Publications by authors named "Paula M O"

13 Publications

  • Page 1 of 1

Activity of silver nanoparticles on prokaryotic cells and Bothrops jararacussu snake venom.

Drug Chem Toxicol 2019 Jan 2;42(1):60-64. Epub 2018 Jul 2.

c Post-Graduate Program in Pharmaceutical Sciences , University of Sorocaba (UNISO) , Sorocaba , Brazil.

Nanoparticle-conjugated venom-toxins of venomous animals and its therapeutic efficacy against emerging or neglecting diseases is a promising strategy. In this study, silver nanoparticles (AgNPs ∼50 nm, 0.081 mg mL) were studied against the neuromuscular blockade, myotoxic effects induced by Bothrops jararacussu venom (60 µg mL) and also against prokaryotic cells. The neurotoxicity was evaluated on ex vivo mouse phrenic nerve-diaphragm using traditional myographic technique, able to obtain functional contractile responses and to check the neurotransmission. The myotoxicity on mammalian cells was evaluated in muscles resulting from pharmacological assays using routine histological techniques and light microscopy. The toxicity to prokaryotic cells was evaluated on Salmonella typhimurium TA100 without metabolic activation. The in vitro preincubation model between AgNPs and venom was enough to abolish toxic effects of B. jararacussu venom, but mammalian cells were highly sensitive to AgNPs more than prokaryotic cells, by acting as dose-independently and dose-dependently parameters, respectively. These results allowed us to conclude that AgNPs showed promising activity as antivenom agent but for its safer use, the toxicity should be evaluated on experimental animals.
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http://dx.doi.org/10.1080/01480545.2018.1478850DOI Listing
January 2019

Characterization of oxidative stress biomarkers in a freshwater anomuran crab.

Braz J Biol 2018 Feb 12;78(1):61-67. Epub 2017 Jun 12.

Universidade Regional Integrada do Alto Uruguai e das Missões, Erechim, RS, Brazil.

In general, environmental responses at level of populations or communities are preceded by alterations at lower biological levels which can be efficiently detected by the analysis of biomarkers. We analyzed the oxidative biomarkers TBARS and Catalase in Aegla singularis, a freshwater crustacean highly sensitive to environmental changes. The objective was to address if are differences in these biomarkers related to the gender as well if they are influenced by seasonal or water physicochemical variables. The results showed differences in biomarkers profile related to the gender. In female crabs were not sensitive to seasonal variations throughout the study period. However, in males the biomarkers evaluated were higher in the winter as compared to remaining seasons and showed tendency of negative correlation with water temperature and pH. This study highlights that gender, seasonal variations and physicochemical variables can influence oxidative stress biomarkers in A. singularis. Female crabs probably are better suited as a model for biomarker application in environmental studies, because their insensibility to seasonal variations can facilitate the observations of responses related specifically to environmental disturbances.
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http://dx.doi.org/10.1590/1519-6984.04816DOI Listing
February 2018

Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients.

Hum Vaccin Immunother 2017 05 6;13(5):1040-1050. Epub 2017 Jan 6.

a Department of Biochemistry and Immunology , Ribeirão Preto Medical School, University of São Paulo , Ribeirão Preto , Brazil.

Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4 or CD8 cell were assessed by flow cytometry. The CD4 cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8 cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.
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http://dx.doi.org/10.1080/21645515.2016.1264547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443371PMC
May 2017

Requirement of MyD88 and Fas pathways for the efficacy of allergen-free immunotherapy.

Allergy 2015 Mar 24;70(3):275-84. Epub 2014 Dec 24.

Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.

Background: We have shown that mycobacterial antigens and CpG oligodeoxynucleotides downmodulate airway allergic inflammation by mechanisms dependent on T-cell activation. Here, we investigated the participation of the innate response, particularly the role of MyD88 adaptor, and Fas molecules in the effectiveness of DNA-HSP65 or CpG/culture filtrated proteins (CFP) immunotherapy.

Methods: Mice sensitized and challenged with Der p 1 allergen were treated with DNA-HSP65, CpG/CFP, or with adoptively transferred cells from immunized mice. The treatment efficacy was assessed by evaluating eosinophil recruitment, antibody, and cytokine production.

Results: In addition to downregulating the Th2 response, DNA-HSP65 and CpG/CFP promoted IL-10 and IFN-γ production. Adoptive transfer of cells from mice immunized with DNA-HSP65 or CpG/CFP to allergic recipients downmodulated the allergic response. Notably, transfer of cells from DNA-HSP65- or CpG/CFP-immunized MyD88(-/-) mice failed to reduce allergy. Additionally, for effective reduction of allergy by cells from CpG/CFP-immunized mice, Fas molecules were required. Although DNA-HSP65 or CpG/CFP immunization stimulated antigen-specific production of IFN-γ and IL-10, the effect of DNA-HSP65 was associated with IL-10 while CpG/CFP was associated with IFN-γ. Moreover, after stimulation with mycobacterial antigens plus Der p 1 allergen, cells from mite-allergic patients with asthma exhibited similar patterns of cytokine production as those found in the lung of treated mice.

Conclusions: This study provides new insights on the mechanisms of allergen-free immunotherapy by showing that both DNA-HSP65 and CpG/CFP downregulated house dust mite-induced allergic airway inflammation via distinct pathways that involve not only induction of mycobacterial-specific adaptive responses but also signaling via MyD88 and Fas molecules.
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http://dx.doi.org/10.1111/all.12555DOI Listing
March 2015

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+) /CD4(+)  Foxp3(+) cells.

Immunology 2012 Nov;137(3):239-48

Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette-Guérin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+) ) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.
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http://dx.doi.org/10.1111/imm.12006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482681PMC
November 2012

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway.

Clin Exp Allergy 2012 Jan 1;42(1):131-43. Epub 2011 Sep 1.

Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Riberão Preto, Brazil.

Background: Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders.

Objective: Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response.

Methods: We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured.

Results: We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response.

Conclusions And Clinical Relevance: Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen- and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.
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http://dx.doi.org/10.1111/j.1365-2222.2011.03845.xDOI Listing
January 2012

IFN-γ-mediated efficacy of allergen-free immunotherapy using mycobacterial antigens and CpG-ODN.

Immunol Cell Biol 2011 Oct 15;89(7):777-85. Epub 2011 Mar 15.

Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Riberão Preto, Brazil.

Epidemiological and experimental evidence supports the notion that microbial infections that are known to induce Th1-type immune responses can suppress Th2 immune responses, which are characteristics of allergic disorders. However, live microbial immunization might not be feasible for human immunotherapy. Here, we evaluated whether induction of Th1 immunity by the immunostimulatory sequences of CpG-oligodeoxynucleotides (CpG-ODN), with or without culture filtrate proteins (CFP), from Mycobacterium tuberculosis would suppress ongoing allergic lung disease. Presensitized and ovalbumin (OVA)-challenged mice were treated subcutaneously with CpG, or CpG in combination with CFP (CpG/CFP). After 15 days of treatment, airway inflammation and specific T- and B-cell responses were determined. Cell transfer experiments were also performed. CpG treatment attenuated airway allergic disease; however, the combination CpG/CFP treatment was significantly more effective in decreasing airway hyperresponsiveness, eosinophilia and Th2 response. When an additional intranasal dose of CFP was given, allergy was even more attenuated. The CpG/CFP therapy also reduced allergen-specific IgG1 and IgE antibodies and increased IgG2a. Transfer of spleen cells from mice immunized with CpG/CFP also reduced allergic lung inflammation. CpG/CFP treatment induced CFP-specific production of IFN-γ and IL-10 by spleen cells and increased production of IFN-γ in response to OVA. The essential role of IFN-γ for the therapeutic effect of CpG/CFP was evidenced in IFN-γ knockout mice. These results show that CpG/CFP treatment reverses established Th2 allergic responses by an IFN-γ-dependent mechanism that seems to act both locally in the lung and systemically to decrease allergen-specific Th2 responses.
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http://dx.doi.org/10.1038/icb.2011.9DOI Listing
October 2011

Host genetic background affects regulatory T-cell activity that influences the magnitude of cellular immune response against Mycobacterium tuberculosis.

Immunol Cell Biol 2011 May 19;89(4):526-34. Epub 2010 Oct 19.

Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, São Poulo, Brazil.

Using two mouse strains with different abilities to generate interferon (IFN)-γ production after Mycobacterium tuberculosis infection, we tested the hypothesis that the frequency and activity of regulatory T (Treg) cells are influenced by genetic background. Our results demonstrated that the suppressive activity of spleen Treg cells from infected or uninfected BALB/c mice was enhanced, inhibiting IFN-γ and interleukin (IL)-2 production. Infected C57BL/6 mice exhibited a decrease in the frequency of lung Treg cells and an increased ratio CD4(+):CD4(+)Foxp3(+) cells compared with infected BALB/c mice and uninfected C57BL/6 mice. Moreover, infected C57BL/6 mice also had a decrease in the immunosuppressive capacity of spleen Treg cells, higher lung IFN-γ and IL-17 production, and restricted the infection better than BALB/c mice. Adoptive transfer of BALB/c Treg cells into BALB/c mice induced an increase in bacterial colony-forming unit (CFU) counts. Furthermore, BALB/c mice treated with anti-CD25 antibody exhibited lung CFU counts significantly lower than mice treated with irrelevant antibody. Our results show that in BALB/c mice, the Treg cells have a stronger influence than that in C57BL/6 mice. These data suggest that BALB/c and C57BL/6 mice may use some different mechanisms to control M. tuberculosis infection. Therefore, the role of Treg cells should be explored during the development of immune modulators, both from the perspective of the pathogen and the host.
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http://dx.doi.org/10.1038/icb.2010.116DOI Listing
May 2011

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection.

Braz J Med Biol Res 2010 Jul 7;43(7):645-50. Epub 2010 Jun 7.

Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 microg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg x kg(-1) x day(-1)) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.
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http://dx.doi.org/10.1590/s0100-879x2010007500053DOI Listing
July 2010

Mycobacterium tuberculosis culture filtrate proteins plus CpG Oligodeoxynucleotides confer protection to Mycobacterium bovis BCG-primed mice by inhibiting interleukin-4 secretion.

Infect Immun 2009 Dec 14;77(12):5311-21. Epub 2009 Sep 14.

Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.
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http://dx.doi.org/10.1128/IAI.00580-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786481PMC
December 2009

Increased levels of interferon-gamma primed by culture filtrate proteins antigen and CpG-ODN immunization do not confer significant protection against Mycobacterium tuberculosis infection.

Immunology 2007 Aug 13;121(4):508-17. Epub 2007 Apr 13.

Tuberculosis Research Centre, Department of Biochemistry and Immunology, Ribeirão Preto School of Medicine, University of São Paulo, Brazil.

The results of various animal model studies of tuberculosis (TB) suggest that culture filtrate proteins (CFPs), which are antigens secreted by Mycobacterium tuberculosis, are largely responsible for improvements in TB vaccines. The great obstacle to developing protein subunit vaccines is that adjuvants are required in order to stimulate relevant protective immune responses. Acting as immune adjuvants, CpG-oligodeoxynucleotides (CpG-ODNs) promote the activation of Th1 cells and of pro-inflammatory cytokines. To evaluate the adjuvant role of CpG-ODNs in conferring enhanced immunogenic capacity and protection against M. tuberculosis, we immunized mice with CFP antigen combined with synthetic CpG-ODNs (CFP/CpG) or with incomplete Freund's adjuvant (IFA) (CFP/IFA). Immunization with CFP/CpG induced a T helper 1 (Th1)-biased response accompanied by a higher immunoglobulin G2a (IgG2a) antibody/IgG1 antibody ratio, elevated production of interferon-gamma (IFN-gamma) by spleen cells and in lungs. However, CFP/IFA-immunized mice presented higher levels of IgG1 antibodies, as well as increased production of IFN-gamma, interleukin (IL)-5, and IL-10 by spleen cells, together with lower levels of IFN-gamma in the lungs. Despite the stronger Th1 response seen in both groups, believed to be necessary for protection against TB, only mice immunized with CFP/IFA were protected after M. tuberculosis infection. Lung histology revealed that lung parenchyma were better preserved in CFP/IFA-immunized mice, which also presented intense lymphocyte recruitment to the lesion, whereas CFP/CpG-immunized mice presented severe pulmonary injury accompanied by necrosis. Based on the data presented, we discuss the widely accepted paradigm that high levels of IFN-gamma are directly correlated with protection against experimental TB.
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http://dx.doi.org/10.1111/j.1365-2567.2007.02597.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265969PMC
August 2007

Hybridization signatures of gamma-irradiated murine fetal thymus organ culture (FTOC) reveal modulation of genes associated with T-cell receptor V(D)J recombination and DNA repair.

Mol Immunol 2006 Feb 13;43(5):464-72. Epub 2005 Apr 13.

Molecular Immunogenetics Group, Department of Genetics, Faculty of Medicine, University of São Paulo (USP), 14040-900 Ribeirão Preto, SP, Brazil.

In this study, we observed the occurrence of TRBV8.1-DB2.1 V(D)J recombination in murine fetal thymus organ culture (FTOC), in which the thymic microenvironment is mimicked. Since ionizing radiation affects T-cell development, we irradiated FTOCs with gamma rays to evaluate the modulation of genes implicated in TRBV8.1-BD2.1 rearrangements. The nylon cDNA microarray method was employed to monitor the expression of 9216 genes, which were organized in coexpression clusters. Clustering analysis showed similar expression profiling of genes implicated in the V(D)J recombination and DNA double strand break (DSB) repair processes such as XRCC4, RAG-2, Artemis and DNA-PK-cs, thus suggesting overlap between the two processes. The RUNX3 gene, whose coded protein binds to the enhancers of TR genes, was also modulated and the DNA cross-linking LR1 gene, which plays a role in the opening of hairpin DNA structures and whose expression pattern is similar to Artemis, may play a role in the control of V(D)J recombination. Furthermore, our data demonstrate that the FTOC model system and cDNA microarray method are useful tools to evidentiate genes that may play a role in both processes V(D)J recombination and DNA repair.
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http://dx.doi.org/10.1016/j.molimm.2005.03.010DOI Listing
February 2006

Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys.

Rev Inst Med Trop Sao Paulo 2003 Jan-Feb;45(1):5-9

School of Biology, Centro Universitário Adventista de São Paulo, São Paulo, SP, Brazil.

Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.
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http://dx.doi.org/10.1590/s0036-46652003000100002DOI Listing
June 2003
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