ABSTRACT
Leptospirosis is a zoonotic disease with significant global impact and a challenging diagnosis. The utilization of adequately validated rapid tests is relevant for the opportune identification of the disease and for reduction in fatality rates. The present study analyzes the accuracy and reliability of the Dual Path Platform (DPP) assay -produced in Brazil by the Oswaldo Cruz Foundation (Fiocruz)- for diagnosing leptospirosis. Firstly, a serological panel was constructed in the Brazilian Reference Laboratory for Leptospirosis using samples routinely handled by reference laboratories of six Brazilian states. It consisted of 150 positive (according to MAT and IgM-ELISA) and 250 negative samples for leptospirosis. Subsequently, the panel samples were distributed to the reference laboratories for the performance of DPP assays in triplicate. Different measures were used in the assessment of diagnostic quality. Predictive values were estimated for different pre-test probability settings. Sensitivities varied between 67.33 % and 74.00 % and specificities between 93.20 % and 98.40 % in the states, and there were adequate agreements between them. Accuracies were lower for the samples of patients with less than 7 days of symptoms. In contexts of prevalence values up to around 25 %, positive and negative predictive values were around 90 %. However, in situations of high pre-test probabilities, NPVs were low. This study improves understanding of the use of DPP in diagnosing leptospirosis, particularly its application in healthcare settings. As long as the time of symptoms onset and clinical and epidemiological contexts are adequately considered for the interpretation of results, DPP is a valid option to be used in the leptospirosis diagnostic routine.
ABSTRACT
Leptospirosis is a globally distributed infectious disease caused by pathogenic spirochetes of the Leptospira genus, often overlooked. It is estimated that the disease affects approximately one million people annually, resulting in more than 58,900 deaths. The gold standard for serodiagnosis of leptospirosis is the Microscopic Agglutination Test (MAT). However, the limitations of this technique necessitate the exploration of alternative diagnostic methods. In this study, we evaluated the ErpY-like recombinant protein (rErpY-like) in the development of a serologic diagnostic assay for human leptospirosis. Eighty-six human sera samples, characterized by MAT, underwent evaluation through indirect IgM-ELISA and IgG-ELISA. The sensitivity and specificity values obtained from IgM-ELISA were 60% and 76%, respectively, while those from IgG-ELISA were 96.4% and 100%, respectively. The use of the rErpY-like protein in both IgM-ELISA and IgG-ELISA proves to be a sensitive and specific method for antibody detection. This could potentially serve as a valuable alternative tool in the diagnosis of human leptospirosis.
ABSTRACT
Leptospirosis diagnosis by MAT requires antibody levels that are typically present only after the first week of symptoms, many days after infection. To improve testing capacity and to develop a fast and reliable solution for the diagnosis of this disease in the first few days after clinical manifestations, the National Reference Laboratory for Leptospirosis/WHO Collaborating Center in Brazil implemented a duplex molecular method by qPCR for human samples for the detection of the gene lipL32, conserved in pathogenic Leptospira spp. In this paper, we describe the overall performance of this protocol in the first 3 months as a standard routine. Detection of pathogenic Leptospira spp. DNA was similar between blood, plasma, and tissue samples, with a limit of detection as low as one cell per sample, and among 391 samples from suspected cases, 174 (44.6%) were positive. The average RNASEP1 control gene detection cycle thresholds (Ct) were 28.4 and 29.8 for positive and negative samples, respectively. The median sample collection interval from the beginning of symptoms was 3 days for positive and 4 days for negative samples, respectively. Neither age, sex, nor the time intervals between sample collection and DNA extraction significantly influenced the results. Surprisingly, positivity was related to the time between DNA extraction and the qPCR reaction. These data support the use of this routine as a diagnostic approach to strengthen the molecular detection of leptospirosis and to develop new strategies.
ABSTRACT
A leptospirose é a zoonose de maior distribuição geográfica, com estimativa de cerca de 60.000 mortes por ano. A doença é causada por bactérias do gênero Leptospira, que possui mais de 300 diferentes sorovares e 64 espécies já identificadas, sendo o ambiente a principal fonte de contaminação. A doença em humanos apresenta manifestações clínicas variadas e caráter bifásico, devendo ser confirmada por meio do diagnóstico laboratorial. O objetivo deste trabalho foi reunir conceitos atualizados sobre a leptospirose humana e as principais técnicas de diagnóstico laboratorial empregadas. A MAT é considerada o padrão-ouro para o diagnóstico da leptospirose, mas devido à baixa sensibilidade na fase inicial da doença é necessário o emprego de técnicas mais sensíveis neste período. Baseado em diversos estudos, as metodologias de PCR, ELISA-IgM e teste rápido apresentaram sensibilidade satisfatória nos primeiros dias após o início dos sintomas. Na segunda semana, a MAT apresentou 100% de sensibilidade, mantendo sua alta especificidade em ambas as fases. No geral, os testes sorológicos de ELISA-IgM e teste rápido apresentaram resultados satisfatórios como métodos de diagnóstico precoce, principalmente tratando-se de locais com pouca infraestrutura, diferente dos laboratórios de referência onde é possível empregar as técnicas de PCR e MAT.
Leptospirosis is the most widespread zoonosis, which has a balance of almost 60,000 deaths per year. Bacteria of Leptospira genus, which has more than 300 different serovars and 64 species already identified, cause the disease, being the environment the main source of contamination. The human disease presents a large set of clinical manifestations, showing biphasic presentation, the reason why leptospirosis must be confirmed by laboratory diagnosis. This study aimed to group recent concepts concerning human leptospirosis and the main diagnosis techniques employed at the laboratory. MAT is considered the gold standard for leptospirosis diagnosis, but has low sensitivity on the onset of disease, leading to the use of techniques with higher sensitivity on this period. Based on several studies, PCR, ELISA-IgM and rapid test presented satisfactory sensitivity on the onset of symptoms. In the second week, MAT showed 100% of sensitivity, maintaining its high specificity in both phases. In general, the ELISA-IgM and rapid serological tests showed satisfactory results as methods for early diagnosis, especially in the case of places with poor infrastructure, different from the reference laboratories where it is possible to use the PCR and MAT techniques.
Subject(s)
Weil Disease , Leptospirosis/diagnosis , Leptospirosis/etiology , Spirochaetales , Polymerase Chain Reaction , Clinical Laboratory Techniques , LeptospiraABSTRACT
Leptospirosis, a zoonotic disease with worldwide distribution, is caused by spirochetes of the genus Leptospira. In dogs, this disease is frequently misdiagnosed. Few studies have attempted to associate the detection of Leptospira spp. infection with clinicopathological and renal histopathological findings using a multidisciplinary approach. The present study isolated and characterized Leptospira spp. obtained from naturally infected dogs and described relevant clinical and histopathological findings. Blood and urine were collected from 57 dogs with clinical symptomatology suggestive of leptospirosis; 38 cases were confirmed by PCR in urine or by culture or microscopic agglutination testing (titers ≥800). A total of 12 strains of pathogenic Leptospira were isolated from the studied dogs (seven in blood, four in urine and one in both blood and urine samples). All isolates were characterized as Leptospira interrogans serogroup Icterohaemorrhagiae. Of the confirmed cases, almost one-third of the animals had been vaccinated. Our analysis of laboratory testing revealed that azotemia and proteinuria were statistically significant predictors of infection. The main histopathological findings seen in kidney tissues were necrosis, degeneration, tubular regeneration, mononuclear inflammatory infiltrate and congestion. A multidisciplinary approach involving clinicopathological and histopathological characterization of renal involvement can aid in the identification of acute leptospirosis infection.
Subject(s)
Dog Diseases , Leptospira interrogans , Leptospira , Leptospirosis , Animals , Dog Diseases/diagnosis , Dogs , Leptospira interrogans/genetics , Leptospirosis/diagnosis , Leptospirosis/veterinary , Prospective Studies , SerogroupABSTRACT
Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers' samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.
Subject(s)
Leptospira/isolation & purification , Leptospirosis/blood , Leptospirosis/microbiology , Water Microbiology , Adult , Blood/microbiology , Blood Culture/methods , DNA, Bacterial , Electrophoresis, Gel, Pulsed-Field , Humans , Leptospira/genetics , Middle Aged , Minisatellite Repeats , Multilocus Sequence Typing , Polymerase Chain Reaction , Reference Values , Rural Population , Serogroup , UruguayABSTRACT
OBJECTIVE: Leptospirosis is one of the most widespread zoonoses in the world and is caused by spirochetes of the genus Leptospira. In Mozambique, the disease is largely ignored and its epidemiology is unknown. The objective of this study was to investigate the occurrence of leptospirosis in febrile patients. METHODS: This cross-sectional study was performed between July 2012 and September 2015 among febrile patients. A total of 373 paired serum samples were drawn from febrile patients; 208 were from Caia District Hospital (rural setting) in Sofala Province and 165 were from Polana Caniço General Hospital (suburban setting) in Maputo City. Samples were initially screened using an in-house ELISA for IgM and IgG antibodies. Double positive samples were confirmed using a microagglutination test (MAT). RESULTS: Of the 373 febrile patients, five (1.3%) had acute leptospirosis (MAT ≥400) and 38 (10.2%) had a presumptive infection (IgM-positive/MAT <400). While most of the patients with a presumptive infection lived in the rural setting (84.2%, 32/38), the majority of patients with acute infections (60%, 3/5) and with negative results (60.3%, 199/330) lived in the suburban setting (p=0.000). Contact with rodents was significantly higher in patient with acute leptospirosis (100%, 5/5) than in those with a presumptive infection (39.5%, 15/38) or negative results (41.8%, 138/330) (p=0.031). Four out of the five patients (80%) with acute leptospirosis were treated with antimalarial drugs although malaria results were negative. The prevailing serogroup, according to MAT results, was Australis (40%; 4/10), followed by Icterohaemorrhagiae (30%, 3/10). CONCLUSIONS: This study found that leptospirosis is prevalent among Mozambicans, and most cases are misdiagnosed as malaria.
Subject(s)
Floods , Leptospirosis/epidemiology , Adult , Animals , Antibodies, Bacterial/blood , Antimalarials/therapeutic use , Cross-Sectional Studies , Enzyme-Linked Immunosorbent Assay , Female , Fever/epidemiology , Fever/parasitology , Humans , Leptospira/classification , Leptospirosis/diagnosis , Leptospirosis/drug therapy , Leptospirosis/physiopathology , Male , Mozambique/epidemiology , Poverty Areas , Prevalence , Rural Population , Seroepidemiologic Studies , Serogroup , Zoonoses/epidemiologyABSTRACT
INTRODUCTION:: Leptospirosis is an important health concern in Brazil. Currently, information on the epidemiology of the disease in the rural areas of the country is lacking. METHODS:: Serological and molecular techniques were used to characterize a clinical isolate of Leptospira. RESULTS:: The strain CLEP 00060, isolated from a 59-year-old man in a rural area of Rio Grande do Sul state, Brazil, was identified as belonging to L. kirschneri serogroup Pomona serovar Mozdok. CONCLUSIONS:: This study contributes to the local epidemiological knowledge of leptospirosis, prevention of the disease by vaccines, and improvements in its diagnosis.
Subject(s)
Leptospira/classification , Leptospirosis/microbiology , Agglutination Tests , Brazil , Electrophoresis, Gel, Pulsed-Field , Humans , Leptospira/genetics , Leptospira/immunology , Leptospirosis/diagnosis , Male , Middle Aged , Multilocus Sequence Typing , Phylogeny , Rural Population , Serogroup , SerotypingABSTRACT
Abstract INTRODUCTION: Leptospirosis is an important health concern in Brazil. Currently, information on the epidemiology of the disease in the rural areas of the country is lacking. METHODS: Serological and molecular techniques were used to characterize a clinical isolate of Leptospira. RESULTS: The strain CLEP 00060, isolated from a 59-year-old man in a rural area of Rio Grande do Sul state, Brazil, was identified as belonging to L. kirschneri serogroup Pomona serovar Mozdok. CONCLUSIONS: This study contributes to the local epidemiological knowledge of leptospirosis, prevention of the disease by vaccines, and improvements in its diagnosis.
Subject(s)
Humans , Male , Leptospira/classification , Leptospirosis/microbiology , Phylogeny , Rural Population , Brazil , Agglutination Tests , Serotyping , Electrophoresis, Gel, Pulsed-Field , Multilocus Sequence Typing , Serogroup , Leptospira/genetics , Leptospira/immunology , Leptospirosis/diagnosis , Middle AgedABSTRACT
INTRODUCTION: Leptospirosis is caused by a bacterium of the genus Leptospira. This study aimed at investigating the seroprevalence of and risk factors for leptospirosis in humans in Manaus, State of Amazonas. METHODS: Interviews were performed, and 1,000 blood serum samples were examined using a microscopic agglutination test. RESULTS: Forty-three cases were positive; there were 10 serotypes, with coagglutination in 8 cases. The most frequently occurring serotypes were Icterohaemorrhagiae (20.7%), Cynopteri (20.7%), Australis (18.8%), and Copenhageni (16.9%), and the Midwest (54.7%) and South (23.8%) had the most cases; these areas lack basic sanitation. CONCLUSIONS: Disease occurrence might be reduced through improved basic infrastructural conditions.
Subject(s)
Antibodies, Bacterial/blood , Leptospira/immunology , Leptospirosis/epidemiology , Adult , Agglutination Tests , Brazil/epidemiology , Female , Humans , Leptospira/classification , Leptospirosis/diagnosis , Male , Risk Factors , Seroepidemiologic Studies , Socioeconomic Factors , Young AdultABSTRACT
Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and phenotypic characterization showed that all strains carried toxins A and B genes, and belonged to PCR-ribotypes 014, 043 and 046. The isolated strains were sensitive to metronidazole and vancomycin, and resistant to ciprofloxacin and levofloxacin. Resistance to moxifloxacin, was present in the strain from PCR-ribotype 014, that showed an amino acid substitution in gyrB gene (Asp 426 â Asn). This is the first time that this mutation in a PCR-ribotype 014 strain has been described in Brazil.
Subject(s)
Anti-Bacterial Agents/pharmacology , Clostridioides difficile/drug effects , Clostridioides difficile/isolation & purification , Clostridium Infections/microbiology , Drug Resistance, Bacterial , Feces/microbiology , Fluoroquinolones/pharmacology , Adult , Bacterial Toxins/analysis , Brazil , Clostridioides difficile/classification , Clostridioides difficile/genetics , Cross Infection/microbiology , Enzyme-Linked Immunosorbent Assay , Female , Hematologic Neoplasms/complications , Humans , Immunocompromised Host , Male , Moxifloxacin , RibotypingABSTRACT
The aim of this study was to develop an immunocapture polymerase chain reaction (IC-PCR) protocol for leptospirosis. For the standardization of IC-PCR, polyclonal (AS) and monoclonal (MAb) antibodies against different serogroups and serovars of Leptospira were coupled to polystyrene plates. Human sera were artificially contaminated with leptospires and incubated on plates. The bacterial DNA was obtained and used in a multiplex PCR. Sensitivity was tested using sera contaminated with crescent concentrations of leptospires, while specificity was established using sera contaminated with different bacterial genera and sera obtained from patients positive for viral infections. IC-PCR using AS was able to recognize specific serogroups, although some cross-reactions have been observed. No cross-reactions were observed when MAbs were used; however, the sensitivity in this case was lower than that of IC-PCR using AS. IC-PCR proved to be specific to Leptospira and is a promising tool for early diagnosis of leptospirosis, providing additional information about the infecting serovar or serogroup.
Subject(s)
Bacteriological Techniques/methods , Leptospira/isolation & purification , Leptospirosis/diagnosis , Molecular Diagnostic Techniques/methods , Polymerase Chain Reaction/methods , Early Diagnosis , Epidemiological Monitoring , Humans , Immunoassay/methods , Leptospira/genetics , Leptospira/immunology , Sensitivity and SpecificityABSTRACT
The aim of this study was to investigate Clostridium difficile-associated diarrhea (CDAD) in an intensive care unit (ICU) of a tertiary hospital in Rio de Janeiro, Brazil, and to characterize epidemiologically C. difficile strains obtained from an outbreak of CDAD. Within almost a 4-year surveillance period, CDAD incidence was determined for the first time in Brazil, and a 3-fold increase was observed in the average rate of CDAD, featuring an outbreak. About 80% of the patients were over 65 years. The main antibiotic that could be probably associated to CDAD was piperacillin/tazobactam. Four toxigenic strains were isolated, 3 from stools and 1 from environmental samples. They were all resistant to clindamycin and fluoroquinolones. Fingerprinting analysis revealed their distribution between 2 different polymerase chain reaction ribotypes, with one of them being exclusively found in Brazil. It was possible to detect cross-infection and environmental contamination in the ICU. Our results highlight the importance of a continuous CDAD surveillance in the hospitals, especially when a risk group is exposed.
Subject(s)
Clostridioides difficile/isolation & purification , Cross Infection/epidemiology , Diarrhea/epidemiology , Disease Outbreaks , Enterocolitis, Pseudomembranous/epidemiology , Intensive Care Units/statistics & numerical data , Aged , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Brazil/epidemiology , Clostridioides difficile/classification , Clostridioides difficile/drug effects , Clostridioides difficile/genetics , Cross Infection/microbiology , Diarrhea/microbiology , Drug Resistance, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Enterocolitis, Pseudomembranous/microbiology , Feces/microbiology , Humans , Incidence , Polymerase Chain Reaction/methods , Population Surveillance/methods , RibotypingABSTRACT
The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies.
Subject(s)
DNA, Bacterial/analysis , Leptospira/isolation & purification , Polymerase Chain Reaction/methods , Water Microbiology , Brazil , Leptospira/genetics , Poverty AreasABSTRACT
INTRODUCTION: Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. METHODS: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. RESULTS: Each strain presented a unique profile that could be considered to be a specific genomic identity, with the exception of the serovars Icterohaemorrhagiae and Copenhageni, whose profiles were indistinguishable. CONCLUSIONS: It was possible to create a database of molecular profiles, which are being used in the Laboratory for comparing and identifying strains isolated from clinical cases.
Subject(s)
Electrophoresis, Gel, Pulsed-Field , Leptospira/classification , Serotyping/methods , Agglutination Tests , DNA, Bacterial/analysis , Deoxyribonucleases, Type II Site-Specific/analysis , Leptospira/enzymology , Leptospira/geneticsABSTRACT
The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies.
Subject(s)
DNA, Bacterial/analysis , Leptospira/isolation & purification , Polymerase Chain Reaction/methods , Water Microbiology , Brazil , Leptospira/genetics , Poverty AreasABSTRACT
INTRODUÇÃO: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. MÉTODOS: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. RESULTADOS: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. CONCLUSÕES: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.
INTRODUCTION: Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. METHODS: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. RESULTS: Each strain presented a unique profile that could be considered to be a specific genomic identity, with the exception of the serovars Icterohaemorrhagiae and Copenhageni, whose profiles were indistinguishable. CONCLUSIONS: It was possible to create a database of molecular profiles, which are being used in the Laboratory for comparing and identifying strains isolated from clinical cases.
Subject(s)
Electrophoresis, Gel, Pulsed-Field , Leptospira/classification , Serotyping/methods , Agglutination Tests , DNA, Bacterial/analysis , Deoxyribonucleases, Type II Site-Specific/analysis , Leptospira/enzymology , Leptospira/geneticsABSTRACT
This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5% of the samples were positive for tetA, and also 37.5% were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.
Subject(s)
Aeromonas/drug effects , Anti-Bacterial Agents/pharmacology , Antiporters/genetics , Bacterial Proteins/genetics , Lactuca/microbiology , Tetracycline Resistance/genetics , Tetracycline/pharmacology , Aeromonas/genetics , Aeromonas/isolation & purification , Chromosomes, Bacterial/genetics , Microbial Sensitivity Tests , Phenotype , Polymerase Chain ReactionABSTRACT
This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5 percent of the samples were positive for tetA, and also 37.5 percent were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.
Subject(s)
Aeromonas/drug effects , Anti-Bacterial Agents/pharmacology , Antiporters/genetics , Bacterial Proteins/genetics , Lactuca/microbiology , Tetracycline Resistance/genetics , Tetracycline/pharmacology , Aeromonas/genetics , Aeromonas/isolation & purification , Chromosomes, Bacterial/genetics , Microbial Sensitivity Tests , Phenotype , Polymerase Chain ReactionABSTRACT
Metastasis is the major process responsible for the death in cancer patients. In the search for more effective antineoplasic drugs, many substances are under investigation, among them lapachol. This study aims to examine the molecular and morphological alterations caused by lapachol treatment, as well as its effects on the intrinsic tissue invasive property of this cell line. HeLa cells were exposed to different concentrations of lapachol, and the resulting alterations on cellular protein profile, morphology and invasiveness property were studied. At 400 microg/ml, cellular viability remains unchanged, but lapachol induces alterations in the protein profile and inhibits the invasiveness of HeLa cells in CAM model. With these results, we can conclude that lapachol has a great potential of application in fighting metastasis.