Progut info letter 1/2005

Transcription

1 info letter 1/25 Comparative Research of Escherichia coli bacteria attachment to and other Saccharomyces products. Justus- Liebig University Giessen 24 (Bauerfeind R., Baljer, G. and Barth,S.)¹ 1. Objective The aim of the present research was to test capability of binding enterotoxic Escherichia coli bacteria with fimbria types F4, F5 and F6 (F4-, F5-, F6- or F4/F6-ETEC) as well as E.coli bacteria without fimbria in comparison to four other yeast cell products (, Med. Hefe, Yea-Sacc and forte) and Saccharomyces cerevisiae reference strain. 2. Material and methods In order to answer this question, the yeast cell products were co-incubated with E.coli bacteria at 37 C. After one hour the suspension was centrifuged at low speed (sedimentation of the bacteria infested yeast cells) and the number of unbound bacteria in the supernatant was determined. Materials and methods are described more exactly in appendix. 3. Results 3.1. Characteristics of the E.coli strains used The characteristics of the used E.coli strains are presented in table 1. According to the PCR results the F4 and the F4/F6 ETEC strains reacted positively with the F4 specific antiserum and the F5 ETEC strain reacted with the F5 specific antiserum during the rapid slide agglutination test. There was no agglutination with the F6 specific antiserum. In case of the E.coli isolate without fimbria no ETEC virulence characteristics could be proven. All the strains except F5 ETEC were motile. Two strains (F4-ETEC and F4/F6-ETEC) were haemolytic. Table 1 Characteristics of the used Escherichia coli strains Aggl. in test Haemolysis Motility serum with Type Strain ETEC gene F4 F5 F6 F4-ETEC P4238/1-2 faeg, elt, estb F5-ETEC P695/1-2 fana, estap F6-ETEC P5416/3-3 fasa, estap F4/F6- ETEC Abbotstown faeg, fasa, elt, estb, estap F-E. coli P5279/2 None Translated and modified from the original report by Juhani Vuorenmaa

2 3.2. Microscopic examination For microscopic analysis 1 million CFU of each E.coli strain were added to the cultures. In 29 out of the 3 cultures analyzed more than 5 % of the microscopically visible bacteria cells were in close association with yeast cells showing that yeasts were capable to adsorb E.coli germs. Number of free bacteria cells was lowest for all E.coli strains in the cultures with showing the best adsorption capacity Attachment of E.coli strains to different yeast products The efficiency of different yeast products to attach E.coli bacteria was evaluated by counting the number of unbound E.coli bacteria in the supernatants after centrifugations. The germ counts in the cultures, that contained Saccharomyces products, were compared to germ counts (1%) determined for the cultures that contained only the relevant E.coli strain. The Saccharomyces products reacted very differently with the E.coli strains. The E.coli count in the supernatants from the cultures with was in every case the lowest (figures 1-5) showing that all the studied E.coli strains attached most efficiently to. In most of the cases this difference was also significant (p<,29). With the exception of the F4-ETEC strain the lowest attachment was seen with cultures. Compared with the control cultures most of the yeasts reduced the germ counts with F4-ETEC and F4/F6 ETEC strains (figures 1-2) but not with the other strains (figure 3-5). In the other cultures the germ count was in some cases even significantly higher than in control cultures. This indicates that with F5-, F6- and F ETEC E.coli strains yeast may even have a growth promoting effect. In comparison to other yeast products had clearly the lowest E.coli counts also with these E.coli strains. It should be noted that the most common pathogenic E.coli strains with piglets are the F4-ETEC types. Figure 1. Unattached bacteria content in the supernatant after one-hour coincubation of F4-ETEC E.coli and different yeast products (mean from triple E.coli % to control F4-ETEC control E.coli F4-ETEC

3 Figure 2. Unattached bacteria content in the supernatant after one-hour coincubation of F4/F6-ETEC E.coli and different yeast products (mean from triple E.coli count % to control F4/F6-ETEC control E.coli F4/F6 ETEC Figure 3. Unattached bacteria content in the supernatant after one-hour coincubation of F5-ETEC E.coli and different yeast products (mean from triple E.coli count % of control E.coli F5-ETEC F5-ETEC control

4 Figure 4. Unattached bacteria content in the supernatant after one-hour coincubation of F6-ETEC E.coli and different yeast products (mean from triple E.coli cunt % to control E.coli F6-ETEC F6-ETEC control Figure 5. Unattached bacteria content in the supernatant after one-hour coincubation of F -ETEC E.coli and different yeast products (mean from triple E.coli count % to control E.coli F' ETEC F'-ETEC control

5 4. Conclusions All the tested Saccharomyces products were capable to adsorb E.coli bacteria Number of free bacteria cells in the microscopic tests was lowest for all E.coli strains in the cultures with showing the best adsorption capacity All the studied E.coli strains attached most efficiently to. In most of the cases the difference to other yeast products was also significant (p<,29) Most of the yeast products reduced the E.coli counts compared to control with F4-ETEC and F4/F6 ETEC strains but not with the other strains. This indicates that with F5-, F6- and F - ETEC E.coli strains yeasts may even have a growth promoting effect. In comparison to other yeast products had clearly the lowest E.coli counts also with F5-, F6- and F ETEC strains Evidently, it can be suggested that the special characteristics of are related to its patent protected production process. Reference: Vergleichende Untersuchung zur Adhäsion von Escherichia coli- Bakterien an und andere Saccharomyces- Produkten (Baljer, G., Barth, S. und Bauerfeind, R. 24)

6 Appendix. Materials and methods used in the trial Table 1 Escherichia coli strains used Type Strain Stereotype Origin F4-ETEC P4238/1-2 O149:K91 Faeces samples from Hessen F5-ETEC P695/1-2 O141:K85ab pigs; isolated during routine F6-ETEC P5416/3-3 n.t.¹ diagnostics at Institute of F -E. coli P5279/2 n.t. 1 Animal Hygiene and Infectious Diseases in Giessen (Institut für Hygiene und Infektionskrankheiten der Tiere, Gießen) F4/F6- ETEC Abbotstown O149:K91, K88ac Prof. Dr. C. Wray, Weybridge, U.K. Before the experiment was started motility of the E.coli strains was tested by homogeneous diffusion of the nutrient medium. Haemolysis was verified after the cultivation of bacteria on the blood agar plates. The examination of ETEC-typical virulence factors as well as heat stable enterotoxins was carried out using Multiplex-PCR according to Bosworth & Casey (1997). The expression of fimbria was tested using rapid slide agglutination with monovalent diagnostic pig E.coli serum K88 (F4), K99 (F5) and 987p (F6) respectively. Co-incubation of E.coli germs and Saccharomyces products and E.coli germ count determination For co-incubation the Saccharomyces products were used at, 3% (/w/v) final concentration and the E.coli germs were added at different germ concentrations. All the tests were carried out in triple replicates. Cultures of respective E.coli strains without the addition of Saccharomyces products were used as controls. The co-incubation took place in overhead shakers for 1 h at 37 ºC. Immediately after that the cultures were kept on ice for at least 1 minutes and subsequently centrifuged (8xg, 2 min, RT). Determination of the unattached E.coli germ counts in the suspensions after the centrifugation was done with plate count method according to Amtliche Sammlung von Untersuchungsverfahren nach 35 LMBG". (An. 1984). The ratio of free and yeast cells associated E.coli bacteria cells was determined under the microscope. Statistics The arithmetical mean and standard deviation of the determined germ counts were calculated for statistical analysis. Mean differences were tested for significance using Student s t-test. 1 n.t. = non-typifiable, i.e. the E.coli serum reacted with no or at least two of the used test serums [O138:K81, O139:K81, O141:K85ac, O141:K85ab, O147:K89, O149:K91, O157:K-].