API ZYM Tests Research Articles

Evaluation of Antifungal Properties of Titania P25.

Commercial titania photocatalyst—P25 was chosen for an antifungal property examination due to it exhibiting one of the highest photocatalytic activities among titania photocatalysts. Titania P25 was homogenized first (HomoP25) and then annealed at different temperatures. Additionally, HomoP25 was modified with 0.5 wt% or 2.0 wt% of platinum by a photodeposition method. The obtained samples were characterized by diffuse-reflectance spectroscopy (DRS), X-ray photoabsorption spectroscopy (XPS), X-ray diffraction (XRD) and Raman spectroscopy. Moreover, photocatalytic activity was tested for methanol dehydrogenation under UV/vis irradiation. The spore-destroying effect of photocatalysts was investigated against two mold fungal species, i.e., Aspergillus fumigatus and Aspergillus niger. Both the mycelium growth and API ZYM (estimation of enzymatic activity) tests were applied for the assessment of antifungal effect. It was found that annealing caused a change of surface properties of the titania samples, i.e., an increase in the noncrystalline part, a growth of particles and enhanced oxygen adsorption on its surface, which resulted in an increase in both the hydrogen evolution rate and the antifungal effect. Titania samples annealed at 300−500 °C were highly active during 60-min UV/vis irradiation, inhibiting the germination of both fungal spores, whereas titania modification with platinum (0.5 and 2.0 wt%) had negligible effect, despite being highly active for hydrogen evolution. The control experiments revealed the lack of titania activity in the dark, as well as high resistance of fungi for applied UV/vis irradiation in the absence of photocatalysts. Moreover, the complete inhibition of 19 hydrolases, secreted by both tested fungi, was noted under UV/vis irradiation on the annealed P25 sample. It is proposed that titania photocatalysts of large particle sizes (>150 nm) and enriched surface with oxygen might efficiently destroy fungal structures under mild irradiation conditions and, thus, be highly promising as covering materials for daily products.

Isolation and phenotypic characterization of Gallibacterium anatis biovar haemolytica from a hen with hemorrhagic ooforitis

Objective: the isolation and phenotypically identification of a Gallibacterium anatis biovar haemolytica strain from a hen with hemorrhagic ooforitis; the antimicrobial susceptibility testing of this isolate. Methods and results: a strain of G. anatis biovar haemolytica, was isolated and phenotypic identified by morphological, cultural and biochemical characters examination, with API 20 E, API 20 NE, API STAPH, API ZYM tests and ABIS online software. The antimicrobial susceptibility of the isolate was performed using the standard disk diffusion method. Conclusions: a strain of G. anatis biovar haemolytica was isolated and phenotypically identified from a hen. From our knowledge, this is the first reporting in Romania of isolation and identification of G. anatis biovar haemolytica. The Gah IDSA 161 strain could be phenotypic identified only by ABIS on line software, Pasteurellaceae Database version, unifying the results of four API kits and other biochemical tests. The isolate showed a multi-drug resistant profile to tetracyclines (tetracycline, oxitetracycline, doxicyclin), floroquinolones (enrofloxacin, ciprofloxacin), ampicillin, trimethoprim, nalidixic acid, clindamycin, and it was susceptible to sulfonamide, sulfomethoxazole/trimethoprim, gentamicin, cephalothin, streptomycin, amoxicilin/clavulanic acid.

Characterisation and bioactivities of an exopolysaccharide from an Antarctic bacterium Shewanella frigidimarina W32–2

The continent of Antarctica breeds numerous special extremophiles that have adapted to the extreme variation of environment with a wide range of novel biodiversity. A cold-adapted bacterium Shewanella frigidimarina W32–2, isolated from Antarctic sediments, was identified to be a promising candidate of exopolysaccharides (EPS) production. In the present study, the physiochemistry and bioactivities of the EPS were investigated to promote its application in aquaculture. Physiological characterisation of this strain was firstly performed by means of combined API 20NE and API ZYM tests. A biochemical analysis revealed that the EPS contained a majority of carbohydrates (56.34 ± 1.29%), uronic acids (8.23 ± 0.55%) and high content of sulphate groups (35.43 ± 1.84%), which were also confirmed by the FTIR spectrum showing various functional groups with potential antioxidant activities. The bioactive properties of the EPS in vitro and in vivo were both evaluated. The effect of the EPS on the non-specific immunity of red tilapia via serum injection exhibited 100% of survival rate. Improvement of activities in different serum enzymes, including AKP, ACP, SOD, CAT and LZM, was observed generally at the early stage of EPS treatment (except LZM at later stage). This study explores novel EPS from an extremophile exhibiting powerful bioactivity with immense potential in aquaculture sustainability.

Reclassification of Bacillus invictae as a later heterotypic synonym of Bacillus altitudinis.

The aim of this study was to reclarify the taxonomic status of strain Bacillus invictae Bi.FFUP1T by performing comparative analyses with the other four type strains within the Bacillus pumilus group. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains B. invictae Bi.FFUP1T ( = DSMZ 26896T = MCCC 1A07089T), B. altitudinis 41KF2bT ( = DSMZ 21631T = MCCC 1A06452T), B. safensis FO-36bT ( = DSMZ 19292T = MCCC 1A6451T), B. pumilus ATCC 7061T ( = DSMZ 27T = MCCC 1A06453T) and B. xiamenensis HYC-10T ( = MCCC 1A00008T) were, respectively, 82.90 % and 98.10 %, which are greater than the thresholds for bacterial species delineation, suggesting that they should belong to the same species, while the dDDH and ANI values between strain B. invictae DSMZ 26896T and the other three type strains within the B. pumilus group were below the respective thresholds of 70 % and 95 %. Meanwhile, B. invictae DSMZ 26896T and B. altitudinis 41KF2bT shared 98.7 % gyrB gene sequence similarity based on resequencing, whereas strain B. invictae DSMZ 26896T shared low similarities ( < 95 %) with the other three type strains. In addition, in comparison with those from the other three type strains, phenotypic data of B. invictae DSMZ 26896T and B. altitudinis 41KF2bT, including API 20NE, API ZYM, Biolog GN2 and API 50CHB tests, showed slight differences. The data from these combined genotypic and phenotypic analyses suggest that Bacillus invictae Branquinho et al. 2014 should be regarded as a later heterotypic synonym of Bacillus altitudinisShivaji et al. 2006.

Roseovarius albus sp. nov., a new Alphaproteobacterium isolated from the Mediterranean Sea

Strain 4SM10(T), an aerobic marine, Gram-negative, heterotrophic and non pigmented bacterium isolated from seawater from Vinaroz in Castellón, Spain, was characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence placed the strain within the Roseobacter clade in the family Rhodobacteraceae. Phylogenetic analyses also showed that strain 4SM10(T) forms a stable clade with species of the genus Roseovarius, being related to Roseovarius nubinhibens ISM(T) and Roseovarius aestuarii SMK-122(T) at 97.5 and 97.4 % 16S rRNA sequence similarity, respectively. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strain 4SM10(T) does not belong to the same species as R. aestuarii CECT 7745(T) and Roseovarius nubinhibens CECT 7750(T) displaying ANI values well below the 95 % boundary for genomic species. Strain 4SM10(T) requires Na(+) plus a divalent cation (either Mg(2+) or Ca(2+)) to grow, reduces nitrate to nitrite and uses a large number of amino acids and organic acids (but no carbohydrates) as sole carbon sources. Enzymatic activities displayed in API ZYM tests are alkaline phosphatase, leucine arylamidase and acid phosphatase. The major cellular fatty acids were identified as C18:1 ω7c and/or C18:1 ω6c (67.1 %). The DNA G+C content was determined to be 54.27 mol%. Based on the genotypic and phenotypic data obtained, the name Roseovarius albus sp. nov. is proposed for this novel taxon, with the type strain 4SM10(T) (=CECT 7450(T) = KCTC 22653(T)).

Grain dust originating from organic and conventional farming as a potential source of biological agents causing respiratory diseases in farmers

IntroductionAgricultural producers are exposed to a number of different health risks associated with their work environment.AimThe objective of the study was to assess the degree of colonization by fungi in terms of quantity and in terms of variety of species the samples taken from the settled dust from combine threshing of rye cultivation from organic and conventional farms in the Province of Lublin.Material and methodsThis paper is a preliminary quantitative assessment of the species of fungi colonizing the samples of settled dust collected during combine threshing from organic and conventional farms in the Province of Lublin. One of the stages of the project was the classification of biosafety BSL (biosafety level) of selected isolates and API ZYM tests to evaluate the potential ability of isolates to cause adverse health effects. To determine the concentration and composition of fungi in collected samples plate dilution method was used with two media: Malt Agar and Potato Dextrose Agar.ResultsMost commonly isolated fungi in settled dust samples collected during combine threshing from organic farms, on PDA medium were: Alternaria alternata and Aureobasidium pullulans. Cultures on MA medium were dominated by Alternaria alternata, Mycelia sterilia and Fusarium poae. In samples of dust from conventional crops, the predominant species was Alternaria alternata on PDA medium and on MA medium.ConclusionsThe obtained results show a potential risk of people involved in agricultural work.

Photobacterium aphoticum sp. nov., isolated from coastal water

A facultatively anaerobic marine gammaproteobacterium, designated strain M46(T), was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. The strain was characterized by using a polyphasic approach and was found to be situated within the genus Photobacterium in the family Vibrionaceae. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain M46(T) was closely related to P. rosenbergii CECT 7644(T), P. halotolerans CECT 5860(T) and P. ganghwense CECT 7641(T), showing sequence similarities of 96.8, 96.4 and 96.2 %, respectively. According to the results of phylogenetic analyses based on recA and gyrB gene sequences, the most closely related taxon was P. ganghwense CECT 7641(T) with 87.4 and 85.0 % sequence similarity, respectively. Regardless of the gene used in phylogenetic analysis, strain M46(T) always formed a separate and stable clade containing these three species of the genus Photobacterium. Strain M46(T) was not luminescent and produced a diffusible brown pigment. It required NaCl to grow, reduced nitrate to nitrite and oxidized a small number of substrates in Biolog GN plates. Strain M46(T) was positive for arginine dihydrolase (ADH), β-galactosidase, aesculin hydrolysis and DNase activity. In API ZYM tests, the novel strain was positive for alkaline phosphatase, leucine arylamidase and acidic phosphatase activities. The major cellular fatty acids were unsaturated C(18) and C(16), as in other members of the genus Photobacterium, but their relative amounts and the presence or absence of other fatty acids differentiated strain M46(T) from its closest relatives. Based on the results of this polyphasic taxonomic study, strain M46(T) represents a novel species of the genus Photobacterium, for which the name Photobacterium aphoticum is proposed. The type strain is M46(T) ( = CECT 7614(T) = KCTC 23057(T)).

Ruegeria pelagia is a later heterotypic synonym of Ruegeria mobilis

The 16S rRNA genes of Ruegeria pelagia NBRC 102038T and Ruegeria mobilis NBRC 101030T were resequenced and the results confirmed that they differ by only one base in their almost full-length sequences (1425 nt). The gyrB gene sequence similarity between the two strains was also high (97.7%). The outcome of API 20NE, API ZYM and antibiotic susceptibility tests showed that the two strains show only one difference, in beta-galactosidase activity, in API tests and five differences in susceptibility among 30 tested antibiotics. In addition, similar BOX-PCR fingerprints were obtained and the DNA-DNA relatedness between the two strains was 91+/-4%. On the basis of these results, it is concluded that Ruegeria pelagia Lee et al. 2007 is a later heterotypic synonym of Ruegeria mobilis Muramatsu et al. 2007.

Inhibitory effect of nicotinamide on enzymatic activity of selected fungal strains causing skin infection

Pathogenicity of fungi is connected with their ability to easily penetrate the host tissues, survive in the infected host organism and use the elements of the host tissues as nutrients. Hence, the co-occurrence of pathogenic properties with the high enzymatic activity, which is manifested through the production of various enzymes including extracellular enzymes, was observed. It can be expected that it is possible to decrease fungal pathogenicity by lowering their enzymatic activity. The aim of the study was to determine the effect of nicotinamide on enzymatic activity of the fungi, which are most frequently isolated in cases of skin infection. Enzymatic activity was analysed using 15 Candida albicans, 15 Trichophyton rubrum and 15 Trichophyton mentagrophytes strains. The strains used for the study were collected from the current diagnostic material. API ZYM tests were used in diagnostic analysis. MICs of nicotinamide were determined by the macrodilution method in liquid medium. In the case of Candida strains, the presence of nicotinamide in the broth had a significant effect on the decrease of enzymatic activity (P < 0.05) of esterase (C4), esterase lipase (C-8), valin-arylamidase, acid phosphatase and alpha-glycosydase. A considerably stronger effect of nicotinamide was observed in the case of dermatophytes (P < 0.005). Its action led to a decrease in the activity of all the enzymes under study except alpha-glucosidase produced by T. rubrum strains. Thus, nicotinamide exhibited biological activity towards C. albicans, T. rubrum and Trichophyton mentagrophytes, which resulted in a decrease in the activity of enzymes produced by the fungi.

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle

BackgroundDigital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further.MethodsTissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline.ResultsSeven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern.ConclusionThis is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.

Exploring diversity among Spanish strains of Erwinia amylovora and possible infection sources

We have examined the intraspecific diversity of a collection of 63 Spanish strains of Erwinia amylovora, isolated from 1995 to 2001, to determine whether or not they could be grouped based on phenotypic or genotypic criteria and to investigate the sources of inoculum for fire blight dissemination in Spain. Several biochemical and molecular techniques, such as miniaturized API 20E, API 50CH, ATB G-5 and API-ZYM tests, BIOLOG metabolic fingerprinting, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), minisatellite-primed PCR (MSP-PCR), random amplified polymorphic DNA (RAPD) analyses and AFLP were used. We report the first identification in Spain of the PFGE pattern Pt1, already described in other European countries, together with Pt3 and Pt4 patterns. Moreover, PFGE, together with MSP-PCR, RAPD analyses and AFLP are, until now, the only techniques that have provided information about the possible infection sources and relationships between the different foci in Spain, with AFLP being the most discriminative. These techniques have allowed grouping of Spanish strains by their geographical origin. Our results support the hypothesis that some fire blight outbreaks have been caused by the introduction in Spain of infected plant material, or other inoculum sources from different European countries.

Actividad enzimática extracelular y serotipo en cepas de Cryptococcus neoformans de pacientes con sida en Brasil

One hundred and fifty-one Cryptococcus neoformans strains isolated from AIDS patients in Brazil and maintained in the Adolfo Lutz Institute (São Paulo, Brazil) were tested for phospholipase, protease and other extracellular enzymatic activities and their serotypes determined. Production of extracellular phospholipase and protease was tested by the agar plate methods. Determination of extracellular enzyme profile of the strains was performed by using the API-ZYM kit system, which can test 19 different enzymes. The serotypes were determined by cell agglutination using the Crypto-check method. Among the 151 strains, 147 were identified as serotype A and four strains were serotype AD. Production of extracellular phospholipase and protease was extensive and observable at early stages of incubation. All of the tested strains were positive for the production of both enzymes. In the API-ZYM tests, more than 90 % of the 151 tested strains were positive for esterase C4 (No. 3), esterase lipase C8 (No. 4), leucine arylamidase (No. 6), phosphatase acid (No. 11), naphtol-AS-BI-phosphohydrolase (No. 12), alpha-glucosidase (No. 16) and beta-glucosidase (No. 17). Differences in enzymatic activities between the Brazilian strains and strains isolated in other countries were observed. The phospholipase, protease and other enzyme activities may play a role in host tissue invasion by C. neoformans.

Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov.

The characteristics of the fusiform species Bacteroides forsythus, isolated from human periodontal pockets, were examined. 165 rDNA sequence analysis confirmed that B. forsythus was not a species within the genus Bacteroides sensu stricto. Although B. forsythus was phylogenetically related to Bacteroides distasonis and Bacteroides merdae in the phylogenetic tree, the ratio of anteiso-15:0 to iso-15:0 in whole-cell methanolysates of B. forsythus was different from those of B. distasonis, B. merdae and other Bacteroides species. B. forsythus did not grow on medium containing 20% bile, but members of the Bacteroides fragilis group did. B. forsythus was the only species tested that was trypsin-positive in API ZYM tests. The dehydrogenase enzyme pattern was of no use for the differentiation of B. forsythus and the B. fragilis group. On the basis of these data, a new genus, Tannerella, is proposed for Bacteroides forsythus, with one species, Tannerella forsythensis corrig., gen. nov., comb. nov. The type strain of Tannerella forsythensis is JCM 10827T (= ATCC 43037T).

Characterization of the Lactic Acid Bacteria in Ewe's Milk and Cheese from Northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.

Changes in microbial flora during manufacture of a traditional fermented milk from ewe's milk

Changes in the microbial flora during the manufacture of a sour milk from raw ewe's milk were studied. Mean log counts of lactic acid bacteria, lactobacilli and streptococci (respective values in milk 4.55, 3.19 and 5.56) increased significantly (P<0.05) to 8.46, 6.98 and 8.46, respectively, at 5 days and at the same time the pH decreased from 6.58 in milk to 4.71 in the final product. Levels of total counts, coliforms and psychrotrophs also increased to 108, 107and 106cfu/ml, respectively. Halotolerants and yeasts were counted at quite low numbers throughout manufacture.Isolates of lactic acid bacteria were classified as lactococci, lactobacilli, leuconostocs and enterococci. Enterococci predominated (58.9% of the isolates) over the other lactic acid bacteria after 1 day meanwhile leuconostocs became predominant (46.9%) after 5 days, followed by lactobacilli (29.7%), lactococci (17.2%) and enterococci (6.2%).Lactobacillus plantarumandLeuconostoc mesenteroidessub-spp.cremoriswere the species most frequently isolated. Enzyme activities (API-ZYM tests) of lactococci and leuconostocs were low, in general, but activities ofLb. plantarumstrains were considerably higher. ThreeLc. lactissub-spp.lactisstrains produced diacetyl, four of them formed acetoin, and one was characterized as good active, concerning acidification ability.Results presented here suggest the necessity of using a starter culture, consisting of selected strains of lactococci, leuconostocs and possibly lactobacilli, and pasteurized milk for the production of a high quality product.

Activity of hydrolytic enzymes of Candida albicans strains isolated from patients with periodontal and membrane mucosae of oral cavity diseases.

Fungi are elements of the ontocenosis of the oral cavity and causal factors of inflammatory lesions in its mucous membrane. The objective of the study was to find differences in the activity of hydrolytic enzymes of Candida albicans isolated from patients with diseases of the periodontium and mucous membrane of the oral cavity. Of 235 patients examined, 31 were diagnosed with gingivitis, 38 with glossitis, 28 with leucoplakia, 37 with adult periodontitis, 25 with juvenile periodontitis, 36 stomatitis prothetica and 40 with stomatitis atrophica. In 196 patients (83.4 +/- 2.4%), fungi belonging to Candida species were detected. In the evaluation of Candida albicans strains (146) properties, bioMerieux API ZYM tests containing substrates for the detection of 19 hydrolases were used. All the investigated strains were characterized by the activity of 14 enzymes, i.e. phosphatase alcaline, esterase (C4), esterase lipase (C8), leucine and valine arylamidase, phosphatase acid, naphthol-AS-BI-phosphohydrolase, alpha galactosidase, beta galactosidase, alpha glucosidase, beta glucosidase, N-acetyl-beta-glucosaminidase, alpha mannosidase and alpha fucosidase. Strains isolated from the oral cavity of patients with disease of periodontium and mucous membrane are characterised by the highest phosphatase acid activity. The greatest enzymatic activity is characteristic of Candida albicans isolated from patients with stomatitis atrophica or stomatitis prothetica, and the lowest in strains from gingivitis or juvenile periodontitis cases. Differences in the activity of hydrolases are statistically significant (p < 0.01) for: esterase (C4), leucine and valine arylamidase, phosphatase acid, naphthol-AS-BI-phosphohydrolase, beta glucosidase, N-acetyl-beta-glucosaminidase, of fungi isolated from patients with particular clinical diagnoses.

Human root caries: microbiota of a limited number of root caries lesions.

The microbiota of root caries lesions of different grades of severity were studied. Fourteen lesions were examined. The experimental design of the study allowed correlation of histopathologically distinguishable stages with specific and distinct microbial populations. Dentin samples were ground in a sterile mortar and cultured anaerobically on nonselective Columbia blood agar plates supplemented with 5% hemolyzed human blood and on media selective for Lactobacillus spp. and streptococci. The cultivable microbiota were quantitatively speciated using Rapid ID 32A, Rapid ID 32 Strep, API 20 Strep, APIZYM, and API50 CH tests and SDS-PAG electrophoresis. In initial as well as in advanced lesions gram-positive bacteria accounted for approximately 90% of the CFUt. The proportion of Actinomyces, and in particular A. naeslundii was significantly higher (p < 0.05) in initial lesions than in advanced lesions. In contrast, the percentage of Streptococcus and especially S. mutans was higher (p < 0.05) in advanced than in initial lesions. Surprisingly low (0.8% of the CFUt) was the percentage of lactobacilli in advanced lesions. Gram-negative bacteria formed a minor part of the microbiota in both initial and advanced lesions. Among the gram-negative isolates, Prevotella, Selenomonas, and Bacteroides spp. were most noticeable. In advanced lesions, only the outermost layer of 0.5 mm thickness was populated by a high number of bacteria; the following segments harbored a negligible number of bacteria only. It is concluded that root caries is a continuous destruction process which is restricted to a subsurface zone of limited depth. The necrotic dentin is successively worn away, leading to a saucer-shaped cavitation which is repopulated by plaque. The creation of cavitations favors an aciduric flora. This might explain the succession of bacterial populations observed during the destruction process.

Enzymatic Relatedness Amongst Phytopathogenic Coryneform Bacteria and its Potential Use for their Identification

Summary API ZYM and other experimental enzymatic API-galleries (aminopeptidases, esterases, osidases) were tested on 197 strains representing 16 phytopathogenic coryneform species. It was our aim to evaluate the utility of these tests in the identification of members of this group. The data were subjected to a numerical analysis. At the 71% similarity level, eight phenons were distinguished. Phenon A consisted of Clavibacter michiganense subsp. sepedonicum , a homogeneous phenon with an internal similarity of 82%. The phena B and C comprised the other subspecies of Clavibacter michiganense i. e. Clavibacter michiganense subsp. insidiosum, Clavibacter michiganense subsp. nebraskense, Clavibacter michiganense subsp. tessellarius and Clavibacter michiganense subsp. michiganense . Phenon C moreover, also comprised Curtobacterium species. The 4 pathovars of Curtobacterium flaccumfaciens were related to some Clavibacter michiganense subspecies; they could not be differentiated by API ZYM tests. Phenon G harboured Rhodococcus and Clavibacter xyli strains. In phenon H, eleven coryneforms isolated from Lolium rigidum clustered together with Clavibacter rathayi and Clavibacter tritici. Rhodococcus species (phenon G) and the group of ‘grass coryneforms’ (phenon H) were only distantly related to the other phena of phytopathogenic coryneforms. According to the enzymatic tests strains of the following taxa were scattered over at least two phenons: Clavibacter michiganense subsp. michiganense , subsp. insidiosum , and subsp. nebraskense; Curtobacterium flaccumfaciens pv. flaccumfaciens and Arthrobacter ilicis . For the differentiation of phytopathogenic coryneforms β-galactosidase, β-xylosidase and α-mannosidase seem to be the best differentiating features.

Ochrobactrum anthropi gen. nov., sp. nov. from Human Clinical Specimens and Previously Known as Group Vd

In this study we examined the taxonomic relationships of strains variously labeled Achromobacter species biotypes 1 and 2, Achromobacter group A, and Centers for Disease Control (CDC) groups Vd-1 and Vd-2. Previous studies of ribosomal ribonucleic acid cistron similarities placed these organisms on the Brucella ribosomal ribonucleic acid branch of ribosomal ribonucleic acid superfamily IV; their closest neighbors were Brucella, Phyllobacterium, and the Agrobacterium-Rhizobium complex. We performed a numerical taxonomic analysis of 284 phenotypic features (69 conventional tests, 147 API assimilation tests, 68 API ZYM tests) carried out on 95 strains. These organisms comprised 56 strains thought to correspond to CDC group Vd (including 3 strains originally labeled “Pseudomonas arsenoxydans”) and 39 strains (included for reference purposes) representing the genera Achromobacter, Agrobacterium, Alcaligenes, Brucella, Mycoplana, Phyllobacterium, and Rhizobium. A phenotypic analysis showed that group Vd bacteria are most similar to Phyllobacterium. However, strains of Group Vd were shown to be distinct by deoxyribonucleic acid (DNA)-DNA hybridization and by several phenotypic tests from Phyllobacterium and other related taxa. The CDC group Vd strains formed essentially a single taxon in the numerical taxonomic analysis of phenotypic characters and as determined by DNA-DNA hybridization. This taxon could be subdivided into three biotypes (biotypes A, C, and D), but none of these corresponded to the two biotypes originally described among the group Vd strains. For CDC group Vd we propose a new genus and new species, Ochrobactrum anthropi: The type strain is strain CIP 82.115 (= CIP 14970 = NCTC 12168 = LMG 3331). O. anthropi strains are rod shaped, aerobic, gram negative, nonpigmented, and motile by means of peritrichous flagella, produce acid from several carbohydrates, and reduce both nitrate and nitrite. The guanine-plus-cytosine contents of the DNAs of 29 strains ranged from 56 to 59 mol%. Almost all 56 group Vd strains were originally isolated from various human clinical specimens, commonly from blood cultures.

Three Systems for Biochemical Characterization of Lactobacilli Associated with Meat Spoilage

Three methods (APIZYM, Minitek and VPI systems) were compared for their ability to characterize some of lactobacillus isolates from anaerobically spoiled meats. Nineteen authentic lactobacillus cultures obtained from various sources were also compared. Greening spoilage isolates from frankfurters were identified as Lactobacillus viridescens by the APIZYM and Minitek tests. Lactobacillus isolates from spoiled veal were identified as Lactobacillus plantarum by one of the three methods. Other isolates which could not be assigned to a generic level appeared to be atypical streptobacteria or betabacteria. Except for some disagreement in the identity of lactobacilli among the three systems, they proved useful in distinguishing between the different lactobacilli.