Cecal Mucus Research Articles

Isolation and characterization of an attenuated strain of Pseudomonas aeruginosa AC869, a 3,5-dichlorobenzoate degrader.

Pseudomonas aeruginosa AC869, a 3,5-dichlorobenzoate degrader, is a mouse pathogen and has a reported 50% lethal dose (LD50) of 1.05 x 10(7) CFU when given intranasally to C3H/HeJ mice (S.E. George, M.J. Kohan, M.I. Gilmour, M.S. Taylor, H.G. Brooks, J.P. Creason, and L.D. Claxton, Appl. Environ, Microbiol. 59:3585-3591, 1993). AC869 was serotyped as O6 when grown in CD-1 mouse cecal and lung mucus but could not be assigned an O serotype when grown in Luria broth (LB). After growth in mouse cecal mucus, a less virulent mutant, AC869-11, was isolated from AC869 by using bacteriophage E79, which adsorbs to the O side chain of lipopolysaccharide (LPS). AC869-11 produced significantly less O antigen on its LPS than AC869 when grown in mouse lung and cecal mucus. The mutant also produced half the amount of exoenzyme S and 16-fold less extracellular protease than AC869 and was more sensitive than its parent to a number of antibiotics when grown either in LB or in mouse lung mucus. AC869-11 had ninefold higher LD50 than AC869 in CD-1 mice when administered intranasally. AC869-11 was found in the lungs, small intestine, cecum, and large intestine in numbers at least 100-fold below AC869, 3 h after intranasal exposure of mice to a sublethal dose of the two strains. Moreover, AC869-11 induced a decreased pulmonary inflammatory response relative to AC869. In contrast to AC869, AC869-11 did not translocate to the mesenteric lymph nodes, liver, and spleen following a sublethal dose. Despite attenuation, AC869-11 grew as well as AC869 with 3,5-dichlorobenzoate as the sole carbon and energy source. However, although AC869-11 survived in 3,5-dichlorobenzoate-contaminated soil as well as AC869 for 1 week, it failed to survive as well thereafter. These results suggest the possibility that mutations that lead to pulmonary attenuation of P. aeruginosa in mice also lead to weakness in the environment, despite such mutants maintaining the ability to degrade toxic substances under laboratory conditions.

Role of lipopolysaccharide in colonization of the mouse intestine by Salmonella typhimurium studied by in situ hybridization.

An avirulent, streptomycin-resistant Salmonella typhimurium strain, SL5319, and its lipopolysaccharide (LPS)-deficient mutant strain, SL5325, differ in their ability to colonize the large intestines of streptomycin-treated mice. When fed to mice independently, the strains colonize equally well, but when fed together, the LPS-deficient mutant is outcompeted by the wild-type strain during establishment in the gut (J.J. Nevola, B.A.D. Stocker, D.C. Laux, and P.S. Cohen, Infect. Immun. 50:152-159, 1985). In the present study, the spatial distribution in the intestinal mucosal layer of the two strains was visualized by specific hybridization to bacterial rRNA in histological sections of mouse colon and cecum. The first day after infection, 9.8% of the smooth SL5319 cells observed in mucus were found to be associated with the mouse epithelial cells, but three days after infection, the corresponding fraction of adhering bacteria was reduced to 2.1%. The LPS-deficient S. typhimurium strain was confined to the part of the mucosal layer closest to the colonic lumen and was not observed to adhere to the epithelium either at day 1 or 3 after infection. Quantitative determinations of the distance from the S. typhimurium cells to the epithelial wall confirmed that the average distance for the rough S. typhimurium SL5325 was much larger than for its smooth counterpart, S. typhimurium SL5319. Quantification of the hybridization signal from bacteria isolated from the cecal mucus revealed that the two strains had the same ribosome concentration, indicating that they have the same potential for growth in the intestinal environment. On the basis of these observations, we suggest that the better colonization ability of the strain carrying wild-type LPS is due to the better abilities to penetrate the intestinal mucosal layer and to subsequently bind to the epithelial cells in vivo.

Escherichia coli F-18 and E. coli K-12 eda mutants do not colonize the streptomycin-treated mouse large intestine.

The Escherichia coli human fecal isolates F-18 and K-12 are excellent colonizers of the streptomycin-treated mouse intestine. E. coli F-18 and E. coli K-12 eda mutants (unable to utilize glucuronate, galacturonate, and gluconate) were constructed by insertional mutagenesis. Neither the E. coli F-18 eda nor the E. coli K-12 eda mutant was able to colonize the streptomycin-treated mouse intestine, whether they were fed to mice together with their respective parental strains or alone. Complementation of the eda mutants with pTC190 (containing a functional E. coli K-12 eda gene) completely restored the colonization ability of both eda mutants. Relative to their parental strains, the E. coli F-18 eda mutant and the E. coli K-12 eda mutant grew poorly in cecal mucus isolated from mice fed either normal mouse chow or a synthetic diet containing sucrose as the sole carbon source, yet the mutants and parental strains demonstrated identical growth rates in minimal medium with glucose as the carbon source. E. coli F-18 edd eda and E. coli K-12 edd eda double mutants colonized the streptomycin-treated intestine when fed to mice alone; however, when fed simultaneously with their respective parental strains, they were poor colonizers. Since the edd gene is involved only in gluconate metabolism via the Entner-Doudoroff pathway, these results implicate the utilization of gluconate and the Entner-Doudoroff pathway as important elements in E. coli colonization of the streptomycin-treated mouse large intestine.

Physiological state of Escherichia coli BJ4 growing in the large intestines of streptomycin-treated mice

Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static.

5] Adhesin-dependent isolation and characterization of bacteria from their natural environment

Understanding the physiology of bacteria grown in vivo in various hosts has been a difficult task. By using the adhesive properties of the microorganism, it is possible to isolate certain bacteria from their natural environment for further investigations. Pure cultures so obtained do not require growth on an artificial laboratory medium. The chapter describes the method for adhesin-dependent isolation and characterization of bacteria from their natural environment. It is performed by using a streptomycin-treated mouse as a host for a human Escherichia coli strain, producing type 1 fimbriae in vivo . Type 1 fimbriae bind specifically to D-mannose moieties. In this context, experiments were performed in which sepharose beads coupled with D-mannose were used for isolating bacterial cells from a fecal suspension, and/or from cecal mucus and cecal contents. Then, the isolated intact bacterial cells can be characterized by techniques such as electron microscopy, flow cytometry, and in situ hybridization. By electron microscopy, cell shape and surface components can be observed. A flow cytometer can be used for determining rapidly and with high precision the cell size of individual bacterial cells and the cell-size distribution in the isolated population. DNA content can be measured and the number of genomes per cell calculated. By in situ hybridization with labeled ribosomal RNA probes, the rRNA can be measured, which will reflect the metabolic activity of the bacterial cell in the host.

Characterization of Salmonella california and S. typhimurium Strains with Reduced Ability to Colonize the Intestinal Tract of Broiler Chicks

This study determined the ability of eight strains of Salmonella and their agarsubcultured variants to colonize the intestinal tract of broiler chicks. Nalidixic-acid (NAL)-resistant and streptomycin-resistant subcultured strains (S. california 1989/A and S. typhimurium 3366/A) that persisted in the ceca of chicks in lower numbers than their NAL-resistant parent strains (1989/O and 3366/O) were selected for additional study S. typhimurium strain 3366/A was present in the ceca of chicks in lower numbers than the parent strain 3366/O when given concomitantly with the parent strain or when the two strains were given separately to different chicks. S. california 1989/A strain was present in the ceca in lower numbers than the parent strain after concomitant oral or intracloacal inoculation. Strains 3366/O and 3366/A of S. typhimurium differed in growth rates in BHI broth and cecal mucus. The lipopolysaccharide (LPS) profile indicated that LPS components present in S. california 1989/O were missing from strain 1989/A. A mutant of 1989/O--2095/R--was also LPS- and colonization-deficient.

Utilization of the mouse large intestine to select an Escherichia coli F-18 DNA sequence that enhances colonizing ability and stimulates synthesis of type 1 fimbriae.

Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18 Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice alone but is eliminated when fed together with E. coli F-18. Random sequences of E. coli F-18 DNA were cloned into pRLB2, a par-B-stabilized derivative of pHC79. The entire gene library was transformed into E. coli F-18 Col- and fed to streptomycin-treated mice. The mouse large intestine selected a predominant clone which contained a recombinant plasmid (pRLB7) that enhanced E. coli F-18 Col- colonizing ability 100-fold but did not stimulate colicin synthesis. Moreover, pRLB7 simultaneously improved the survival of E. coli F-18 Col- in stationary phase in vitro, utilizing nutrients derived from mouse cecal mucus, and stimulated synthesis of both type 1 fimbriae and three E. coli F-18 Col- outer membrane proteins (74, 71, and 69 kDa). The 6.5-kb E. coli F-18 DNA sequence in pRLB7 does not contain either the fim operon or pilG (hns), both known to be involved in type 1 fimbrial synthesis. The sequence encodes six proteins, all smaller than the three E. coli F-18 Col- outer membrane proteins whose synthesis it stimulates. Collectively, the results suggest that the cloned E. coli F-18 DNA sequence contains one or more regulators of E. coli F-18 Col- operons expressed in the mouse large intestine in vivo and in isolated mouse cecal mucus in vitro.

Escherichia coli F-18 phase locked 'on' for expression of type 1 fimbriae is a poor colonizer of the streptomycin-treated mouse large intestine

Escherichia coli F-18, a human fecal isolate, makes type 1 fimbriae in vitro and in the streptomycin-treated mouse large intestine in vivo, and is an excellent colonizer of the cecal mucus layer in the streptomycin-treated mouse large intestine. E. coli F-18(pPKL91) harbors an extra fimB gene on a parB stabilized pPBR322 plasmid and is therefore phase-locked 'on' such that all cells express type 1 fimbriae. E. coli F-18(pPR633) contains essentially the same plasmid minus the fim B gene and in L-broth about 30% of the cells express type 1 fimbriae. When fed alone to streptomycin-treated mice, E. coli F-18(pPKL91) colonized the large intestine at about 107 cfu/g of feces. However, when simultaneously fed with E. coli F-18(pPR633) at either high (1010 cfu), or low doses (104 cfu), E. coli F-18(pPKL91) was a poor colonizer dropping to a level of between 102 and 103 cfu/g of feces. When given enough time to establish the state of colonization (10 days), E. coli F-18(pPKL91 ) persisted in feces in high numbers despite subsequent challenge by E. coli F-18(pPR633). Moreover, although both E. coli F-18(pPR633) and E. coli F-18(pPKL91) grew equally well in cecal mucus in vitro, E. coli F-18(pPR633) traveled through a layer of cecal mucus in vitro much faster than E. coli F-18(pPKL91). Together, the data suggest that type 1 fimbriated cells are at a disadvantage in initiating the colonization state because they have difficulty entering the mucus layer of the intestine as rapidly as non-fimbriated cells. The data also point to the possible biological significance of type 1 fimbrial phase-variation in the mouse large intestine.

Phosphatidylserine found in intestinal mucus serves as a sole source of carbon and nitrogen for salmonellae and Escherichia coli.

Salmonella choleraesuis (a pig pathogen), Salmonella typhimurium (a virulent strain in mice), and three strains of Escherichia coli (including a human enterohemorrhagic strain, a human urinary tract isolate, and a human fecal isolate) grew as well in vitro utilizing the lipids derived from mouse cecal mucus as the sole source of carbon and nitrogen as they did in mouse crude cecal mucus. Further analysis of the total lipid extracts of mucus dialysates showed that the acidic lipid fraction supported growth nearly as well as the total lipid fraction. Interestingly, among the many purified acidic lipids from mucus which were tested and analyzed, including several phospholipids, only phosphatidylserine was found to support the growth of all of these enteric bacteria, including Salmonella milwaukee, a human pathogen. The possible role of growth on pure phosphatidylserine in the pathogenesis of salmonellae is discussed.

Expression of Escherichia coli F-18 type 1 fimbriae in the streptomycin-treated mouse large intestine.

Escherichia coli F-18, isolated from the feces of a healthy human, makes type 1 fimbriae and is an excellent colonizer of the streptomycin-treated mouse large intestine. Recently, it was shown that the inability to produce type 1 fimbriae had no effect on the ability of E. coli F-18 to colonize the streptomycin-treated mouse large intestine, suggesting the possibility that E. coli F-18 does not express type 1 fimbriae in vivo. However, we show here that E. coli F-18 does express type 1 fimbriae in mouse cecal mucus in vivo and, in fact, appears to express substantially more type 1 fimbriae in cecal mucus in vivo than in L broth in vitro.

Growth of Salmonella typhimurium SL5319 and Escherichia coli F-18 in mouse cecal mucus: role of peptides and iron

Escherichia coli F-18, a normal human fecal isolate, and Salmonella typhimurium SL5319, an avirulent strain, are known to colonize the streptomycin-treated CD-1 mouse large intestine by utilizing nutrients present in intestinal mucus for growth. Moreover, previous experiments suggested the possibility that E. coli F-18 and S. typhimurium SL5319 utilized different mucus nutrients. Therefore, mouse cecal mucus was fractionated into high and low molecular weight components, and each fraction was inoculated either simultaneously or separately with E. coli F-18 and S. typhimurium SL5319. A 50 kd fraction was found in which the growth of S. typhimurium SL5319 suppressed growth of E. coli F-18. Evidence is presented that in this fraction S. typhimurium SL5319 utilizes peptides, presumably generated by mucus proteases, as a source of amino acids for growth. Furthermore, it is shown that S. typhimurium SL5319 grows in this 50 kd fraction with a generation time of 27 min in the presence of at most 7 μg of carbohydrate per ml and 2.2 μg of peptide per ml, and that S. typhimurium SL5319 suppresses E. coli F-18 growth in this fraction by sequestering iron. The data are discussed with respect to the role of peptide utilization and iron sequestration in the ability of S. typhimurium SL5319 to colonize the mouse large intestine.

Growth of Salmonella typhimurium SL5319 and Escherichia coli F-18 in mouse cecal mucus: role of peptides and iron

Growth of Salmonella typhimurium SL5319 and Escherichia coli F-18 in mouse cecal mucus: role of peptides and iron

Growth ofSalmonella typhimuriumSL5319 andEscherichia coliF-18 in mouse cecal mucus: role of peptides and iron

Escherichia coli F-18, a normal human fecal isolate, and Salmonella typhimurium SL5319, an avirulent strain, are known to colonize the streptomycin-treated CD-1 mouse large intestine by utilizing nutrients present in intestinal mucus for growth. Moreover, previous experiments suggested the possibility that E. coli F-18 and S. typhimurium SL5319 utilized different mucus nutrients. Therefore, mouse cecal mucus was fractionated into high and low molecular weight components, and each fraction was inoculated either simultaneously or separately with E. coli F-18 and S. typhimurium SL5319. A 50 kd fraction was found in which the growth of S. typhimurium SL5319 suppressed growth of E. coli F-18. Evidence is presented that in this fraction S. typhimurium SL5319 utilizes peptides, presumably generated by mucus proteases, as a source of amino acids for growth. Furthermore, it is shown that S. typhimurium SL5319 grows in this 50 kd fraction with a generation time of 27 min in the presence of at most 7 μg of carbohydrate per ml and 2.2 μg of peptide per ml, and that S. typhimurium SL5319 suppresses E. coli F-18 growth in this fraction by sequestering iron. The data are discussed with respect to the role of peptide utilization and iron sequestration in the ability of S. typhimurium SL5319 to colonize the mouse large intestine.

Neither motility nor chemotaxis plays a role in the ability of Escherichia coli F-18 to colonize the streptomycin-treated mouse large intestine

Escherichia coli F-18, isolated from the feces of a healthy human in 1977, is an excellent colonizer of the streptomycin-treated mouse large intestine and displays normal motility and chemotaxis ability. A chemotaxis-defective derivative of E. coli F-18, E, coli F-18 CheA-, and a nonflagellated derivative, E. coli F-18 Fla-, were constructed. These strains were found to colonize the streptomycin-treated mouse large intestine as well as E. coli F-18 when mice were fed both E. coli F-18 and either the CheA- or Fla- derivative at high levels (10(10) CFU of each strain per mouse) or low levels (10(4) CFU of each strain per mouse). Furthermore, E. coli F-18 lost motility and chemotaxis ability when grown in colonic or cecal mucus in vitro despite retaining the ability to synthesize flagella. Thus, it appears that neither motility nor chemotaxis plays a role in the ability of E. coli F-18 to colonize because this strain becomes functionally nonmotile upon growth in the streptomycin-treated mouse large intestine.

Movement of Entamoeba histolytica trophozoites in rat cecum and colon intact mucus blankets and harvested mucus gels.

Entamoeba histolytica trophozoites were centrifuged into mucus gels harvested from in vivo loops of rat cecum and proximal colon. Frank ameba movement was not detected in the colonic mucus, but attenuated motility was measured in the cecal mucus. The harvested rat cecal mucus had significantly lower apparent viscosity and neutral glycoprotein concentration values than the colonic mucus. A shape factor method was developed to assess the motility of amebae in mucus gels and intact mucus blankets. Shape factor data obtained from harvested mucus gel and intact mucus blanket experiments indicated that such mucus severely attenuated trophozoite movement with the attenuation being greater with colonic than with cecal mucus. Entamoeba trophozoites are known to be able to generate a pseudopod force of 3.3 x 10(-6) Newtons. Latex microspheres of the size range of Entamoeba trophozoites were forced through cecal and colonic mucus gels under gravity. Colonic mucus gels could withstand a force of 3.3 x 10(-6) Newtons while cecal mucus could not, suggesting that the ameba movement that was observed in cecal mucus involved mechanical penetration of the mucus by the ameba pseudopodia and did not require prior gel dissolution by Entamoeba enzymes.

Roles of motility, chemotaxis, and penetration through and growth in intestinal mucus in the ability of an avirulent strain of Salmonella typhimurium to colonize the large intestine of streptomycin-treated mice.

Previously, it had been shown that an avirulent strain of Salmonella typhimurium, SL5316, with wild-type lipopolysaccharide (LPS) was a far better colonizer of the streptomycin-treated CD-1 mouse large intestine, was far more motile, did not bind to mouse intestinal mucus nearly as well as, but penetrated through a layer of intestinal mucus in vitro far better than an almost isogenic LPS-deficient transductant, SL5325. In the present investigation, a nonflagellated transductant, SL5681, and a nonchemotactic transductant, SL5784, were isolated from SL5316 and tested for relative colonizing ability versus SL5316 (smooth) and SL5325 (rough) in streptomycin-treated mice. In addition, the Salmonella strains were tested for their ability to grow together in cecal intestinal mucus and in cecal luminal contents, for their tumbling and swimming activities after growth in cecal mucus, and for their ability to adhere to and travel through cecal mucus in vitro. The data show that the nonflagellated and nonchemotactic derivatives colonized large intestine nearly as well as their parent and were far better colonizers than the LPS-deficient mutant, that all the strains grew equally well in cecal mucus but did not grow in cecal luminal contents, and that cecal mucus-grown strains lost tumbling and swimming activities. Furthermore, the LPS-deficient strain adhered to cecal mucus far better but penetrated mucus far worse than did the nonflagellated transductant, the nonchemotactic transductant, and the parent. Thus, motility and chemotaxis do not appear to play a major role in the ability of the avirulent S. typhimurium strains to colonize the mouse large intestine, colonization may require growth in cecal mucus but does not depend on growth in cecal luminal contents, growth in cecal mucus inhibits S. typhimurium motility, and increased adhesion of the LPS-deficient mutant to cecal mucus and its poor ability to penetrate cecal mucus may play a role in its poor intestine-colonizing ability.

Colonization of the streptomycin-treated mouse large intestine by a human fecal Escherichia coli strain: role of growth in mucus

The relative colonizing abilities of Escherichia coli F-18, isolated from the feces of a healthy human, and E. coli F-18col-, a strain derived from it which does not make the E. coli F-18 colicin, were studied. In a previous report, it was shown that when each strain was fed individually to streptomycin-treated mice, at approximately 10(10) CFU per mouse, each colonized the large intestine at between 10(7) and 10(8) CFU/g of feces indefinitely. However, when simultaneously fed to mice, although E. coli F-18 colonized at about 10(8) CFU/g of feces, E. coli F-18col- dropped to a level of 10(3) CFU/g of feces within 3 to 5 days. In the present investigation, we show that when given enough time to establish a state of colonization, E. coli F-18col- persists in feces in high numbers despite subsequent challenge by E. coli F-18. Therefore, a major defect in the ability of E. coli F-18col- to colonize in the presence of E. coli F-18 appears to be in initiating that state. In addition, when mucus was scraped from the cecal wall and, without further treatment, was inoculated with E. coli F-18 or F-18col-, both strains grew well. However, when cecal mucus was inoculated with both strains simultaneously, E. coli F-18 grew far more rapidly than E. coli F-18col-. Moreover, neither strain grew in cecal luminal contents. Together, these data suggest the possibility that both E. coli F-18 and F-18col- must grow in mucus to colonize the streptomycin-treated mouse large intestine, that E. coli F-18col- is eliminated by E. coli F-18 because it does not grow in mucus as well as E. coli F-18, and that E. coli F-18col- can resist elimination by E. coli F-18 if it is allowed enough time to establish itself within the mucus layer.

Colonization of the streptomycin-treated mouse large intestine by a human fecal Escherichia coli strain: role of adhesion to mucosal receptors.

Escherichia coli F-18, a normal fecal isolate, was previously shown to be an excellent colonizer of the streptomycin-treated CD-1 mouse large intestine, whereas E. coli F-18col-, a derivative of E. coli F-18 that no longer makes the E. coli F-18 colicin, was shown to be a poor mouse colonizer. It was also shown that E. coli F-18 bound two to three times more soluble colonic mucus protein than did E. coli F-18col- and that a major receptor in CD-1 mouse colonic mucus was a 50.5-kilodalton glycoprotein. In the present investigation, an additional E. coli F-18 colonic mucus glycoprotein receptor (66 kilodaltons) and three cecal mucus glycoprotein receptors (94, 73, and 66 kilodaltons) were identified. Numerous colonic and cecal brush border protein receptors specific for E. coli F-18 were also identified. Furthermore, E. coli F-18col- was found to bind to the same mucus and brush border receptors as E. coli F-18, although to a far lesser extent. Adhesion of both E. coli F-18 and F-18col- was inhibited by D-mannose and alpha-methyl-D-mannoside, and both strains were shown to bind specifically to the mannose moiety of a mannose-bovine serum albumin glycoconjugate, although again E. coli F-18col- bound to a lesser extent. Finally, both E. coli F-18 and F-18col- were shown to be piliated. The possible role of pilus mediated adhesion in E. coli F-18 colonization of the streptomycin-treated mouse large intestine is discussed.