API 50CH Systems Research Articles

Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolate.

A strain of lactic acid bacteria from cheese was isolated, that showed strong growth inhibitory effects on Streptococcus mutans. The API 50CH system and 16S rDNA sequencing verified that this was a novel strain, and was named Lacticaseibacillus rhamnosus VHProbi M14. The strain inhibited the growth of S. mutans and Fusobacterium nucleatum under mixed culture conditions, coaggregated with S. mutans and F. nucleatum, and reduced the adhesion of S. mutans and F. nucleatum on cultured human primary gingival epithelial (HPGE) cells. The pH, peroxidase and protease sensitivity testing found antibacterial substances of protein- and peptide-like structures in addition to organic acids. The antimicrobial substances were sensitive to hydrolysis with trypsin, papain and pineapple protease and were inactived at temperatures above 100°C. Ammonium sulphate-precipitated proteins from the M14 strain retained the ability to inhibit the growth of S. mutans and F. nucleatum. The M14 strain contained 23 bacteriocin-related genes encoding for metabolites, belonging to class II bacteriocins. The M14 strain also showed inhibitory effects on 8 other pathogenic strains (A. actinomycetemcomitans, C. albicans, E. coli, G. vaginalis, P. acnes, P. gingivalis, S. aureus, S. enteritids), and thus has a broad spectrum of bacterial inhibition. This new isolate has been identified as having potential to be used as a probiotic bacterium in clinical applications.

Identification of a new probiotic strain, Lactiplantibacillus plantarum VHProbi® V38, and its use as an oral health agent.

Probiotics can be used to treat oral diseases such as dental caries, gingivitis, periodontitis, and halitosis. This study screened for strains capable of inhibiting Streptococcus mutans,one of the primary pathogenic bacteria responsible for dental caries by agar diffusion in different samples. Strain identification was performed by 16S rDNA sequencing and the API 50CH system. The potential functions of the strains in terms of oral health properties were also tested by agglutination assays, growth inhibition assays, adhesion assays, biofilm removal assays and inhibition of adhesion in human primary gingival epithelial (HPGE) cells assays. This study identified a probiotic strain from fermented cabbages that has a strong inhibitory effect on Streptococcus mutans. The API 50CH system and 16S rDNA sequencing verified that this was a new strain and it was given the name, Lactiplantibacillus plantarum VHProbi®V38. Agglutination, growth inhibition and adhesion, and biofilm removal tests indicated that L. plantarum VHProbi® V38 inhibited and reduced S. mutans. This probiotic was shown to have a broad antibacterial spectrum, simultaneously inhibiting the growth of periodontal pathogenic bacteria such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. After 2 hours of co-cultivation with these pathogens, L. plantarum VHProbi® V38 was able to significantly reduce pathogens adhesion on human primary gingival epithelial (HPGE) cells. These findings suggest that L. plantarum VHProbi® V38 could potentially prevent and treat periodontal diseases caused by these pathogenic bacteria. L. plantarum VHProbi® V38 also adheres strongly to HPGE cells and thus has potential as an oral probiotic. This study describes new methods that can be used to aid the screening and identification of oral probiotics.

Isolation, Identification and Screening of Potential Probiotic Bacteria in Milk from South African Saanen Goats.

This study aimed to evaluate lactic acid bacteria isolates from Saanen goats' milk for probiotic attributes, thereby determining their potential as direct-fed microbials for goats. Isolates were identified using API 50CH system, 16S rDNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry. All 17 isolates obtained were identified as Lactobacillus plantarum except one identified as Pediococcus acidilactici. Four isolates identified as L. plantarum (Accession numbers KJ026587.1, KM207826.1, KC83663.1 and KJ958428.1) by at least two of the techniques used and isolate 17 differently identified by all the methods used were selected as representatives and then screened for probiotic properties. These isolates displayed phenotypic probiotic attributes including tolerance to acid and bile salts, ability to adhere to intestines and possession of antagonistic activities against Proteus vulgaris, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa and Escherichia coli. The lactic acid bacteria isolated from Saanen goats' milk showed potential to be used as sustainable probiotics in goats' industry. Successful use of probiotics in animals depends upon availability of appropriate isolates originating from the specific host animal. This study is a positive contribution towards identification of isolates with potential for formulation as direct-fed microbials for South African Saanen goats.

Isolation of a chitinolytic Bacillus licheniformis S213 strain exerting a biological control against Phoma medicaginis infection.

Among nine chitinase-producing strains isolated from Tunisian soil, one isolate called S213 exhibited a potent chitinolytic activity. S213 strain was identified as Bacillus licheniformis by API 50CH system and sequence analysis of its partial 16S ribosomal DNA. Chitinolytic activity was induced either by colloidal chitin or fungal cell walls, and the highest chitinase activity reached at the late stationary phase exhibiting optimal temperature and pH of 50-60 °C and pH 6.0, respectively. SDS-PAGE analysis of the secreted colloidal chitin-induced proteins showed a major protein of about 65 kDa. This protein was identified as chitinase on the basis of its peptide sequences which displayed high homology with chitinase sequence of B. licheniformis ATCC 14580. Moreover, chitinolytic activity containing supernatant inhibited the growth of several phytopathogenic fungi including Phoma medicaginis. Interestingly, S213 strain reduced efficiently the damping-off disease caused by P. medicaginis in Medicago truncatula and should be envisaged in enzyme-based biopesticides against phytopathogen application.

Isolation and characterization of native Bacillus thuringiensis isolates from Saudi Arabia

A total of 500 soil samples were collected from different localities across the kingdom to isolate native Bacillus thuringiensis (Bt) strains. Sodium acetate- (0.25 M) -selection heat-pasteurization, and 50% ethanol treatment methods were used for Bt isolation. Phenotypic characterization and identification of recovered Bt isolates was accomplished on the basis of morphological characteristics of colonies, parasporal crystal, and spores, as well as hemolytic activity, motility, susceptibility to antibiotics, and carbohydrate utilization (API 50CH system). Analysis of 16S rRNA gene was performed to further confirm Bt species identity and differentiate the spore-forming bacilli. Bioassay tests were conducted on larvae of Aedes caspius and Culex pipiens . Based on results, a total of 64 Bt isolates were recovered and the overall Bt index corresponding to the whole sampling areas was 0.25. Most of Bt isolates showed spherical crystals (56%), while, irregular, bipyramidal, and attached crystal to the spores constituted 14, 10 and 6% respectively. The most common biochemical type (31%) was as that of the reference Bt subsp. kurstaki (Btk). The 16S rRNA gene analysis, using the universal primer, provided an efficient identification of Bt isolates and their discrimination from the closely related Bacillus cereus , as well as from other spore forming bacilli. Bioassay showed that only one Bt isolate exhibited anti-diptera activity similar to Btk strain while most of the native Bt isolates exhibited weak (<30% mortality) or non insecticidal activity. However, results of motility, hemolytic activity, antibiotic-susceptibility patterns, and crystal shape, seem to suggest that many of our Bt isolates may exhibit parasporins activity. Key words: Bacillus thuringiensis , parasporal crystal, isolation, biochemical type, insecticidal, cry gene, mosquitoes.

Reclassification of the members of the genus Tetrathiobacter Ghosh et al. 2005 to the genus Advenella Coenye et al. 2005

The taxonomic position of the genera Advenella and Tetrathiobacter was examined. 16S rRNA gene sequence analysis revealed that the two genera are closely related, representing a monophyletic cluster with high sequence similarity (98.1-99.7%) within the family Alcaligenaceae. The phenotypic characteristics of the type strains of Advenella incenata, Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis were re-examined using the API 20NE, API ZYM and API 50CH systems. Phylogenetic data together with similarities in phenotypic characteristics, G+C content and cellular acid composition suggest that they should be classified in the same genus. On the basis of the data presented, the two species of the genus Tetrathiobacter should be transferred to the genus Advenella, since this genus has nomenclatural priority. Therefore, Tetrathiobacter kashmirensis and Tetrathiobacter mimigardefordensis should be transferred to the genus Advenella as Advenella kashmirensis comb. nov. (type strain WT001T=LMG 22695T=MTCC7002T) and Advenella mimigardefordensis comb. nov. (type strain DPN7T=DSM 17166T=LMG 22922T). Emended descriptions of Advenella incenata and the genus Advenella are also presented.

Antagonistic Potential against Pathogenic Microorganisms and Hydrogen Peroxide Production of Indigenous Lactobacilli Isolated from Vagina of Chinese Pregnant Women

To investigate the indigenous lactobacilli from the vagina of pregnant women and to screen the isolates with antagonistic potential against pathogenic microorganisms. The strains were isolated from pregnant women's vagina and identified using the API50CH system. The ability of the isolates to produce hydrogen peroxide was analyzed semi-quantitatively using the TMB-HRP-MRS agar. The antagonistic effects of the isolates on pathogenic microorganisms were determined with a double layer agar plate. One hundred and three lactobacilli strains were isolated from 60 samples of vaginal secretion from healthy pregnant women. Among them, 78 strains could produce hydrogen peroxide, in which 68%, 80%, 80%, and 88% had antagonistic effects against Candida albicans CMCC98001, Staphylococcus aureus CMCC26003, Escherichia coli CMCC44113, and Pseudomonas aeruginosa CMCC10110, respectively. The recovery of hydrogen peroxide-producing lactobacilli decreases with the increasing pregnant age and time. The most commonly isolated species from vagina of Chinese pregnant women are Lactobacillus acidophilus and Lactobacillus crispatus. Most of L. acidophilus and L. crispatus produce a high H2O2 level.

First Report of Bacterial Deep Bark Canker of Walnut Caused by Brenneria (Erwinia) rubrifaciens in Europe.

During the summer of 1995 and subsequent years, bark cankers were observed in walnut trees (cv. Hartley grafted on Juglans hindsii) imported from California in 1978 growing in Badajoz, Spain. Two foci were found in an orchard of 200 ha where 80 walnut trees were affected. Cankers were observed on trunks and branches, and dark exudates staining the bark appeared mainly in summer. Isolations were performed from affected tissue using King's B medium, and Brenneria (Erwinia)-like colonies (1) were purified and characterized. Gram reaction, Kovacs' oxidase, O/F metabolism, aesculin hydrolysis, urease activity, and levan production were assayed for five isolates (1). Biochemical characterization was performed by the miniaturized API 20E, API 20NE, and API 50CH systems (BioMérieux, Marcy-l'Etoile, France) as recommended, except for incubations that were made at 25°C for 48 h. Analyses of the cellular fatty acids of selected isolates were performed as described by Sasser (2). They were also tested in indirect enzyme-linked immunosorbent assay (ELISA) using antisera obtained against the reference strain CFBP 1284 and one Spanish isolate. When compared to the reference strain from California, isolates were identified as Brenneria rubrifaciens (1,3) on the basis of physiological and biochemical characteristics, fatty acid profiles, and ELISA. Pathogenicity of two selected Spanish isolates was confirmed using three 2-year-old walnut trees per bacterial isolate by inoculating 108 CFU of each isolate in deep wounds made in the trunk at 40 and 80 cm from the crown. The reference strain and water were also inoculated as controls. Two months later, removal of the outer bark of walnut revealed typical dark lesions in the inner bark at all the inoculation sites on trees inoculated with the Spanish and reference strains, but no external cankers were observed. Four years later, these plants showed internal lesions (20 to 80 cm), from which B. rubrifaciens was reisolated far away from the inoculation site. To our knowledge this is the first report of this bacterium in Europe.

Phenotypic and genetic diversity of Paenibacillus azotofixans strains isolated from the rhizoplane or rhizosphere soil of different grasses

Fifty-three strains identified as Paenibacillus azotofixans were isolated from the rhizoplane and rhizosphere of different grasses and from soil. To study the diversity within this species, four approaches were used: assessment of homology with a nifKDH probe in hybridization experiments; use of a selected 20-mer primer to produce RAPD profiles and of BOX- PCR to generate genomic fingerprintings; and phenotypic tests using the API50CH system. The API tests performed with the 53 P. azotofixans strains showed that all strains produced acid from 15 carbohydrates; using six other carbohydrates (sorbitol, dulcitol, tagarose, starch, glycogen and D- arabitol), the strains could be divided in five groups of related strains. All strains tested showed homology to Klebsiella pneumoniae nifKDH genes, resulting in 14 different hybridization patterns with this probe. Using RAPD- fingerprinting with one appropriate primer, 23 different amplification patterns were observed. The BOX-PCR approach confirmed the grouping suggested by the RAPD fingerprinting. A comparison of the 53 strains by similarity matrix analysis using the data obtained in all approaches resulted in a phenogram, grouping them into five broad groups at 74% similarity and into 27 subgroups at 94% similarity. At 100% similarity, 31 groups of strains could be formed, indicating a high degree of diversity among the strains tested. Overall, the diversity was independent from the origin of strains, since a variety of different groups was isolated from each plant studied. However, some clusters were dominant in wheat and sugarcane samples. The results indicated that the methods used here are sensitive indicators of diversity among the strains studied and can be applied as efficient and reliable means for further ecological and biogeographical studies.

Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species

Summary: One hundred and nineteen strains of alkaliphilic and alkalitolerant, aerobic endospore-forming bacteria were examined for 47 physiological and biochemical characters, and DNA base composition. Numerical analysis (S J and S SM/UPGMA clustering) revealed 11 clusters that comprised three or more strains. Most of the phena were further characterized by analysis of carbohydrate utilization profiles using the API 50CH system, but strains of two taxa could not be cultured by this method. DNA reassociation studies showed that nine of the phena were homogeneous, but strains of phenon 4 and phenon 8 were each subdivided into two DNA hydridization groups. The strains could therefore be classified into 13 taxa plus a number of unassigned single-membered clusters. Two taxa were equated with Bacillus cohnii and B. alcalophilus and nine of the remainder are proposed as new species with the following names: B. agaradhaerens sp. nov., B. clarkii sp. nov., B. clausii sp. nov., B. gibsonii sp. nov., B. halmapalus sp. nov., B. halodurans comb, nov., B. horikoshii sp. nov., B. pseudalcalophilus sp. nov. and B. pseudofirmus sp. nov. Two taxa were insufficiently distinct to allow confident identification and these have therefore not been proposed as new species.

Characterization of Metarhizium anisopliae Strains by Carbohydrate Utilization (API50CH)

The utilization of 49 carbohydrates was examined for 134 isolates of Metarhizium anisopliae covering 16 characterized strains, seven isolates of Metarhizium flavoviride var. flavoviride, nine isolates of Microhilum oncoperae , and 30 isolates of Beauveria bassiana using the API50CH system. Using canonical variate analysis five major groupings were discernible: B. bassiana, M. oncoperae, M. flavoviride, and two M. anisopliae groups. The two M. anisopliae groups consisted of a large cluster of M. anisopliae var. anisopliae strains and a group of cold-active M. anisopliae isolates able to germinate at 5°C. Based on this and other data, we suggest that the cold-active group forms a distinct variety of M. anisopliae which we propose to call var. frigidum. Differentiation of the 16 strains of M. anisopliae was possible using the API50CH system and we conclude that carbohydrate utilization is a useful and biologically relevant taxonomic criteria for the separation of Metarhizium strains and other entomogenous fungi.

Evaluation of the Mast ID and API 50CH systems for identification of Listeria spp

A multipoint inoculation technique (Mast ID) for the identification and species determination of Listeria monocytogenes (sensu strictu) and six other species of the genus Listeria was evaluated. This was compared with the commercially available API 50CH system. Both methods successfully identified all 123 strains tested. The Mast ID system is inexpensive and utilizing a multipoint inoculation technique permits the screening of up to 21 isolates per 9-cm petri dish. The API 50CH system was more expensive and time consuming and is therefore suitable only for the examination of smaller numbers of strains.