New Strategies on Molecular Biology Applied to Microbial Systematics

NEW STRATEGIES ON MOLECULAR BIOLOGY APPLIED TO MICROBIAL SYSTEMATICS

José F. HÖFLING(¹), Edvaldo A. R. ROSA(¹), Mirian J. BAPTISTA(²) & Denise M. P. SPOLIDÓRIO(¹)

SUMMARY

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.

KEYWORDS: Microbial systematics; New strategies; Molecular biology.

INTRODUCTION

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification⁴¹. The term classification or taxonomy means the arrangement of the organisms in groups (rate). Nomenclature is the attribution of correct international scientific names to organisms. Identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification is required previously.

The majority of modern bacterial taxonomies are based on global similarity. These taxonomies are, at times, called phenetic classifications, once they derive from similarities and differences of the phenotypic characters. Contrasting to phylogenetics, the word phenetic has no evolutionary implication, except in the context of showing the end product of evolution⁷³. Phylogenetic classifications are expressions of the evolutionary relationships among organisms. They convey the degree of changing in the evolutionary lines.

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 was achieved using numerical taxonomy and molecular taxonomy.

The conventional numerical taxonomy is an efficient means used to establish phylogenetic relationships in lower levels to genre⁵⁰. Principle of numerical classification involves the generation of a great deal of data that are grouped in clusters (taxospecies) based on similarity index⁷². Firstly, equal values are attributed to all characters and, after successive confirmatory repetitions, a similarity matrix is generated or a phenogram that will allow the numerical identification of the isolated⁸⁴. Among the samples being studied standard strains are included and their origin and classification are known what will allow the numerical comparison.

Numerical classification is based on phenetic data that are analyzed two by two. The main methods include serology contrasted with antibodies, total proteins and isoenzymes. As one could notice, these methods searched only the presence or absence of proteins in the samples confirming that numerical taxonomy evaluates phenetic data.

Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria^{65, 66}, 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 near of others.

In this review we will show some aspects and applications of the following Molecular Biology techniques: One-Dimensional SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Isoenzymes, Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphism of DNA (RAPD), Ribossomic RNA Polymorphism, and Pulsed Field Gel Electrophoresis (PFGE).

ONE-DIMENSIONAL SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)

The one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has emerged as one of the most efficient versatile and inexpensive techniques for the separation and resolution of bacterial proteins²² that may be used for the establishment of the phenotypic variability and relationship within species^{16, 26, 69}. The usage of SDS promotes modification of total electric charge of proteins annulling the electric positive charges and favoring differential migration because of molecular mass differences⁵⁴. Once electrophoresis finished, that is conducted in discontinuous buffer system⁴⁸, gels may be dyed using protein dyes²² or silver⁸⁷.

Dispositions of protein bands, along the gel, are converted into numerical values based on their relative mobilities (Rf values), that are compared 2 by 2, concerning presence or absence of such bands. These values generate a binary data matrix that, by specialized computer programs⁴⁶, provide similarity matrix that may be converted to similarity phenograms^{40, 72}.

In the last decades, many researches have applied this technique in the identification of clinically significant strains at the species level and its use has found a higher application nowadays. Protein patterns offer considerable potential for typing strains of clinical interest and for taxonomic purposes, especially for the studies of the qualification of biodiversity among microorganisms^{8, 10, 11, 57}. Several entities could be better classified using this technique. COSTAS et al.²⁷ analyzed 31 isolated of Providencia rettgeri of different origins (feces, urine, spittle, insect) and classified them in 13 clusters, with a minimum of 84% of similarity. COSTAS et al.²⁸ assessed levels of similarity among 20 samples of Enterobacter cloacae obtained from 2 British hospitals and classified in 11 clusters with a minimum of 90% of similarity. TANNER et al.⁷⁸ issued an article in which identified, through SDS-PAGE and DNA-DNA hybridization, 12 strains of Bacterioides isolated from advanced periodontal lesions (with 90% of similarity, among them) and due to the low similarity to other species, Bacteriodes forsytus was suggested to this new specie.

This technique is still employed because of its low costs, simplicity and possibility of reducing the number of isolates that would be analyzed by other molecular methods more complex (VANCANNEYT et al.⁸¹, 1991).

ISOENZYMES

In Bacteriology, the usage of isoenzymes in organisms classification refer to articles issued in the 60’s and has assisted Systematics and Taxonomy of several genera and species⁶. Strains of Escherichia coli were intensively assessed concerning genetic diversity, in isolated of urinary infection²⁰ and other sources^{18, 19}. Haemophilus influenzae⁵⁵, Neisseria¹⁷, Pseudomonas of medical interest⁴⁹ and of agronomic interest⁷, Rhizobium⁸⁸, among others.

FOTTREL³² reports that Market & Møller in 1959 were the first ones who observed the occurrence of multiple forms of an enzyme converting the same substratum to which the denomination of isozyme (or isoenzyme). HARRIS⁴² explains isoenzymes as being variations of the structure of an enzyme, genetically determined, occurring within the same specie. It may be due to the fact that more than one genic locus codifies separate versions of one enzyme or due to the fact of the existence of multiple alleles occurring in the same locus (allelic polymorphism). PRAKASH et al.⁶⁰ designated allelic variants of codified enzymes in the same genic locus as allozymes (or alloenzymes) attributing the isoenzyme designation to those that are codified in different loci. Biochemistry Nomenclature Commission of IUPAC-IUB suggested a classification to multiple enzymatic forms in 1977, divided in seven groups: 1) genetically independent proteins; 2) polypeptide chains, not linked in a covalent way; 3) allelic genetic variants; 4) proteins conjugated to other groups; 5) proteins derived from a polypeptide chain; 6) polymers of one sole sub-unit, and 7) different structure forms. Analyzing this classification, DIXON & WEBB³⁰ conclude that groups 1, 2 and 3 are true isoenzymes, while groups 4, 5 and 6 are secondary isoenzymes derived from posttranscriptional modifications of the products of the same gene. Genetically independent proteins (group 1) are products of separated genes, but in some cases these genes may be originated from the duplication of a common ancestor, followed by independent variants. The authors above quote publications of Holmes & Scopes in 1974, Masters & Holmes in 1975 and the review of Whitt, Shaklee & Markert in 1975, developed from variations of lactate dehydrogenase enzyme (E.C. 1.1.1.27) in different animal species, became confirmatory of this theory. Isoenzymes may be evidenced after extraction and not denaturant electrophoresis of the bacterial protean content, in gels of starch or polyacrylamide (ALFENAS et al.⁴) that are "developed" using solutions of substratum and color indicatives (SELANDER et al.⁶⁸).

RESTRICTION FRAGMENT LENGHT POLYMORPHISM (RFLP)

The technique of Restriction Fragment Length Polymorphism (RFLP) is a variation of the technique of DNA-DNA hybridization that is based in the capacity of two complementary DNA strands recognize and recombine by means homology on their sequences of nucleotides, with great applicability in the bacterial systematics. The polymorphism observed in the RFLP technique occurs because the DNA of genetically separate individuals differ in the sequence of nucleotides along the DNA³¹. The presence or absence of specific sequences from 4 to 8 pairs of bases, recognized and cleaved by restriction enzymes, may vary among different individuals, generating polymorphism. Differences in the individuals DNA sequence may also result from insertions, deletions or from other rearrangements (translocations, inversions) that alter the distance among pairs of restriction sites. Undergoing a cleavage by a restriction enzyme, the DNA of genetically separate individuals is cut in the restriction sites generating fragments of different sizes. This way, the genetic base of the observed polymorphism results from mutations in the restriction sites or from deletions, insertions and other rearrangements among these sites.

The basic principle of this technique consists of enzymatic digestion of the DNA, by endonucleases, followed by electrophoresis in gel of agarose that will be subjected to a transference to nylon membrane (Southern Blot). As restriction enzymes cut the DNA in many fragments (thousands), if we process a development of the membrane using an ordinary indicator, as ethidium bromide or the like, we observe a electrophoretic track proceeding from enzymatic digestion in several points of the length of genome. Portions of the DNA radioactively marked or chemiluminescent agent (probes), may hybridize themselves with homologous segments of the immobilized DNA in the membrane. Once taken out the excess of probe that didn’t go through hybridization, the exposition of the membrane to a radiographic or photographic film, it will sensitize the radiographic film in portions where the probe settles appearing bands that may represent genetic polymorphism. RFLP technique allows obtaining a great quantity of electromorphic profiles varying restriction enzymes and probes. RFPL markers show genetic variation in the nucleotide sequence of the region that codify genic products and, on a smaller scale, in non-transcriptable regions, while isoenzymes are restrict to transcriptable regions.

Unlike isoenzymes, the number of RFLP markers is practically unlimited and allelic polymorphism is much higher in each locus³³. COOK et al.²⁴ used RFLP technique in order to analyze phylogenetic relationships among 62 strains of Pseudomonas solanacearum, phytopathogenic bacteria. Genomic DNA was digested by restriction enzymes EcoR I and BamH I and the fragments were hybridized by Southern Blot with DNA probes. The bands obtained were analyzed and allowed the calculation of similarity coefficient among different strains. Two separate groups were identified: the first one includes race 01 (biotypes 3, 4 and 5) and the second one includes race 01 (biotype 01) and races 02 and 03. The average similarity coefficients were 78% and 62% respectively in each group and among the groups 13.5%. ALMEIDA et al.⁵ used sequences of a bacteriophage as probe in order to characterize Vibrio cholerae after digestion with Hind III, obtaining ten RFLP patterns among 58 isolated. This kind of study can also be carried on with a specific segment of DNA, for instance, the DNA of the 16S rRNA, previously enlarged through PCR technique. NORMAND et al.⁵⁶ used enlargement of codifier genes segment of 16S rRNA of species of Frankia sp, an actinomycete fixer of nitrogen, in order to establish phylogenetic relationships among strains of these organisms. The phylogenetic tree was established which show the great existing diversity among physiological races within the genre and alterations were proposed within the Frankiacea family.

The ability in to associate with other techniques, as amplification by PCR or use of rRNA probes, seems to be the main advantage of the RFLP allowing the increasing of sensibility and applicability in researches of microbial variability.

RANDOM AMPLIFIED POLYMORPHISM OF DNA (RAPD)

Random Amplified Polymorphism of DNA (RAPD) technique is an evolution of using markers based on Polymerase Chain Reaction (PCR). PCR is a powerful technique that involves enzymatic synthesis in vitro (conduced in apparatus called" thermal cyclers") of millions of copies of a specific segment of DNA in the presence of DNA polymerase enzyme. PCR reaction is based on enzymatic amplification after annealing of "primers" (starters, from the edges 5) that limited the sequence of the double strand which is the aim of enlargement⁶³. These primers are artificially synthesized in a way that the nucleotide sequences are complementary to specific sequences which go along the aim region. In the RAPD technique only one" primer" is used, arbitrary, while in the PCR classic reaction two primers are used with known aim sequences⁸¹. In order to occur enlargement of a RAPD fragment in the genome in question, two sequences of complementary DNA to the arbitrary primer must be near enough (more than 4,000 base pairs) and in opposite orientation, in a way to allow exponential enlargement of the aim segment through Taq polymerase. Once finished the enlargement (after several cycles), electrophoresis is processed in agarose gel with later development through ethidium bromide, in ultraviolet light or polyacrylamide gel of high resolution, and visualization through radioautograph¹³. The appearing of electrophoretic bands allow the assessment of the molecular nature of the polymorphism to RAPD loci. Experimental evidences lead to the fact that differences of only one pair of bases (point mutations) is enough to cause primer non-completeness with the linking site and, this, way, impede the enlargement of a segment⁸³. Other sources of polymorphism may include deletions of starting sites or insertions that place two starting sites in an adjacent position, in a greater distance which Taq polymerase is able to running. This way, the genetic polymorphism detected by RAPD markers has a binary nature, that is, the enlarged segment (expressed by band in the gel) may be present or absent.

Markers based on RAPD fingerprints has permitted the identification or classification of numberless bacterial entities in several fields: in agriculture, BROUSSAU et al.¹² could identify several strains of Bacillus thuringiensis, an entomopathogenic bacteria used as biological insecticide. In odontology, MENARD et al.⁵³ established patterns to the subspecific identification and classifiction of Porphyromas gengivalis, anaerobic bacteria implied in the development of the periodontal infections. In medicine, several genera were assessed. Helycobacter pylori, main organism involved with appearing and development of gastric ulcers, was studied what concerns its diversity in samples of clinical material by AKOPYANZ et al.¹. Campylobacter isolates from diverse anatomic sites were analyzed by MAZURIER et al.⁵², using such technique. In this same year, MAZURIER et al.⁵¹ issued an article in which they compared RAPD profiles to other methods of lineage classification for isolates of Listeria from clinical material.

Nowadays, the development of more efficient thermal cyclers and better reagents and PCR primers, have increased the quality of the RAPD analyses, and this technique has shown be a powerful tool for applications in taxonomic studies and epidemiological investigations.

RIBOSSOMIC RNA (rRNA) POLYMORPHISM

Special attention has been given to ribotyping. This methodology is based on the idea that the ribossomic RNA (rRNA) alters very slowly as time goes by and this property makes it an extraordinary "biological clock" or "molecular chronometer" through which different genera present different DNA sequences, they may be compared in phylogenetic studies^{44, 84}. In the Escherichia coli DNA, there are seven separate "operons" that collaborate in the rRNA synthesis that represents 70% to 80% of the total RNA of this species. This nucleic acid is composed of three sub-units: 16S, 23S and 5S. Sub-unit 16S is synthesized from a sequence of nearly 1,500 nucleotides. Sub-unit 23S is synthesized from a sequence of nearly 3,000 nucleotides. Sub-unit 5S is synthesized from a sequence of nearly 120 nucleotides. Among these three segments there are intergenic spacer regions. These sub-units bigger and smaller are conserved regions, at subspecific level and highly variable, at subgeneric level. rRNA genes were largely used in order to detect polymorphism among bacteria given the fact that they represent about 0.1% of the genome and are highly conserved⁷⁵. This property was first observed by GRIMONT & GRIMONT³⁷ to identify sub-group within a species, but the word "ribotyping" was used by STULL et al.⁷⁵ in order to describe a method of typing by RFLP in studies of molecular epidemiology for a wide range of bacterial species.

Probes may be rRNA molecules prepared from a representative strain of the study group or, in some cases in which there is enough similarity of sequences, rRNA obtained commercially from E. coli, may be used as probe^{3, 39}.

Concerning ribotyping technique, DNA is extracted from bacterial cells and digested by restriction endonucleases. Fragments are separated by electrophoresis in agarose gel and transferred to the nylon membrane or cellulose acetate ("Southern Blot" technique). These fragments are complementary to the rRNA probe that are observed then, after hybridization, by radioactivity or chemiluminescence^{38, 39}.

Usage of ribotyping has been helping genotypic characterization of streptococci of medical and odontological importance^{2, 62}. TEE et al.⁷⁹ have observed the occurrence of 77 different patterns within 126 clinical isolated of Helicobacter pylori. In a study searching for evaluating differences at subspecific level to clinical isolated of Staphylococus epidermitis, IZARD et al.⁴³ obtained 11 ribotypes from 86 isolated. SMITH & CALLIHAN⁷⁰, in a study about subspecific variability of the Bacterioides genus, used ribotyping to compare 7 species of medical and veterinary.

Ribossomic RNA (rRNA) Polymorphism method has been used mainly in phylogenetic studies, when the researchers want to establish the relationship among some groups of microorganisms. With the emergence of techniques more sensible to genetic variation, as RAPD technology, the studies involving epidemiological interest have not more employed the use of ribossomic RNA as genetic marker.

PULSED FIELD GEL ELECTROPHORESIS (PFGE)

With similar relevance, the Pulsed Field Gel Electrophoresis (PFGE) has been providing a very important taxonomic knowledge. This technique relies on the concept that the limit of the size of DNA fragments separable by conventional electrophoresis in agarose (about 50 kb) may be increased by introducing pulses or changes in the direction of the electric field⁴⁵. PIZZIRANI-KLEINER & AZEVEDO⁵⁹ mention that over 50-60 kb DNA molecules have electrophoretic mobility that are independent of their respective sizes, that is, they all migrate together in the gel, once they present greater sizes than the gel pore diameters so that they dispose themselves longitudinally to the pores.

It was SCHWARTZ & CANTOR⁶⁷ who solved the matter of electrophoretic separation of big DNA segments. These researchers observed that alternating the direction of the electric field, they could cause the molecules to migrate differentially. Applying a variable electric field (here derives the word‘ pulsed’), the DNA molecules move diagonally, in "zigzag", this way, being able of isolating themselves one from another because of their molecular mass. Several electrode configurations were tested aiming improvement of the technique: OFAGE– Orthogonal Field Alternation Gel Electrophoresis¹⁴; FIGE – Field Inversion Gel Electrophoresis¹⁵; CHEF – Contour-Clamped Homogeneous Electric Field²³, as well as variations of each of these methods (i.e. usage of vertical gels). According to BIRREN & LAI⁹ there are other configurations: TAFE (Transverse Alternating Field Electrophoresis), RGE (Rotating Gel Electrophoresis), PACE (Programmable Autonomously Controlled Eletrodes), ZIFE (Zero Integrated Field Electrophoresis) and ST/RIDE (Simultaneously Tangential/Rectangular Inversion Decussate Electrophoresis).

That technique allows obtaining electrophoretic profiles of high molecular mass DNA, for instance chromosomes, allowing the elaboration of eucariotic karyotype whose chromosomes are very small (fungus, yeasts, protozoon etc.). Still, it allows long fragment separation from bacterial chromosome recently digested by restriction endonucleases of rare cleavage sites⁴⁴. Using this technique, it’s feasible to make physical maps to several bacterial genera, once endonucleases which provides great restriction fragments are used⁴⁷. These physic maps may provide important information as size of the genome^{71, 76}, chromosome conformation^{29, 58, 77} or genetic maps when hybridization is done with "cloned" probes from restriction fragments^{41, 80}.

Electrophoresis in pulsed field has been used in epidemiological research of strains of Escherichia coli apart from health professionals²⁵. GORDILLO et al.³⁶ could assess the variability of isolated of Escherichia coli of an epidemic outbreak comparing to an invasive lineage (EIEC), after a previous digestion by Xba I. CHANG & TAYLOR²¹ have made restriction maps and determined genome size of Campylobacter hyointestinalis in clinical isolated. Both publications used Scal I endonuclease, showing the flexibility of usage of this technique. GOERING & DUENSING³⁴ used PFGE associated with hybridization through probes to rRNA genes in order to classify isolates of Staphylococcus of epidemiological interest. PREVOST et al.⁶¹, working with Staphylococcus aureus methicilin-resistent, obtained 26 separate electrophoretic patterns in 239 isolated, after digestion with Sma I. Still, these authors have preconized the superiority of this PFGE technique to the characterization of these organisms when compared to the ribotyping technique.

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. GOMES³⁵, however, points out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a unique technique separately may not provide significant information from Bacterial Systematics viewpoint.

RESUMO

Novas estratégias em biologia molecular aplicadas à sistemática microbiana

Sistemática é o estudo da diversidade dos organismos e suas relações, compreendendo classificação, nomenclatura e identificação. O termo classificação ou taxonomia relaciona-se ao arranjo dos organismos em grupos, nomenclatura é a atribuição de nomes científicos internacionais corretos aos organismos e identificação é a inclusão de linhagens desconhecidas em grupos derivados da classificação. Portanto, a classificação requer previamente, uma nomenclatura estável e uma perfeita identificação. O limiar da nova era da Sistemática bacteriana, pode ser atribuído à introdução e aplicação dos novos conceitos taxonômicos, a partir das décadas de 1950 e 1960. Progressos importantes foram conseguidos empregando-se recursos de taxonomia numérica e a taxonomia molecular. A taxonomia molecular surgiu com a emergência dos recursos da Biologia molecular, provendo conhecimentos que abrangem a Sistemática de bactérias, na qual existe grande interesse evolutivo, ou onde é observada a necessidade de eliminação de quaisquer interferências ambientais. Quando estudamos a composição e disposição de nucleotídeos em determinadas posições do material genético, estamos procurando conhecer seu genoma, muito menos susceptível às alterações ambientais que as proteínas codificadas por ele. Na taxonomia molecular, pode-se pesquisar tanto o DNA quanto o RNA, e as principais técnicas que tem sido empregadas na Sistemática compreendem a construção de mapas de restrição, hibridação DNA-DNA e DNA-RNA, seqüenciamento do DNA, seqüenciamento das subunidades 16S e 23S do rRNA, RAPD, RFLP, PFGE, etc. Técnicas baseadas em seqüenciamento, embora extremamente sensíveis e de grande precisão, são relativamente caras e impraticáveis para a grande maioria dos laboratórios de taxonomia bacteriana. Algumas outras técnicas tem sido aplicadas ao estudo de microrganismos, nos últimos anos elas incluíram preliminarmente, a análise eletroforética de proteínas solúveis e isoenzimas e subseqüentemente a determinação da composição das bases do DNA e a determinação da homologia das seqüências de bases através de experimentos com hibridação DNA-RNA, entre outros. Essas várias técnicas, tem mostrado a carência de informação taxonômica na Sistemática bacteriana. Existem inúmeras técnicas e metodologias que tornam possíveis a identificação e classificação bacteriana – parte delas descritas nessa revisão – permitindo o estabelecimento dos diferentes graus de similaridade em níveis subespecífico e interespecífico, através da análise do polimorfismo fenético. Contudo foi apontada a necessidade do emprego de mais de uma técnica para melhor se estabelecer graus de similaridade entre microrganismos. Dados obtidos à partir de uma única técnica isolada, podem não prover informação suficiente para a Sistemática bacteriana.

REFERENCES

1. AKOPYANZ, N.; BUCANOV, N. O.; WESTBLOM, T. U.; KRESOVICH, S. & BERG, D. E. – DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res., 20: 5137-5142, 1992.

2. ALALUUSUA, S.; ALALUUSUA, S. J.; KARJALAINEN, J. et al. – The demonstration by ribotyping of the stability of oral Streptococcus mutans infection over 5 to 7 years in children. Arch. oral Biol., 39: 467-471, 1995.

3. ALALUUSUA, S.; MÄTTÖ, J.; GRÖNROOS, M. et al. – Oral colonization by more than one clonal type of mutans streptococcus in children with nursing-bottle dental caries. Arch. oral Biol., 41: 167-173, 1996.

4. ALFENAS, A. C.; PETERS, I.; BRUNE, W. & PASSADOR, G. C. – Eletroforese de proteínas e isoenzimas de fungos e essências florestais. Viçosa, Universidade Federal de Viçosa, 1991.

5. ALMEIDA, R. J.; CAMERON, D. N.; COOK, W. L. & WACHSMUTH, I. K.– Vibriophage VcA-3 as an epidemic strain marker for the U. S. gulf coast Vibrio cholerae 01 clone. J. clin. Microbiol., 30: 300-304, 1992.

6. BAPTIST, J. N.; SHAW, C. R. & MANDEL, M. – Zone electrophoresis of enzymes in bacterial taxonomy. J. Bact., 99: 180-188, 1969.

7. BAPTIST, J. N.; SHAW, C. R. & MANDEL, M. – Comparative zone electrophoresis of enzymes of Pseudomonas solanacearum and Pseudomonas cepacia. J. Bact., 108: 799-803, 1971.

8. BARNS, S. M.; LANE, D. J.; SOGIN, M. L.; BIBEAU, C. & WEISBURG, W. G. – Evolutionary relationships among pathogenic Candida species and relatives. J. Bact., 173: 2250-2255, 1991.

9. BIRREN, B. & LAI, L. – Pulsed field gel electrophoresis. A pratical guide. New York. Academic Press, 1993.

10. BLIGNAUT, E. & KOCH, J. L. F. – The presence of yeasts on carious and non-carious teeth. J. dent. Res., 71: 961, 1992.

11. BOOTH, T. – Strategies for study of fungi in marine and marine influenced ecosystems. Rev. Microbiol.(S. Paulo), 10: 123-138, 1979.

12. BROUSSEAU, R.; SAINTONGE, A.; PREFONTAINE, G.; MASSON, L. & CABANA, J. – Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains. Appl. environ. Microbiol., 59: 114-119, 1993.

13. CANCILLA, M. R.; POWELL, I. B.; HILLIER, A. J. & DAVIDSON, B. E. – Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with P32 and fluorescent labels. Appl. environ. Microbiol., 58: 1772-1775, 1992.

14. CARLE, G. F. & ORSON, M. V. – Separation of chromosomal DNA molecules from yeast by orthogonal-field-alteration gel electrophoresis. Nucleic Acids Res., 12: 5647-5664, 1984.

15. CARLE, G. F.; FRANK, M. & ORSON, M. V. – Electrophoretic separations of large DNA molecules by periodic inversion of electric field. Science, 232: 65-68, 1986.

16. CATO, E. P.; HASH, D. E.; HOLDEMAN, L. V. & MOORE, W. E. C.– Electrophoretic study of Clostridium species. J. clin. Microbiol., 15: 688-702, 1982.

17. CAUGANT, D. A.; BØVRE, K.; GAUSTAD, P. et al. – Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers. J. gen. Microbiol., 132: 641-652, 1986.

18. CAUGANT, D. A.; LEVIN, B. R. & SELANDER, R. K. – Genetic diversity and temporal variation in the E. coli population of a human host. Genetics, 98: 467-490, 1981.

19. CAUGANT, D. A.; LEVIN, B. R. & SELANDER, R. K. – Distribution of multilocus genotypes of Escherichia coli within and between families. J. Hyg. (Lond.), 92: 377-384, 1984.

20. CAUGANT, D. A.; LEVIN, D. R.; LIDIN-JANSON, G. et al. – Genetic diversity and relationships among strains of Escherichia coli in the intestine and those causing urinary tract infections. Progr. Allergy, 33: 203-227, 1983.

21. CHANG, N. & TAYLOR, D. E. – Use of pulsed field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a Sal I map of Campylobacter jejuni UA580. J. Bact., 172: 5211-5217, 1990.

22. CHART, H. – Sodium dodecyl sulfate-polyacrylamid gel electrophoresis for the separation and resolution of bacterial components. In: CHART, H. Methods in practical laboratory bacteriology. London, CRC Press, 1994.

23. CHU, G.; VOLLRATH, D. & DAVIS, R. W. – Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science, 234: 1582-1585, 1986.

24. COOK, D.; BARLOW, E. & SEQUEIRA, L. – Genetic diversity of Pseudomonas solanacearum: detection of restriction fragment length polymorphisms with DNA probes that specify virulence and the hypersensitive response. Molec. Plant-Microbe Interact., 2: 113-121, 1989.

25. CORRÊA, I. – Avaliação do procedimento da lavagem das mãos no plano assistencial à criança portadora de diarréia aguda bacteriana. Piracicaba, 1995. (Tese de Doutoramento – Faculdade de Odontologia de Piracicaba– Universidade Estadual de Campinas).

26. COSTAS, M.; HOLMES, B. & SLOSS, H. H. – Numerical analysis of electrophoretic protein patterns of Providencia rustigianii strains from human diarrhoea and other sources. J. appl. Bact., 63: 319-328, 1987.

27. COSTAS, M.; HOLMES, B.; WOOD, A. C. & ON, S. L. W. – Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human feces, urine and other specimens. J. appl. Bact., 67: 441-452, 1989a.

28. COSTAS, M.; SLOSS, L. L.; OWEN, R. J. & GASTON, M. A. – Evaluation of numerical analysis of SDS-PAGE of protein patterns for typing Enterobacter cloacae. Epidem. Infect., 103: 265-274, 1989b.

29. DAVIDSON, B. E.; MacDOUGALL, J. & SAINT-GIRONS, I. – Physical map of the linear chromosome of the bacterium Borrelia burgdorferi 212, a causative agent of Lyme disease, and localization of rRNA genes. J. Bact., 174: 3766-3774, 1992.

30. DIXON, M. & WEBB, E. C. – Enzymes 3. ed. New York, Academic Press, 1979.

31. FERREIRA, M. E. & GRATTAPAGLIA, D. – Introdução ao uso de marcadores RAPD e RFLP em análise genética. Brasília. Embrapa-Cenargen, 1995. (Documento 20).

32. FOTTRELL, P. F. – Functions and applications of isoenzymes. Sci. Prog. Oxf., 55: 543-559, 1967.

33. GABRIEL, D. W. & FEYTER, R. – RFLP analysis and gene tagging for bacterial identification and taxonomic. In: GURR, S. J.; McPHERSON, M. J. & BOWLES, D. J. – Molecular plant pathology. A practical approach. Oxford, University Press, 1992. v. 1, p. 216.

34. GOERING, R. V. & DUENSING, T. D. – Rapid field inversion gel electrophoresis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci. J. clin. Microbiol., 28: 426-429, 1990.

35. GOMES, L. H. – Avaliação de quatro métodos para a caracterização de leveduras. Piracicaba, 1995. (Tese de Mestrado – Escola Superior de Agricultura Luiz de Queiróz da Universidade de São Paulo).

36. GORDILLO, M. E.; REEVE, G. R.; PAPPAS, J. et al. – Molecular characterization of strains of enteroenvasive Escherichia coli 0143, including isolates from a large outbreak in Houston, Texas. J. clin. Microbiol., 30: 889-893, 1992.

37. GRIMONT, F. & GRIMONT, P. A. D. – Ribosomal ribonucleic acid gene restriction patterns as potential taxonomical tools. Ann. Inst. Pasteur Microbiol., 137B: 165-170, 1986.

38. GUNDERSEN, D. E.; LEE, I. M.; SCHAFF, D. A.; HARRISON, D. A. et al. – Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (Asteryellows and related Phytoplasmas) and III (X-disease and related Phytoplasmas). Int. J. system. Bact., 46: 64-75, 1996.

39. GUSTAFERRO, C. A. & PERSING, D. H. – Chemiluminescent universal probe for bacterial ribotyping. J. clin. Microbiol., 30: 1039-1041, 1992.

40. MENDONÇA-HAGLER, L. C. & HAGLER, A. N. – Taxonomia de microrganismos. In: ROITMAN, I.; TRAVASSOS, C. R. & AZEVEDO, J. L. – Tratado de microbiologia. São Paulo. Manole, 1991. v. 2.

41. HANTMAN, M. J.; SUN, S.; PIGGOT, P. J. & DANEO-MOORE, L. – Chromosome organization of Streptococcus mutans GS-5. J. gen. Microbiol., 139: 67-77, 1993.

42. HARRIS, H. – Isoenzymes. New York. Academic Press, 1975. vol. 4.

43. IZARD, N. C.; HAECHLER, H.; GREHN, M. & KAYSER, F. H. – Ribotyping of coagulase-negative Staphylococci with special emphasis on intraspecific typing of Staphylococcus epidermidis. J. clin. Microbiol., 30: 817-823, 1992.

44. KAUFMANN, M. E. & PITT, T. L. – Pulsed-field gel electrophoresis of bacterial DNA. In: CHART, H. – Practical laboratory bacteriology. London. CRC Press, 1994.

45. KAUFMANN, M. E.; PITCHER, D. G. & PITT, T. L. – Ribotyping of bacterial genomes. In: CHART, H. – Practical laboratory bacteriology. London, CRC Press, 1994.

46. KERSTERS, K. – Numerical methods in the classification of bacteria by protein electrophoresis. In: GOODFELLOW, M.; JONES, D. & PRIEST, F. G.– Computer assisted bacterial systematics. New York, Academic Press, 1985.

47. KRAWIEC, S. & RILEY, M. – Organization of the bacterial chromosome. Microbiol. Rev., 54: 502-539, 1990.

48. LAEMMLI, U. K. – Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.

49. LEVIN, M. H.; WEINSTEIN, R. A.; NATHAN, C. et al. – Association of infection caused by Pseudomonas aeruginosa serotype 011 with intravenous abuse of pentazocine mixed with tripelennamine. J. clin. Microbiol., 20: 758-762, 1984.

50. MacDONELL, M. T. & COLWELL, R. R. – The contribution of numerical taxonomy to the systematics of Gram-negative bacteria. In: GOODFELLOW, M.; JONES, D. PRIEST, F. G. – Computer-assisted bacterial systematics. New York, Academic Press, 1985.

51. MAZURIER, S. I.; AUDURIER, A.; MARQET-VAN DER MEE, N.; NOTERMANS, S. & WERNARS, K. A. – A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemic studies of Listeria monocytogenes isolates. Res. Microbiol., 143: 507-512, 1992b.

52. MAZURIER, S. I.; VAN DE GIESSEN, A.; HEUVELMAN, K. & WERNARS, K. A. – Rapid analysis of Campylobacter isolates – DNA fingerprinting without the need to purify DNA. Lett. appl. Microbiol., 14: 260-262, 1992a.

53. MENARD, C.; BROUSSEAU, R. & MOUTON, C. – Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis. FEMS Microbiol. Lett., 95: 163-168, 1992.

54. MOORE, W. E. C.; HASH, D. E.; HOLDEMAN, L. V. & CATO, E. P.– Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl. environ. Microbiol., 39: 900-907, 1980.

55. MUSSER, J. M.; HEWLETT, E. L.; PEPPLER, M. S. & SELANDER, R. K.– Genetic diversity and relationships in populations of Bordetella spp. J. Bact., 166: 230-237, 1986.

56. NORMAND, P.; ORSO, R. J.; COURNOYER, B. et al. – Molecular phylogeny of the genus Frankia and related genera and emendation of the family Frankiaceae. Int. J. system. Bact., 46: 1-9, 1996.

57. O’DONNELL, A. G.; GOODFELLOW, M. & HAWKSWORTH, D. L.– Theoretical and practical aspects of the quantification of biodiversity among microorganisms. Philos. Trans. roy. Soc. Lond. Biol. Sci., 348: 65-73, 1994.

58. OLD, I. G.; MacDOUGALL, J.; SAINT-GIRONS, I. & DAVIDSON, B. E.– Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: possible localization for its origin of replication. FEMS Microbiol. Lett., 78: 245-250, 1992.

59. PIZZIRANI-KLINE, A. A. & AZEVEDO, J. L. – Técnicas eletroforéticas para separação de cromossomos de microrganismos. Piracicaba, Manual Técnico, FEALQ, 1989.

60. PRAKASH, S.; LEWONTIN, R. C. & HUBBY, J. L. – A molecular approach to the study of genic heterozigosity in natural populations. IV. Patterns of genetic variation in central, marginal and isolated populations of Drosophila pseudobscura. Genetics, 61: 841-858, 1969.

61. PREVOST, G.; JAULHAC, B. & PIEMONT, Y. – DNA fingerprinting by pulsed-field gel electrophoresis is more effective than ribotyping in distinguishing among methicillin-resistant Staphylococcus aureus isolates. J. clin. Microbiol., 30: 967-973, 1992.

62. SAARELA, M.; ALALUUSUA, S.; TAKEI, T. & ASIKAINEN, S. – Genetic diversity within isolates of mutans streptococci recognized by an rRNA gene probe. J. clin. Microbiol., 31: 584-587, 1993.

63. SAIKI, R. K.; GELFAND, D. H.; STOFFEL, S. J.; HIGUCHI, R. & HORN, G. T. – Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491, 1988.

64. SALAMA, S. M.; TABOR, H.; RICHTER, M. & TAYLOR, D. E. – Pulsed-field gel electrophoresis for epidemiologic studies of Campylobacter hyointestinalis isolates. J. clin. Mycrobiol., 30: 1982-1984, 1992.

65. SCHLEIFER, K. H. & STACKEBRANDT, E. – Molecular systematics of prokaryotes. Ann. Rev. Microbiol., 37: 143-187, 1983.

66. SCHLEIFER, K. H. & KILPPER-BÄLZ, R. – Molecular and chemotaxonomic approaches to the classification of streptococci, enterococci and lactococci: a review. System. appl. Microbiol., 10: 1-19, 1987.

67. SCHWARTZ, D. C. & CANTOR, C. R. – Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell, 37: 67-75, 1984.

68. SELANDER, R. K.; CAUGANT, D. A. & OCHMAN, H. – Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. environ. Microbiol., 51: 873-884, 1986.

69. SIMONSON, L. G. & SHKLAIR, I. L. – Gel electrophoresis of some cariogenic streptococci. J. dent. Res., 51: 488-491, 1972.

70. SMITH, C. J. & CALLIHAN, D. R. – Analysis of rRNA restriction fragment length polymorphisms from Bacteroides sp. and Bacteroides fragilis isolates associated with diarrhea in humans and animals. J. clin. Microbiol., 30: 806-812, 1992.

71. SMITH, C. L. & CONDEMINE, G. – New approaches for physical mapping of small genomes. J. Bact., 172: 1167-1172, 1990.

72. SNEATH, P. H. A. – Bacterial classification. II. Numerical taxonomy. In: STALEY, J. T.; BRYANT, M. P.; PFENNIG, N. & HOLT, J. G. – Bergey’s manual of systematic bacteriology. Baltimore, Williams and Wilkins, 1989. v. 3. p. 5-7.

73. STACKEBRANDT, E. & GOODFELLOW, M. – Nucleic acid techniques in bacterial systematics. London, John Willey, 1991.

74. STALEY, J. T. & KRIEG, N. R. – Classification of procaryotic organisms: an overview. In: KRIEG, N. R. & HOLT, J. G. – Bergey’s manual of systematic bacteriology. Baltimore, Williams & Wilkins, 1984. v. 1. p. 1-4.

75. STULL, T.; LIPUMA, J. & EDLIND, T. D. – A broad-spectrum probe for molecular epidemiology of bacteria: ribosomal RNA. J. infect. Dis., 157: 280-288, 1988.

76. SUWANTO, A. & KAPLAN, S. – Physical and genetic mapping of the Rhodobacter sphaerodes 2.4.1 genome: genome size, fragment identification, and gene localization. J. Bact., 171: 5840-5849, 1989a.

77. SUWANTO, A. & KAPLAN, S. – Physical and genetic mapping of the Rhodobacter sphaerodes 2.4.1 genome: presence of two unique circular chromosomes. J. Bact., 171: 5850-5859, 1989b.

78. TANNER, A. C. R.; LISTGARTEN, M. A.; EBERSOLE, J. L. & STRZEMPKO, S. – Bacteroides forsythus sp. nov; a slow growing, fusiform Bacteroides sp. from the human oral cavity. Int. J. system. Bact., 36: 213-221, 1986.

79. TEE, W.; LAMBERT, J.; SMALLWOOD, R. et al. – Ribotyping of Helicobacter pylori from clinical specimens. J. clin. Microbiol., 30: 1562-1567, 1992.

80. TULLOCH, D. L.; FINCH, L. R.; HILLIER, A. J. & DAVIDSON, B. E.– Physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 and localization of six putative rRNA operons. J. Bact., 173: 2768-2775, 1991.

81. VANCANNEYT, M.; POT, B.; HENNEBERT, G. & KERSTERS, K. – Differentiation of yeast species based on electrophoretic whole-cell protein patterns. System. appl. Microbiol., 14: 23-32, 1991.

82. WELSH, J. & McCLELLAND, M. – Fingerprinting genomes using PCR with random primers. Nucleic Acids Res., 18: 7213-7218, 1990.

83. WILLIAMS, J. G.; KUBELIK, A. R.; LIVAK, K. J.; RAFALSKI, L. A. & TINGEY, S. V. – DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535, 1990.

84. WILLIANS, S. T.; VICKERS, J. C. A. & GOODFELLOW, M. – Application of new theoretical concepts to the identification of streptomycetes. In: GOODFELLOW, M.; JONES, D. & PRIEST, F. G. – Computer-assisted bacterial systematics. New York, Academic Press, 1985. p. 289-306.

85. WOESE, C. R. – Bacterial evolution. Microbiol. Rev., 51: 221-271, 1987.

86. WRAY, W.; BOULIKAS, T.; WRAY, V. P. & HANCOCK, R. – Silver staining of proteins in polyacrylamide gels. Analyt. Biochem., 118: 197-203, 1981.

87. YOUNG, J. P. W. – Rhizobium population genetics: enzyme polymorphism in isolates from peas, clover, beans, and lucern grown at the same site. J. gen. Microbiol., 131: 2399-2408, 1985.

Recebido para publicação em 18/08/1997

Aceito para publicação em 04/12/1997

References

• 1. AKOPYANZ, N.; BUCANOV, N. O.; WESTBLOM, T. U.; KRESOVICH, S. & BERG, D. E. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res., 20: 5137-5142, 1992.
• 2. ALALUUSUA, S.; ALALUUSUA, S. J.; KARJALAINEN, J. et al. The demonstration by ribotyping of the stability of oral Streptococcus mutans infection over 5 to 7 years in children. Arch. oral Biol., 39: 467-471, 1995.
• 3. ALALUUSUA, S.; MÄTTÖ, J.; GRÖNROOS, M. et al. Oral colonization by more than one clonal type of mutans streptococcus in children with nursing-bottle dental caries. Arch. oral Biol., 41: 167-173, 1996.
• 4. ALFENAS, A. C.; PETERS, I.; BRUNE, W. & PASSADOR, G. C. Eletroforese de proteínas e isoenzimas de fungos e essęncias florestais Viçosa, Universidade Federal de Viçosa, 1991.
• 5. ALMEIDA, R. J.; CAMERON, D. N.; COOK, W. L. & WACHSMUTH, I. K. Vibriophage VcA-3 as an epidemic strain marker for the U. S. gulf coast Vibrio cholerae 01 clone. J. clin. Microbiol., 30: 300-304, 1992.
• 6. BAPTIST, J. N.; SHAW, C. R. & MANDEL, M. Zone electrophoresis of enzymes in bacterial taxonomy. J. Bact., 99: 180-188, 1969.
• 7. BAPTIST, J. N.; SHAW, C. R. & MANDEL, M. Comparative zone electrophoresis of enzymes of Pseudomonas solanacearum and Pseudomonas cepacia J. Bact., 108: 799-803, 1971.
• 8. BARNS, S. M.; LANE, D. J.; SOGIN, M. L.; BIBEAU, C. & WEISBURG, W. G. Evolutionary relationships among pathogenic Candida species and relatives. J. Bact., 173: 2250-2255, 1991.
• 9. BIRREN, B. & LAI, L. Pulsed field gel electrophoresis A pratical guide. New York. Academic Press, 1993.
• 10. BLIGNAUT, E. & KOCH, J. L. F. The presence of yeasts on carious and non-carious teeth. J. dent. Res., 71: 961, 1992.
• 11. BOOTH, T. Strategies for study of fungi in marine and marine influenced ecosystems. Rev. Microbiol.(S. Paulo), 10: 123-138, 1979.
• 12. BROUSSEAU, R.; SAINTONGE, A.; PREFONTAINE, G.; MASSON, L. & CABANA, J. Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains. Appl. environ. Microbiol., 59: 114-119, 1993.
• 13. CANCILLA, M. R.; POWELL, I. B.; HILLIER, A. J. & DAVIDSON, B. E. Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with P32 and fluorescent labels. Appl. environ. Microbiol., 58: 1772-1775, 1992.
• 14. CARLE, G. F. & ORSON, M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alteration gel electrophoresis. Nucleic Acids Res., 12: 5647-5664, 1984.
• 15. CARLE, G. F.; FRANK, M. & ORSON, M. V. Electrophoretic separations of large DNA molecules by periodic inversion of electric field. Science, 232: 65-68, 1986.
• 16. CATO, E. P.; HASH, D. E.; HOLDEMAN, L. V. & MOORE, W. E. C. Electrophoretic study of Clostridium species. J. clin. Microbiol., 15: 688-702, 1982.
• 17. CAUGANT, D. A.; BŘVRE, K.; GAUSTAD, P. et al. Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers. J. gen. Microbiol., 132: 641-652, 1986.
• 18. CAUGANT, D. A.; LEVIN, B. R. & SELANDER, R. K. Genetic diversity and temporal variation in the E. coli population of a human host. Genetics, 98: 467-490, 1981.
• 19. CAUGANT, D. A.; LEVIN, B. R. & SELANDER, R. K. Distribution of multilocus genotypes of Escherichia coli within and between families. J. Hyg. (Lond.), 92: 377-384, 1984.
• 20. CAUGANT, D. A.; LEVIN, D. R.; LIDIN-JANSON, G. et al. Genetic diversity and relationships among strains of Escherichia coli in the intestine and those causing urinary tract infections. Progr. Allergy, 33: 203-227, 1983.
• 21. CHANG, N. & TAYLOR, D. E. Use of pulsed field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a Sal I map of Campylobacter jejuni UA580. J. Bact., 172: 5211-5217, 1990.
• 22. CHART, H. Sodium dodecyl sulfate-polyacrylamid gel electrophoresis for the separation and resolution of bacterial components. In: CHART, H. Methods in practical laboratory bacteriology London, CRC Press, 1994.
• 23. CHU, G.; VOLLRATH, D. & DAVIS, R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science, 234: 1582-1585, 1986.
• 24. COOK, D.; BARLOW, E. & SEQUEIRA, L. Genetic diversity of Pseudomonas solanacearum: detection of restriction fragment length polymorphisms with DNA probes that specify virulence and the hypersensitive response. Molec. Plant-Microbe Interact., 2: 113-121, 1989.
• 25. CORRĘA, I. Avaliaçăo do procedimento da lavagem das măos no plano assistencial ŕ criança portadora de diarréia aguda bacteriana Piracicaba, 1995. (Tese de Doutoramento Faculdade de Odontologia de Piracicaba Universidade Estadual de Campinas).
• 26. COSTAS, M.; HOLMES, B. & SLOSS, H. H. Numerical analysis of electrophoretic protein patterns of Providencia rustigianii strains from human diarrhoea and other sources. J. appl. Bact., 63: 319-328, 1987.
• 27. COSTAS, M.; HOLMES, B.; WOOD, A. C. & ON, S. L. W. Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human feces, urine and other specimens. J. appl. Bact., 67: 441-452, 1989a.
• 28. COSTAS, M.; SLOSS, L. L.; OWEN, R. J. & GASTON, M. A. Evaluation of numerical analysis of SDS-PAGE of protein patterns for typing Enterobacter cloacae Epidem. Infect., 103: 265-274, 1989b.
• 29. DAVIDSON, B. E.; MacDOUGALL, J. & SAINT-GIRONS, I. Physical map of the linear chromosome of the bacterium Borrelia burgdorferi 212, a causative agent of Lyme disease, and localization of rRNA genes. J. Bact., 174: 3766-3774, 1992.
• 30. DIXON, M. & WEBB, E. C. Enzymes 3. ed. New York, Academic Press, 1979.
• 31. FERREIRA, M. E. & GRATTAPAGLIA, D. Introduçăo ao uso de marcadores RAPD e RFLP em análise genética Brasília. Embrapa-Cenargen, 1995. (Documento 20).
• 32. FOTTRELL, P. F. Functions and applications of isoenzymes. Sci. Prog. Oxf., 55: 543-559, 1967.
• 33. GABRIEL, D. W. & FEYTER, R. RFLP analysis and gene tagging for bacterial identification and taxonomic. In: GURR, S. J.; McPHERSON, M. J. & BOWLES, D. J. Molecular plant pathology. A practical approach Oxford, University Press, 1992. v. 1, p. 216.
• 34. GOERING, R. V. & DUENSING, T. D. Rapid field inversion gel electrophoresis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci. J. clin. Microbiol., 28: 426-429, 1990.
• 35. GOMES, L. H. Avaliaçăo de quatro métodos para a caracterizaçăo de leveduras Piracicaba, 1995. (Tese de Mestrado Escola Superior de Agricultura Luiz de Queiróz da Universidade de Săo Paulo).
• 36. GORDILLO, M. E.; REEVE, G. R.; PAPPAS, J. et al. Molecular characterization of strains of enteroenvasive Escherichia coli 0143, including isolates from a large outbreak in Houston, Texas. J. clin. Microbiol., 30: 889-893, 1992.
• 37. GRIMONT, F. & GRIMONT, P. A. D. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomical tools. Ann. Inst. Pasteur Microbiol., 137B: 165-170, 1986.
• 38. GUNDERSEN, D. E.; LEE, I. M.; SCHAFF, D. A.; HARRISON, D. A. et al. Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (Asteryellows and related Phytoplasmas) and III (X-disease and related Phytoplasmas). Int. J. system. Bact., 46: 64-75, 1996.
• 39. GUSTAFERRO, C. A. & PERSING, D. H. Chemiluminescent universal probe for bacterial ribotyping. J. clin. Microbiol., 30: 1039-1041, 1992.
• 40. MENDONÇA-HAGLER, L. C. & HAGLER, A. N. Taxonomia de microrganismos. In: ROITMAN, I.; TRAVASSOS, C. R. & AZEVEDO, J. L. Tratado de microbiologia Săo Paulo. Manole, 1991. v. 2.
• 41. HANTMAN, M. J.; SUN, S.; PIGGOT, P. J. & DANEO-MOORE, L. Chromosome organization of Streptococcus mutans GS-5. J. gen. Microbiol., 139: 67-77, 1993.
• 42. HARRIS, H. Isoenzymes New York. Academic Press, 1975. vol. 4.
• 43. IZARD, N. C.; HAECHLER, H.; GREHN, M. & KAYSER, F. H. Ribotyping of coagulase-negative Staphylococci with special emphasis on intraspecific typing of Staphylococcus epidermidis J. clin. Microbiol., 30: 817-823, 1992.
• 44. KAUFMANN, M. E. & PITT, T. L. Pulsed-field gel electrophoresis of bacterial DNA. In: CHART, H. Practical laboratory bacteriology London. CRC Press, 1994.
• 45. KAUFMANN, M. E.; PITCHER, D. G. & PITT, T. L. Ribotyping of bacterial genomes. In: CHART, H. Practical laboratory bacteriology London, CRC Press, 1994.
• 46. KERSTERS, K. Numerical methods in the classification of bacteria by protein electrophoresis. In: GOODFELLOW, M.; JONES, D. & PRIEST, F. G. Computer assisted bacterial systematics New York, Academic Press, 1985.
• 47. KRAWIEC, S. & RILEY, M. Organization of the bacterial chromosome. Microbiol. Rev., 54: 502-539, 1990.
• 48. LAEMMLI, U. K. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.
• 49. LEVIN, M. H.; WEINSTEIN, R. A.; NATHAN, C. et al. Association of infection caused by Pseudomonas aeruginosa serotype 011 with intravenous abuse of pentazocine mixed with tripelennamine. J. clin. Microbiol., 20: 758-762, 1984.
• 50. MacDONELL, M. T. & COLWELL, R. R. The contribution of numerical taxonomy to the systematics of Gram-negative bacteria. In: GOODFELLOW, M.; JONES, D. PRIEST, F. G. Computer-assisted bacterial systematics New York, Academic Press, 1985.
• 51. MAZURIER, S. I.; AUDURIER, A.; MARQET-VAN DER MEE, N.; NOTERMANS, S. & WERNARS, K. A. A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemic studies of Listeria monocytogenes isolates. Res. Microbiol., 143: 507-512, 1992b.
• 52. MAZURIER, S. I.; VAN DE GIESSEN, A.; HEUVELMAN, K. & WERNARS, K. A. Rapid analysis of Campylobacter isolates DNA fingerprinting without the need to purify DNA. Lett. appl. Microbiol., 14: 260-262, 1992a.
• 53. MENARD, C.; BROUSSEAU, R. & MOUTON, C. Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis FEMS Microbiol. Lett., 95: 163-168, 1992.
• 54. MOORE, W. E. C.; HASH, D. E.; HOLDEMAN, L. V. & CATO, E. P. Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl. environ. Microbiol., 39: 900-907, 1980.
• 55. MUSSER, J. M.; HEWLETT, E. L.; PEPPLER, M. S. & SELANDER, R. K. Genetic diversity and relationships in populations of Bordetella spp. J. Bact., 166: 230-237, 1986.
• 56. NORMAND, P.; ORSO, R. J.; COURNOYER, B. et al. Molecular phylogeny of the genus Frankia and related genera and emendation of the family Frankiaceae Int. J. system. Bact., 46: 1-9, 1996.
• 57. ODONNELL, A. G.; GOODFELLOW, M. & HAWKSWORTH, D. L. Theoretical and practical aspects of the quantification of biodiversity among microorganisms. Philos. Trans. roy. Soc. Lond. Biol. Sci., 348: 65-73, 1994.
• 58. OLD, I. G.; MacDOUGALL, J.; SAINT-GIRONS, I. & DAVIDSON, B. E. Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: possible localization for its origin of replication. FEMS Microbiol. Lett., 78: 245-250, 1992.
• 59. PIZZIRANI-KLINE, A. A. & AZEVEDO, J. L. Técnicas eletroforéticas para separaçăo de cromossomos de microrganismos Piracicaba, Manual Técnico, FEALQ, 1989.
• 60. PRAKASH, S.; LEWONTIN, R. C. & HUBBY, J. L. A molecular approach to the study of genic heterozigosity in natural populations. IV. Patterns of genetic variation in central, marginal and isolated populations of Drosophila pseudobscura Genetics, 61: 841-858, 1969.
• 61. PREVOST, G.; JAULHAC, B. & PIEMONT, Y. DNA fingerprinting by pulsed-field gel electrophoresis is more effective than ribotyping in distinguishing among methicillin-resistant Staphylococcus aureus isolates. J. clin. Microbiol., 30: 967-973, 1992.
• 62. SAARELA, M.; ALALUUSUA, S.; TAKEI, T. & ASIKAINEN, S. Genetic diversity within isolates of mutans streptococci recognized by an rRNA gene probe. J. clin. Microbiol., 31: 584-587, 1993.
• 63. SAIKI, R. K.; GELFAND, D. H.; STOFFEL, S. J.; HIGUCHI, R. & HORN, G. T. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491, 1988.
• 64. SALAMA, S. M.; TABOR, H.; RICHTER, M. & TAYLOR, D. E. Pulsed-field gel electrophoresis for epidemiologic studies of Campylobacter hyointestinalis isolates. J. clin. Mycrobiol., 30: 1982-1984, 1992.
• 65. SCHLEIFER, K. H. & STACKEBRANDT, E. Molecular systematics of prokaryotes. Ann. Rev. Microbiol., 37: 143-187, 1983.
• 66. SCHLEIFER, K. H. & KILPPER-BÄLZ, R. Molecular and chemotaxonomic approaches to the classification of streptococci, enterococci and lactococci: a review. System. appl. Microbiol., 10: 1-19, 1987.
• 67. SCHWARTZ, D. C. & CANTOR, C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell, 37: 67-75, 1984.
• 68. SELANDER, R. K.; CAUGANT, D. A. & OCHMAN, H. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. environ. Microbiol., 51: 873-884, 1986.
• 69. SIMONSON, L. G. & SHKLAIR, I. L. Gel electrophoresis of some cariogenic streptococci. J. dent. Res., 51: 488-491, 1972.
• 70. SMITH, C. J. & CALLIHAN, D. R. Analysis of rRNA restriction fragment length polymorphisms from Bacteroides sp. and Bacteroides fragilis isolates associated with diarrhea in humans and animals. J. clin. Microbiol., 30: 806-812, 1992.
• 71. SMITH, C. L. & CONDEMINE, G. New approaches for physical mapping of small genomes. J. Bact., 172: 1167-1172, 1990.
• 72. SNEATH, P. H. A. Bacterial classification. II. Numerical taxonomy. In: STALEY, J. T.; BRYANT, M. P.; PFENNIG, N. & HOLT, J. G. Bergeys manual of systematic bacteriology Baltimore, Williams and Wilkins, 1989. v. 3. p. 5-7.
• 73. STACKEBRANDT, E. & GOODFELLOW, M. Nucleic acid techniques in bacterial systematics London, John Willey, 1991.
• 75. STULL, T.; LIPUMA, J. & EDLIND, T. D. A broad-spectrum probe for molecular epidemiology of bacteria: ribosomal RNA. J. infect. Dis., 157: 280-288, 1988.
• 76. SUWANTO, A. & KAPLAN, S. Physical and genetic mapping of the Rhodobacter sphaerodes 2.4.1 genome: genome size, fragment identification, and gene localization. J. Bact., 171: 5840-5849, 1989a.
• 77. SUWANTO, A. & KAPLAN, S. Physical and genetic mapping of the Rhodobacter sphaerodes 2.4.1 genome: presence of two unique circular chromosomes. J. Bact., 171: 5850-5859, 1989b.
• 78. TANNER, A. C. R.; LISTGARTEN, M. A.; EBERSOLE, J. L. & STRZEMPKO, S. Bacteroides forsythus sp. nov; a slow growing, fusiform Bacteroides sp. from the human oral cavity. Int. J. system. Bact., 36: 213-221, 1986.
• 79. TEE, W.; LAMBERT, J.; SMALLWOOD, R. et al. Ribotyping of Helicobacter pylori from clinical specimens. J. clin. Microbiol., 30: 1562-1567, 1992.
• 80. TULLOCH, D. L.; FINCH, L. R.; HILLIER, A. J. & DAVIDSON, B. E. Physical map of the chromosome of Lactococcus lactis subsp. lactis DL11 and localization of six putative rRNA operons. J. Bact., 173: 2768-2775, 1991.
• 81. VANCANNEYT, M.; POT, B.; HENNEBERT, G. & KERSTERS, K. Differentiation of yeast species based on electrophoretic whole-cell protein patterns. System. appl. Microbiol., 14: 23-32, 1991.
• 82. WELSH, J. & McCLELLAND, M. Fingerprinting genomes using PCR with random primers. Nucleic Acids Res., 18: 7213-7218, 1990.
• 83. WILLIAMS, J. G.; KUBELIK, A. R.; LIVAK, K. J.; RAFALSKI, L. A. & TINGEY, S. V. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535, 1990.
• 84. WILLIANS, S. T.; VICKERS, J. C. A. & GOODFELLOW, M. Application of new theoretical concepts to the identification of streptomycetes. In: GOODFELLOW, M.; JONES, D. & PRIEST, F. G. Computer-assisted bacterial systematics New York, Academic Press, 1985. p. 289-306.
• 85. WOESE, C. R. Bacterial evolution. Microbiol. Rev., 51: 221-271, 1987.
• 86. WRAY, W.; BOULIKAS, T.; WRAY, V. P. & HANCOCK, R. Silver staining of proteins in polyacrylamide gels. Analyt. Biochem., 118: 197-203, 1981.
• 87. YOUNG, J. P. W. Rhizobium population genetics: enzyme polymorphism in isolates from peas, clover, beans, and lucern grown at the same site. J. gen. Microbiol., 131: 2399-2408, 1985.

(1

) Oral Diagnosis Department, Laboratory of Microbiology and Immunology, School of Dentistry, University of Campinas, Piracicaba, SP, Brasil.

(2

) Phytopathology Department, ESALQ, University of São Paulo, Piracicaba, SP, Brasil.

Correspondence to: Dr. José F. Höfling. Laboratório de Microbiologia e Imunologia, Faculdade de Odontologia de Piracicaba, CP 52, 13414-900 Piracicaba, SP, Brasil. e-mail: [e-mail]

Publication Dates

• Publication in this collection
  09 Oct 1998
• Date of issue
  Nov 1997

History

• Accepted
  04 Dec 1997
• Received
  18 Aug 1997