Genotypic and Phenotypic Diversity of phlD-Containing Pseudomonas Strains Isolated from the Rhizosphere of Wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2,4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

Probiotic Potential of Bacillus Strains Isolated from an Acidic Fermented Food Idli

Present study is intended to assess the probiotic properties of Bacillus spp. isolated from idli batter, a traditional fermented food of Southern India and Sri Lanka. A total of 32 isolates were screened for potential pathogenic behaviour through haemolysis assay, DNase activity and antibiotics sensitivity. Two of the isolates were found to be potentially safe and identified as Bacillus spp. These strains were characterized for in vitro probiotic attributes and antioxidant activity. Both the strains showed strong acid and bile tolerance, transit tolerance, lysozyme tolerance, cell surface hydrophobicity, auto-aggregation, co-aggregation, biofilm formation potential and adhesion to human colon adenocarcinoma (HT 29) cell line demonstrating potential probiotic ability. These strains also exhibited considerable cholesterol binding, thermostability, β-galactosidase production, proteolytic, amylolytic and lipolytic activity. Cell-free supernatant inhibited the biofilm formation by Pseudomonas aeruginosa (KT266804) to 90%. Intact cells showed significant DPPH (41%), hydroxyl (31%), radical scavenging activity and lipid peroxidation inhibition (20.38%), while cell-free extracts exhibited significant superoxide anion radical scavenging activity (16.25%). Results revealed that isolates could be potential probiotic candidate after further assessment of in vivo probiotic properties and safety evaluation and could be utilised as starter cultures in functional foods.

     

雪貂葡萄球菌的分离与鉴定

目的对雪貂分离菌株进行鉴定。方法通过菌落、菌体形态、生化特征等细菌学检测方法和药敏实验对所分离菌株进行鉴定。结果根据生物学特性和药敏实验确定所分离菌株为金黄色葡萄球菌和中间葡萄球菌。结论分离菌株为金黄色葡萄球菌和中间葡萄球菌。     

Phenotypic and Genotypic Characterizations of Campylobacter jejuni Isolated from the Broiler Meat Production Process

A set of C. jejuni isolates of different origins and flaA-genotypes obtained throughout the broiler meat production chain was tested in this study for a possible correlation of their origin, phylogenetic relationship, and phenotypic properties. Interestingly, the results showed a correlation of the origin and the phylogenetic relationship between the C. jejuni isolates and their ability to form biofilm, but not in their ability to survive at -18, 5, 20, and 48?°C. Two strains, a broiler cloacae isolate and a broiler fillet isolate, were unable to develop biofilm, while most of the C. jejuni isolates originating from meat and surfaces of the slaughterhouse readily formed biofilms after both 24, 48, and 72?h. Interestingly, these biofilm-forming strains were closely related. Furthermore, two strains that were isolated after disinfection developed significantly more biofilms after 24?h of incubation than the remaining strains. A comparative genomic analysis using DNA microarrays showed that the gene contents of strains that efficiently formed biofilms were different from those that did not. The study suggests that biofilm formation might be a lineage specific property, allowing C. jejuni to both survive environmental stress at the slaughterhouse and to attach to the surface of meat.     

Plant-Dependent Genotypic and Phenotypic Diversity of Antagonistic Rhizobacteria Isolated from Different Verticillium Host Plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards Verticillium. The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

Genotypic and Phenotypic Diversity within Species of Purple Nonsulfur Bacteria Isolated from Aquatic Sediments

To assess the extent of genotypic and phenotypic diversity within species of purple nonsulfur bacteria found in aquatic sediments, a total of 128 strains were directly isolated from agar plates that had been inoculated with sediment samples from Haren and De Biesbosch in The Netherlands. All isolates were initially characterized by BOX-PCR genomic DNA fingerprinting, and 60 distinct genotypes were identified. Analyses of 16S rRNA gene sequences of representatives of each genotype showed that five and eight different phylotypes of purple nonsulfur bacteria were obtained from the Haren and De Biesbosch sites, respectively. At the Haren site, 80.5% of the clones were Rhodopseudomonas palustris, whereas Rhodoferax fermentans and Rhodopseudomonas palustris were numerically dominant at the De Biesbosch site and constituted 45.9 and 34.4% of the isolates obtained, respectively. BOX-PCR genomic fingerprints showed that there was a high level of genotypic diversity within each of these species. The genomic fingerprints of Rhodopseudomonas palustris isolates were significantly different for isolates from the two sampling sites, suggesting that certain strains may be endemic to each sampling site. Not all Rhodopseudomonas palustris isolates could degrade benzoate, a feature that has previously been thought to be characteristic of the species. There were differences in the BOX-PCR genomic fingerprints and restriction fragment length polymorphisms of benzoate-coenzyme A ligase genes and form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes between benzoate-degrading and non-benzoate-degrading genotypes. The ability to distinguish these two Rhodopseudomonas palustris groups based on multiple genetic differences may reflect an incipient speciation event resulting from adaptive evolution to local environmental conditions.     

Bacteriocinogenic LAB Strains for Fermented Meat Preservation: Perspectives,Challenges, and Limitations

Over the last decades, much research has focused on lactic acid bacteria (LAB) bacteriocins because of their potential as biopreservatives and their action against the growth of spoilage microbes. Meat and fermented meat products are prone to microbial contamination, causing health risks, as well as economic losses in the meat industry. The use of bacteriocin-producing LAB starter or protective cultures is suitable for fermented meats. However, although bacteriocins can be produced during meat processing, their levels are usually much lower than those achieved during in vitro fermentations under optimal environmental conditions. Thus, the direct addition of a bacteriocin food additive would be desirable. Moreover, safety and technological characteristics of the bacteriocinogenic LAB must be considered before their widespread applications. This review describes the perspectives and challenges toward the complete disclosure of new bacteriocins as effective preservatives in the production of safe and “healthy” fermented meat products.     

我国新分离乙型脑炎病毒株毒力特征研究

将分离自不同年代的17株乙脑毒株在小鼠脑内传代,然后将病毒在BHK21细胞单层上观察不同毒株的空斑形成大小形态,小鼠脑内和皮下途径接种观察病毒的毒力,结果显示不同毒株在BHK21细胞上形成的噬斑大小不尽相同,减毒株形成的噬斑最小。所有毒株对小鼠的脑内毒力都很强,病毒滴度高达lg8.0/mL以上,毒株间无明显差异。毒株对9～11g小日龄小鼠的皮下毒力有一定差异但不明显,但对14～16g较大日龄小鼠则差别明显。PFU/LD50的对数值差异除一株(M47株)为8.44外,其余各株差别在3.94～0.45间。本研究结果证明自然界乙脑毒株存在明显的神经外毒力差异,毒力差异与分离年代和病毒基因型无关,但从人体分离到的毒株毒力表现较强。     

Genotypic and Phenotypic Diversity among Induced, stx2-Carrying Bacteriophages from Environmental Escherichia coli Strains

Shiga toxin 2 (stx₂) gene-carrying bacteriophages have been shown to convert Escherichia coli strains to Shiga toxin-producing Escherichia coli (STEC). In this study, 79 E. coli strains belonging to 35 serotypes isolated from wastewaters of both human and animal origin were examined for the presence of stx₂-carrying bacteriophages in their genomes. The lytic cycle of the bacteriophages was induced by mitomycin, and the bacteriophage fraction was isolated and used for morphological and genetic characterization. The induced bacteriophages showed morphological diversity, as well as restriction fragment length polymorphism variation, in the different strains belonging to different serotypes. The ability to infect new hosts was highly variable, although most of the induced phages infected Shigella sonnei host strain 866. In summary, in spite of carrying either the same or different stx₂ variants and in spite of the fact that they were isolated from strains belonging to the same or different serotypes, the induced bacteriophages were highly variable. The high level of diversity and the great infectious capacity of these phages could enhance the spread of the stx₂ gene and variants of this gene among different bacterial populations in environments to which humans may be exposed.     

Phenotypic and Genotypic Characteristics of Neisseria meningitidis Disease-Causing Strains in Argentina, 2010

Phenotypic and genotypic characterization of 133 isolates of Neisseria meningitidis obtained from meningococcal disease cases in Argentina during 2010 were performed by the National Reference Laboratory as part of a project coordinated by the PAHO within the SIREVA II network. Serogroup, serotype, serosubtype and MLST characterization were performed. Minimum Inhibitory Concentration to penicillin, ampicillin, ceftriaxone, rifampin, chloramphenicol, tetracycline and ciprofloxacin were determined and interpreted according to CLSI guidelines. Almost 49% of isolates were W135, and two serotype:serosubtype combinations, W135∶2a:P1.5,2:ST-11 and W135∶2a:P1.2:ST-11 accounted for 78% of all W135 isolates. Serogroup B accounted for 42.1% of isolates, and was both phenotypically and genotypically diverse. Serogroup C isolates represented 5.3% of the dataset, and one isolate belonging to the ST-198 complex was non-groupable. Isolates belonged mainly to the ST-11 complex (48%) and to a lesser extent to the ST-865 (18%), ST-32 (9,8%) and the ST-35 complexes (9%). Intermediate resistance to penicillin and ampicillin was detected in 35.4% and 33.1% of isolates respectively. Two W135∶2a:P1.5,2:ST-11:ST-11 isolates presented resistance to ciprofloxacin associated with a mutation in the QRDR of gyrA gene Thr91-Ile. These data show serogroup W135 was the first cause of disease in Argentina in 2010, and was strongly associated with the W135∶2a:P1.5,2:ST-11 epidemic clone. Serogroup B was the second cause of disease and isolates belonging to this serogroup were phenotypically and genotypically diverse. The presence of isolates with intermediate resistance to penicillin and the presence of fluorquinolone-resistant isolates highlight the necessity and importance of maintaining and strengthening National Surveillance Programs.     

Relationship Between Phenotypic and Genotypic Characteristics of <Emphasis Type="Italic">Trichophyton mentagrophytes</Emphasis> Strains Isolated from Patients with Dermatophytosis

According to epidemiological, clinical and mycological criteria, it has long been admitted that the Trichophyton mentagrophytes species includes two varieties: a zoophilic variety (var. mentagrophytes) and an anthropophilic variety (var. interdigitale) that involve the upper and the lower part of the body, respectively. The further application of molecular techniques to the characterization of dermatophyte strains showed that this classification is unreliable. The aim of our study was to assess the usefulness of PCR–RFLP (restriction fragment length polymorphism) and sequencing in the characterization of T. mentagrophytes strains taken from Tunisian patients. The study was carried out in 2008 in the laboratory of Parasitology–Mycology of Farhat Hached University Hospital, Sousse, Tunisia. A total of 133 strains were isolated from 133 patients addressed to the laboratory for dermatological lesions very evocative of dermatomycosis. Eighty strains were isolated from lesions located on the lower part of the body (onychomycosis, tinea pedis) and 53 strains from the upper part of the body (tinea capitis, tinea corporis). All strains were submitted to mycological examination (direct microscopic examination and culture on Sabouraud medium) and further investigated by using RFLP analysis of the PCR-amplified ITS1-5.8 s-ITS2 region of the ribosomal DNA and the MvaI restriction enzyme. In addition, 62 strains were further submitted to a sequencing of the ITS1-5.8 s-ITS2 region. On the basis of mycological criteria, all strains were diagnosed as T. mentagrophytes. All strains produced the same RFLP pattern and were identified as T. mentagrophytes interdigitale regardless of the location of lesions. Out of the 62 sequenced strains, 16 were found anthropophilic and 46 were zoophilic. In conclusion, all strains provisionally diagnosed as T. mentagrophytes on the basis of mycological criteria were shown to belong to T. interdigitale by using PCR–RFLP and sequencing irrespective of the site of lesions. The predominance of zoophilic strains needs further investigation.     

Biochemical and Genetic Evidence of Enterocin P Production by Two Enterococcus faecium-Like Strains Isolated from Fermented Sausages

Two bacteriocin-producing Enterococcus faecium-like strains were independently isolated from fermented sausages. Bacteriocins were purified to homogeneity by ammonium sulfate precipitation, gel filtration, cationic exchange, hydrophobic interaction, and reverse-phase liquid chromatography. Two peptide inhibitory fractions were purified from each strain, denominated A and B for E. faecium AA13, and C and D for E. faecium G16. Fraction B was blocked for amino acid sequencing by Edman degradation, while the amino acid sequences obtained from peptides A, C, and D contained the YGNGV consensus motif in positions 5 to 9, and the ATRS sequence in positions 1 to 4. By use of PCR techniques and nucleotide sequencing, the structural gene of enterocin P was found both in E. faecium AA13 and E. faecium G16. Metabolic and genetic features of the two strains suggest that they are slightly different, they may produce more than one bacteriocin, and both produce enterocin P. Received: 13 May 1999 / Accepted: 22 June 1999     

Borrelia japonica in Nature: Genotypic Identification of Spirochetes Isolated from Japanese Small Mammals

Spirochetes were isolated from earlobe tissues of shrews (Sorex unguiculatus, Sorex caecutiens, and Crocidura dsinezumi), voles (Clethrionomys rufocanus), and mice (Apodemus argenteus and Apodemus speciosus) captured in various localities in Japan. The isolates were identified as Borrelia japonica by rRNA gene restriction fragment length polymorphism analysis. The data suggest that these small mammals are candidates of reservoir hosts for B. japonica.     

Identification and Characterization of Leuconostoc fallax Strains Isolated from an Industrial Sauerkraut Fermentation

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.     

Phenotypic and Genotypic Characterization of Biofilm Forming Capabilities in Non-O157 Shiga Toxin-Producing Escherichia coli Strains

The biofilm life style helps bacteria resist oxidative stress, desiccation, antibiotic treatment, and starvation. Biofilm formation involves a complex regulatory gene network controlled by various environmental signals. It was previously shown that prophage insertions in mlrA and heterogeneous mutations in rpoS constituted major obstacles limiting biofilm formation and the expression of extracellular curli fibers in strains of Escherichia coli serotype O157:H7. The purpose of this study was to test strains from other important serotypes of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O113, O121, and O145) for similar regulatory restrictions. In a small but diverse collection of biofilm-forming and non-forming strains, mlrA prophage insertions were identified in only 4 of the 19 strains (serotypes O103, O113, and O145). Only the STEC O103 and O113 strains could be complemented by a trans-copy of mlrA to restore curli production and Congo red (CR) dye affinity. RpoS mutations were found in 5 strains (4 serotypes), each with low CR affinity, and the defects were moderately restored by a wild-type copy of rpoS in 2 of the 3 strains attempted. Fourteen strains in this study showed no or weak biofilm formation, of which 9 could be explained by prophage insertions or rpoS mutations. However, each of the remaining five biofilm-deficient strains, as well as the two O145 strains that could not be complemented by mlrA, showed complete or nearly complete lack of motility. This study indicates that mlrA prophage insertions and rpoS mutations do limit biofilm and curli expression in the non-serotype O157:H7 STEC but prophage insertions may not be as common as in serotype O157:H7 strains. The results also suggest that lack of motility provides a third major factor limiting biofilm formation in the non-O157:H7 STEC. Understanding biofilm regulatory mechanisms will prove beneficial in reducing pathogen survival and enhancing food safety.