Numerical classification of some Rhodococci, Corynebacteria and related organisms

Nineteen strains of Corynebacterium sensu stricto, 23 received as Corynebacterium equi or Rhodococcus equi, marker cultures of Arthrobacter, Brevibacterium, Bacterionema matruchotii, Cellulomonas flavigena, Kurthia zopfii, Listeria denitrificans, Microbacterium lacticum, Rhodococcus rubropertinctus and 88 representatives of Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon were the subject of numerical phenetic analyses using 92 characters. The data were examined using the simple matching (SSM) and Jaccard (SJ) coefficients and clustering was achieved using the average linkage algorithm. With a single exception, strains containing meso-diaminopimelic acid, arabinose, galactose and mycolic acids were recovered in five aggregate clusters corresponding to Corynebacterium sensu stricto, Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon. Most of the Corynebacterium (Rhodococcus) equi strains formed a good taxospecies which included the type strain of Corynebacterium hoagii. The numerical data, and the results of earlier chemical and genetical studies, also provide sufficient evidence for the transfer of Bacterionema matruchotii to Corynebacterium sensu stricto as Corynebacterium matruchotii comb.nov. and for the recognition of Rhodococcus globerulus sp.nov. for some strains previously classified as Rhodococcus rubropertinctus (Hefferan) Goodfellow & Alderson. The classification of the remaining marker strains correlates well with other major developments in coryneform taxonomy.     

Numerical taxonomy of alpha-amylase producing Bacillus species

A total of 134 alpha-amylase producing Bacillus isolates and 21 reference strains were divided into 12 groups according to their similarities (% SSM). Phenotypic characteristics determined by the API 20E and API 50CHB galleries, other biochemical tests and morphological characteristics were used for the numerical analysis. The API Computer Service identified 45% of the isolates. The amylase yields of 16 alpha-amylase hyperproducing (AHP) isolates were compared with those of seven amylolytic reference and type strains. The AHP isolates were related to Bacillus subtilis, B. licheniformis and 'B. amyloliquefaciens'.     

Numerical classification and identification of Aeromonas genospecies.

A total of 176 Aeromonas strains representing all currently characterized genospecies were tested for 329 biochemical characters. Overall similarities of all strains were determined by numerical taxonomic techniques, the UPGMA algorithm and the SSM and the SJ coefficients as measures of similarity. Sixteen clusters (two or more strains) and seven unclustered strains were recovered at the 93.5% similarity level (SSM). Genospecies 1, 4, 5, 6, 7, 9, 12 and 13 were largely represented by single phena, whereas strains of genospecies 2 and 3 were found in closely-related phena. Strains belonging to genospecies 8 formed two distinct biotypes. Strains belonging to genospecies 11 formed a subcluster within a cluster representing different genospecies. In general, similar groupings were obtained with the Jaccard coefficient at a similarity level of 80.0% (SJ) with minor changes in the definition of clusters. The phenetic data showed good correlation with the taxa defined by DNA/DNA hybridization and those obtained by multilocus enzyme analysis. For all genospecies (independent from cluster assignment) 30 diagnostic characters were selected to construct a matrix for probabilistic identification. The correct identification rate of the matrix was 71.51% taking a Willcox probability greater than 0.99, and 83.7% taking a Willcox probability greater than 0.9 as identification threshold levels.     

Numerical taxonomy of heavy metal-tolerant bacteria isolated from an estuary.

A total of 230 strains of metal-tolerant bacteria from water and sediment samples collected in Chesapeake Bay were isolated on a medium containing cobalt, lead, mercury, or molybdenum. In addition, a set of 71 cultures were simultaneously isolated on glucose tryptone yeast extract agar medium without metals. Twenty-three reference strains were also included in the numerical taxonomy study of these bacteria, bringing the grand total of strains examined to 324. All strains were examined for 112 biochemical, cultural, morphological, and physiological characters. The taxonomic data obtained were analyzed by computer and the simple matching (SSM) and Jaccard (SJ) coefficients were calculated. Clustering achieved by unweighted average linkage is presented and, from sorted similarity matrices and dendrograms, 294 strains, i.e., 97% of the total, were recovered in 12 phenetic groups defined at the 75 to 80% similarity level. Among the strains there were nine phena presumptively identified as Bacillus, Erwinia, Mycobacterium, Pseudomonas, and coryneforms. From the results of the taxonomic study, it is concluded that metal tolerance in estuarine water and sediment bacteria occurs among a restricted range of taxa distributed throughout the estuarine environment.     

Numerical taxonomy of heavy metal-tolerant nonhalophilic bacteria isolated from hypersaline environments.

A total of 232 metal-tolerant bacterial strains were isolated from water and sediment samples collected in different hypersaline environments located in Cádiz, Huelva and Morón de la Frontera (Spain). They were isolated on a medium containing mercury, chromium, cadmium, copper or zinc. These halotolerant isolates were analyzed by numerical taxonomy techniques by using the simple matching (SSM) and Jaccard (SJ) coefficients; clustering was achieved using the unweighted pair group method with averages (UPGMA) algorithm. At the 81% and 83% similarity level, different numbers of phenons were obtained for Gram-negative and Gram-positive halotolerant microorganisms. Most of the 48 Gram-negative metal-tolerant strains studied were grouped into nine phenons, representing the genera Pseudoalteromonas, Alteromonas, Xanthomonas, Pseudomonas, Alcaligenes and Enterobacteria. The 72 Gram-positive metal-tolerant strains grouped into eight phenons, with only 15 strains left unassigned. Most of the isolates were assigned to the genus Bacillus (seven phenons), and one phenon comprised microorganisms with phenotypic characteristics similar to those of the genus Celullomonas.     

Numerical taxonomy of α-amylase producing Bacillus species

A total of 134 α-amylase producing Bacillus isolates and 21 reference strains were divided into 12 groups according to their similarities (% S_SM). Phenotypic characteristics determined by the API 20E and API 50CHB galleries, other biochemical tests and morphological characteristics were used for the numerical analysis. The API Computer Service identified 45% of the isolates. The amylase yields of 16 α-amylase hyperproducing (AHP) isolates were compared with those of seven amylolytic reference and type strains. The AHP isolates were related to Bacillus subtilis, B. licheniformis and 'B. amyloliquefaciens' .     

Numerical taxonomy and laboratory identification of Actinomyces and Arachnia and some related bacteria.

A numerical taxonomic study was made on 49 facultative anaerobic Gram-positive filamentous and/or diphtheroidal organisms isolated from dental plaques, carious dentin and faeces, together with 63 reference strains belonging to the genera Actinomyces, Arachnia, Bifidobacterium, Actinobacterium, Propionibacterium, Eubacterium and Lactobacillus. They were examined for 90 unit characters covering a wide range of tests and properties. The data were subjected to computer analysis in which the simple matching coefficient (SSM) and the similarity index (SJ) were calculated, and the results of single linkage techniques and an unweighted average linkage cluster analysis technique were compared. The strains fell into six major groups (phena). The Actinomyces strains were recovered in two phena; the first contained Actinomyces israelii and the other facultative anaerobic Actinomyces, including subclusters equal to taxospecies of A. odontolyticus and A. viscosus/A. naeslundii, while the other phenon corresponded to the genera Arachnia, Actinobacterium, Bifidobacterium and Propionibacterium. The groups of Arachnia and Actinobacterium each contained one species, representing taxospecies of Arachnia propionica and Actinobacterium meyerii. Taxonomic criteria, both constant and discriminative, were selected to form a diagnostic table useful for laboratory identification of this group of organisms. Immunofluorescence supported the numerical data.     

Numerical taxonomy of Bacillus isolated from orally administered drugs

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (SSM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (SSM = 93.13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (SSM = 84.35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (SSM = 80.14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.     

A numerical taxonomic study of anaerobic gram-negative bacilli classified as Bacteroides ureolyticus isolated from patients with non-gonococcal urethritis

A numerical taxonomic study of 64 strains of anaerobic Gram-negative bacilli isolated from men with non-gonococcal urethritis, two unclassified laboratory strains of 'corroding bacilli', and 12 other strains of anaerobic Gram-negative bacilli, including nine received as anaerobic curved rods and three as 'Bacteroides corrodens' (B. ureolyticus), isolated from women with bacterial vaginosis, was undertaken. Seventeen reference anaerobic strains belonging to the genera Bacteroides, Fusobacterium, Mobiluncus, Mitsuokella and Wolinella were included. Morphological, biochemical and physiological characteristics were examined in 103 tests. The resemblance between the 95 strains was calculated using the SSM, SJ and DP coefficients for cluster analyses based on the UPGMA method. All three approaches gave similar groupings, and the estimated average probability of test error was 2.46%. The strains fell into 10 phenons. The unclassified strains from men and three from women with lower genital-tract infections, and the laboratory strains of 'corroding bacilli' clustered in one phenon with the reference strains of B. ureolyticus, indicating that they correspond to B. ureolyticus. The other unclassified strains of anaerobic curved rods clustered as a distinct phenon. They correspond to species of the newly described genus Mobiluncus. The taxonomic data and the compilation of diagnostic tables serve as a useful guide for the laboratory identification of clinical isolates regarded as B. ureolyticus.     

Classification of the spoilage flora of fish, with special reference to Shewanella putrefaciens

One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes, were phenotypically classified using 124 unit characters. Data were processed by the Simple Matching (SSM) and Jaccard (SJ) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% SJ similarity level which correspond to the SSM level of 86%. SJ-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps. lundensis (cluster 2; 2% of the field strains), Ps. fluorescens biovar III (cluster 4; 4% of the field strains), Ps. putida biovar A (cluster 5; 3% of the field strains), Ps. fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer. sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish). Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10(8) cfu/g. Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071T and reference strains from sources other than fish, did not group in any of the clusters. The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.     

Biosystematics and diversity amongst novel carboxydotrophic actinomycetes

Fifty-four carboxydotrophic actinomycetes isolated from soils and composts were compared through 119 unit characters with representative mesophilic and thermophilic streptomycetes. The data were examined using the Jaccard, pattern and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. Acceptable cophenetic correlation and test error values allowed confidence to be placed in the resultant numerical taxonomies. The carboxydotrophic actinomycetes, which were distinct from cluster-groups corresponding to the mesophilic and thermophilic streptomycetes, formed two major cluster-groups the members of which were examined for the presence of diagnostic chemical markers. All but two of the carboxydotrophic actinomycetes had a profile of chemical properties consistent with their assignment to the genusStreptomyces. Quantitative fatty acid data were examined using the SIMCA package and the two statistically significant groups obtained corresponded with the cluster-groups circumscribed in the numerical phenetic analysis. Members of the two groups were also distinguished on the basis of their phospholipid composition. The two strains that containedmeso- as opposed to LL-diaminopimelic acid in their peptidoglycan also showed a distinct chemotaxonomic profile. It was concluded that the carboxydotrophic actinomycetes form a novel and taxonomically diverse group.     

Proposal of two subspecies of Fusobacterium necrophorum (Flügge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898)

The biological and biochemical properties, DNA base compositions, and levels of DNA-DNA homology of two biovars of Fusobacterium necrophorum were examined. Some differences were found between the two biovars in biological and biochemical properties. The G + C contents of DNAs from biovar A strains VPI 2891T (T = type strain), NCTC 10576, N167, Fn47, and Fn43, were 32, 30, 29, 28, and 31 mol%, respectively. The G + C contents of DNAs from biovar B strains Fn524T, 606, Fn49, Fn45, and 1260 were 30, 31, 27, 31, and 30 mol%, respectively. Labeled DNA from biovar A strain VPI 2891T exhibited 100 to 80% relatedness to DNAs from biovar A strains and 59 to 51% relatedness to DNAs from biovar B strains. Labeled DNA from biovar B strain Fn524T exhibited 100 to 81% relatedness to DNAs from biovar B strains and 71 to 60% relatedness to DNAs from biovar A strains. Therefore, the names Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898), are proposed for Fusobacterium necrophorum biovars A and B, respectively. The type strain of F. necrophorum subsp. necrophorum is strain VPI 2891 (= JCM 3718 = ATCC 25286), and the type strain of F. necrophorum subsp. funduliforme is strain Fn524 (= JCM 3724).     

A polyphasic taxonomic study of thermophilic bacilli from shallow, marine vents

Eighty-seven thermophilic, aerobic, spore-forming bacteria were isolated from shallow, marine, thermal vents of the Eolian Islands (Italy) and tested for a broad spectrum of phenotypic characteristics. A numerical taxonomy study was performed on these isolates and 8 thermophilic Bacillus and Geobacillus reference strains by 89 selected features. Results from cluster analysis showed the formation of nine clusters. Most of the isolates (83%) fell into several phenetically well distinguished clusters, loosely related to Geobacillus thermodenitrificans. The remaining isolates grouped together with different reference strains. Eighteen isolates, representative of the different clusters, were selected for subsequent genotypic characterisation, including partial 16S rDNA sequence analysis of 18 strains and almost complete 16S rDNA sequences of 9 strains. Subsequent DNA/DNA reassociation studies and determination of the base composition of DNA identified seven isolates as Geobacillus thermodenitrificans, two isolates as G. thermoleovorans and one isolate as Bacillus pallidus. Four isolates represented two novel species of Bacillus. The remaining four represented novel Geobacillus species, one of which has recently been described as Bacillus vulcani DSMZ 13174 T.     

Lactobacillus intestinalis (ex Hemme 1974) sp. nov., nom. rev., isolated from the intestines of mice and rats

The genetic and phenotypic properties of 10 strains identified as Lactobacillus intestinalis sp. nov. were examined. These strains constitute a distinct species which can be differentiated from all of the previously described homofermentative species in the genus Lactobacillus by their carbohydrate fermentation pattern. The guanine-plus-cytosine contents of their DNAs are 33 to 35 mol%. DNAs from 10 other Lactobacillus species did not exhibit significant levels of relatedness to representative strains of the new species. The name Lactobacillus intestinalis (ex Hemme) sp. nov., nom. rev. is proposed for these isolates, and strain Th4 (= ATCC 49335 = JCM 7548) is the type strain.     

Numerical taxonomy and ecology of petroleum-degrading bacteria.

A total of 99 strains of petroleum-degrading bacteria isolated from Chesapeake Bay water and sediment were identified by using numerical taxonomy procedures. The isolates, together with 33 reference cultures, were examined for 48 biochemical, cultural, morphological, and physiological characters. The data were analyzed by computer, using both the simple matching and the Jaccard coefficients. Clustering was achieved by the unweighted average linkage method. From the sorted similarity matrix and dendrogram, 14 phenetic groups, comprising 85 of the petroleum-degrading bacteria, were defined at the 80 to 85% similarity level. These groups were identified as actinomycetes (mycelial forms, four clusters), coryneforms, Enterobacteriaceae, Klebsiella aerogenes, Micrococcus spp. (two clusters), Nocardia species (two clusters), Pseudomonas spp. (two clusters), and Sphaerotilus natans. It is concluded that the degradation of petroleum is accomplished by a diverse range of bacterial taxa, some of which were isolated only at given sampling stations and, more specifically, from sediment collected at a given station.     

A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains, B. cereus (8), including two of var. mycoides, and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus. Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.     

A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains), B. cereus (8), including two of var. mycoides , and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus . Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.     

Identification of thermophilic bacteria in solid-waste composting.

The thermophilic microbiota of solid-waste composting, with major emphasis on Bacillus spp., was examined with Trypticase soy broth (BBL Microbiology Systems) with 2% agar as the initial plating medium. Five 4.5-liter laboratory units at 49 to 69 degrees C were fed a mixture of dried table scraps and shredded newspaper. The composting plants treating refuse at Altoona, Pa., and refuse-sludge at Leicester, England, were also sampled. Of 652 randomly picked colonies, 87% were identified as Bacillus spp. Other isolates included two genera of unidentified nonsporeforming bacteria (one of gram-negative small rods and the other of gram-variable coccobacilli), the actinomycetes Streptomyces spp. and Thermoactinomyces sp., and the fungus Aspergillus fumigatus. Among the Bacillus isolates, the following, in order of decreasing frequency, were observed: B. circulans complex, B. stearothermophilus, B. coagulans types A and B, B. licheniformis, B. brevis, B. sphaericus, Bacillus spp. types i and ii, and B. subtilis. About 15% of the Bacillus isolates could be assigned to species only by allowing for greater variability in one or more characteristics than has been reported by other authors for their strains. In particular, growth at higher temperatures than previously reported was found for strains of several species. A small number of Bacillus isolates (less than 2%) could not be assigned to any recognized species.     

Identification of thermophilic bacteria in solid-waste composting

The thermophilic microbiota of solid-waste composting, with major emphasis on Bacillus spp., was examined with Trypticase soy broth (BBL Microbiology Systems) with 2% agar as the initial plating medium. Five 4.5-liter laboratory units at 49 to 69 degrees C were fed a mixture of dried table scraps and shredded newspaper. The composting plants treating refuse at Altoona, Pa., and refuse-sludge at Leicester, England, were also sampled. Of 652 randomly picked colonies, 87% were identified as Bacillus spp. Other isolates included two genera of unidentified nonsporeforming bacteria (one of gram-negative small rods and the other of gram-variable coccobacilli), the actinomycetes Streptomyces spp. and Thermoactinomyces sp., and the fungus Aspergillus fumigatus. Among the Bacillus isolates, the following, in order of decreasing frequency, were observed: B. circulans complex, B. stearothermophilus, B. coagulans types A and B, B. licheniformis, B. brevis, B. sphaericus, Bacillus spp. types i and ii, and B. subtilis. About 15% of the Bacillus isolates could be assigned to species only by allowing for greater variability in one or more characteristics than has been reported by other authors for their strains. In particular, growth at higher temperatures than previously reported was found for strains of several species. A small number of Bacillus isolates (less than 2%) could not be assigned to any recognized species.     

Characterization of bacterial pectinolytic strains involved in the water retting process

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.