Viviana Klose

In vitro antagonistic activities of animal intestinal strains against swine-associated pathogens.

A wide range of enteropathogens cause costly diarrhoeal diseases in fattening piglets and account for food-related infections in humans. The objective of this study was to screen beneficial bacterial strains from the gastrointestinal tract of various animal sources for antagonistic activity against diverse pathogens associated with hazardous pig production times. Using agar spot assays, 15 well-characterized strains belonging to Lactobacillus, Enterococcus, Bifidobacterium and Bacillus were studied for inhibition of Clostridium perfringens type A, various serovars of enterotoxigenic Escherichia coli and Salmonella enterica, as well as Brachyspira pilosicoli. Strong antagonists were further analyzed by studying their cell-free supernatants with and without pH neutralization, proteinase K and catalase treatment. Enterobacteriaceae were effectively inhibited by Lactobacillus salivarius and Lactobacillus reuteri strains, independent from the animal source, and on a lower level by single strains belonging to Lactobacillus mucosae, Lactobacillus amylovorus and Bifidobacterium thermophilum, due to organic acid production. The Bacillus subtilis strain was found to produce an anti-clostridial and anti-Brachyspira metabolite of proteinaceous nature. Homofermentative lactobacilli and B. thermophilum could suppress the growth of B. pilosicoli, the causative agent of intestinal spirochaetosis, whereas heterofermentative strains belonging to L. reuteri and L. mucosae had no effect. The lactic acid bacteria exerted their activity primarily by organic acid release, except one Enterococcus faecium and L. amylovorus strain, which exhibited antagonism through joint activity of lactate and hydrogen peroxide. The findings of this study provide a basis for further in vitro studies and encourage feeding studies to evaluate the antagonistic potential of promising strains in pig production.

Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation

Anaerobic biodegradation of petroleum hydrocarbons (PHC) to methane has been recognized to occur in oil reservoirs and contaminated surface sites alike. This process could be employed efficiently for the treatment of contaminated materials, including petrochemical wastes and PHC-contaminated soil, since no external electron acceptor is required. Moreover, the controlled production of methane in digestion plants, similarly to the anaerobic digestion (AD) of energy crops or organic residues, would enable for energy recovery from these wastes. At present, little is known about the bacterial communities involved in and responsible for hydrocarbon fermentation, the initial step in PHC conversion to methane. In the present study, the fate of two different methanogenic communities derived from the AD of wastewater (WWT) and of biowaste, mixed with PHC-contaminated soil (SWT), was monitored during incubation with PHC using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA genes amplified with Bacteria-specific primers. During 11 months of incubation, slight but significant degradation of PHC occurred in both sludges and distinct bacterial communities were developing. In both sludges, Bacteroidetes were found. In addition, in WWT, the bacterial community was found to be dominated by Synergistetes and Proteobacteria, while Firmicutes and unidentified members were abundant in SWT. These results indicate that bacterial communities from anaerobic digesters can adapt to and degrade petroleum hydrocarbons. The decontamination of PHC-containing waste via fermentative treatment appears possible.

Development of a strain-specific real-time PCR assay for enumeration of a probiotic Lactobacillus reuteri in chicken feed and intestine.

A strain-specific real-time PCR assay was developed for quantification of a probiotic Lactobacillus reuteri (DSM 16350) in poultry feed and intestine. The specific primers were designed based on a genomic sequence of the strain derived from suppression subtractive hybridization with the type strain L. reuteri DSM 20016. Specificity was tested using a set of non-target strains from several sources. Applicability of the real-time PCR assay was evaluated in a controlled broiler feeding trial by using standard curves specific for feed and intestinal matrices. The amount of the probiotic L. reuteri was determined in feed from three feeding phases and in intestinal samples of the jejunum, ileum, and caecum of three, 14, and 39 day old birds. L. reuteri DSM 16350 cells were enumerated in all feeds supplemented with the probiotic close to the inclusion rate of 7.0×103 cfu/g, however, were not detected in L. reuteri DSM 16350 free feed. In three day old birds L. reuteri DSM 16350 was only detected in intestinal samples from probiotic fed animals ranging from 8.2±7.8×105 cfu/g in the jejunum, 1.0±1.1×107 cfu/g in the ileum, and 2.5±5.7×105 cfu/g in the caecum. Similar results were obtained for intestinal samples of older birds (14 and 39 days). With increasing age of the animals the amount of L. reuteri signals in the control animals, however, also increased, indicating the appearance of highly similar bacterial genomes in the gut microbiota. The L. reuteri DSM 16350 qPCR assay could be used in future for feeding trials to assure the accurate inclusion of the supplement to the feed and to monitor its uptake into the GIT of young chicken.

Identification and antimicrobial susceptibility of porcine bacteria that inhibit the growth of Brachyspira hyodysenteriae in vitro.

Aims:  To identify bacilli, lactic acid bacteria and bifidobacteria that inhibit the growth of Brachyspira hyodysenteriae.

Suppression subtractive hybridisation and real-time PCR for strain-specific quantification of the probiotic Bifidobacterium animalis BAN in broiler feed

To ensure quality management during the production processes of probiotics and for efficacy testing in vivo, accurate tools are needed for the identification and quantification of probiotic strains. In this study, a strain-specific qPCR assay based on Suppression Subtractive Hybridisation (SSH) for identifying unique sequences, was developed to quantify the strain Bifidobacterium animalis BAN in broiler feed. Seventy potential BAN specific sequences were obtained after SSH of the BAN genome, with a pool of closely related strain genomes and subsequent differential screening by dot blot hybridisation. Primers were designed for 30 sequences which showed no match with any sequence database entry, using BLAST and FASTA. Primer specificity was assessed by qPCR using 45 non-target strains and species in a stepwise approach. Primer T39_S2 was the only primer pair without any unspecific binding properties and it showed a PCR efficiency of 80% with a Cq value of 17.32 for 20 ng BAN DNA. Optimised feed-matrix dependent calibration curve for the quantification of BAN was generated, ranging from 6.28 × 10(3)cfu g(-1) to 1.61 × 10(6)cfu g(-1). Limit of detection of the qPCR assay was 2 × 10(1)cfu g(-1) BAN. Applicability of the strain-specific qPCR assay was confirmed in a spiking experiment which added BAN to the feed in two concentrations, 2 × 10(6)cfu g(-1) and 2 × 10(4)cfu g(-1). Results showed BAN mean recovery rates in feed of 1.44 × 10(6) ± 4.39 × 10(5)cfu g(-1) and 1.59 × 10(4) ± 1.69 × 10(4)cfu g(-1), respectively. The presented BAN-specific qPCR assay can be applied in animal feeding trials, in order to control the correct inclusion rates of the probiotic to the feed, and it could further be adapted, to monitor the uptake of the probiotic into the gastrointestinal tract of broiler chickens.

Microbial Antagonists in Animal Health Promotion and Plant Protection

The use of agrochemicals in animal husbandry and crop cultivation is well established, but the public acceptance is generally low and in some cases, substances have already been legally banned because their application poses risks for public health. Microbes that are able to suppress the growth of pathogens have been shown to be an effective alternative to maintain animal or plant health. Isolation and screening of potent strains as well as the characterization of their mode of action and the assessment of potential risks play an important role in order to obtain a safe and acceptable biological product. The development of a commercial production process, product formulation, and the requirements for the registration process are further critical items, which will determine over the commercial success of the final product.