Three strains of enteropathogenic Escherichia coli (EPEC), originally isolated from humans and previously shown to cause diarrhea in human volunteers by unknown mechanisms, and one rabbit EPEC strain were shown to attach intimately to and efface microvilli and cytoplasm from intestinal epithelial cells in both the pig and rabbit intestine. The attaching and effacing activities of these EPEC were demonstrable by light microscopic examination of routine histological sections and by transmission electron microscopy. It was suggested that intact colostrum-deprived newborn pigs and ligated intestinal loops in pigs and rabbits may be useful systems to detect EPEC that have attaching and effacing activities and for studying the pathogenesis of such infections. The lesions (attachment and effacement) produced by EPEC in these systems were multifocal, with considerable animal-to-animal variation in response to the same strain of EPEC. The EPEC strains also varied in the frequency and extent of lesion production. For example, three human EPEC strains usually caused extensive lesions in rabbit intestinal loops, whereas two other human EPEC strains usually did not produce lesions in this system.

In chloride-secretory epithelia, the basolateral Na-K2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl ؊ and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K ؉ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1 ؊/؊ ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl ؊ channel, were reduced in jejunum, cecum, and trachea of Nkcc1 ؊/؊ mice, indicating that NKCC1 contributes to cAMP-induced Cl ؊ secretion. In contrast, secretion of gastric acid in adult Nkcc1 ؊/؊ stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1 ؊/؊ mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K ؉ movements involved in generation of the K ؉ -rich endolymph and the endocochlear potential.

SUMMARY Reassessment of the 'gastric bactericidal barrier' to enteric bacteria in man included studies of the bactericidal activity of (1) the normal and achlorhydric stomach in vivo and (2) normal and achlorhydric gastric juice and other media in vitro. Within 30 minutes virtually all bacteria (Serratia marcescens) were eliminated in the normal stomach whereas no reduction occurred in the achlorhydric stomach in one hour. In vitro, identical bactericidal activity was observed at the same pH (from 2.0 to 7.0) in normal gastric juice, achlorhydric gastric juice, aqueous HCI, and nutrient broth. At pH less than 4.0, 99.9 % of the bacteria were killed within 30 minutes. The presence of profuse bacterial flora, including coliforms, found in markedly acid-deficient but not in normal stomachs, correlates well with the absence of bactericidal activity. Thus, the 'gastric bactericidal barrier' is primarily pH-hydrochloric acid dependent, with other constituents of gastric juice contributing little, if any, detectable effect on the destruction of microorganisms.A relationship between gastric acid secretion and bacterial diarrhoeas has been suspected for the past 100 years since enteric bacteria do not survive in an acidic environment (Bartle and Harkins, 1925;Garrod, 1939). Furthermore, gastric bactericidal factors, in addition to acid, have been postulated (Gregersen, 1916;Scheer, 1919;Knott, 1923;Goldsworthy and Florey, 1930;Sebastianelli, 1937;Garrod, 1939;Thompson, 1940;Balazs, 1962). In the latter part of the 19th and early 20th centuries, Hewetson (1904), Knott (1923), Arnold (1927), Camps (1933, Hurst (1934), Garrod (1939, and others (Gregersen, 1916;Scheer, 1919;Bartle and Harkins, 1925;Sebastianelli, 1937) believed that patients with reduced or absent gastric acid secretion were more susceptible to bacterial dysenteries and enunciated the concept of the 'gastric bactericidal barrier' (Hewetson, 1904;Gregersen, 1916;Scheer, 1919;Knott, 1923;Bartle and Harkins, 1925;Arnold, 1927;Camps, 1933;Hurst, 1934;Teale, 1934;Sebastianelli, 1937;Garrod, 1939). Today, despite a century of study, the relationship of gastric acidity to the pathogenesis of enteric infec-'Please address reprint requests to Dr Norman Zamcheck, Mallory Gastrointestinal Laboratory, Boston City Hospital, Boston, Mass. 02118.Received for publication 16 December 1971. tions and the particular relevance of gastric anacidity in this regard are still insufficiently appreciated.It is the purpose of this paper (a) to reassess the bactericidal activity of the normal and achlorhydric stomach and of normal and achlorhydric gastric juice; (b) to enumerate the bacterial flora of the normal and acid-deficient stomach; and (c) to update the clinical relevance of the 'gastric bactericidal barrier' in the light of these findings and those of other workers. Materials and Methods PATIENTSThree groups were studied: (1)

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Although the suckling mouse assay is widely used for the detection of heatstable Escherichia coli enterotoxin (ST), few data have been published concerning the reproducibility, optimal growth, and test conditions of this assay. Four strains of toxigenic E. coli known to elaborate both heat-labile enterotoxin and ST or ST alone were used to study these parameters. ST activity after heat treatment and the effect of purified choleragen were also examined. ST production was optimal in Casamino Acids-yeast extract media, but both Trypticase soy and brain heart infusion broths resulted in several false negative reactions. Growing cultures in roller tubes was the most reliable method of ST production. Shaking-flask cultures and stationary-grown cultures resulted in suboptimal ST production in several strains. Optimal mouse incubation time was 3 h, and fluid secretion did not rise thereafter. Adequate toxin production occurred after 16 to 24 h of incubation. The coefficient of variation of various toxins tested on many occasions varied between 10.5 and 15.7%. Toxin activity was stable for 6 months when frozen at-20 C. There was no decrease in ST activity when heated at 65 C for 15 min, but a small decrease was observed in two of four strains after heating at 100 C for 30 min. Choleragen, tested at various doses and at multiple times, gave uniformly negative results. These studies indicate that when done under the proper conditions, the suckling mouse assay is a simple, rapid, and reproducible assay for E. coli ST.