Transmission and infectious dose of Escherichia coli O157:H7 in swine.

Escherichia coli O157:H7 is only occasionally isolated from healthy swine, but some experimentally infected animals will shed the organism in their feces for at least 2 months. Potential explanations for the paucity of naturally occurring infections in swine, as compared to cattle, include a lack of animal-to-animal transmission so that the organism cannot be maintained within a herd, a high infectious dose, or herd management practices that prevent the maintenance of the organism in the gastrointestinal tract. We hypothesized that donor pigs infected with E. coli O157:H7 would transmit the organism to naïve pigs. We also determined the infectious dose and whether housing pigs individually on grated floors would decrease the magnitude or duration of fecal shedding. Infected donor pigs shedding <10(4) CFU of E. coli O157:H7 per g transmitted the organism to 6 of 12 naïve pigs exposed to them. The infectious dose of E. coli O157:H7 for 3-month-old pigs was approximately 6 x 10(3) CFU. There was no difference in the magnitude and duration of fecal shedding by pigs housed individually on grates compared to those housed two per pen on cement floors. These results suggest that swine do not have an innate resistance to colonization by E. coli O157:H7 and that they could serve as a reservoir host under suitable conditions.

Persistence of Escherichia coli O157:H7 in experimentally infected swine.

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.

In vitro detection of Shiga toxin using porcine alveolar macrophages.

Porcine alveolar macrophages were found to be highly susceptible to the cytolytic effects of a toxin (Shiga toxin [Stx]) produced by certain strains of Escherichia coli and sometimes associated with clinical disease in pigs and other animals. In comparison with the cells that are most commonly used for Stx detection and titration in vitro (namely, Vero cells), porcine alveolar macrophages appeared to be generally more sensitive and test results could be obtained in less time. Moreover, unlike Vero cells, porcine alveolar macrophages need not be continuously propagated to ensure immediate availability. They can simply be removed from a low-temperature repository, thawed, seeded, and shortly thereafter exposed to the sample in question. These characteristics suggest that porcine alveolar macrophages may be useful in developing a highly sensitive and timely diagnostic test for Stx.

Intervention with Shiga toxin (Stx) antibody after infection by Stx-producing Escherichia coli.

Shiga toxins (Stxs) produced by Escherichia coli (STEC) cause systemic vascular damage, manifested as hemolytic uremic syndrome in humans and as edema disease in pigs. Edema disease, a naturally occurring disease of pigs, was used to determine whether Stx antibodies, administered after infection and after the onset of Stx production, could prevent the systemic vascular damage and clinical disease caused by Stxs. A total of 119 STEC-infected pigs were treated with low, medium, or high doses of Stx antibody or with placebo. After inoculation with STEC, antibodies or placebo was injected intraperitoneally at 2 days postinoculation (DPI; low dose) or 4 DPI (medium and high doses). Edema disease was prevented with the low- and high-dose Stx antibody treatments administered at 2 and 4 DPI, respectively. High-dose antibody treatment also reduced the incidence and extent of vascular lesions. The degree of protection depended on the dose of antibody and the time of administration.

Persistent colonization of sheep by Escherichia coli O157:H7 and other E. coli pathotypes.

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10(7) or 10(10) CFU/strain/animal. The other strains were given only at 10(10) CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10(7) or 10(10) CFU. One of the ETEC strains also persisted when inoculated at 10(10) CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10(7) CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.

Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins.

Escherichia coli O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans. These infections are often foodborne, and healthy carrier cattle are a major reservoir of E. coli O157:H7. We were interested in knowing why cattle are tolerant to infection with E. coli O157:H7. Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb(3)), for receptivity to Stx binding in vitro, and for susceptibility to the enterotoxic effects of Stx in vivo. TLC was used to detect Gb(3) in tissues from a newborn calf. Gb(3) was detected by TLC in kidney and brain, but not in the gastrointestinal tract. Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues. Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (n = 3) and adult cattle (n = 3). Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined. The lack of Gb(3) and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine. We found that neither viable E. coli O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves. The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for E. coli O157:H7.

Shiga toxin-producing Escherichia coli infection: temporal and quantitative relationships among colonization, toxin production, and systemic disease.

Edema disease, a naturally occurring disease of swine caused by Shiga toxin-producing Escherichia coli (STEC), was used as a model for the sequence of events that occur in the pathogenesis of STEC infection. The mean time from production of levels of Shiga toxin 2e (Stx2e) detectable in the feces (day 1) to the onset of clinical disease (neurologic disturbances or death) was 5 days (range, 3-9). Bacterial colonization and titers of Stx2e in the ileum peaked at 4 days after inoculation in pigs without signs of clinical disease and at 6 days after inoculation in clinically affected pigs. Animals with the greatest risk of progressing to clinical disease tended to have the highest fecal toxin titers (>/=1:4096). Stx2e was detected in the red cell fraction from blood of some pigs showing clinical signs of edema disease but was not detected in the serum or cerebrospinal fluid.

Edema disease as a model for systemic disease induced by Shiga toxin-producing E. coli.

Edema disease (ED) is a naturally occurring disease of weaned pigs caused by host adapted strains of E. coli that produce Shiga toxin (STEC). We determined the temporal and quantitative relationships between intestinal colonization by STEC, levels of Shiga toxin (Stx2e) in the gut, in the blood, and clinical manifestations of ED. Bacterial colonization (10(8) CFU/cm ileum) was highest 4 days post inoculation (pi) in animals that did not develop clinical disease and 6 days pi in animals with clinical signs of ED. The mean time for the development of clinical signs of ED was 6 days pi (range 4-10). Average peak titers of Stx2e in the ileum were 1:16,384 in asymptomatic animals and 1:32,768 in clinical animals. Titers of Stx2e in the feces reflected the toxin titers in the ileum but were lower. Intestinal titers of Stx2e and the density of bacterial colonization were predictive of clinical ED for a group of animals but not for individuals. Approximately 50% of the pigs that had Stx2e titers of > or = 1:4096 and a bacterial density of > or = 10(6) CFU/cm in their ileum, had clinical ED. Pigs that had intestinal Stx2e titers < 1:4096 were asymptomatic. Stx2e was detected in the red cell fraction of blood from some of the pigs with clinical ED and in some that were asymptomatic. Stx2e was not detected in the serum of any animals. ED may be a useful model for predicting the temporal and quantitative relationships between bacterial colonization, Stx levels in the gut and blood and systemic disease for STEC in other species.

Assimilation of oxalate, acetate, and CO2 by Oxalobacter formigenes.

Oxalobacter formigenes is the only well-documented oxalate-degrading bacterium isolated from the gastrointestinal tract of animals. The production of ATP by Oxalobacter formigenes is centered around oxalate metabolism and oxalate is required for growth. A small amount of acetate (0.5 mM) is also required. Oxalate is decarboxylated to formate plus CO2 in nearly equimolar amounts. Experiments were conducted to determine which potential carbon sources (oxalate, acetate, formate, CO2) were assimilated by Oxalobacter formigenes and which metabolic pathways were operative in carbon assimilation. Measurements of the specific activities of total cell carbon after growth with different 14C-labeled precursors indicated that at least 54% of the total cell carbon was derived from oxalate and at least 7% was derived from acetate. Carbonate was also assimilated, but formate was not a significant source of cell carbon. Labeling patterns in amino acids from cells grown in [14C]oxalate or 14CO3 were different; however, in both cases 14C was widely distributed into most cellular amino acids. Carbon from [14C]acetate was less widely distributed and detected mainly in those amino acids known to be derived from alpha-ketoglutarate, oxaloacetate, and pyruvate. Cell-free extracts contained citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities. The labeling observed in amino acids derived from acetate is in agreement with the function of these enzymes in biosynthesis and indicates that the majority of acetate carbon entered into amino acid biosynthesis via well-known pathways.

Anabolic Incorporation of Oxalate by Oxalobacter formigenes.

Cell-free lysates of the strict anaerobe Oxalobacter formigenes contained the following enzymatic activities: oxalyl coenzyme A reductase, glyoxylate carboligase, tartronic semialdehyde reductase, and glycerate kinase. NAD(P)-linked formate dehydrogenase, serine-glyoxylate aminotransferase, and NAD(P) transhydrogenase activities were not detected. These results support the hypothesis that O. formigenes assimilates carbon from oxalate by using the glycerate pathway, whereby oxalate is reduced to 3-phosphoglycerate before entering common biosynthetic pathways.

Lachnospira pectinoschiza sp. nov., an anaerobic pectinophile from the pig intestine.

Pectinophiles are bacteria that utilize pectin and only a few related compounds as substrates. Obligately anaerobic pectinophiles have been isolated from the intestinal tracts and gingivae of humans and from the rumina of cattle. We isolated three strains of pectinophilic bacteria from colonic contents of pigs but were unable to isolate pectinophiles from the rumen contents of four sheep, even when the animals were fed a high-pectin diet. The pectinophiles isolated from pigs were strictly anaerobic, motile, gram-positive rods (0.36 to 0.56 by 2.4 to 3.1 microns). Pectin, polygalacturonic acid, and gluconate were the only substrates that supported rapid growth. All three strains grew slowly on either lactose or cellobiose and fermented fructose after a lag of several days. Pectin was degraded by means of an extracellular pectin methylesterase and a Ca(2+)-dependent exopectate lyase. A comparison of the 16S rRNA sequences of these isolates with the 16S rRNA sequences of other gram-positive bacteria revealed a specific relationship with Lachnospira multipara (level of similarity, 94%). The Gram reaction, formation of spore-like structures, and the utilization of lactose and cellobiose differentiated the pig isolates from previously described pectinophiles. The pig isolates represent a previously undescribed species of the genus Lachnospira, for which we propose the name Lachnospira pectinoschiza.

The antimicrobial susceptibility patterns of the Bacteroides fragilis group in the United States, 1987.

A nationwide survey to monitor the susceptibility of the Bacteroides fragilis group, which began in 1981, was continued during 1987. In addition to the eleven drugs evaluated in 1986, sulbactam, a potent beta-lactamase inhibitor, was tested alone and in combination with ampicillin and cefoperazone. Imipenem, ampicillin/sulbactam, cefoperazone/sulbactam, and ticarcillin/clavulanic acid were the most active newer drugs tested, with less than 1% resistance rates. Chloramphenicol, metronidazole and clindamycin also had excellent activity with resistance rates of 0%, 0%, and 3% respectively. Resistance rates to cefoxitin remained stable at 8%. Ceftizoxime and cefotetan had resistance rates of 26% and 29%, respectively. Rates of resistance varied among different institutions and between the various species.

Effect of bismuth subsalicylate on fecal microflora.

Several studies have documented the efficacy of bismuth subsalicylate (BSS) for the prophylaxis and treatment of bacterial diarrhea. It is not known what effect, if any, BSS has on the normal bowel flora. We quantitated the fecal flora in healthy volunteers before and after they took BSS. In the first group, 8 ounces of BSS was given on two successive days. In the second group, a standard oral intestinal lavage preparation (GoLYTELY) was used to clean the colon and then 8 ounces of BSS was given during a 4-hour period. There were no changes in total microbial counts or in counts of individual groups such as enteric bacteria, Pseudomonas, Staphylococcus, Bacteroides, or Clostridium difficile. On day 2 the yeast counts rose 2.4 log10 colony-forming units/g in the group taking 16 ounces of BSS but did not increase in the group taking 8 ounces of BSS plus GoLYTELY. The counts returned to pretreatment levels by day 7. These results show that a 1- or 2-day course of BSS, even with an oral intestinal lavage preparation, did not have significant effects on the normal microbial populations in the fecal microflora.

In vitro antibacterial activity of bismuth subsalicylate.

This study was undertaken to determine the in vitro activity of bismuth subsalicylate (BSS) and sodium salicylate (SS) against various groups of pathogenic bacteria. BSS had the greatest activity against Clostridium difficile, which had a minimal inhibitory concentration for 90% of the strains (MIC90) of 128 micrograms/mL. The Bacteroides fragilis group also had a relatively low MIC90 of 512 micrograms/mL. BSS had the least activity against Pseudomonas (MIC90, 6,144 micrograms/mL). SS was as active as BSS against aerobic bacteria but was less active against anaerobic bacteria. The MIC90 values of SS for C. difficile and the B. fragilis group were greater than 8,192 and 4,096 micrograms/mL, respectively. This study demonstrates that BSS has antibacterial activity in vitro at levels that should be achievable in the gastrointestinal tract.

Suppression of colonic microflora by cefoperazone and evaluation of the drug as potential prophylaxis in bowel surgery.

We evaluated the activity of cefoperazone (CPZ) on the intestinal flora in terms of its use as a single prophylactic drug in colon surgery. Twenty-four healthy male volunteers between the ages of 20 and 40 were assigned to receive either CPZ, oral neomycin-erythromycin, or no antibiotics. A mechanical bowel preparation, Golytely, was also given to each of the subjects. With intravenous CPZ, antibiotic levels in the stool ranged from less than 2 to 649 micrograms/ml and the total fecal bacterial counts dropped 3 to 4 log10 CFU/g. Higher levels of CPZ were detected in the stools when an oral dose was added, 1,446 to 5,445 micrograms/ml, and the bacterial counts were reduced maximally 4 to 6 log10 CFU/g. The combination of the oral and intravenous doses produced suppression of the microflora and high levels in blood, all with a single antibiotic.

Campylobacter.

Campylobacter is now known to be a major cause of gastrointestinal illness throughout the world. There are seven species known to be associated with enteritis, and it is likely that more will be described. Campylobacter jejuni is responsible for more than 95 per cent of the cases of diarrhea due to Campylobacter. Contaminated food products are the major source of infection. The clinical illness usually involves diarrhea, abdominal pain, and other constitutional symptoms. An accurate diagnosis of Campylobacter enteritis is made when the organism is cultured from the patient's stool. Selective media are widely available, and the isolation of C. jejuni is not difficult. Although erythromycin and the quinolones have good in vitro activity against C. jejuni, antibiotic therapy generally is not indicated.

Synergistic activity of cefotaxime and desacetylcefotaxime against the Bacteroides fragilis group.

Sensitivities to cefotaxime (CTX) and its metabolite, desacetylcefotaxime (DCTX), alone and in combination, were determined for 77 organisms of the B. fragilis group. Synergy was observed in 23 (30%) of the strains, of which 21 were those that had a MIC to CTX alone of less than or equal to 16 micrograms/ml. In the remaining two strains, which were resistant to CTX alone, one strain was susceptible to the combination, while the second strain was not. In one strain of B. fragilis, the drug combination increased the MIC of CTX, but both the MIC and MBC remained in the susceptible range.

Activity of cefmetazole against anaerobic bacteria.

The in vitro activity of cefmetazole versus that of other antimicrobial drugs was assessed against 374 clinical isolates of Bacteroides spp., Clostridium spp., and anaerobic gram-positive cocci. Compared with cefoxitin, cefmetazole showed good activity against Bacteroides fragilis, other Bacteroides species, and anaerobic cocci. It was somewhat less active than cefoxitin against Bacteroides thetaiotaomicron, B. ovatus, B. distasonis, and B. vulgatus and somewhat more active against Clostridium spp.