New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

Meningococcal Disease Caused by Neisseria meningitidis Serogroup B Serotype 4 in São Paulo, Brazil, 1990 to 1996

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988.21 A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Molecular typing of dengue virus type 2 in Brazil

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).

Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods

Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related) by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP) PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE). Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998.

Adenoviruses isolated from civilian and military personnel in the city of Rio de Janeiro, Brazil

Adenovirus are important pathogen primarily associated to respiratory infections of children and military personnel, even though it is also associated to cases of conjunctivitis and keratoconjunctivitis. We analyzed respiratory secretion collected from subjects with and without respiratory infection symptoms, being 181 civilians and 221 military subjects. The samples were inoculated in HEp-2 and/or A549 tissue cultures for viral isolation. Samples presenting cytopathogenic effect (CPE) in any tissue culture were tested by a polymerase chain reaction (PCR) assay to confirm adenovirus isolation. The isolates confirmed as adenovirus were further analyzed by restriction endonuclease assay for determination of viral species. Three isolates were identified as specie A (two from civilian and one from military), one isolate from military was identified as specie C, and one isolate from civilian was identified as specie D. For two isolates the specie could not be identified.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

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.

Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification

Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.

Toxoplasma gondii genotyping in a dog co-infected with distemper virus and ehrlichiosis rickettsia

This paper reports a toxoplasmosis, erhlichiosis and distemper co-infection in a dog with an exuberant neuropathological clinical picture. Primary involvement was discussed based on information collected in the analysis of the clinical case, such as neurological impairment, epidemiological data, poor immunoprophylactic scheme of the dog affected and the role of these diseases on immunosuppression. Canine distemper and ehrlichiosis were diagnosed based on epidemiologic data, clinical signs, hematological and cytological evaluation. Toxoplasma gondii was isolated and genetically characterized as Type I using restriction analysis (RFLP) with SAG-2 genes. Immunosuppression features of both dogs and human beings are discussed, as well as implications on animal and public health. This is the first report on toxoplasmosis, ehrlichiosis and distemper co-infection in a dog in Brazil, associated with genotyping determination of the T. gondii strain involved.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

Difficulties in the diagnosis of HTLV-2 infection in HIV/AIDS patients from Brazil: comparative performances of serologic and molecular assays, and detection of HTLV-2b subtype

The current diagnosis of human T-lymphotropic virus type-2 (HTLV-2) infection is based on the search of specific antibodies; nevertheless, several studies conducted in Brazil pointed deficiencies of the commercially available kits in detecting HTLV-2, mostly in HIV/AIDS patients. This study searched for the presence of HTLV-1 and -2 in 758 HIV/AIDS patients from Londrina, Paraná, Brazil. Serum samples were screened for HTLV-1/2 antibodies using two EIA kits (Vironostika and Murex), and confirmed by WB (HTLV Blot 2.4, Genelabs). The results obtained by EIA disclosed 49 (6.5%) reactive sera: 43 positive by both EIA kits, and six with discordant results. WB confirmed HTLV-1 infection in seven samples (0.9%) and HTLV-2 in 21 sera (2.8%). Negative and indeterminate results were detected in four (0.5%) and 16 (2.1%) sera, respectively. Blood from 47 out of 49 HTLV seroreactive patients were collected and analyzed for the presence of env, LTR and tax genomic segments of HTLVs by PCR. PCR confirmed six cases of HTLV-1 and 37 cases of HTLV-2 infection (14 out of 16 that were found to be WB indeterminate). Restriction analysis of the env PCR products of HTLV-2 disclosed 36 isolates of HTLV-2a/c subtype, and one of HTLV-2b subtype. These results emphasize the need of improving serologic tests for detecting truly HTLV-2 infected patients from Brazil, and confirm the presence of HTLV-2b subtype in the South of this country.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Molecular characterization of environmental Cryptococcus neoformans isolated in Vitoria, ES, Brazil

Cryptococcus neoformans is the major cause of fungal meningitis, a potentially lethal mycosis. Bird excreta can be considered a significant environmental reservoir of this species in urban areas, thirty-three samples of pigeon excreta were collected within the city of Vitoria, Brazil. Cryptococcus neoformans was isolated and identified using standard biochemical assays in ten samples. PCR amplification with primer M13 and orotidine monophosphate pyrophosphorylase (URA5) gene-restriction fragment length polymorphism (RFLP) analysis discerned serotypes and genotypes within this species. All isolates were serotype A (C. neoformans var. grubii) and genotype VNI. The two alternative alleles a and α at the mating type locus were determined by PCR amplification and mating assays performed on V8 medium. All isolates were MAT α mating type but only 50% were able to mate in vitro with the opposite mating type MAT a tester strains (JEC20, KN99a and Bt63). This study adds information on the ecology and molecular characterization of C. neoformans in the Southeast region of Brazil.