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J F Höfling

Publications and source records attributed to J F Höfling.

17 recordsLinked to original sources

Mutacin production in Streptococcus mutans genotypes isolated from caries-affected and caries-free individuals.

Relationships between genetic diversity and mutacin production in Streptococcus mutans were evaluated in 319 clinical isolates from eight caries-affected and eight caries-free individuals. The isolates were submitted to mutacin typing and AP-PCR (arbitrarily primed polymerase chain reaction) assay. The mutacin production was detected for 12 Streptococcus sp. indicator strains. Results showed significant variations in the mutacin production profiles and the inhibitory spectra of both groups. A possible association was seen between mutacin activity and the distinct patterns of Streptococcus sp. colonization in the two groups. Genotyping by AP-PCR using the primers OPA-02 and OPA-13 revealed 101 distinct genotypes against 48 phenotypes identified by mutacin typing. No correlation was observed between the inhibitory spectra of mutacin and genotypic similarities based on AP-PCR analyses. According to our results, strains of the same S. mutans genotype showed different mutacin profiles, suggesting a high degree of interstrain diversity. In conclusion, mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species.

Adolescent↗

Evaluation of biochemical and serological methods to identify and clustering yeast cells of oral Candida species by CHROMagar test, SDS-PAGE and ELISA.

The purpose of this work was to evaluate biochemical and serological methods to characterize and identify Candida species from the oral cavity. The strains used were five Candida species previously identified: C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. tropicalis, and Kluyveromyces marxianus, as a negative control. The analyses were conducted through the SDS-PAGE associated with statistical analysis using software, chromogenic medium, and CHROMagar Candida (CA), as a differential medium for the isolation and presumptive identification of clinically important yeasts and an enzyme-linked immunoabsorbent assay (ELISA), using antisera produced against antigens from two C. albicans strains. This method enabled the screening of the three Candida species: C. albicans, C. tropicalis, and C. krusei, with 100% of specificity. The ELISA using purified immunoglobulin G showed a high level of cross-reaction against protein extracts of Candida species. The SDS-PAGE method allowed the clustering of species-specific isolates using the Simple Matching coefficient, S(SM) = 1.0. The protein profile analysis by SDS-PAGE increases what is known about the taxonomic relationships among oral yeasts. This methodology showed good reproducibility and allows collection of useful information for numerical analysis on information relevant to clinical application, and epidemiological and systematical studies.

Animals↗

Differentiation and numerical analysis of oral yeasts based on SDS-Page profiles. Influence of the culture media on the whole-cell protein extracts.

The application of gel electrophoresis and numerical analysis of yeast soluble proteins analysis to the investigation of 12 oral yeast strains belonging to five species is described. It involves one-dimensional electrophoresis of SDS-solubilized whole-cell proteins using different culture media for the cultivation of the cells, integration densitometries in the areas of the gels and percentages of the proteins extraction. These extracts were prepared from four isolates of Candida albicans, two of C. tropicalis, C. guilliermondii, C. parapsilosis and C. krusei. The extracts from whole-cells proteins using different culture media for the cultivation of the cells were fractionated by slab electrophoresis using a discontinuous buffer system. The corresponding patterns showed at least 36 polypeptides in the range of 14.4-200 kDa. Different isolates of each species were clearly different in each of the five species. The data obtained suggest that different nutritional compositions led to the expression of different proteins derived from alternatives metabolic pathways expressed by the electrophoretic profiles. The construction of a database of protein fingerprints and numerical analysis based on such data, may have some implications in the classification and identification of such species with epidemiological, ecological and taxonomic purposes. A well defined or synthetic culture media seems to be much properly.

Candida↗

Inter and intra-specific genetic variability of oral Candida species.

In this report, strains of five different Candida species (Candida albicans, Candida guilliermondii, Candida tropicalis, Candida krusei, and Candida parapsilosis) isolated from healthy human oral cavities as well as their respective type-strains were used in order to establish the genetic diversity existing among the different species and within a certain species, by the analysis of their electrophoretic alloenzyme patterns. These profiles were analyzed for their band positions in the gels, which allowed to group the strains of the same species in species-specific clusters and to treat them as conspecific populations. A total of thirteen enzymatic loci were obtained (ACO, ADH1, ADH2, CAT, G6PDH, GDH, GOT, IDH1, IDH2, LAP, LDH, PER, and SOD). The allelic frequencies (p) and the heterozygosity (h) for all the thirteen loci were determined by diversity index formulas. The GST index is the estimated proportion of genetic diversity that was applied in order to establish inter and intra populational diversity, which, for our results, indicated that 37.75% of total genetic diversity was attributable to differences among the species and the remaining 62.25% was attributable to differences within these populations. An Euclidian distance dendrogram for the different conspecific populations was built, showing that C. guilliermondii grouped first with C. tropicalis and thus formed a expanded cluster with C. albicans. This cluster combined later with another one composed by C. parapsilosis and C. krusei. Comparing our results to the others that were obtained by different molecular techniques, we have observed that the clustering hierarchies follow different paths of organization, varying according to the methodology employed.

Journal Article↗

Clonal variability among oral Candida albicans assessed by allozyme electrophoresis analysis.

A total of 49 Candida albicans strains were isolated from the saliva of 11 healthy children in Piracicaba, Brazil and were analyzed according to their alloenzymatic patterns. Among eight loci assayed, seven were polymorphic and allowed to determine allelic and genotype frequencies, in order to establish the genetic variables for this fungal population. Some children showed just one genetic type, whereas other harbored two or more clones of such yeast, in a multiclonal manner of colonization by C. albicans.

Brazil↗

Grouping oral Candida species by multilocus enzyme electrophoresis.

Multilocus enzyme electrophoresis (MLEE) and numerical taxonomic methods were used to establish the degrees of relatedness among five Candida species commonly isolated from humans oral cavities. Of twenty enzymic systems assayed, five showed no enzymic activity (aspartate dehydrogenase, mannitol dehydrogenase, sorbitol dehydrogenase, glucosyl transferase and alpha-amylase). The obtained data revealed that some of these enzymes are capable of distinguishing strains of different species, but most of them could not organize all strains in their respective species-specific clusters. Numerical classification based on MLEE polymorphism must be regarded for surveys involving just one Candida species.

Candida↗

Degradation of albumin, haemopexin, haptoglobin and transferrin, by black-pigmented Bacteroides species.

Strains of six black-pigmented Bacteroides species and one un-named strain were examined for their ability to degrade the plasma proteins albumin, haemopexin, haptoglobin and transferrin. Strains of B. gingivalis were most effective, degrading all four plasma proteins at different rates. Strains of B. intermedius and B. asaccharolyticus showed intermediate activities, degrading different individual plasma proteins; strains of B. melaninogenicus, B. loeschei and B. denticola were least active, degrading only haemopexin. These findings are discussed in relation to the availability in tissue fluids of iron for bacterial growth.

Bacteroides↗

Degradation in vivo of the C3 protein of guinea-pig complement by a pathogenic strain of Bacteroides gingivalis.

The pathogenicity of five black-pigmented strains of Bacteroides was tested in subcutaneously implanted Teflon cages in guinea pigs. The tissue reaction around the cages was registered and the contents of the fluid of the cages were analyzed. Two strains of B. intermedius produced a localized abscess around the cages, while one strain (381) of B. gingivalis and an asaccharolytic strain (BN11a-f) different from B. gingivalis did not induce any signs of abscess formation. One strain (W83) of B. gingivalis caused extensive purulent breakdown of the tissues. When the inoculum of strain W83 contained more than 10(9) cells, the animals were killed. Strain W83 was the only strain that increased in number in the cage. The fluid of cages inoculated with strain W83 was also remarkably different from the fluid of cages inoculated with the other strains. The fluid had a high proteolytic activity. No C3 protein of complement and only traces of immunoglobulins could be detected in the fluid. Both strain W83 and strain 381 had a high proteolytic activity against whole guinea-pig serum and when bacteria of these two strains were incubated with guinea-pig serum for 24 h, almost all serum proteins, including the C3 protein, were degraded. These two strains might thus have similar capacity in perturbing the host defence when inoculated into the tissue cages. The actual difference in pathogenicity between the strains might be explained by a recent finding that the pathogenic strain W83, but not strain 381, requires complement in activating polymorphonuclear leukocytes. The degradation of the C3 protein by the pathogenic strain W83 of B. gingivalis thus may be the crucial event in its perturbation of the host defence. A degradation of the C3 protein by strain 381 would be of no help in eluding the host defence, since this strain activates polymorphonuclear leukocytes in the absence of complement.

Animals↗

Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis.

Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin.

Bacteroides↗

Streptococcus mutans genotypes isolated from root and coronal caries.

The aim of the present study was to evaluate the colonization profile and clonal distribution of Streptococcus mutans isolated from oral cavities that presented coronal and root caries lesions. The isolation and biochemical identification of mutans streptococci were carried out by using saliva samples, dental plaque, and tissue from the caries lesions. In order to confirm their molecular identity, S. mutans and Streptococcus sobrinus were submitted to the PCR method, using specific primers for portions of the glucosyltransferase genes (gtfB and gtfI, respectively). The AP-PCR method was used to detect the genetic polymorphism of S. mutans strains. Among the isolated and identified species, S. mutans showed a significantly greater frequency of isolation (59.2%) than the other mutans streptococci. Each of the subjects harbored two to ten genotypes of S. mutans, randomly distributed in different sites. S. mutans genotypes showed no evidence of variability in colonizing noncarious and carious surfaces within the same individual, nor evidence of etiologic differences between coronal and root caries. This study showed that no particular genotype of S. mutans is uniquely associated with the initiation and progression of caries, and that root and coronal caries can emerge in the presence of a broad spectrum of S. mutans clones.

Aged↗

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.

Bacteria↗

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species.

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved.

Candida↗

Candida spp. biotypes in the oral cavity of school children from different socioeconomic categories in Piracicaba-SP, Brazil.

Two hundred and thirty-nine (239) Brazilian children, distributed into five distinct socioeconomic categories (A to E) were studied. Saliva samples were analyzed as to flow rate, pH, buffer capacity and microbial parameters. The results revealed the presence of Candida spp. in 47.3% of the samples. The most commonly isolated species was C. albicans, in all socioeconomic categories, followed by C. tropicalis, C. krusei and C. parapsilosis. There was no statistical correlation between secretion rate, buffer capacity and Candida spp. CFU/ml. The prevalence of Candida spp. did not differ substantially among the groups; however the microorganisms were more detected in categories B and C. Among all species, C. albicans was the most prevalent. Only 5% of the sample presented more than one species--C. albicans associated with C. tropicalis, C. parapsilosis or C. krusei. It was possible to detect a significant correlation between caries indices and the socioeconomic categories. All categories presented increased caries indices; however the studied population was considered of low caries risk. There was no positive correlation between the presence of Candida and caries risk in the analyzed population.

Brazil↗

[Quantitative evaluation of Streptococcus mutans and Candida species and salivary factors in the oral cavity of patient undergoing radiotherapy].

The aim of this study was to quantify the microorganisms Streptococcus mutans and Candida sp in the oral cavity of patients with oropharynx carcinoma, before, during and after radiotherapy, and to correlate the results with salivary factors such as pH, buffer capacity (CT) and flow rate (FS). Saliva samples were collected, diluted and inoculated in SB-20 agar and in Sabouraud agar, for Streptococcus mutans and Candida sp, respectively. Previously to dilution, the concentrated saliva was analyzed, and the salivary factors were determined. After the growth of colonies, the number of microorganisms was determined in CFU/ml. The analysis of the results allowed to conclude that the salivary factors are related to the presence of microorganisms, and that the number of CFU/ml increased as salivary flow rate decreased. The effects of radiation compromised salivary homeostasis and favored the increase of infection by yeasts and bacteria.

Analysis of Variance↗

Evaluation of different dehydrogenases to recognize Candida species commonly isolated from human oral cavities.

Electrophoresis of some dehydrogenases were carried out in order to establish relatedness degrees among five Candida species commonly isolated from oral cavity of humans, by numerical taxonomy methods. The obtained data revealed that some dehydrogenases are capable of distinguishing strains of different species, but most of these enzymes could not organize all strains in their respective clusters. Numerical classifications based on dehydrogenase polymorphism must be considered for surveys involving just one species of yeast genus, where this resource had already shown to be useful.

Candida↗

Criteria for Candida albicans numerical analysis based on electrophoretic protein patterns.

Even in normalized gels, some problems with the choice and position of protein bands always have hindered the processing of electrophoretic data. We have developed a way to establish which criterion best fits the necessities in order to maximize the similarity indexes for numerical analysis. Some repetitions of a Candida albicans strain were carried out in eleven different gels. After staining, the bands were scored in numbers within ranges of +/- n values with increases of one quarter steps (+/- 0.25 kDa, +/- 0.5 kDa, +/- 0.75 kDa, +/- 1.0 kDa, +/- 1.25 kDa) that will limit the possibility of variation for the same bands that will appear on the repetitions. Using this criterion, we have determined that values scored within +/- 1.25 kDa could optimize the minimum limiting value of similarity in different dendrograms built.

Candida albicans↗

Oral Candida albicans and Candida dubliniensis differentiation by multilocus enzyme electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis.

Five oral strains of Candida albicans and five C. dubliniensis, as well as their respective type-strains, were analyzed by multilocus enzyme electrophoresis (MLEE) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoreses and numerical analyses, we obtained two distinct species-specific taxa, which may justify the use of MLEE and SDS-PAGE as reliable methods for differentiation and complementary identification of C. dubliniensis.

Alcohol Dehydrogenase↗