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An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody to Campylobacter jejuni and Campylobacter coli. Heat-stable antigen, a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verified Campylobacter enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter species or C. jejuni serotype, with the important exception of response to C. jejuni O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (P = 0.006) and IgA (P = 0.06) compared with other C. jejuni and C. coli serotypes.

Pseudomonas aeruginosa PAO1 became considerably more sensitive to the action of ampicillin when grown in the presence of certain phospholipids. Only phospholipids capable of forming lipid bilayers or micelles proved to be capable of enhancing ampicillin activity. Of the phospholipids tested, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, also called monopalmitoylphosphatidic acid (MPPA), was the best enhancer. In the absence of MPPA, the MIC and MBC of ampicillin for P. aeruginosa PAO1 were 1 and 2 g/L, respectively. In the presence of MPPA, the MIC and MBC were 20 and 40 mg/L, respectively. MPPA was shown to enhance ampicillin activity by binding both Ca(2+) and Mg(2+), suggesting that the mechanism of enhancement is similar to that previously reported for Ca(2+) and Mg(2+) chelators. Surprisingly, MPPA by itself slowed the growth of four mucoid multiply antibiotic-resistant strains of P. aeruginosa recently isolated from the sputum of cystic fibrosis patients, and enhanced their sensitivity to piperacillin. It also increased the sensitivity of two ceftazidime-resistant P. aeruginosa cystic fibrosis strains to ceftazidime.

A minitransposon mutant of Salmonella enterica serovar Typhimurium SR-11, SR-11 Fad(-), is unable to utilize gluconeogenic substrates as carbon sources and is avirulent and immunogenic when administered perorally to BALB/c mice (M. J. Utley et al., FEMS Microbiol. Lett., 163:129-134, 1998). Here, evidence is presented that the mutation in SR-11 Fad(-) that renders the strain avirulent is in the cra gene, which encodes the Cra protein, a regulator of central carbon metabolism.

The ability of Escherichia coli to survive stress during growth in different environments is, in large part, dependent on rpoS and the genes that comprise the rpoS regulon. E. coli BJ4 and an isogenic BJ4 rpoS mutant were used to examine the influence of the rpoS gene on E. coli colonization of the streptomycin-treated mouse large intestine. Colonization experiments in which the wild-type E. coli BJ4 and its rpoS mutant were fed individually as well as simultaneously to mice suggested that E. coli BJ4 does not face prolonged periods of nutrient starvation in the mouse large intestine and that the rpoS regulon is not expressed during long-term colonization after adaptation of the bacteria to the gut environment.

The role of the Klebsiella pneumoniae capsular polysaccharide (K antigen) during colonization of the mouse large intestine was assessed with wild-type K. pneumoniae LM21 and its isogenic capsule-defective mutant. When bacterial strains were fed alone to mice, the capsulated bacteria persisted in the intestinal tract at levels of 10(8) CFU/g of feces while the capsule-defective strain colonized at low levels, 10(4) CFU/g of feces. In mixed-infection experiments, the mutant was rapidly outcompeted by the wild type. In situ hybridization on colonic sections revealed that bacterial cells of both strains were evenly distributed in the mucus layer at day 1 after infection, while at day 20 the wild type remained dispersed and the capsule-defective strain was seen in clusters in the mucus layer. These results suggest that capsular polysaccharide plays an important role in the gut colonization ability of K. pneumoniae.

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, 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.

Type 1 fimbriae of Escherichia coli are surface organelles which mediate binding to D-mannose-containing structures. By direct binding of FimH to D-mannose attached to a carrier protein, we demonstrated that this protein was uniquely responsible for the receptor specificity. Furthermore, we show by receptor immunoelectron microscopy that the FimH protein is located laterally in the structure of the type 1 fimbriae.

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 BJ4, a rat isolate, was used to examine the growth and differentiation of the microorganism in its natural habitat, the intestine. Growth of E. coli BJ4 in the large intestine of streptomycin-treated mice was compared with its growth in laboratory media. By a number of methods, it was shown that E. coli BJ4 differentiates, during growth in the intestine, into two distinct populations, one that has the characteristics of the laboratory-grown strain and one that appears as a coccoid cell. Furthermore, it was shown that there is a natural selection for the coccoid-type cell in the intestine, while in laboratory media growth of rod-shaped E. coli BJ4 is enhanced.

The ability of Escherichia coli K-12(K88ab) to adhere to immobilized porcine small intestine mucus was examined. E. coli K-12(K88ab) but not the isogenic E. coli K-12 strain was found to adhere readily to immobilized crude mucus but not to bovine serum albumin. The adhesion of E. coli K-12(K88ab) was inhibited in a specific fashion by anti-K88 antiserum. Adhesion was also inhibited by pretreatment of receptor-containing crude mucus preparations with sodium metaperiodate or proteolytic enzymes. Removal of glycolipids from crude mucus by chloroform-methanol extraction did not affect the ability of E. coli K-12(K88ab) to bind to mucus preparations. Adsorption of crude mucus preparations with K88ab fimbriae but not type 1 fimbriae resulted in the removal of K88-specific receptors. Analysis of the pelleted fimbriae-receptor complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, together with gel filtration chromatography of crude mucus preparations, suggest that the K88-specific receptor present in porcine small intestine mucus is a 40- to 42-kDa glycoprotein.

Fluorescent oligonucleotide probes targeting rRNA were used to develop an in situ hybridization technique by which the spatial distribution of Escherichia coli in the large intestines of streptomycin-treated mice was determined. Single E. coli cells were identified in thin frozen sections from the large intestines by the use of a probe specific for E. coli 23S rRNA. Furthermore, the total bacterial population was visualized with an rRNA probe targeting the domain Bacteria. By this technique, all E. coli cells were seen embedded in the mucosal material overlying the epithelial cells of the large intestine, and no direct attachment to the epithelium was observed.

The growth physiology of Escherichia coli during colonization of the intestinal tract was studied with four animal models: the streptomycin-treated mouse carrying a reduced microflora, the monoassociated mouse with no other microflora than the introduced strain, the conventionalized streptomycin-treated mouse, and the conventionalized monoassociated mouse harboring a full microflora. A 23S rRNA fluorescent oligonucleotide probe was used for hybridization to whole E. coli cells fixed directly after being taken from the animals, and the respective growth rates of E. coli BJ4 in the four animal models were estimated by correlating the cellular concentrations of ribosomes with the growth rate of the strain. The growth rates thus estimated from the ribosomal content of E. coli BJ4 in vivo did not differ in the streptomycin-treated and the monoassociated mice. After conventionalization there was a slight decrease of the bacterial growth rates in both animal models.

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.

<it>Objectives</it>: It has been suggested recently that intracellular bacteria surviving antibiotic treatment might serve as a reservoir for recurrent infection. The purpose of this study was to directly examine the location of <it>Escherichia coli</it> bacteria in the mouse bladder after treatment with mecillinam. <it>Methods</it>: The bladders were studied by use of colony counts, <it>in situ</it> hybridization and electron microscopy. <it>Results</it>: The bacterial counts in the bladder remained ∼103–4 cfu/bladder even after mecillinam treatment had finished, and re-growth in the urine was observed. In the bladder epithelium from treated mice, bacteria cells were occasionally seen, presumably representing intracellularly located bacteria. <it>Conclusions</it>: This is the first <it>in vivo</it> study indicating that during mecillinam treatment <it>E. coli</it> cells can penetrate the mouse bladder epithelium and persist.