key: cord-0711757-u1ylvtld authors: Bomba, A.; Kravjanský, I.; Kaštel', R.; Herich, R.; Juhásová, Z.; Čížek, M.; Kapitančík, B. title: Inhibitory effects of Lactobacillus casei upon the adhesion of enterotoxigenic Escherichia coli K99 to the intestinal mucosa in gnotobiotic lambs date: 1997-01-03 journal: Small Ruminant Research DOI: 10.1016/s0921-4488(96)00905-4 sha: 35540a705727d5efb767afb086b9f86fed0bd4b7 doc_id: 711757 cord_uid: u1ylvtld Abstract Observations were carried out of the interactions between Lactobacillus casei 294/89 and enterotoxigenic Escherichia coli CCM 612 (O101:K99) in vivo. In gnotobiotic lambs, inoculation with enterotoxigenic E. coli (ETEC) resulted in diarrhea with a typical clinical picture and patho-anatomical findings. E. coli adhered to the mucosa of the digestive tract at counts amounting to 105 per cm2. In these lambs, disturbances of intestinal biochemical processes became evident; proteolytic enzyme activity was significantly reduced. Preventive administration of Lactobacillus casei inhibited the negative effects of ETEC in gnotobiotic lambs, minimized the clinical signs to those of a very moderate diarrhea in the first 12 h after inoculation and significantly reduced the patho-anatomical findings. Enterotoxigenic E. coli counts decreased by 99.1 and 76% on days 2 and 4 after inoculation respectively, and amounted to 103 per cm2. The inhibitory effects of L. casei against E. coli were most obvious in the jejunum and ileum. The numbers of adhering E. coli increased from the duodenum with the length of the gut. ETEC counts in the digestive tract of lambs that had been preventively treated with L. casei amounted to 107 ml−1. It can be assumed that, in addition to competitive exclusion, the inhibitory effect of L. casei upon ETEC adherence was also mediated by a Lactobacillus-produced substance that inhibited E. coli adhesion to the gut mucosa. When rearing young animals, diarrhoic diseases present a serious health and economic problem. These may be caused by different infectious agents acting either separately or in association with other microorganisms and essential factors. Escherichia coli, salmonellae, rota-and coronaviruses as well as cryptosporidia play an important part in the etiology of the diarrhoic syndrome. In the young, enterotoxigenic E. coli appear to be the most frequent diarrhea-causing agents (Tzipori, 1981) ; they can be found in stables with a reduced level of husbandry where morbidity and mortality rates are high. Macrobacilli are a component of the natural microflora of the gut; they colonize the latter as early as within the first hours of life. They have rather beneficial effects upon the macroorganism and inhibit several pathogens. Raccach et al. (1989) and Sudirman et al. (1993) reported Lactobacilli to inhibit Lisreriae while and Chateau et al. (1993) observed these microorganisms to inhibit Staphylococci. According to Watkins et al. (19821, Foumiat et al (1992) and Lidbeck et al. (1987) Lactubacilli also inhibit pathogenic E. coli. Employing Lactobacilli in the form of probiotics that would be based on their inhibitory effects against pathogens seems to be a very efficacious method of preventing and treating diseases caused by pathogenic microorganisms, mainly the diarrhoeic syndrome in the young of farm animals (Gilliland et al., 1980) . The administration of Lactobacilli containing probiotics is a biotechnological method having not only beneficial effects upon the health state of the young but also a positive impact on the environment. Lactobacillus acidophilus, L. casei, L. lactis. L. reuteri, L. plantarum, L. fermentum, L. brevis and L. delbruecki (Fuller, 1989; Jonsson and Conway, 1992) are those strains that are most widely incorporated in probiotic preparations. Some authors claim the antibacterial effects of probiotics to result from the production of lactic acid and a decrease in pH, the production of hydrogen peroxide and the antibacterial properties of the latter, the production of natural antibiotic substancesbacteriocines, the antienterotoxic activity (mainly against the E. coli enterotoxin) and the inhibition of pathogen adherence to the wall of the intestinal tract (Vandenbergh, 1993; Chauviere et al., 1992) . The pathogenicity of E. coli is conditional on two factors: the ability to produce enterotoxin (Smith and Gyles, 1970) and the presence of colonization factors enabling the carrier to colonize the mucosa of the small intestine (Bertschinger et al., 1972; Jones and Rutter, 1972) . The ability of enterotoxigenic E. coli to colonize the gut presents the primary and decisive pathogenic factor since it is inevitable for the second virulence factor -the enterotoxin -to exert its effects. It was the aim of our work to observe the effects of L. cusei upon the adherence of enterotoxigenic E. coli in the digestive tract of gnotobiotic lambs and the effects of mutual interaction upon intestinal biochemistry. In the experiment six germ-free Improved Wallachian lambs were included. They were obtained by hysterectomy, divided into two groups (E and L-E, respectively) counting three animals each and reared in two isolators according to Bomba et al. (1993) . Four times a day the animals had ad libitum access to a commercial milk mixture. The group E lambs were inoculated at 1 day of age with enterotoxigenic E. coli CCM 612 (0 101: K99). The group L-E lambs were inoculated with L. cusei 294/89 at the age of 1,2 and 3 days. On day 4 this group was inoculated with E. culi similar to Group E. Each inoculum contained 1 X lo8 germs in 1 ml. The inocula (2 ml> were given once a day. The 294/89 strain of L. cusei was isolated from the rectal swab of a calf aged 2 days, chosen of a total of 324 strains of Lactobacilli observed and tested by routine biochemical methods. In each of the groups one lamb was killed 2 and 4 days after inoculation, respectively. In the L-E group one lamb was killed also 7 days after inoculation, but to do the same in Group E was impossible since one lamb had died as early as 12 h after inoculation. Immediately after slaughter samples were obtained of the duodenum, jejunum, ileum and colon (10 cm*) and their contents. Tissue samples designed for estimation of counts of microorganisms adhering to the intestinal mucosa were 3 X washed with 0.15 M PBS (pH 7.2) at moderate stirring, then the mucosa was scraped with a covering slide. The material yielded was placed into 10 ml 0.15 M PBS (pH 7.2) containing 1% Tween and decadic dilutions were prepared. Selective Rogosa agar and MacConkey agar were used to state Lactobacillus and E. coli counts, respectively. Lactic, acetic and propionic acids were determined using the method of capillary isotachophoresis on a capillary isotachophoresis analyzer (Radioecological Institute, KoSice, Slovakia). As conductive and final electrolyte 0.01 mmol 1-l HCl (pH 4.25) and mmol 1-l capronic acid (pH 4.5) were used, respectively. For alpha-amylase determination the Spofa-test alfa-amylaza (Slovakofarma Hlohovec, Slovakia) was employed; trypsin and chymotrypsin were assayed using the test by Boehringer (Mannheim, Germany). haemorrhagic abomasitis and enteritis were found. In the remaining two lambs acute diffuse catarrhal abomasitis and enteritis were seen. In the lambs preventively inoculated with lactobacilli a very mild acute catarrhal enteritis was found, the sites of inflammation being circumscribed and mainly localized in the duodenum and jejunum. In the lamb killed 7 days after E. coli inoculation the acute catarrhal inflammation in the duodenum in part passed into a circumscribed haemorrhagic inflammation. However, the ileum and the colon did not reveal pathological changes. Statistical analysis. The results are arithmetic means. For statistical evaluation Student's t-test was used. In the group of animals inoculated with enterotoxigenic E. coli CCM 612 (Group E) profuse diarrhoea occurred in two lambs as early as during the first 12 h after inoculation. Their faeces were aqueous and of a yellow-green colour. In the third lamb faeces were thinner, without apparent diarrhea. One of the lambs died 12 h following inoculation. All lambs manifested clinical signs such as dehydration, apathy, general weakness, inappetence and decreased skin turgor. In the animals who received a preventive dose of L. casei 294/89 and subsequently enterotoxigenic E. coli CCM 612 (Group L-E), a very moderate diarrhoea occurred with more watery faeces of a yellow-brown colour. Mild weakness and indistinct apathy were seen. Up to 24 h after inoculation the clinical state of all lambs apparently improved and no inappetence was present. No health disturbances could be observed later and the lambs drank milk showing good appetite. The faeces became condensed and of chocolate-brown colour. Two and four days after inoculation the numbers of enterotoxigenic E. coli CCM 612 (0 101: K 99) adhering to the intestinal mucosa of group E lambs counted 5.1 + 1.0 and 5.0 + 0.93 log 10 germs per cm'. In the L-E group the number of E. co/i CCM 612 ranged between 3.4 + 0.52 and 3.6 + 1.3 log10 germs per cme2 (Fig. 1) . The differences between the groups were significant 2 days after inoculation (P < 0.05). The counts of enteropathogenic E. co/i CCM 612 adhering to the digestive tract mucosa of group E lambs increased from the duodenum (3.8 f 0.32 log10cm-2) to the colon (5.7 + 0.73 log10cm-2). In the digestive tract of group L-E lambs, the minimum and maximum counts of enterotoxigenic E. coli CCM 612 (Fig. 2) were observed in the jejunum (2.8 + 0.15 loglOcm-*) and colon (4.7 f 1.2 log10cm-2), respectively. Preventive administration of L. casei 294/89 to lambs of the L-E group decreased the counts of enterotoxigenic E. coli by 99.1 and 76% on days 2 and 4 after inoculation, respectively. In animals of the L-E group the numbers of adhering enterotoxigenic E. coli were decreased by 23, 99.7, 99.8 and 75.8% in the duodenum, jejunum, ileum and colon, respectively. Four and seven days after inoculation the mean counts of enterotoxigenic E. coli in the gut contents of the L-E lambs ranged between 7.1 f 1 .l and 7.5 + 0.63 1oglOmll reaching minimum and maximum values in the duodenum (5.5 f 0.46 log1 Oml-' > and jejunum (8.1 f 0.24 loglO.ml-'), respectively; in the ileum and the colon 7.7 f 0.37 and 7.5 + 0.03 log 1 Oml-' of E. coli were present, respectively. In the dead lamb of the group of animals inocu- The mean counts of enterotoxigenic E. coli in the lated only with enterotoxigenic E. coli acute diffuse gut contents of the E lambs were very similar. In L-E group, the numbers of mucosa-adherent L. casei 294/89 following E. coli inoculation varied between 1.9 and 2.7 log10.cm-2. Comparable numbers of mucosa-adherent L. casei (2.2 f 0.86 and 2.5 f 0.15 log10.cm-2) were counted in the individual gut segments of L-E lambs, revealing a minimum increase between the duodenum and the colon whereas in the digesta an increase could be seen between the duodenum (4.9 + 0.92 loglOml-'1 and the ileum (6.6 + 0.1 loglOml-' >. The difference between the counts of mucosa-adherent Lactobacilli and the counts of Luctobacilli in the digesta in the duodenum, ileum and colon showed to be significant at the level of P < 0.05, P < 0.01 and P < 0.001, respectively. Lactic acid (LA) levels in the duodenal and ileal digesta of L-E lambs were twice those of the E lambs (Table 1 ). In both groups, maximum LA levels were observed in the duodenum (3.6 and 6.7 mmol.l-', respectively). Acetic acid levels were prevailingly increased in the digesta of the L-E group, but without marked differences. In comparison to group E, propionic acid levels in the small intestine of L-E lambs were 2-4-times increased. Inoculation of enterotoxigenic E. coli markedly reduced the enzyme activity of the digesta, mainly that of the proteolytic enzymes. Preventive administration of L. casei inhibited the abovementioned effect of E. co/i. The activity of alpha-amylase (E.C.3.2.1 .l) in the digesta of L-E lambs was increased, however, the differences in comparison to E lambs (except of the duodenum) were negligible. Trypsin activity of the digesta in the jejunum and ileum of L-E lambs revealed a 4-and 5-fold increase, respectively, when compared to that of the E group. Chymotrypsin activity in the jejunum and ileum of the L-E lambs was twice that of the E group. Inoculation of ETEC CCM 612 (OlOl:K99) to lambs induced diarrhoea with complex symptoms, a typical clinical picture and patho-anatomical findings. E. cofi colonized the mucosa of the digestive tract in numbers approximating 100000 germs per cm2. The biochemistry of the gut was disturbed, the activity of the proteolytic enzymes was markedly reduced. Preventive administration of L. cusei 294/89 inhibited the negative effects of ETEC upon germ-free lambs, minimizing the clinical symtpoms to a very mild diarrhea in the first 12 h after inoculation and markedly reducing the patho-anatomical findings. Two and four days after ETEC inoculation the numbers of ETEC adhering to the mucosa of the digestive tract decreased by 99.1 and 76%, respectively, and counted 1000 germs per cm'. The inhibitory effects of L. cusei upon the addherence of ETEC were most obvious in the jejunum and ileum. The results obtained show that L. cusei 294/89, though it could not fully inhibit the adherence of ETEC to the mucosa of the digestive tract, remarkably reduced the number of adhering E. co/i, thus preventing the development of diarrhoea with its clinical symptoms and patbo-anatomical picture. A similar effect was observed by Underdahl et al. (1982) who preventively administered Streprococcus fuecium C-68 to germ-free suckling piglets in order to inhibit the adherence of ETEC. Several authors have reported Luctobucilli to have inhibitory effects upon the adherence of E. coli in the digestive tract. Muralidhara et al. (1973) observed the administration of Luctobucilli to fully inhibit the adherence of ETEC in the first three segments of the small intes-tine that had been divided into nine segments. Fourniat et al. (1992) described inhibition of enterotoxigenie E. coli B 41 (OlO:K99:F41:ST+) to HeLa cells by heat-killed (1 OO-105°C) L. ucidophihs. also point to the marked inhibitory effects of L. ucidophilus upon pathogenic E. coli in germ-free fowl. The mechanism of the effects of probiotics is widely discussed. It is very important that an essential stage in the pathogenesis of intestinal infections, especially diarrhoea of bacterial origin, involves the adhesion of the microorganisms concerned to the intestinal epithelial cells. In principle, bacterial diarrhoea could be treated by preventing adhesion of pathogenic bacteria (Foumiat et al., 1992) . The following methods are in use: 1. Vaccination against bacterial adhesion factors (Levine et al., 1983; Runnels et al., 1987) . 2. Administration of antibiotics which inhibit the expression of adhesion factors (Deneke et al., 1985; Chopra and Hacker, 1986) . 3. Oral administration of substances containing structures similar to those of the adhesion factors of pathogens (Neeser et al., 1988) or structures that mimic receptors of the intestinal mucosa (Mouricout et al., 1986) . Oral administration of certain strains of Luctobucilli has also been found to inhibit implantation of various pathogens in the digestive tract. This action may be due to nonspecific competitive inhibition of the adhesion of pathogenic strains to epithelial cells of the digestive tract (Barrow et al., 1980) . As can be seen from the results obtained, it was not only competitive inhibition that caused the inhibitory effect of L. cusei. This is testified by the low counts of L. cusei adhering to the epithelium of the digestive tract and the unreduced counts of E. coli in the gut contents. It can be concluded that L. cusei 294/89 produced unknown substance which inhibited the adhesion of ETEC either through blockage of receptors in the intestinal mucosa of lambs, or through disturbance of the adhesion factors or inhibition of their expression in E. coli. Similarly Blomberg et al. (1993) observed L. fermenturn 104R to produce a proteinaceous component detectable in spent culture fluid during growth in both complex and defined media; this component inhibited the adhesion of K88ab and K88ac fimbriae to ileal mucus by interacting with mucus components. Keeping in mind the prevention of clinical manifestation of the disease an anti-enterotoxic effect may also be considered. The anti-enterotoxic activity of Lactobacilli was also reported by Mitchell and Kenworthy (1976) . The administration of L. casei 294/89 proved to have rather beneficial effects upon gut biochemistry and to prevent a decrease in proteolytic enzyme activity observed after the inoculation of ETEC CCM 612. Further experiments will be conducted in order to study factors stimulating the inhibitory effects of Luctobacilli against ETEC and factors selectively supporting the adhesion of Lactobacilli. This may lead to the development of potentiated probioticsnew preparations for prevention and treatment. The attachment of bacteria to the gastric epithelium of the pig and its importance in the microecology of the intestine Association of Escherichiu coli with the small intestinal epithelium. I. Comparison of enteropathogenic and nonenteropathogenic porcine strains in pigs Inhibition of adhesion of Escherichia coli K 88 to piglet ileal mucus by Lactobacillus spp The recovery and rearing of gnotobiotic lambs and their use in veterinary medicine Distribution of pathogen inhibition in the LactobaciNus isolates of a commercial probiotic consortium Competetive exclusion of diarrheagenic Escherichia coli (ETIX) by heat killed Lactobacillus Inhibition of K 88 -mediated adhesion of Escherichia coli to mammalian receptors by antibiotics that affect bacterial protein synthesis Effect of tetracycline on the attachment of K 88+ enterotoxigenic Escherichia coli to porcine small -intestinal cells l992 Heat killed Luctobacillus acidophilus inhibits adhesion of Escherichia coli B 41 to HeLa cells Probiotics in man and animals Comparison of two strains of Lactobacillus acidophilus as dietary adjuncts for yong calves Role of the K 88 antigen in the pathogenesis of neonatal diarrhea caused by Escherichia coli in piglets Probiotics the scientific basis New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development Impact of Lactobacillus acidophilus supplements on the human oropharyngeal and intestinal microflora. Stand Investigations on a metabolite form Lactobacillus bulgaricus which neutralizes the effect of enterotoxin from Escherichia coli pathogenic for pigs Mode d'action d'agenst inhibititeurs de l'adhtsion de Escherichia coli enterotoxigenes (ECET) aux glycoproteines de la mugueuse intestinale bovine. Mode of action of inhibitors upon the adhesion of enterotoxigenic Escherichia coli (ETEC) IO the glycoproteins of intestinal bovine mucosa Screening for complex carbohydrates inhibiting hemagglutinations by CFA/I-and CFA/IIexpressing enterotoxigenie Escherichia coli strains Inhibiosis of some lactic acid bacteria including Lactobacillus acidophilus toward Listeria monocytogenes F 41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F 41, K 99, or both pilus antigens Lactic acid bacteria, their metabolic teropathogenic strains of Escherichia coli of porcine origin. J. products and interference with microbial growth Detection and properties of curvaticin 13, a bacteriocin-like substance produced by Lactobacillus cmvatus SB 13 In vivo inhibitory effect of Lactobacillus acidophilus against pathogenic Escherichia coli in gnotobiotic chicks The aetiology and diagnosis of calf diarrhoea Effect of Streptococcus faecium C 68 in control of Escherichia coli induced diarrhea in gnotobiotic pigs Competetive gut exclusion of avian pathogens by Lactobacillus acidophilus in gnotobiotic chicks The authors wish to thank G. Gre'serova-TkEikova, MA, for translating the manuscript into English.