Peyer's patch lymphoid follicle epithelial adherence of a rabbit enteropathogenic Escherichia coli (strain RDEC-1). Role of plasmid-mediated pili in initial adherence.

Escherichia coli (strain RDEC-1) adheres to M cells of rabbit Peyer's patch lymphoid follicle epithelium. The RDEC-1 strain contains an 85 X 10(6) D plasmid that codes for pili, which, when purified, adhere to gut absorptive epithelium. This study compared the in vivo lymphoid follicle adherence of the RDEC-1 strain with that of a Shigella flexneri (ShD15) that contained the 85 X 10(6) D plasmid and expressed the RDEC-1 pili, a control E. coli, and a control S. flexneri (ShD12). The bacteria were given in a dose of 10(10) to 0.7-1.1 kg rabbits. The rabbits were sacrificed at 2, 4, 6, and 12 h postinoculation. Peyer's patch tissue was examined by electronmicroscopy and direct fluorescence microscopy. The piliated ShD15 and RDEC-1 bacteria adhered in large numbers at 2 and 4 h postinoculation, but only the RDEC-1 strain persisted and increased in numbers past that time. Control strains did not adhere. The ShD15 strain adhered to and was rapidly taken into M cells, precipitating an acute inflammatory reaction within the follicle and adjacent lumen. Initial lymphoid follicle M cell adherence of the ShD15 strain was associated with the possession of the adherence pilus plasmid. The failure of the ShD15 strain to survive and colonize the lymph follicle epithelium contrasts with the success of the RDEC-1 strain and indicates that the RDEC-1 strain possesses virulence factors in addition to pili.

Antibacterial mechanisms of the lower respiratory tract. I. Immunoglobulin synthesis and secretion.

Immunoglobulin synthesis and secretion have been studied in the rabbit lower respiratory tract, both in the normal state and after infection with Diplococcus pneumoniae or Listeria monocytogenes. In vitro synthesis of immunoglobulin and specific antibody was assessed by incorporation of 14C-labeled amino acids into protein. Lower respiratory tract secretions and serum were analyzed for immunoglobulin and antibody against the infecting organism. Normal respiratory tract produced small quantities of immunoglobulin, most of which was IgG. After bacterial infection of the lower respiratory tract, there was a marked increase in local synthesis of immunoglobulin, especially IgG. Specific antibody of IgG class was produced in all lungs infected with listeria by the 11th day, and in lungs infected with pneumococcus by the 8th day. Secretions from all normal and infected lower respiratory tracts contained IgA and IgG. The IgA to IgG ratios in secretions of normal animals, and animals infected with listeria or pneumococcus, were 2.3, 2.5, and 2.6, respectively. Sera of animals infected with L. monocytogenes contained specific antibody of IgG class but lacked IgA antibody, whereas secretions had both IgA and IgG class antibody against listeria. Similarly, sera of animals infected with D. pneumoniae had IgG class antibody but no IgA antibody, whereas only IgA antibody was found in secretions. The evidence that locally synthesized immunoglobulin (especially IgA), including specific antibody, is secreted into the lower respiratory tract lumen is discussed. Further definition of the role of "local" antibacterial antibody in the respiratory tract is of considerable importance.

Specific adherence of Escherichia coli (strain RDEC-1) to membranous (M) cells of the Peyer's patch in Escherichia coli diarrhea in the rabbit.

The RDEC-1 strain Escherichia coli is an enteroadherent bacterium that produces diarrhea in the rabbit. A histopathologically similar disease has been described in humans. The RDEC-1 bacterium adheres to the epithelium of lymphoid follicles in rabbit ileal Peyer's patches by 4 h postinoculation, 3-4 d before its adherence to absorptive epithelium. The purpose of this study was to determine whether the RDEC-1 bacterium adheres to a specific cell type in the lymphoid follicle epithelium. RDEC-1 bacteria were given in a dose of 2 X 10(6) by the orogastric route to postweanling rabbits. The distal ileal Peyer's patch, taken from 5 control rabbits and 43 rabbits at intervals in the first 24 h postinoculation, was examined by routine and high-voltage electron microscopy. The RDEC-1 bacterium adhered specifically to M (membranous) rather than absorptive epithelial cells of the lymphoid follicle epithelium. Further understanding of how the bacterium attaches to M cells, which transport antigens to intraepithelial lymphocytes, could be useful in designing vaccines to protect mucosal surfaces.

Cell-mediated immunity after bacterial infection of the lower respiratory tract.

Lower respiratory tract and systemic cell-mediated immunity have been studied in rabbits after infection with Listeria monocytogenes or Diplococcus pneumoniae. Respiratory tract cell-mediated immunity was evaluated by direct and indirect assays of migration inhibitory factor (MIF) production. Systemic delayed hypersensitivity was determined by means of intradermal testing with appropriate antigens. Aerosol exposure to listeria was followed by markedly increased numbers of free lower respiratory tract cells. These cells manifested antigen-stimulated inhibition of migration (mean inhibition of migration = 30.4%). Pneumococcal pneumonia was associated with similar but less dramatic changes. Intravenous administration of organisms was uncommonly followed by inhibition of lower respiratory tract cells in direct migration assays. Fractionated MIF, as well as crude supernates of antigen-stimulated lower respiratory tract and lymph node lymphocytes from animals exposed to listeria aerosols, caused inhibition of normal alveolar macrophage migration. MIF, produced by lymph node lymphocytes, has a molecular weight of approximately 65,000 and is inactivated by chymotrypsin or neuraminidase. Delayed dermal hypersensitivity to listeria antigen was observed in 54 of 55 animals exposed to listeria aerosols and in all 9 animals infected by the intravenous route. Delayed dermal reactions to pneumococcal sonicate antigen (but not capsular polysaccharide) followed D. pneumoniae respiratory tract infection in 19 of 28 animals, and was elicited in 5 of 6 animals after intravenous infection. Both local (macrophage migration inhibition) and systemic delayed hypersensitivity followed bacterial infection of the lower respiratory tract. MIF activity was shown to be one mechanism for inhibition of alveolar macrophage migration.

Infectious diarrhea. Pathogenesis and risk factors.

Our understanding of the pathogenesis of infectious, especially bacterial, diarrhea has increased dramatically. New etiologic agents, mechanisms, and diseases have become known. For example, Escherichia coli serogroup O157 is now known to cause acute hemorrhagic colitis. Also, E. coli serogroups that produce Shiga toxin are recognized as etiologic agents in the hemolytic-uremic syndrome. The production of bacterial diarrhea has two major facets, bacterial-mucosal interaction and the induction of intestinal fluid loss by enterotoxins. Bacterial-mucosal interaction can be described in stages: (1) adherence to epithelial cell microvilli, which is often promoted by or associated with pili; (2) close adherence (enteroadherence), usually by classic enteropathogenic E. coli, to mucosal epithelial cells lacking microvilli; and (3) mucosal invasion, as with Shigella and Salmonella infections. Further large strides in understanding infectious diarrhea are likely with the cloning of virulence genes if additional host-specific animal pathogens become available for study.

Methicillin-resistant Staphylococcus aureus outbreak at a Veterans' Affairs Medical Center: importance of carriage of the organism by hospital personnel.

The reported prevalence of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) by hospital personnel averages 2.5%. From August 1985 to September 1987, 155 patients at our Veterans' Affairs Medical Center (VAMC) were colonized or infected with MRSA. In December 1986, only two (2.1%) of 94 healthcare workers were identified as nasal carriers. Prompted by a sharp increase in the number of patients with MRSA in early 1987, contact tracing identified 450 employees, of whom 36 (8%) were nasal carriers. Thirty-five percent of surgical residents (7 of 20) were nasal carriers. Prior to being identified as a nasal carrier, one surgical resident was associated with the inter-hospital spread of the VAMC MRSA strain to the burn unit of the affiliated university hospital. Three family members of two employee carriers were also found to harbor the epidemic strain. All 36 carriers were decolonized with various antimicrobial combinations. Vigorous infection control measures were effective in controlling the epidemic. The frequency of MRSA carriage by hospital personnel at our medical center during the epidemic proved higher than previously appreciated. Thus, healthcare workers may comprise a sizable MRSA reservoir. During an MRSA epidemic, infection control should attempt to identify and decolonize this hospital reservoir, as these individuals can disseminate MRSA both within the hospital as well as into the community.

Inhibition of Entamoeba histolytica cytotoxin by alpha 1 antiprotease and alpha 2 macroglobulin.

We previously reported partial purification of a proteinaceous substance with cytotoxic and enterotoxic activity isolated from the soluble fraction of sonicated axenically cultivated Entamoeba histolytica trophozoites. Demonstration of cytotoxic activity of the preparation (amebal toxin) was dependent on removal of serum from the tissue culture assay system. The objective of the present study was to identify the factor(s) in non-immune sera responsible for producing in vitro inhibition of amebal toxin cytotoxicity on HeLa cells. Gel filtration of non-immune sera from adult humans or bovines demonstrated that two portions of the eluate had significant inhibitory against the toxin. A high molecular weight inhibitory fraction was identified as predominantly alpha-2 macroglobulin and a low molecular weight inhibitory fraction was identified as predominantly alpha-1 antiprotease. Preparative isoelectric focusing of human serum isolated inhibitory fractions containing these same alpha globulins. Alpha-2 macroglobulin was purified and alpha-1 antiprotease was partially purified from human serum by other methods and shown to have high inhibitory activity against the amebal cytotoxin. Substances that were inhibitory to the cytotoxic activity of the amebal toxin also mediated reattachment of toxin treated HeLa cells. We conclude that the characteristics of the serum inhibitors, especially their ability to reverse the cytotoxic effects of amebal toxin on HeLa cells, suggests that the amebal toxin has protease activity.

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions.

A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, Hafnia alvei, a strain of Citrobacter freundii, and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:116, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii, EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii. EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eueA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.

Emergence of resistance to clarithromycin during treatment of disseminated cutaneous Mycobacterium chelonae infection: case report and literature review.

Results of in vitro susceptibility studies and one clinical trial have led to recommendations of clarithromycin monotherapy for the treatment of disseminated cutaneous Mycobacterium chelonae infections. We describe the case of a 65-year-old woman, immunocompromised by the use of chronic steroid therapy, who developed disseminated cutaneous infection with M. chelonae and failed clarithromycin monotherapy due to the development of drug resistance. In the relapse isolate we document the presence of a single point mutation at position 2058 in the gene coding for 23S rRNA peptidyltransferase regions, a mutation previously implicated in the development of resistance to clarithromycin. Two susceptible control isolates lacked the mutation. Three additional reports in the literature of patients developing recurrent skin lesions with clarithromycin-resistant M. chelonae following initial response to monotherapy are summarized. We demonstrate that clarithromycin monotherapy in patients with disseminated cutaneous infections can lead to clarithromycin resistance and therapeutic failure associated with a single point mutation at position 2058 of 23S rRNA.

Prevention of bacterial infections of mucosal surfaces by immune secretory IgA.

1) Colonization and diarrhea in the rabbit due to RDEC-1 strain E. coli elicits an immune response that prevents subsequent infection with the same bacterium. 2) RDEC-1 and InvEC strain colonization of the gut of pregnant rabbits is followed by the appearance, post-delivery, of anti-InvEC or RDEC-1 strain s-IgA in the milk, some of which was able to agglutinate as many as 3.4 X 10(11) bacteria per mg of s-IgA. 3) Immune s-IgA, given by the orogastric route to rabbits, is able to survive the digestive enzymes of the gastrointestinal tract to prevent the colonization and diarrhea that follows the inoculation of InvEC and RDEC-1 strains. One possible explanation for the protective effect of s-IgA is the in vivo agglutination of bacteria in the lumen of the gastrointestinal tract.

Escherichia coli diarrhea.

E. coli diarrheal disease is becoming ever more complicated as more and more pathogenic mechanisms are identified. E. coli strains remain the major causes of infectious diarrhea worldwide. Presumptive diagnosis based on clinical and laboratory criteria is practical for strains known to be important in the United States. Specific diagnosis is not currently feasible outside of research centers. Therapy, when indicated, shortens the duration of illness. Research is proceeding rapidly at the molecular level and may lead to new diagnostic and therapeutic approaches in the near future.

In-vitro activity of cefpirome compared with that of other agents.

The in-vitro activity of cefpirome, cefotaxime, ticarcillin, piperacillin, and three aminoglycosides was compared by the broth dilution method against 248 consecutive clinical isolates of Pseudomonas aeruginosa and 35 aminoglycoside-resistant isolates of the Enterobacteriaceae. Cefpirome was more active against Ps. aeruginosa than cefotaxime and gentamicin, and as active as piperacillin, ticarcillin, tobramycin and amikacin. Ps. aeruginosa isolates resistant to a given aminoglycoside were significantly more resistant to cefpirome than isolates susceptible to that aminoglycoside. Other beta-lactam antibiotics were also less active against aminoglycoside-resistant strains of Ps. aeruginosa. Cefpirome was more potent than cefotaxime and the penicillins against aminoglycoside-resistant Enterobacteriaceae.

Characterization of colonization of the rabbit gastrointestinal tract by Escherichia coli RDEC-1.

We have reported previously the isolation of an Escherichia coli strain (RDEC-1) that in rabbits is able to colonize the gut, adhere to mucosal epithelial cells of the ileum, cecum, and colon, and cause diarrhea by a novel mechanism. The purpose of the present study was to characterize more fully the colonization of the rabbit gut by strain RDEC-1. Colonization reached a maximum 3 to 4 days post-inoculation with strain RDEC-1 and did not decrease until 15 days post-inoculation. Diarrhea occurred 3 to 4 days after colonization reached its maximum intensity. Semiquantitative rectal swab cultures were found to be correlated with counts of colony-forming units of strain RDEC-1 per gram of ileum, cecum, and colon and their contents and were used to chart the course of colonization of the rabbit gut. The actual number of colony-forming units per gram was dependent on the stage of colonization and ranged from 4.0 X 10(3) to 2.4 X 20(6) in the ileum to 3.1 X 10(5) to 3.6 X 10(7) in the cecum. The number of colony-forming units per gram was not affected by the presence of diarrhea. E. coli RDEC-1 colonizes the ileum, cecum, and colon of rabbits heavily for a relatively long period of time.

Diarrhea due to Escherichia coli strain RDEC-1 in the rabbit: the peyer's patch as the initial site of attachment and colonization.

A strain of Escherichia coli (RDEC-1) has been described that in rabbits colonizes the intestine; adheres to mucosal epithelial cells of the ileum, cecum, and colon; and causes diarrhea by an unknown mechanism. This study attempted to determine the location of the bacteria in the rabbit intestine during the unexplained six- to seven-day interval between bacterial inoculation and onset of diarrhea. Specimens of ileum, cecum, colon, ileal Peyer's patches, sacculus rotundus, and appendix from control rabbits and from rabbits killed at intervals after inoculation with RDEC-1 bacteria were examined by light and direct fluorescence microscopy. Bacteria in large numbers attach to the tips of the Peyer's patch lymphoid follicles by 24 hr, but they did not attached to ileal, cecal, or colonic mucosa until three days after inoculation. The lag time between inoculation and onset of diarrhea was probably due to the need for the bacteria first to attach to and then colonize the Peyer's patch lymphoid follicles. The intestinal mucosa was probably colonized by bacteria shed from the Peyer's patches.

HeLa cell adherence, actin aggregation, and invasion by nonenteropathogenic Escherichia coli possessing the eae gene.

Enteropathogenic Escherichia coli (EPEC) produce diarrhea in humans by a mechanism that involves close adherence to epithelial cells in the intestine and colon. Close adherence is associated with effacement of microvilli and condensation of actin beneath the bacteria, a process termed attaching/effacing adherence. Attaching/effacing adherence of EPEC occurs in vitro in tissue culture, simplifying the study of the molecular genetics of this process. An EPEC gene (eae) necessary for attaching/effacing adherence was recently characterized. Enterohemorrhagic E. coli and the rabbit-specific RDEC-1 strain adhere in a like fashion in vivo and hybridize with eae. However, these strains adhere poorly to tissue culture cells, complicating the in vitro study of attaching/effacing adherence. In order to develop an in vitro model for the study of attaching/effacing activity of non-EPEC bacteria, a plasmid encoding the F1845 adhesin of an E. coli strain (C1845) isolated from a patient with diarrhea was transformed into RDEC-1 and enterohemorrhagic E. coli. The transformed strains adhered in a diffuse pattern to HeLa cells, and they aggregated HeLa cell actin at points of adherence in the fluorescein-isothiocyanate-labeled phalloidin assay. They also invaded HeLa cells in a gentamicin invasion assay, although not to the extent seen with EPEC. The construction of adherent non-EPEC strains facilitates the molecular study of the attaching/effacing properties and invasiveness of these strains in tissue culture models.

Diarrhea due to Escherichia coli in the rabbit: a novel mechanism.

A strain of Escherichia coli O15 (RDEC-1) isolated from several rabbits with diarrhea was examined to determine (1) whether the strain could produce diarrhea when administered by the orogastric route to other rabbits and (2) whether this strain was invasive or enterotoxigenic. Strain RDEC-1 produced diarrhea in 48 of 62 rabbits when given by the orogastric route in doses that ranged from 1.5 X 10(2) to 4 X 10(10) bacteria. The organism did not give a positive result in the 18-hr ileal loop or Serény tests and did not invade HeLa cells. Culture supernatants of strain RDEC-1 did not give positive results in the 6- or 18-hr rabbit ileal loop, suckling mouse, Y-1 adrenal cell, or Chinese hamster ovary cell assays. Fluorescent antibody staining of sections of intestine prepared in a cryostat revealed great numbers of E. coli strain RDEC-1 that adhered to the epithelial surface. It is evident that E. coli can produce diarrhea without being able to invade the mucosa or synthesize enterotoxin. Strains of E. coli similar to RDEC-1 may account for some of the E. coli-associated diarrhea that occurs in humans.