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Twenty percent of bacteria isolated on simple, defined anaerobic media produced H2. Eighty-four percent of these were isolated on glucose. All were Enterobacteriaceae, primarily Citrobacter.
The isolation and identification of 2,220 Enterobacteriaceae from meats indicated that Escherichia coli biotype I, Enterobacter agglomerans, and Serratia liquefaciens were the principal types to be differentiated in meats. Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter hafniae were also commonly identified. Identification of isolates by the Encise II (Roche Diagnostics Inc., Nutley, N.J.) and Minitek (BBL Microbiology Systems, Cockeysville, Md.) coding systems gave similar results with only 255 (11.5%) discrepancies in identity, but both systems required large numbers of supplementary tests for identification of the isolates. Not only the distribution of Enterobacteriaceae types isolated from meats but also some of the biochemical reactions of the isolates differed from those of clinical isolates. The Minitek technique is recommended because of its versatility. However, with the addition of cellobiose and salicin disks and the inclusion of methyl red to the Minitek test and the use of the Voges-Proskauer test and gas production in EC medium at elevated temperature as standard tests, the identification of these Enterobacteriaceae from meats would be greatly facilitated. The inclusion of the motility test, for example, using nitrate motility agar, would also be of value to Enterobacteriaceae identification.
The species of total coliform bacteria isolated from drinking water and untreated surface water by the membrane filter (MF), the standard most-probable-number (S-MPN), and modified most-probable-number (M-MPN) techniques were compared. Each coliform detection technique selected for a different profile of coliform species from both types of water samples. The MF technique indicated that Citrobacter freundii was the most common coliform species in water samples. However, the fermentation tube techniques displayed selectivity towards the isolation of Escherichia coli and Klebsiella. The M-MPN technique selected for more C. freundii and Enterobacter spp. from untreated surface water samples and for more Enterobacter and Klebsiella spp. from drinking water samples than did the S-MPN technique. The lack of agreement between the number of coliforms detected in a water sample by the S-MPN, M-MPN, and MF techniques was a result of the selection for different coliform species by the various techniques.
Pure cultures of Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter freundii were injured ( greater than 90%) in water from a dead-end section of the Bozeman, Montana, distribution system. The effects of the following laboratory variables on the enumeration efficiency of injured and undamaged control cells were examined: (i) diluent composition, temperature, and time of exposure; (ii) media, using various formulations employed in enumerating gram-negative bacteria; and (iii) surface pore morphology of membrane filters. The addition of peptone or milk solids to diluents and low temperature (4 degrees C) maximized the recovery of injured cells, but had little effect on undamaged cells. Control cells were recovered with high efficiencies on most media tested, but recoveries of injured cells ranged from 0 to near 100%. Most of the media commonly used in water analysis recovered less than 30% of injured cells. This was explained in part by the sensitivity of injured bacteria to deoxycholate concentrations greater than 0.01%, whereas control cells were unaffected by 0.1%. Membrane filter surface pore morphology (at 35 degrees C) had a negligible effect on total coliform recoveries. Recommendations are made regarding procedures to improve the recovery of injured coliforms by routine laboratory practices.
Coliforms, primarily Citrobacter freundii, gave negative verification results in the total-coliform membrane filtration test. The organisms produced gas from lactose in brilliant green bile broth but not in lauryl tryptose broth. The discrepancy was related to the peptone sources used in the media.
A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.
An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.
mT7 medium performed no better than m-Endo medium in enumerating cells of Escherichia coli and Citrobacter freundii exposed to ozone. Also, there was no difference in the plate count of heterotrophic bacteria in ozonated raw water determined on modified Henrici agar or R2A agar. Statistically significant differences were seen between bacteria and the type of water in which they were suspended during ozonation.
Campylobacter enteritis in humans has been linked to consumption of chicken. Reducing Campylobacter jejuni colonization in chickens can potentially reduce Campylobacter infections in humans. In this study, the reduction of C. jejuni colonization in chicks by oral administration of defined competitive exclusion (CE) cultures, 2.5% dietary carbohydrates, or CE cultures and carbohydrates was examined. Prevention, elimination, or direct challenge of Campylobacter infection was simulated by administering treatments before, after, or at the same time as that of the Campylobacter inoculation. Additionally, the effect of maintaining high levels of protective bacteria was evaluated by administering CE cultures on days 1 and 4 (booster treatment). All treatments reduced C. jejuni colonization. Protection by aerobically grown CE cultures was not statistically different from that by anaerobically grown CE cultures. A combination of Citrobacter diversus 22, Klebsiella pneumoniae 23, and Escherichia coli 25 (CE 3) was the most effective CE treatment. Maintaining high numbers of CE isolates by administering CE boosters did not increase protection. The greatest reduction of Campylobacter colonization was observed in schemes to prevent or eliminate C. jejuni infection. C. jejuni was not detected in the ceca of birds receiving the prevention treatment, CE 3 with mannose, representing a 62% reduction in the colonization rate. The protection factor (PF), a value combining the colonization rate and the level of infection, for CE 3 with mannose was high (> 13.2). Fructo-oligosaccharides alone strongly prevented Campylobacter colonization. Only 8% of the chicks in this group were colonized, with a PF of > 14.3.(ABSTRACT TRUNCATED AT 250 WORDS)
Glycerol fermentation by Enterobacter agglomerans revealed that both growth and 1,3-propanediol production ceased after consumption of about 430 mM glycerol, irrespective of the initial glycerol content. This phenomenon was assigned to the production of 3-hydroxypropionaldehyde, which was identified by proton nuclear magnetic resonance and which showed a bacteriostatic effect. The accumulation during glycerol fermentation was also observed with two other enterobacterial species, i.e., Klebsiella pneumoniae and Citrobacter freundii.
The fate and impact of elemental mercury in closed bacterial cultures were examined. The quantity of elemental mercury oxidized by bacteria ranged from small amounts for Pseudomonas aeruginosa, P. fluorescens, Escherichia coli, and Citrobacter to essentially all of the added elemental mercury for Bacillus subtilis and B. megaterium. The percentage of the total mercury in the system associated with bacterial cells ranged from 18.6 to 43.2%. Growth of the two Pseudomonas species was inhibited by elemental mercury, whereas growth of the other cultures was not distinguishable from that in mercury-free controls. No methylmercury was formed by the six cultures within 48 h.
It is widely accepted that tight junctions are altered during infections by attaching and effacing (A/E) pathogens. These disruptions have been demonstrated both in vitro and more recently in vivo. For in vivo experiments, the murine model of A/E infection with Citrobacter rodentium is the animal model of choice. In addition to effects on tight junctions, these bacteria also colonize the colon at high levels, efface colonocyte microvilli, and cause hyperplasia and inflammation. Although we have recently demonstrated that tight junctions are disrupted by C. rodentium, the issue of direct effects of bacteria on epithelial cell junctions versus the indirect effects of inflammation still remains to be clarified. Here, we demonstrate that during the C. rodentium infections, inflammation plays no discernible role in the alteration of tight junctions. The distribution of the tight junction proteins, claudin-1, -3, and -5, are unaffected in inflamed colon, and junctions appear morphologically unaltered when viewed by electron microscopy. Additionally, tracer molecules are not capable of penetrating the inflamed colonic epithelium of infected mice that have cleared the bacteria. Finally, infected colonocytes from mice exposed to C. rodentium for 14 days, which have high levels of bacterial attachment to colonocytes as well as inflammation, have characteristic, altered claudin localization whereas cells adjacent to infected colonocytes retain their normal claudin distribution. We conclude that inflammation plays no discernible role in tight junction alteration during A/E pathogenesis and that tight junction disruption in vivo appears dependent only on the direct intimate attachment of the pathogenic bacteria to the cells.
Several species of bacteria from the family Enterobacteriaceae formed crystalline materials containing calcium when grown in a defined culture medium. Enterobacter aerogenes, Proteus vulgaris, Citrobacter freundii, and C. intermedius produced calcium pyrophosphate crystals. Edwardsiella tarda and Escherichia coli formed calcite III crystals, whereas Proteus mirabilis, Klebsiella pneumoniae, Providencia stuartii, and Serratia marcescens produced hydroxyapatite crystals. Several of these bacteria have been isolated from the kidneys of patients with kidney stones, indicating that microorganisms may be involved in the enucleation process of kidney stone formation.
Lipopolysaccharides from three gram-negative bacteria isolated from bale cotton and piggery air were analyzed for their chemical composition, and their pulmonary toxicity for guinea pigs, lethal toxicity for mice, and pyrogenicity for rabbits were measured. Lipopolysaccharides from Enterobacter agglomerans and Citrobacter freundii had closely related chemical compositions; both were pyrogenic for rabbits and caused a dose-dependent influx of polymorphonuclear leukocytes into the airways of guinea pigs. The lethal toxicities of these lipopolysaccharides in mice were comparable to that of Salmonella typhimurium lipopolysaccharide, which was used as a reference. Lipopolysaccharide from Agrobacterium sp. was chemically different from those of E. agglomerans and C. freundii, did not induce any influx of polymorphonuclear leukocytes, and was only weakly toxic or pyrogenic. The low biological activity of the agrobacterial lipopolysaccharide may be due to its different chemical composition.
The toxicity by inhalation of various gram-negative bacteria, isolated from settings associated with inhalation disease, was studied by a variety of means. These microorganisms were not equally toxic. Citrobacter freundii aerosol challenges of rabbits provoked significant (up to fivefold) increases in plasma haptoglobin 24 to 48 h after inhalation. Other strains tested failed to provoke such statistically consistent increases. Measurements of C-reactive protein in these same animals did not lead to as reliable results, due to the variability of the responses. Mice responded differently to inhalation in that haptoglobin responses were either unaffected or depressed. When a strain of mice was used that exhibits more severe inflammatory responses to endotoxin (C3H/HeJ), C. freundii and Escherichia coli aerosols provoked significant haptoglobin increases. Free lung cell analyses demonstrated that the macrophage and neutrophil responses differed depending on the strain of bacteria used. Again, C. freundii induced the greatest responses. When murine B lymphocytes were stimulated with lipopolysaccharide preparations from different gram-negative bacteria, distinctly different dose-response curves were obtained. The types of responses obtained indicate that (i) brief inhalation of bacterial aerosols previously thought to be innocuous may lead to pulmonary inflammation, and (ii) that these bacteria differ in their toxicity, with C. freundii being the most toxic organism of the five studied.
The Vi capsular polysaccharide (Vi) is both a virulence factor and a protective antigen of Salmonella typhi; its pathogenic role for Salmonella paratyphi C is less well understood. We found no differences between the antigenic and immunogenic properties and the structure of the Vi from representative strains of S. paratyphi C, S. typhi, and Citrobacter freundii. There were, however, differences in both the amount produced per cell and the degree of association with the cell among the Vi from the three species of Enterobacteriaceae. S. paratyphi C produced less Vi than both the wild-type S. typhi and C. freundii did, and it showed the fastest release of Vi into the media. These findings may provide an explanation for the inability of the Vi to inhibit completely the agglutination of S. paratyphi C by anti-O sera. In an outbreak of enteric fever caused by S. paratyphi C, 66 of 78 isolates (85%) were Vi positive.
Previously we showed that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated human peripheral blood and lamina propria mononuclear cells. The aims of the present study were to determine whether EPEC-inhibitory factors have similar effects on murine lymphoid populations in order to further delineate the mechanisms of alteration of cytokine production. Preexposure to EPEC lysates inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) production by murine spleen cells, but IL-10 production was increased. The inhibition was not due to increased apoptosis and was not blocked by neutralizating antibodies against IL-10 or transforming growth factor beta (TGF-beta). EPEC lysates also inhibited mitogen-stimulated IL-2 and IFN-gamma production by CD11b-depleted spleen cells, IL-2 and IL-4 production by intraepithelial and Peyer's patch lymphocytes, IL-2 production by the human T-cell line Jurkat, and antigen-stimulated IL-2 production by murine spleen cells. Lysates obtained from Shiga-like toxin-producing E. coli, E. coli RDEC-1, Citrobacter rodentium, and an EPEC espB insertion mutant all inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid cells. In conclusion, lysates of EPEC and related bacteria directly inhibit cytokine production by lymphoid cells from multiple sites by a mechanism that does not increase apoptosis or result from secondary effects of IL-10 or TGF-beta.
Enteropathogenic Escherichia coli (EPEC) is an extracellular bacterial pathogen that infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. EPEC belongs to the group of attaching and effacing (A/E) pathogens. It uses a type III secretion system to deliver proteins into the host cell that mediate signal transduction events in host cells. We used gene array technology to study epithelial cell responses to EPEC infection at the level of gene expression. We found that EPEC induces the expression of several genes in infected HeLa cells by a lipopolysaccharide (LPS)-independent mechanism, including cytokines and early growth response factor 1 (Egr-1). The transcription factor Egr-1 is an immediate-early-induced gene that is activated in most cell types in response to stress. EPEC-induced upregulation of egr-1 is mediated by the activation of the MEK/extracellular signal-regulated kinase signal transduction pathway and is dependent on the type III secretion system. egr-1 is also induced during infection of mice by the A/E pathogen Citrobacter rodentium, suggesting that both Egr-1 and the activation of this mitogen-activated protein kinase signal transduction pathway may play a role in disease.