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 classification and identification of Acinetobacter genomic species

A total of 211 Acinetobacter strains (representing all currently recognized genomic species) were tested for 329 biochemical characters. Overall similarities of all strains were determined for 145 characters by numerical taxonomic techniques, the UPGMA algorithm and the S _SM and the S _J coefficients as measures of similarity. Seven clusters (two or more strains) and three unclustered strains were recovered at a similarity level of 80.0% ( S _SM). At this level a complete correspondence between phenotypic cluster and genomic species was found only for genomic species 12 ( Ac. radioresistens ). At higher similarity levels (84.0% to 84.6% ( S _SM)), however, several subclusters were found, each representing a single genomic species. An exception were the strains belonging to the genetically closely related species of the Acinetobacter calcoaceticus-baumannii complex. These were recovered scattered in several subclusters. The degree of genomic relatedness between some DNA groups correlated with phenotypic similarities, especially for DNA group 8 ( Ac. lwoffii ) and 15 of Tjernberg and Ursing, and for DNA group 4 ( Ac. haemolyticus ) and 6.
For the majority of genomic species, two identification matrices were constructed consisting of 22 and 10 diagnostic characters, respectively. The correct identification rates for the matrices were 98.0% (22 tests) and 90.8% (10 tests) taking a Willcox probability >0.9. For unambiguous identification of some genomic species, however, additional methods (preferably DNA-DNA hybridization or ribotyping) should be used.     

Numerical taxonomy of Micrococcaceae isolated from Spanish sheep's milk cheeses

Fifty-three strains of Micrococcaceae isolated from Spanish sheep's milk cheeses were subjected to numerical analysis using 43 unit characters. At the 80% similarity level ( S _SM) nine clusters (49 strains) were observed. At the 53% similarity level ( S _J) nine clusters were also found. Cluster composition was not markedly affected by the coefficient used. Novobiocin-resistant, coagulase-negative staphylococci formed clusters of closely related species. However, novobiocin-sensitive, coagulase-negative strains fell into heterogeneous groups. The test that best separated the nine groups formed with both coefficients ( S _SM and S _J) was urease production. Aesculin hydrolysis, haemolytic activity ( S _SM), oxidation of mannose and phosphatase production were also useful characters.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

Numerical classification of Rhodococcus equi and related actinomycetes

Eighty-three strains received as Corynebacterium or Rhodococcus equi and marker cultures of Corynebacterium and Rhodococcus species were subjected to numerical phenetic analyses using 112 unit characters. The data were examined using the simple matching ( S _SM) and Jaccard ( S _J) coefficients and clustering was achieved using the average linkage algorithm. Cluster composition was not markedly affected by the coefficient used or by test error, estimated at 2.9%. Most of the strains received as R. (C.) equi formed a distinct and homogeneous cluster in the aggregate Rhodococcus taxon, the strains of which were sharply separated from marker cultures of C. pseudotuberculosis and C. renale. The taxon R. equi was redescribed and Nocardia calcarea Metcalf & Brown reduced to a subjective synonym of R. erythropolis (Gray & Thornton) Goodfellow & Alderson.     

Isolation and characterization of moderately halophilic bacteria from fully cured salted cod (bachalao)

A total of 128 Strains of moderately halophilic bacteria were isolated from wet, dry and desalted bachalao (salted codfish) as well as from fresh cod and curing salt. The viable count of these bacteria in fully cured wet and dry bachalao ranged from 10³ to 10⁷ per g. All strains were characterized with 40 phenotypic tests and clustered using the S _SM coefficient and UPGMA linking analysis. The strains clustered into five phena at 75% similarity, with 77 strains in phenon A and 37 in phenon E. Two main colony types, smooth and rough, were observed and correspond to phena A, B and C on one hand and phenon E on the other. These two types seem to represent the dominating bacterial flora in fully cured, wet and dry bachalao, respectively. Representative strains of the smooth colony type were characterized further and found to grow well in 0.1–4.5 mol I⁻¹ NaCl and at 15–37°C. They have not, at present, been assigned to any known bacterial species.     

Species of Pseudomonas obtained at 7°C and 30°C during aerobic storage of lamb carcasses

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30°C were Ps.fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7°C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% S _SM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group ( Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (>74% S _SM) and Ps. lundensis (>80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Classification of the spoilage flora offish, 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 (S_SM) and Jaccard (S_J) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% S_J similarity level which correspond to the S_SM level of 86%. S_J-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 10⁸ cfu/g.
Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071^T 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.     

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 (S_SM). 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 (S_SM= 93·13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (S_SM= 84·35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (S_SM= 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.     

Gape morphology of cod Gadus morhua L., haddock Melanogrammus aeglefinus (L.) and whiting Merlangius merlangus (L.) through metamorphosis from larvae to juveniles in the western Irish Sea

Variations in standard length ( L _S), gape size ( S _G) and jaw length ( L _J) were studied in larval and juvenile gadoids (cod Gadus morhua , haddock Melanogrammus aeglefinus and whiting Merlangius merlangus ) from 4 to 70 mm. The increase in S _G and L _J was not linear with respect to L _S. The relationship was best described by segmented regression lines in all three species, with an inflection point at c . 10·5 mm. The S _G and L _J increased more rapidly in relation to larval L _S for individuals smaller than this inflection point size. The rates of increase slowed significantly post-inflection, an effect more noticeable in S _G data compared to L _J data. In each case, the inflection point fell in the intermediate period of development between the larval and juvenile stages, which could be considered as metamorphosis. Published equations that have been used to predict S _G from L _J lead to the overestimation of gape. New relationships are presented, which may be used to predict S _G from measurements of either L _S or upper jaw length in cod, haddock and whiting.     

Numerical taxonomy and identification of lactic acid bacteria from spoiled, vacuum-packaged vienna sausages

Sixty-one lactic acid bacteria from spoiled vacuum-packaged vienna sausages and 15 reference strains were tested for 72 phenotypic characteristics. An identification key and a computer data base, both specific for lactic acid bacteria from meat sources, were used for identification and the results were compared. There was a high correlation (86.9%) between the two procedures in the identification of strains to genus level. However, only a 54.8% correlation was obtained in identifying strains to the species level. With numerical taxonomy ( S _sm matching coefficient with average linkage clustering) 60 strains were recovered in six clusters at the 89% similarity level. While most Leuconostoc strains clustered separately from the Lactobacillus strains, the identity of many leuconostocs was not clarified. The presence of a heterogeneous cluster containing typical and 'atypical' strains of the Lactobacillus saké/curvatus group and a separate homogeneous Lact. curvatus cluster was noted. Closer examination of the data suggested that the 'atypical' lactobacilli were all strains of Lact. saké.     

Taxonomy of bacteria isolated from a coastal, marine fish-rearing unit

Phenetic data on almost 600 aerobic, heterotrophic bacteria from a marine fishrearing unit were collected and analysed using numerical taxonomic techniques. Reference strains, representing 42 taxa were included in the analyses. At similarity levels of 85% or above, with analyses prepared from the simple matching coefficient (S_SM), 81% of the isolates were recovered in eight major and 43 minor phena. Five of the major phena were equated with Acinetobacter calcoaceticus, Photobacterium phosphoreum and Vibrio spp. (three groups); the three unidentified phena contained Gram negative rods with polar flagella which were considered to be intermediate between Cytophaga/Flexibacter and Flavobacterium (two phena), and Gram variable rods. The surface of healthy turbot ( Scophthalmus maximus L. ) was populated by a diverse array of bacteria, including Alcaligenes faecalis, Bacillus firmus, Photobacterium angustum,'Photobacterium logei' and Pseudomonas fluorescens. Taxa, isolated as pure culture growth from within the lesions of moribund animals, included Alteromonas haloplanktis and unidentified Gram negative, budding bacteria. Vibrio anguillarum was not recovered from any turbot suspected of suffering from 'vibriosis'.     

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.     

Characterization of monoclonal antibodies for rapid identification of Actinomyces naeslundii in clinical samples

The purpose of this study was to generate highly specific serological reagents for the quantitative identification of Actinomyces naeslundii in clinical samples, in particular dental plaque. Balb/c mice were immunized with pasteurized human A. naeslundii strains representing different genospecies and serotypes. Ten hybrid cell lines secreting monoclonal antibodies reactive with A. naeslundii were isolated and characterized. Antibody specificity was determined by indirect immunofluorescence and enzyme-linked immunosorbent assay using strains from 59 species and by immunofluorescence analyses of supragingival plaque from 10 gingivitis patients. Nine monoclonal antibodies reacted selectively with A. naeslundii, whereas one additionally bound to Actinomyces israelii. They recognized at least nine different epitopes with characteristic expression patterns among the test strains. Six clusters of antigenically unique or closely related strains could be distinguished. Clusters 1, 4, and 5 represented by 12, 18, and 5 strains, respectively, comprised over 80% of the A. naeslundii strains tested. All reference strains for genospecies 1 grouped with cluster 1. Strains associated with genospecies 2 fell into clusters 4 and 5. Tests with mutant strains indicated that three monoclonal antibodies recognize type 2 and one type 1 fimbriae of genospecies 2. Only four isolates grouped with clusters 2 and 3 characterized by the expression of cluster-specific antigens. Interestingly, cluster 2 and 3 bacteria were markedly more abundant in vivo than indicated by their sparse representation in our strain collection. Overall, all but one of the new monoclonal antibodies should prove of value for the serological classification and rapid quantitative determination of A. naeslundii in clinical samples.     

Taxonomic study of extreme halophilic archaea isolated from the "Salar de Atacama", Chile

A large number of halophilic bacteria were isolated in 1984-1992 from the Atacama Saltern (North of Chile). For this study 82 strains of extreme halophilic archaea were selected. The characterization was performed by using the phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility test. The results, together with those from reference strains, were subjected to numerical analysis, using the Simple Matching (S(SM)) coefficient and clustered by the unweighted pair group method of association (UPGMA). Fifteen phena were obtained at an 70% similarity level. The results obtained reveal a high diversity among the halophilic archaea isolated. Representative strains from the phena were chosen to determine their DNA base composition and the percentage of DNA-DNA similarity compared to reference strains. The 16S rRNA studies showed that some of these strains constitutes a new taxa of extreme halophilic archaea.     

Bacterial whole cell protein profiles of the rRNA group II pseudomonads

Studies on bacterial whole cell protein profiles showed that members of the rRNA group II pseudomonads were distinct from other non-fluorescent and fluorescent pseudomonads, including Pseudomonas aeruginosa, the type species of the genus Pseudomonas. Strains of Ps. andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli, Ps. pickettii, Ps. pseudomallei and Ps. rubrisubalbicans showed uniform and distinct protein patterns, while strains of Ps. solanacearum and Ps. cepacia displayed differences within species. Numerical analysis of their protein profiles with GelManager and Taxan programs generated dendrograms comprising 16 clusters at 89% similarity. Each cluster included strains belonging to the same species with the exception of Ps. solanacearum, which fragmented into three clusters. Pseudomonas solanacearum showed different protein patterns correlating with different biovars and the two divisions of Cook et al. (1989), as well as the results of 16S rRNA gene sequencing. The whole cell protein profiles of a total of 83 strains belonging to 14 bacterial species were numerically analysed.     

Phenotypic identification of non-clinical isolates of Acinetobacter species

Five different phenotypic identification systems were used in attempts to place 164 Acinetobacter strains obtained from a biological nutrient removal plant and 16 reference cultures into the genospecies of Bouvet and Grimont, and Tjernberg and Ursing. Of these strains only four, including two of the reference strains, were identified at a Willcox probability level of >0·95 and a modal likelihood fraction of > 0·0001 by all five systems. These different identification schemes were compared for their usefulness with such non-clinical isolates.     

In most Bradyrhizobium groups sequence comparison of 16S-23S rDNA internal transcribed spacer regions corroborates DNA-DNA hybridizations

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.     

New probability matrices for identification of Streptomyces

The character state data obtained for clusters defined in a previous phenetic classification were used to construct two probabilistic matrices for Streptomyces species. These superseded an original published identification matrix by exclusion of other genera and the inclusion of more Streptomyces species. Separate matrices were constructed for major and minor clusters. The minimum number of diagnostic characters for each matrix was selected by computer programs for determination of character separation indices (CHARSEP) and a selection of group diagnostic properties (DIACHAR). The resulting matrices consisted of 26 phena x 50 characters (major clusters) and 28 phena x 39 characters (minor clusters). Cluster overlap (OVERMAT program) was small in both matrices. Identification scores were used to evaluate both matrices. The theoretically best scores for the most typical example of each cluster (MOSTTYP program) were all satisfactory. Input of test data for randomly selected cluster representatives resulted in correct identification with high scores. The major cluster matrix was shown to be practically sound by its application to 35 unknown soil isolates, 77% of which were clearly identified. The minor cluster matrix provides tentative probabilistic identifications as the small number of strains in each cluster reduces its ability to withstand test variation. A diagnostic table for single-membered clusters, constructed using the CHARSEP and DIACHAR programs, was also produced.     

Numerical taxonomy of Vibrionaceae isolated from cultured amberjack (Seriola dumerili) and surrounding water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S(SM) and S(J) coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S(SM) values > or = 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish.