando los procesos tecnológicos para que garanticen un desarrollo sostenible. De esta forma, se conseguiría también reducir la dependencia energética de los países productores de los recursos fósiles más importantes y a su vez, ayudar a otros sectores a diversificar su negocio y mejorar así la economía de las áreas colindantes a las centrales de producción térmica. Gracias a estos programas de incentivo o, también llamados mecanismos de flexibilidad, las empresas productoras de energía, al acometer inversiones en tecnologia limpia, dejan de emitir gases de efecto invernadero a la atmósfera. Por tanto, gracias al comercio de emisiones y al mercado voluntario, las empresas pueden vender dichas emisiones aumentando la rentabilidad de sus proyectos, haciendo más atractivo de por sí el hecho de invertir en tecnología limpia. En el proyecto desarrollado, se podrá comprobar de una forma más extensa todo lo anteriormente citado. Para ello, se desarrollará una herramienta de cálculo que nos permitirá analizar los beneficios obtenidos por la sustitución de un combustible fósil, no renovable, por otro renovable y sostenible, como es la biomasa. En esta herramienta se calcularán, de forma estimada, las reducciones de las emisiones de CO2 que supone dicha sustitución y se hallará, en función del valor de las cotizaciones de los bonos de carbono en los diferentes mercados, cuál será el beneficio económico obtenido por la venta de las emisiones no emitidas que supone esta sustitución. Por último, dicho beneficio será insertado en un balance económico de la central donde se tendrán en cuenta otras variables como el precio del combustible o las fluctuaciones del precio de la electricidad, para hallar finalmente la rentabilidad que supondría la inversión de esta adaptación en la central. Con el fin de complementar y aplicar la herramienta de cálculo, se analizarán dos casos prácticos de una central de carbón, en los cuales se decide su suscripción dentro del contexto de los mecanismos de flexibilidad creados en los acuerdos internacionales.

Genetic diversity and primary resistance among HIV-1-positive patients from Maringá, Paraná, Brazil

The objective of this study is to identify subtypes of Human Immunodeficiency Virus type 1 (HIV-1) and to analyze the presence of mutations associated to antiretroviral resistance in the protease (PR) and reverse transcriptase (RT) regions from 48 HIV-1 positive treatment naïve patients from an outpatient clinic in Maringá, Paraná, Brazil. Sequencing was conducted using PR, partial RT and group-specific antigen gene (gag) nested PCR products from retrotranscribed RNA. Transmitted resistance was determined according to the Surveillance Drug Resistance Mutation List (SDRM) algorithm. Phylogenetic and SimPlot analysis of concatenated genetic segments classified sequences as subtype B 19/48 (39.6%), subtype C 12/48 (25%), subtype F 4/48 (8.3%), with 13/48 (27.1%) recombinant forms. Most recombinant forms were B mosaics (B/F 12.5%, B/C 10.4%), with one C/F (2.1%) and one complex B/C/F mosaic (2.1%). Low levels of transmitted resistance were found in this study, 2/48 (2.1% to NRTIs and 2.1% for PI). This preliminary data may subsidize the monitoring of the HIV evolution in the region.

RESISTANCE TO AMOXICILLIN, CLARITHROMYCIN AND CIPROFLOXACIN OF Helicobacter pylori ISOLATED FROM SOUTHERN BRAZIL PATIENTS

Introduction: Helicobacter pylori is a bacteria which infects half the world population and is an important cause of gastric cancer. The eradication therapy is not always effective because resistance to antimicrobials may occur. The aim of this study was to determine the susceptibility profile of H. pylori to amoxicillin, clarithromycin and ciprofloxacin in the population of Southern Brazil. Material and methods: Fifty four samples of H. pylori were evaluated. The antibiotics susceptibility was determined according to the guidelines of the British Society for Antimicrobial Chemotherapy and the Comité de l'Antibiogramme de la Société Française de Microbiologie. Results: Six (11.1%) H. pylori isolates were resistant to clarithromycin, one (1.9%) to amoxicillin and three (5.5%) to ciprofloxacin. These indices of resistance are considered satisfactory and show that all of these antibiotics can be used in the empirical therapy. Conclusion: The antibiotics amoxicillin and clarithromycin are still a good option for first line anti-H. pylori treatment in the population of Southern Brazil.

ANTIMICROBIAL DRUG RESISTANCE IN STRAINS OF Escherichia coli ISOLATED FROM FOOD SOURCES

A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3%) followed by vegetable salad (20%), raw meat (13.3%), raw egg-surface (10%) and unpasteurized milk (6.7%). The overall incidence of drug resistant E. coli was 14.7%. A total of six (4%) Extended Spectrum β-Lactamase (ESBL) producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.

PREVALENCE OF DRUG RESISTANCE AND VIRULENCE FEATURES IN Salmonella spp. ISOLATED FROM FOODS ASSOCIATED OR NOT WITH SALMONELLOSIS IN BRAZIL

Salmonella is the most common etiological agent of cases and outbreaks of foodborne diarrheal illnesses. The emergence and spread of Salmonella spp., which has become multi-drug resistant and potentially more pathogenic, have increased the concern with this pathogen. In this study, 237 Salmonella spp., associated or not with foodborne salmonellosis in Brazil, belonging mainly to serotype Enteritidis, were tested for antimicrobial susceptibility and the presence of the virulence genes spvC, invA, sefA and pefA. Of the isolates, 46.8% were sensitive to all antimicrobials and 51.9% were resistant to at least one antimicrobial agent. Resistance to more than one antimicrobial agent was observed in 10.5% of the strains. The highest rates of resistance were observed for streptomycin (35.9%) and nalidixic acid (16.9%). No strain was resistant to cefoxitin, cephalothin, cefotaxime, amikacin, ciprofloxacin and imipenem. The invA gene was detected in all strains. Genes spvC and pefA were found in 48.1% and 44.3% of strains, respectively. The gene sefA was detected in 31.6% of the strains and only among S. Enteritidis. Resistance and virulence determinants were detected in Salmonella strains belonging to several serotypes. The high rates of antibiotic-resistance in strains isolated from poultry products demonstrate the potential risk associated with the consumption of these products and the need to ensure good food hygiene practices from farm to table to reduce the spread of pathogens relevant to public health.