Validation of an official control method for enumeration of authorised probiotic yeast in animal feed

An official control method in the framework of Council Directive 70/524/EEC for probiotic yeast used as feed additives was validated in a collaborative study by twenty laboratories in 12 European Countries. A pour plate method following ISO 7954 using chloramphenicol glucose yeast extract (CGYE) and a plate count method using CHROMagar Candida were used. Precision data in terms of repeatability (r) and reproducibility (R) of the method using different feeding stuffs and three inoculation levels were determined. Yeast was present in the samples in mixtures with other probiotic feed additives at a lower, a higher concentration or not present. The enumeration of yeast on CGYE agar showed for the lower and higher concentration a RSD(r) of 2.4-4.9% and a RSD(R) of 7.7-8%, respectively and was preferred by the majority of labs. CHROMagar Candida had a RSD(r) of 1.9-2.8% and a RSD(R) of 1.9-5.9%. For routine analysis the use of the pour plate technique is recommended. CHROMagar Candida can be used for confirmation of the species Saccharomyces cerevisiae. The methods are not recommended for mineral feeds. The results from this study are intended for consideration for adoption as CEN and ISO standards.     

The evaluation of a mupirocin-based selective medium for the enumeration of bifidobacteria from probiotic animal feed

In this study, MRS medium supplemented with cysteine hydrochloride and mupirocin, termed Bifidobacterium selective medium (BSM) was found to be elective for bifidobacteria but inhibitory to a wide range of non-bifidobacteria strains commonly included in probiotic animal feed. Bacilli, lactobacilli, lactococci and streptococci failed to form colonies on BSM and enterococci, pediococci and propionibacteria formed colonies <0.5 mm in diameter. Bifidobacteria formed colonies >1 mm in size and could be readily distinguished. The addition of nystatin to BSM further inhibited Saccharomyces cerevisiae. BSM was successfully used to enumerate the bifidobacteria components, confirmed through fructose-6-phophate-phosphoketolase detection, present in two commercial probiotic feeds. The medium is recommended for the enumeration of bifidobacteria from animal feeds especially when not a numerically dominant component.     

Differentiation of probiotic and environmental Saccharomyces cerevisiae strains in animal feed

Aims: To establish an identification system for probiotic Saccharomyces cerevisiae strains based on artificial neural network (ANN)–assisted Fourier‐transform infrared (FTIR) spectroscopy to improve quality control of animal feed. Methods and Results: The ANN‐based system for differentiating environmental from probiotic S. cerevisiae strains comprises five authorized feed additive strains plus environmental strains isolated from different habitats. A total of 108 isolates were used as reference strains to create the ANN. DHPLC analysis and δ‐PCR were used as reference methods to type probiotic yeast isolates. The performance of the FTIR‐ANN was tested in an internal validation using unknown spectra of each reference strain. This validation step yielded a classification rate of 99·1 %. For an external validation, a test data set comprising 965 spectra of 63 probiotic and environmental S. cerevisiae isolates unknown to the ANN was used, resulting in a classification rate of 98·2 %. Conclusions: Our results demonstrate that probiotic S. cerevisiae strains in feed can be differentiated successfully from environmental isolates using both genotypic approaches and ANN‐based FTIR spectroscopy. Significance and Impact of the Study: FTIR‐based artificial neural network analysis provides a rapid and inexpensive technique for yeast identification both at the species and at the strain level in routine diagnostic laboratories, using a single sample preparation.     

Evaluation of Enterolert for enumeration of enterococci in recreational waters.

Enterolert (IDEXX Laboratories Inc., Westbrook, Maine), a semiautomated, most probable number method for enumeration of enterococci, was compared with the standard membrane filter method by parallel testing of 138 marine and freshwater recreational bathing water samples. No statistically significant difference and a strong linear correlation were found between methods. Culturing of 501 Enterolert test wells resulted in false-positive and false-negative rates of 5.1 and 0.4%, respectively. Less time for setup, incubation (24 versus 48 h), and reading of Enterolert permits more efficient monitoring of recreational bathing areas.     

Improvement of the membrane filter technique for enumeration of enterococci in water

The membrane filter technique with AC agar medium supplemented with 0.04% NaN3 and 0.00015% 2,3,5-triphenyltetrazolium chloride for enumeration of enterococci in water is described. An appropriate volume of a water sample was filtered through the membrane filter. The membrane filter was put on an AC agar plate (designated as the AC.MF technique), which was incubated at 37 C for 18 hr and further at 45 C for 24 hr. By this AC.MF technique, all the colonies grown on the membrane filters were identified as enterococci, and the count of enterococci obtained by the AC.MF technique was similar to that by the AC.MPN technique. The AC.MF technique may be useful for accurate and rapid enumeration of enterococci in water and serve as a simple method for determining the sanitary quality of water.     

Validation of the official control method based on Polymerase Chain Reaction (PCR) for identification of authorised probiotic yeast in animal feed

Council Directive 70/524/EEC regulates the application of probiotic (microorganisms) additives in feeding stuffs. In the present study a method for the differentiation and strain identification of authorised probiotic Saccharomyces cereviseae strains in feeding stuffs by Polymerase Chain Reaction (PCR) was validated. Four different samples of animal feeding stuffs containing yeast at levels between 10(5) to 10(7) CFU/g were examined. Samples were enumerated on chloramphenicol glucose yeast extract agar and colonies were selected from these plates for DNA extraction and subsequent analysis. The PCR method using delta sequence primers produced an 'amplified sequence polymorphism' characteristic for the test strain. Feeds supplemented with one of four probiotic yeast strains each were analysed by seven of nine invited laboratories. All laboratories returned valid results with the exception of one laboratory that had insufficiently separated bands on the gel. The method had a good reproducibility for probiotic yeast isolates from feed of all four authorised probiotic yeast strains (APYS) CBS 493.94, APYS CNCM 1-1079, APYS CNCM 1-1077, APYS NCYC SC47 and of a commercially available yeast reference strain, NCYC 81. The PCR method is to be considered by CEN and ISO as official control method for identification of authorised probiotic Saccharomyces cerevisiae strains from feeding stuffs.     

Characterization of faecal enterococci from rabbits for the selection of probiotic strains

AIMS: To characterize the facultative anaerobic intestinal microbiota of healthy rabbits, especially enterococci, for the selection of potential probiotic strains. METHODS AND RESULTS: Phenotypic and molecular methods were used to identify enterococcal isolates. Results obtained indicated that enterococcal microbiota widely varied among individuals both in size and in composition. Antibacterial and haemolytic activities, and resistance to acid and bile salts were determined. A small group of strains produced bacteriocins active against listeriae and indigenous clostridia and therefore they were selected as potential probiotics. One such strain, 8G, was assayed for colonization capacity. Results obtained suggested that the fate of the introduced strain depended on the composition of the enterococcal indigenous microbiota. CONCLUSIONS: Enterococcus faecalis and Ent. faecium are the predominant enterococcal species in the gut of rabbits. Other species of lactic acid bacteria were not recovered. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal intestinal microbiota of healthy rabbits has been characterized in detail. Monitoring the fate of an introduced probiotic in vivo is required in order to evaluate potential probiotic strains.     

A method for the enumeration of male-specific bacteriophages in sewage

H avelaar , A.H. & H ogeboom , W.M. 1984. A method for the enumeration of male-specific bacteriophages in sewage. Journal of Applied Bacteriology 56 , 439–447.
Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F^--salmonellas—usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimu-rium phage type 3, plaque counts in secondary effluent were found to be in the range of 60–8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiona carrying F'42 lac . A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F^- E. coli but not F^- Salmonella were isolated using this host strain.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

The survival of streptococci and enterococci in animal feed and on straw with particular reference to Streptococcus uberis

The survival of Streptococcus uberis, Strep. bovis, Enterococcus faecalis spp. faecalis and Ent. faecium in animal feed and on wheat straw was studied for one month. Streptococcus bovis survived the best in feed and Strep. uberis the worst while on straw Ent. faecium was the most resilient species and there was little to choose between the other three. The recovery of all species, excepting Strep. bovis , on selective and non-selective agar was comparable. The growth of Strep. bovis , on the other hand, was inhibited on both Edward's medium and inulin agar.     

The survival of streptococci and enterococci in animal feed and on straw with particular reference to Streptococcus uberis

The survival of Streptococcus uberis, Strep. bovis, Enterococcus faecalis spp. faecalis and Ent. faecium in animal feed and on wheat straw was studied for one month. Streptococcus bovis survived the best in feed and Strep. uberis the worst while on straw Ent. faecium was the most resilient species and there was little to choose between the other three. The recovery of all species, excepting Strep. bovis, on selective and non-selective agar was comparable. The growth of Strep. bovis, on the other hand, was inhibited on both Edward's medium and inulin agar.     

Usefulness of epifluorescence for quantitative analysis of lactobacilli in probiotic feed

AIMS: Enumeration of total, active or viable probiotic micro-organisms from liquid or solid commercial feedstuffs was studied during processing and storage. METHODS AND RESULTS: After sample preparation, an epifluorescence microscopy technique and a plating method were investigated comparatively. It was shown that (i) on the day of manufacture, active or viable bacteria were in equivalent amounts and that viable numbers then decreased, depending on the different processing and storage factors enhancing ABNC production, (ii) the amount of total and active lactobacilli remained close and quite stable for months at a high level (>10(8) active fluorescent units). CONCLUSIONS: Processing and storage promoted ABNC cells in the products tested. Consequently, both techniques should be used to evaluate the viable-dead-active status of bacteria for which functional properties are claimed. SIGNIFICANCE AND IMPACT OF THE STUDY: Enumeration of the whole probiotic bacterial population should be take into account for guidelines and labelling since non-viable bacteria could have a probiotic effect.     

Influence of probiotic enterococci on the growth of Streptococcus agalactiae

Individual features of sensitivity of some strains of group B streptococci (GBS) to influence of 2 probiotic cultures of Enterococcus faecium (SF68 and L3) have been studied by double agar test. E. faecium L3 strain had higher antagonistic activity to GBS. Two genes encoding enterocins A and B as well as genes responsible for the expression of the former two genes were found in the genome of this strain. The supernatant and peptide extract of E. faecium L3 contained thermostable low molecular weight peptides which inhibited growth of listeria and GBS but at lesser extent compared with native enterococci. Obtained data allow to suggest that antagonistic activity of enterococci against GBS may be affiliated with production of enterocins A and B and can be increased by the presence of other metabolites.     

A method for the enumeration of Aeromonas bacteriophage in aquatic environments

K.P. FLINT. 1996. Bacteriophage were isolated against type strains and environmental isolates of Aeromonas hydrophila, Aeromonas sobria and Aeromonas caviae . A most probable number method for estimating the number of bacteriophage in a water sample was devised and tested using some of these isolates. The maximum number of bacteriophage against all three type strains were found in water from below a sewage effluent outfall. This corresponds to the increased numbers of each species of bacterium also found in this water sample. High numbers of bacteriophage against Aer. hydrophila were also found in the lake sample examined. Bacteriophage against Aer. caviae were rare in water samples other than those contaminated with sewage effluent.     

Probiotic potential of enterococci isolated from canine feed

Enterococci isolated from 28 different commercially available feeds (10-1000 CFU/mL) were identified and their probiotic potential was determined. Species identification of 22 selected strains was performed by intergenic length-polymorphism analysis (tRNA-PCR); PCR products were analyzed using capillary electrophoresis. Six strains were allotted to the species Enterococcus faecium, four to E. faecalis, one to E. hirae; the remaining strains were not classed. The strains were sensitive to vancomycin, ampicillin, tetracycline and rifampicin. They were able to adhere to human as well as canine intestinal mucus. They produced lactic acid (0.99-1.04 mmol/L) and most of them were urease-positive with sufficient survival in 5 % Oxgall-bile. They did not show any inhibitory activity due to antimicrobial substances. Plasmid DNA was detected in 8 strains, the bands responding to small molecular size (10 kbp). Considering all probiotically important properties, E. faecium strain EE3 was suggested as potential feed additive.