Nilma Cintra Leal

Fundação Oswaldo Cruz, Rio de Janeiro, Rio de Janeiro, Brazil

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Publications (63)81.34 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Since its identification as the causative agent of plague in 1894, thousands of Yersinia pestis strains have been isolated and stored. Here, we report the ability of Y. pestis to survive up to 47 years in agar-stabs, in rubber-stoppered tubes, under refrigeration (+4 to +10°C), although overall subculture recovery rates were poor and inversely related to the length of time stored. Genetic characterization of virulence gene presence among these subcultures was suggestive of significant variation in the genomic stability of Y. pestis subcultures stored under these conditions. Specifically, we found variation in the presence of plasmid and chromosomal virulence markers (genes pla, lcrV, caf1, and irp2) among multiple subcultures of Y. pestis strains in the "Collection of Yersinia pestis" (Fiocruz-CYP) maintained by the SRP of FIOCRUZ-PE in Brazil. This variation, together with all of the inherent temporal, geographic, and other genetic variation represented by all of the recoverable strains in this historical collection was preserved in new frozen culture stocks stored at -70°C as a result of this study. These frozen culture stocks represent a valuable resource for future comparative studies of Y. pestis. This article is protected by copyright. All rights reserved.
    No preview · Article · Nov 2015 · Letters in Applied Microbiology
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    ABSTRACT: Introduction: Listeria monocytogenes is an important foodborne pathogen and the 4b serotype is responsible for many cases of human listeriosis reported in Brazil. Several listeriosis outbreaks worldwide have involved a small number of well-defined clonal groups, designated as epidemic clones (ECs). Methodology: We studied 71 strains of serotype 4b, including 25 isolates from human cases of listeriosis and 46 from meat-based foods, collected in Brazil between 1977 and 2010. The presence of ECs (I and II) markers and virulence genes (inlA, inlB, ilnC, inlJ and actA) were evaluated by PCR assay. The genetic relationship of ECs-positive strains was assessed by pulsed field gel electrophoresis. Results: ECI and ECII markers were found both in human and food strains, with 19.7% positive for the ECI marker and 40.8% for ECII. Most strains (97.2%) were positive for the virulence genes that were studied. Nevertheless, the actA gene amplicons showed two distinct sizes, with all ECI positive strains exhibiting a 105bp deletion. Pulsed field gel electrophoresis (PFGE) analysis allowed the recognition of highly related strains, particularly from two outbreaks of neonatal listeriosis in São Paulo State occurred in 1992 and 1997, both ECII-positive; and two ECI strains from a human case (1982) and from bovine meat (2009). Conclusions: The presence of ECs among clinical samples and beef isolates of serotype 4b from some regions of Brazil highlights the need for rigorous control of production procedures. Furthermore, the association of ECII with two nosocomial outbreaks suggests its ability to spread in these settings.
    Full-text · Article · Sep 2015 · The Journal of Infection in Developing Countries
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    ABSTRACT: Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) strains have been responsible for many nosocomial outbreaks. Within hospitals, colonized employees often act as reservoirs for the spread of this organism. This study collected clinical samples of 91 patients admitted to the intensive care unit (ICU), hemodialysis/nephrology service and surgical clinic, and biological samples from the nasal cavities of 120 professionals working in those environments, of a University Hospital in Recife, in the State of Pernambuco, Brazil. The main objective of this study was to determine the occurrence and dissemination of methicillin- and vancomycin-resistant Staphylococcus spp. Methods: The isolates obtained were tested for susceptibility to oxacillin and vancomycin and detection of the mecA gene. In addition, the isolates were evaluated for the presence of clones by ribotyping-polymerase chain reaction (PCR). Results: MRSA occurrence, as detected by the presence of the mecA gene, was more prevalent among nursing technicians; 48.1% (13/27) and 40.7% (11/27) of the isolates were from health professionals of the surgical clinic. In patients, the most frequent occurrence of mecA-positive isolates was among the samples from catheter tips (33.3%; 3/9), obtained mostly from the hemodialysis/nephrology service. Eight vancomycin-resistant strains were found among the MRSA isolates through vancomycin screening. Based on the amplification patterns, 17 ribotypes were identified, with some distributed between patients and professionals. Conclusions: Despite the great diversity of clones, which makes it difficult to trace the source of the infection, knowledge of the molecular and phenotypic profiles of Staphylococcus samples can contribute towards guiding therapeutic approaches in the treatment and control of nosocomial infections.
    Preview · Article · Jul 2014 · Revista da Sociedade Brasileira de Medicina Tropical
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    Full-text · Article · Jan 2014 · American Journal of Analytical Chemistry
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    Full-text · Article · Jan 2014

  • No preview · Article · Jan 2014 · American Journal of Analytical Chemistry
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    ABSTRACT: Abstract The marine clam Anomalocardia brasiliana is a candidate as a sentinel animal to monitor the contamination levels of coliforms in shellfish-harvesting areas of Brazil's northeastern region. The aim of the present study was to search enterotoxin-encoding genes plus the mecA gene among coagulase-negative staphylococci (CNS) isolates from shellfish meats of A. brasiliana. The specimen clam (n=48; 40 clams per sample) was collected during low tide in the bay area of Mangue Seco from April through June 2009, and random samples of chilled and frozen shelled clam meat (n=33; 250 g per sample) were obtained from retail shops from January through March 2012. Seventy-nine CNS isolates were identified, including Staphylococcus xylosus, S. cohnii spp. urealyticus, S. sciuri, and S. lentus. A high percentage of isolates resistant to erythromycin (58.5%), penicillin (51.2%), and tetracycline (43.9%), and the fluoroquinolones levofloxacin (39%) and ciprofloxacin (34.1%) were recorded from those environmental samples. Isolates from retail shops were particularly resistant to oxacillin (55.3%) and penicillin (36.8%). All CNS resistant to oxacillin and/or cefoxitin were positive for the presence of the mecA gene, but phenotypically susceptible to vancomycin. Also, the enterotoxin-encoding genes seg and seh were detected through multiplex-polymerase chain reaction in 77.7% and 88.8% of the isolates from environmental samples, versus 90.5% and 100% of the isolates from retail shops, respectively. The data reveal the risk to public health due to consuming raw or undercooked shellfish containing enterotoxigenic plus methicillin-resistant CNS.
    No preview · Article · Oct 2013 · Foodborne Pathogens and Disease

  • No preview · Article · Sep 2013
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    ABSTRACT: Introduction: The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods: A duplex-polymerase chain reaction (PCR) was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results: The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions: This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.
    Full-text · Article · May 2013 · Revista da Sociedade Brasileira de Medicina Tropical
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    ABSTRACT: Introduction: Staphylococcus spp. is an important healthcare-Associated pathogen and the identification of methicillin-resistant strains in samples of colonization may provide data to assist in the antimicrobial therapy success. Objectives: To determine the occurrence of colonization by methicillin-resistant Staphylococcus spp. (MRS), through the detection of the mecA gene and to evaluate different phenotypic methods for the presumptive detection of methicillin resistance in samples of the anterior nasal cavity and hands of the health care personnel of a university hospital in the state of Pernambuco, Brazil. Methods: We selected the 28 isolates of Staphylococcus spp., which showed an intermediate or resistant phenotypic profile for oxacillin, detected by the Kirby Bauer technique. The methods used were disk-diffusion tests for cefoxitin, minimal inhibitory concentration by E-test for oxacillin, screening for oxacillin resistance and mecA gene detection by polymerase chain reaction (PCR). Results: About the phenotypic methods utilized, only the E-test of oxacillin did not show a statistically significant difference in relation to PCR for the mecA gene detection, considered the gold standard. Conclusion: The E-test of oxacillin was the best of the phenotypic methods utilized. It is necessary to correctly detect MRS in healthy individuals, because they can act as carriers and can therefore be a potential source of microorganisms involved in hospital infections.
    Preview · Article · Apr 2013 · Jornal Brasileiro de Patologia e Medicina Laboratorial
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    ABSTRACT: After the worldwide cholera epidemic in 1993, permanent environmental monitoring of hydrographic basins was established in Pernambuco, Brazil, where cholera is endemic. After a quiescent period, 4 rfb N (serogroup O1) positive water samples that were culture negative were detected by multiplex single-tube nested PCR (MSTNPCR); 2 of these were also ctx A (cholera toxin) positive. From May to June 2012, 30 V. cholerae O1 isolates were obtained by culturing samples. These isolates were analyzed for the presence of virulence genes by PCR, intergenic spacer region 16S-23S PCR (ISR-PCR), and pulsed field gel electrophoresis (PFGE). The isolates were positive for the rfb N gene and negative for the assessed pathogenic genes and were classified into 2 groups by ISR and the same profile by PFGE. Close genetic similarity was observed between them (2012) and environmental strains from 2004 to 2005, indicating the permanence of endemic V. cholerae O1 in the region.
    Full-text · Article · Feb 2013 · The Scientific World Journal
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    ABSTRACT: This work aimed to assess pathogenic potential and clonal relatedness of Aeromonas sp. and Vibrio cholerae isolates recovered during a diarrhea outbreak in Brazil. Clinical and environmental isolates were investigated for the presence of known pathogenic genes and clonal relatedness was assessed by intergenic spacer region (ISR) 16S-23S amplification. Four Aeromonas genes (lip, exu, gcat, flaA/B) were found at high overall frequency in both clinical and environmental isolates although the lip gene was specifically absent from selected species. A fifth gene, aerA, was rarely found in A. caviae, the most abundant species. The ISR profile revealed high heterogeneity among the Aeromonas isolates and no correlation with species identification. In contrast, in all the V. cholerae isolates the four genes investigated (ctxA, tcpA, zot and ace) were amplified and revealed homogeneous ISR and RAPD profiles. Although Aeromonas isolates were the major enteric pathogen recovered, their ISR profiles are not compatible with a unique cause for the diarrhea events, while the clonal relationship clearly implicates V. cholerae in those cases from which it was isolated. These results reinforce the need for a better definition of the role of aeromonads in diarrhea and whether they benefit from co-infection with V. cholerae.
    No preview · Article · Dec 2012 · Revista do Instituto de Medicina Tropical de São Paulo
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    ABSTRACT: Plague (Yersinia pestis infection) can constitute an international public health emergency, requiring notification of the World Health Organization (WHO) under the International Health Regulations (IHR). To comply with the IHR and to prevent the spread of the disease, rapid, accurate, and efficient diagnosis is necessary. In this work, we carried out studies on two diagnostic tests: single-tube nested PCR (STNPCR) and multiplex PCR (MPCR). These tests allow the discrimination of Y. pestis strains based on the presence or absence of known pathogenicity markers. Two versions of each test were produced: (1) consists of a set of tubes containing premixed reagents for one reaction; (2) consists of a pool of reagents for 10 reactions. The reproducibility of the reactions and stability of the reagents were also evaluated under time and storage conditions. Both versions of the kits were shown to be stable during a 12-month evaluation period of storage at -20°C. The DNA detection limit after 12 months of storage at -20°C was 10 pg for both versions of the STNPCR and MPCR kits. Finally, we simulated two shipment conditions for the version 1 of the kits with liquid reagents in dry ice and also with freeze-dried reagents at room temperature (RT). Liquid kits were viable and produced the expected amplification results after 4 days in dry ice and freeze-dried kits can be shipped at RT over a time period of 30 days. The use of the preformulated test tubes evaluated in the present study could reduce errors in the reaction preparation and improves the efficiency of plague monitoring and control programs.
    No preview · Article · Jul 2012 · Advances in Experimental Medicine and Biology
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    ABSTRACT: In Brazil, plague is a greatly neglected disease. It received some attention when it was first introduced in 1899 and again during the first decades of the twenty century, when it spread to important cities. Plague was forgotten as soon as it became restricted to isolated and poor areas, but it received renewed attention in the 1960s, when the lack of control resulted in increased plague-related morbidity and mortality. Records of this zoonosis are lacking, and the biotic and abiotic factors in the epidemiological chain are virtually unknown by the public health services and universities. However, the systematic detection of Yersinia pestis antibodies in sentinel animals has provided evidence of its continued presence and the possibility of its reemergence. In this paper, some aspects of plague epidemiology and plague control from 1899 to 2011 are described and analyzed. This information could support new studies of the natural history of plague in Brazil.
    Full-text · Article · Jul 2012 · Advances in Experimental Medicine and Biology
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    ABSTRACT: Plague outbreaks are occasionally reported in Brazil. Unfortunately, due to great genetic similarity, molecular subtyping of Yersinia pestis strains is difficult. Analysis of multiple-locus variable number of tandem repeats (VNTR), also known as MLVA, has been found to be a valuable tool to discriminate among strains. To check for genetic differences, strains obtained from two different ecological complexes in Brazil collected during two different epidemiological events, an epizootic in Sítio Alagoinha in 1967 and an outbreak in Planalto da Borborema in 1986, were subtyped through MLVA using 12 VNTR loci. Three clusters (A, B and C) were observed. Of the 20 strains from the epizootic, 18 fit into cluster A. Cluster A was divided into two subgroups: A(1) (15 strains) and A(2) (3 strains). Of the 17 strains from the outbreak, 15 fit into cluster B. Cluster B was divided into three subgroups: B(1) (4 strains), B(2) (4 strains) and B(3) (7 strains). Cluster C is a singleton with one epizootic strain. The external standards, Y. pestis CO92 and Y. pseudotuberculosis IP32953, formed two clusters of singletons. The stability of 12 VNTR loci of three unrelated cultures included in this study was assessed. The 12 VNTR loci were stable through multiple serial subcultures in the laboratory. MLVA revealed that Y. pestis populations in Brazil are not monomorphic, and that there is intraspecific genetic diversity among Brazilian plague strains. We conclude that there is some correlation among genetic groups of this species, related to the temporal and geographic origin of isolates.
    Full-text · Article · Jul 2012 · Genetics and molecular research: GMR
  • Alzira Maria Paiva de Almeida · Nilma Cintra Leal

    No preview · Conference Paper · Jan 2012

  • No preview · Article · Jan 2010
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    ABSTRACT: Traditional methods of typing Vibrio cholerae define virulent strains according to their recognition by sera directed against the known epidemic serogroups O1 and O139, overlooking potentially virulent non-O1/non-O139 strains. Here, we have undertaken the characterization of eight clinical isolates of non-O1/non-O139 V. cholerae, collected during cholera outbreaks in Brazil. Seven of these were typed as O26 and one, 17155, was defined as non-typable. A PCR-based approach has previously detected in these strains several virulence genes derived from the CTXvarphi prophage and generally associated with pathogenic strains. Here, the presence of the O1-specific wbeN gene was investigated through PCR and found to be restricted to strain 17155, as well as one of the O26 strains, 4756, although neither strain was recognized by O1-specific antisera. The same two isolates were the only strains able to express the cholera toxin in culture, assayed by western blotting. They also possessed four repeats of the heptanucleotide TTTTGAT upstream of the ctxAB genes encoding the cholera toxin. The remaining strains possessed only two intact repeats, whereas pathogenic O1 possessed four to six repeats. To define their evolutionary relationships, selected 16S-23S intergenic rRNA spacer regions were sequenced from the various strains and the resulting sequences used to build phylogenetic trees. Strains 4756 and 17155 always clustered with control O1 strains, whereas the remaining O26 strains clustered separately. These results confirm that, despite their serological phenotype, these two strains are genotypically related to O1 strains and potentially able to produce epidemic cholera.
    Preview · Article · Jun 2009 · Clinical Microbiology and Infection
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    ABSTRACT: Through a continuous bacteriological monitoring programme carried out by the Health Secretariat of the State of Pernambuco, Brazil, two isolates of Vibrio cholerae O1 El Tor Ogawa were discovered in an endemic area in 2001, during a cholera inactive period, along with six V. cholerae non-O1/non-O139 strains and two Aeromonas veronii biovar sobria strains showing an unusual characteristic of agglutination with O1 antiserum. Between that time and 2005, eight other O1 isolates were found. The virulence genes present in the V. cholerae differed among strains, with only three O1 strains harboring the ctxA gene. The O1 and some non-O1/non-O139 strains displayed identical patterns of amplification of the 16S-23S intergenic spacer region. RAPD of the 10 V. cholerae O1 strains, with the two primers used, revealed heterogeneity. The presence of V. cholerae carrying virulence genes in the aquatic basins examined confirms that they constitute a vibrio reservoir during a cholera inactive period, thus strengthening the argument for a continuous monitoring programme and preventative measures for cholera, mainly in the areas where the supply of drinking water is deficient.
    Full-text · Article · Apr 2008 · Transactions of the Royal Society of Tropical Medicine and Hygiene
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    ABSTRACT: To examine the virulence factors and the genetic relationship isolates of the serogroup O3 of Vibrio parahaemolyticus in outbreaks of diarrhoea in the northeast region of Brazil. Eighteen samples of the O3:K6 and O3:KUT serotypes of V. parahaemolyticus were analysed by multiplex polymerase chain reaction (m-PCR) for detection of the tl, tdh and trh genes, by random-amplified polymorphic DNA (RAPD) using two primers, and by amplification of the rDNA 16S-23S region. The gene tl was amplified in all the samples, tdh in 16 while trh in none; amplification of rDNA 16S-23S generated only one profile; each RAPD primer produced two amplification patterns allowing grouping two tdh(-) Kanagawa-negative isolates. V. parahaemolyticus with characteristics of the pandemic clone appears to be widely disseminated in the studied region. Because of the genetic uniformity of the isolates, elucidation of outbreaks or tracking the source of contamination by the present molecular techniques seems useless. Detection of V. parahaemolyticus with virulence potential of pandemic clone from two outbreaks and from several isolated gastroenteritis cases points out the need for inclusion of this micro-organism in the Brazilian routine monitoring of the diarrhoeas for elucidation of their aetiology.
    Preview · Article · Apr 2008 · Journal of Applied Microbiology

Publication Stats

387 Citations
81.34 Total Impact Points

Institutions

  • 1989-2014
    • Fundação Oswaldo Cruz
      • • Departamento de Bacteriologia (INCQS)
      • • Departamento de Microbiologia (CPqAM)
      Rio de Janeiro, Rio de Janeiro, Brazil
  • 2004
    • Federal University of Pernambuco
      Arrecife, Pernambuco, Brazil
  • 2002
    • Universidade Federal Rural de Pernambuco
      Arrecife, Pernambuco, Brazil
    • Universidade Federal da Paraíba
      Frederícia, Paraíba, Brazil
  • 1994
    • Institut Pasteur
      Lutetia Parisorum, Île-de-France, France