Antagonistic activities of selected fungal isolates against Armillaria mellea

The antagonistic activities of six selected fungal isolates against Armilloria mellea were studied on two different concentrations of three media, on fungicides-amended malt extract agar (MEA) medium, and in glasshouse pots filled with John Innes No.2 compost and natural field soil. Trichoderma hamatum isolate Tham1 was found the most effective in reducing Armillaria growths on both the low and high concentrations of malt extract, potato dextrose and V-8 juice in MEA, potato dextrose agar (PDA) and V-8 juice agar (VJA), respectively, followed by T. harzianum isolate Th2 and T. viride isolate Tv3. Neither dose rate (200 or 2000 mg l(-1)) of fenpropidin allowed any growth of Armillaria on MEA, while that of the antagonists was also completely inhibited or greatly restricted. However, both dose rates of fosetyl-A1 allowed the growth of Armillaria and almost all the antagonists. Data on colony diameters of Armillaria showed Tham1 as the most effective antagonist along with Th2, Th23 and Tv3. Tham1 was also found the most effective in protecting hazel billets from colonization by Armillaria, followed by Th2 and Th23. Compared with 7.1 colonized billets in the inoculated controls, only 1.3, 2.6 and 2.7 billets (out of ten) were colonized, respectively, when protected with these antagonists. The results indicate that the Trichoderma isolates are able to maintain their antagonistic effects on A. mellea under a variety of nutritional, chemical and edaphic regimes. More investigations are needed to develop a system of control for the disease with these potential antagonists.

Characterization of some Actinomyces-like isolates from human clinical sources: description of Varibaculum cambriensis gen. nov., sp nov

Fifteen strains of an anaerobic, catalase-negative, gram-positive diphtheroid-shaped bacterium recovered from human sources were characterized by phenotypic and molecular chemical and molecular genetic methods. The unidentified bacterium showed some resemblance to Actinomyces species and related taxa, but biochemical testing, polyacrylamide gel electrophoresis analysis of whole-cell proteins, and amplified 16S ribosomal DNA restriction analysis indicated the strains were distinct from all currently named Actinomyces species and related taxa. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto-unknown phylogenetic line that is related to but distinct from Actinomyces, Actinobaculum, Arcanobacterium, and Mobiluncus. We propose, on the basis of phenotypic and phylogenetic evidence, that the unknown bacterium from human clinical specimens should be classified as a new genus and species, Varibaculum cambriensis gen. nov., sp. nov. The type strain of Varibaculum cambriensis sp. nov. is CCUG 44998(T) = CIP 107344(T).

Some functional properties of fractionated soy protein isolates obtained by microfiltration

Five soy proteins isolate (SPI) fractions were produced using two microfiltration membranes with different pore sizes. Fractionation was carried out on SPI produced by isoelectric precipitation of a crude protein extract. The five fractions were two retentates and two permeates from the two membranes, the fifth fraction was obtained as the retentate on the smaller-po re- sized membrane fed with the permeate from the larger-pore-sized membrane. Solubility, foaming and emulsifying properties of the collected fractionates were investigated. It was observed that in the pH range 3-8 the retentates featured superior solubility compared with permeates. There was no significant difference (p > 0.0 1) in solubility between the retentates and SPI at pH >= 6. Foaming characteristics of the fractions followed the same trend as solubility with regard to foam expansion. There was, however, no particular trend observed with regards to foam stability. Emulsions stabilised by the retentates exhibited higher values (p<0.01) of emulsion stability index (ESI) and emulsifying activity index (EAI) than those stabilised with permeates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles indicated that the fractions exhibiting high functionality in terms of solubility, foaming and emulsifying properties were also richer in 7S globulin soy protein subunits. Isoelectric focussing (IEF) profiles showed that retentates were richer in species with isoelectric points (pl) between 5.2 and 5.6 while permeates featured more prominently at pis between 4.5 and 4.8. (C) 2006 Elsevier Ltd. All rights reserved.

Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine

Aims: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. Methods and Results: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. Conclusions: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. Significance and impact of the Study: Selected strains could be used as adjunct starters to make high quality Feta cheese.

Characterization of Actinomyces isolates from infected root canals of teeth: description of Actinomyces radicidentis sp. nov.

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733.

Characterization of Actinomyces isolates from samples from the human urogenital tract: description of Actinomyces urogenitalis sp. nov.

Three strains of a previously undescribed Actinomyces-like bacterium were isolated from human clinical sources (urine, urethra and vaginal secretion). Biochemical testing and PAGE analysis of whole-cell proteins indicated that the strains were phenotypically homogeneous and distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium from humans be classified as Actinomyces urogenitalis sp. nov. The type strain of Actinomyces urogenitalis is CCUG 38702T (= CIP 106421T).

Lactobacilli antagonize the growth, motility, and adherence of brachyspira pilosicoli: a potential intervention against avian intestinal spirochetosis

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic Brachyspira species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of Brachyspira they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two Lactobacillus strains, Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2, suppressed the growth of Brachyspira pilosicoli B2904 in a pH-dependent manner. In in vitro adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal in vitro organ culture (IVOC) model, the adherence and invasion of B. pilosicoli in epithelial cells were reduced significantly by the presence of lactobacilli (P < 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of B. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli and Brachyspira was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for B. pilosicoli to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

Characterisation of Escherichia fergusonii isolates from farm animals using an Escherichia coli virulence gene array and tissue culture adherence assays

Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusonii prfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a 'localised adherence' or 'diffuse adherence' phenotype, and they proved to be moderately invasive. The E fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E coli, and support existing evidence that strains of E fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

Genetic diversity among Escherichia coli O157 : H7 isolates and identification of genes linked to human infections

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:117 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or H. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be 117) were significantly different from other Stx-positive and -negative E. coli O157:117 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:117 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent.

Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages

Escherichia coli, the most common cause of bacteraemia in humans in the UK, can also cause serious diseases in animals. However the population structure, virulence and antimicrobial resistance genes of those from extraintestinal organs of livestock animals are poorly characterised. The aims of this study were to investigate the diversity of these isolates from livestock animals and to understand if there was any correlation between the virulence and antimicrobial resistance genes and the genetic backbone of the bacteria and if these isolates were similar to those isolated from humans. Here 39 E. coli isolates from liver (n=31), spleen (n=5) and blood (n=3) of cattle (n=34), sheep (n=3), chicken (n=1) and pig (n=1) were assigned to 19 serogroups with O8 being the most common (n=7), followed by O101, O20 (both n=3) and O153 (n=2). They belong to 29 multi-locus sequence types, 20 clonal complexes with ST23 (n=7), ST10 (n=6), ST117 and ST155 (both n=3) being most common and were distributed among phylogenetic group A (n=16), B1 (n=12), B2 (n=2) and D (n=9). The pattern of a subset of putative virulence genes was different in almost all isolates. No correlation between serogroups, animal hosts, MLST types, virulence and antimicrobial resistance genes was identified. The distributions of clonal complexes and virulence genes were similar to other extraintestinal or commensal E. coli from humans and other animals, suggesting a zoonotic potential. The diverse and various combinations of virulence genes implied that the infections were caused by different mechanisms and infection control will be challenging.

Contingency locus in ctsR of Listeria monocytogenes Scott A: a strategy for occurrence of abundant piezotolerant isolates within clonal populations

In a recent study we demonstrated that a high-hydrostatic-pressure-tolerant isolate of Listeria monocytogenes lacks a codon in the class 3 heat shock regulator gene ctsR. This mutation in the region that encodes four consecutive glycines was directly responsible for the observed piezotolerance, increased stress resistance, and reduced virulence. The aim of the present study was to determine whether mutations in ctsR are frequently associated with piezotolerance in L. monocytogenes. Wild-type cultures of L. monocytogenes were therefore exposed to 350 MPa for 20 min, and the piezotolerance of individual surviving isolates was assessed. This rendered 33 isolates with a stable piezotolerant phenotype from a total of 84 survivors. Stable piezotolerant mutants were estimated to be present in the initial wild-type population at frequencies of >10�5. Subsequent sequencing of the ctsR gene of all stable piezotolerant isolates revealed that two-thirds of the strains (i.e., n � 21) had mutations in this gene. The majority of the mutations (16 of 21 strains) consisted of a triplet deletion in the glycine-encoding region of ctsR, identical to what was found in our previous study. Interestingly, 2 of 21 mutants contained a codon insertion in this repeat region. The remaining three stable piezotolerant strains showed a 19-bp insertion in the glycine repeat region, a 16-bp insertion downstream of the glycine repeat area (both leading to frameshifts and a truncated ctsR), and an in-frame 114-bp deletion encoding a drastically shortened carboxy terminus of CtsR. In four instances it was not possible to generate a PCR product. A piezotolerant phenotype could not be linked to mutations in ctsR in 8 of 33 isolates, indicating that other thus-far-unknown mechanisms also lead to stable piezotolerance. The present study highlights the importance of ctsR in piezotolerance and stress tolerance of L. monocytogenes, and it demonstrates that short-sequence repeat regions contribute significantly to the occurrence of a piezotolerant and stress-tolerant subpopulation within L. monocytogenes cultures, thus playing an important role in survival.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

Background. The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. Results. Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. Conclusions. The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

Isolation of Actinomyces hyovaginalis from sheep and comparison with isolates obtained from pigs

Actinomyces hyovaginalis, an organism initially described from pigs, was recovered from nine sheep and a moufflon. Further strains of A. hyovaginalis were recovered from five samples from pigs over the same period. 16S rRNA sequencing and extensive phenotyping demonstrated high similarity between the ovine and porcine isolates; however differences with respect to erythritol, adonitol and l-arabitol fermentation were detected. Ovine isolates were made from various sample sites including abscesses and highlight the importance of the accurate identification of the various coryneform isolates which affect sheep. A. hyovaginalis can be added to the growing list of coryneforms which can cause disease in sheep including Corynebacterium pseudotuberculosis, Trueperella pyogenes and Arcanobacterium pluranimalium.