Preventing the spread of vancomycin-resistant enterococci in a long-term care facility.

OBJECTIVES: To test the hypothesis that infection control practices can prevent the spread of vancomycin-resistant enterococci (VRE) to residents of a long-term care facility (LCF) from an affiliated acute care facility with a high endemic rate of colonization. DESIGN: Point prevalence study of the rate of rectal colonization. SETTING: A state-supported veterans nursing home and an acute care veterans hospital. PARTICIPANTS: Residents in a state veterans home. INTERVENTIONS: Identification of patients with rectal colonization by VRE before transfer to the state veterans home, contact isolation for colonized veterans, use of oral bacitracin to eliminate colonization. MEASUREMENTS: Rectal swab and culture for VRE, review of clinical records and recording of presumptive risk factors for VRE colonization. The risk factors were age, gender, length of stay at nursing home, treatment with vancomycin or oral antibiotics, prior hospitalization at the acute care facility during the prior year, use of indwelling urethral catheters, presence of diarrhea, and fecal or urinary incontinence. RESULTS: Sixty-nine of 200 residents were cultured in the first study (1996) and 130 of 230 residents were cultured in the second study (1998). Residents who consented to culture differed from those who did not only with regards to gender (2 vs 7, P = .012). In neither study were any residents found to be colonized with VRE who had not already been identified as positive on admission. CONCLUSIONS: Adherence to infection control practices by the patient care staff of the LTCF was associated with the absence of transmission of VRE colonization among its residents. The presence of rectal colonization with VRE in an acute care patient should not be a barrier to acceptance in a nursing home.

Autoradiographic study of tobramycin uptake by proximal and distal tubules of normal and pyelonephritic rats.

Multiple factors may modify the pharmacokinetics of aminoglycosides and affect their nephrotoxic potential. In the present study, the influence of Escherichia coli pyelonephritis on the renal handling of [3H]tobramycin was investigated. The accumulation of [3H]tobramycin in proximal and distal tubules in both normal and infected rats was compared. Following induction of pyelonephritis, disturbed intrarenal localization of the drug was noted. Grain counts were affected in both proximal and distal tubules. Decreased labeling was observed at all time intervals in the proximal tubules. Electron microscopy showed that radioactivity was associated mostly with lysosomes in both normal and infected rats 1 and 24 h following the injection of the drug. We could detect significantly higher amounts of drug in the distal tubules of the pyelonephritic kidney than the normal levels at 10 min and 24 h postinjection. The drug did not seem to be associated with any particular organelle and was evenly distributed within the distal tubular cells. The present study shows that the transport of tobramycin within the infected nephron is disturbed. These data might shed some light on the influence of infection on the intrarenal pharmacology of aminoglycosides.

Stimulation of human polymorphonuclear leukocyte oxidative metabolism by type 1 pili from Escherichia coli.

We compared the degree to which Escherichia coli phase variants which do (T1P+ E. coli) or do not (T1P- E. coli) express type 1 pili (T1P) stimulate human polymorphonuclear leukocyte (PMN) oxidative activity. Unopsonized T1P+ E. coli stimulated the release of 0.20 to 0.24 nmol of H2O2 per 10(6) PMN per min and the consumption of 1.4 to 4.0 nmol of O2 per 10(6) PMN per min; no measurable PMN oxidative activity was stimulated by unopsonized T1P- E. coli. In the presence of serum opsonins, T1P+ E. coli stimulated the release of 1.12 to 1.16 nmol of H2O2 per 10(6) PMN per min and the consumption of 5.0 to 6.0 nmol of O2 per 10(6) PMN per min, whereas T1P- E. coli stimulated the release of 0.42 to 0.43 nmol of H2O2 per 10(6) PMN per min and the consumption of 0.6 to 2.0 nmol of O2 per 10(6) PMN per min. Although unaggregated T1P did not stimulate PMN, latex beads coated with T1P (T1P-latex) stimulated alpha-methylmannoside-inhibitable, opsonin-independent PMN oxidative activity. The activity stimulated by either T1P+ E. coli or T1P-latex was susceptible to inhibition by cytochalasin B. Latex particles coated with bovine serum albumin or mannose-resistant pili did not stimulate PMN. These data indicate that T1P+ E. coli stimulate PMN oxidative metabolism more effectively than do T1P- E. coli and that a similar PMN oxidative response follows cellular stimulation by either unopsonized T1P+ or opsonized T1P- E. coli. Furthermore, T1P-latex faithfully mimics the ability of T1P+ E. coli to stimulate PMN oxidative metabolism. Such particles may be useful in further analyses of cellular responses to T1P+ E. coli.

An accurate method to obtain urine for culture in men with external catheters.

Results of urine cultures from 26 male nursing home patients wearing external catheters, collected by a simple standardized technique, were compared with culture results from the same patients obtained by sterile in-and-out catheterization. The culture results were the same in 22 (85%) of the matched specimens, and specimens collected by the standardized technique were 100% sensitive and 94% specific in detecting significant growth of pathogenic organisms. In contrast, 13 (57%) of 23 specimens collected from patients with external catheters by the nursing home staff using their routine technique yielded three or more organisms and were considered contaminated. These results suggest that it is possible to obtain a urine specimen that reflects bladder urine in the vast majority of patients with external catheters, and thus potentially avoid the need for in-and-out catheterization when diagnosing and planning treatment for urinary tract infections in this population.

Change in degree of type 1 piliation of Escherichia coli during experimental peritonitis in the mouse.

To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.

The surface of virulent Treponema pallidum: resistance to antibody binding in the absence of complement and surface association of recombinant antigen 4D.

The binding of immunoglobulin G present in syphilitic immune rabbit serum, syphilitic human serum, and rabbit antiserum to purified recombinant Treponema pallidum antigen 4D by T. pallidum, Nichols strain, was studied by immunoelectron microscopy. Treponemes were incubated with antiserum under the conditions of the T. pallidum immobilization test, in which T. pallidum-specific antibody renders the organism nonmotile and avirulent only in the presence of complement after a 16-h incubation period in an anaerobic environment. Antibody was not demonstrable on the surface of T. pallidum incubated with nonimmune rabbit serum or normal human serum in the presence of complement. Similarly, in the absence of complement, little or no antibody was found on the treponemal surface after incubation with syphilitic immune rabbit serum, syphilitic human serum, or rabbit antiserum directed against the recombinant 4D antigen. The addition of complement to syphilitic immune rabbit serum, syphilitic human serum, and anti-4D antibody resulted in immobilization and the deposition of antibody on the entire surface of the immobilized organisms. These results corroborate earlier work by other investigators demonstrating the resistance of freshly isolated T. pallidum to antibody binding in a variety of serological tests. Detection of 4D antigen on the surface of immobilized T. pallidum strongly implies that the use of T. pallidum immobilization test conditions provides a means to demonstrate the association of individual surface antigens on virulent T. pallidum. The resistance of T. pallidum to antibody binding may be relevant to the pathogenesis of syphilis.

Effect of Tamm-Horsfall urinary glycoprotein on phagocytosis and killing of type I-fimbriated Escherichia coli.

Human polymorphonuclear leukocytes (PMN) ingest type I (mannose sensitive) fimbriated Escherichia coli even in the absence of antibody, complement, or other serum opsonins. Our studies suggest that the Tamm-Horsfall urinary glycoprotein (THP) interferes with serum-independent ingestion. Electron micrographs showed that dissolved THP adhered to type I fimbriae and formed a pseudocapsule around bacteria bearing type I fimbriae. Phase-variant bacteria grown on blood agar neither expressed fimbriae nor bound THP. Affinity column chromatography demonstrated mannose-sensitive binding between purified type I fimbriae and purified THP. The ability of human PMN to bind and ingest type I-fimbriated E. coli was diminished if the bacteria had been coated by exposure to THP at physiologic concentrations. At 1 h, PMN were associated with an average of 2.62 uncoated bacteria, but with only 0.18 coated bacteria (P less than 0.001). alpha-Methyl mannoside blocked the observed effect of THP on binding and phagocytosis in a dose-dependent fashion: increased mannoside led to increased blocking. PMN preincubated with THP were able to bind and phagocytose normally. There did not appear to be any significant clumping of bacteria in suspension to account for these effects. Bactericidal assays with leukocytes in suspension demonstrated protection of THP-coated bacteria. At 1 h, PMN killed 42% of noncoated E. coli (a decrease of 0.24 log), but the number of THP-coated bacteria increased by 75% (an increase of 0.24 log). These observations may partially explain the virulence of E. coli in the bladder and kidney, where serum activity is low and THP is abundant.

Interaction of bacterial pili and leukocytes.

Since Duguid and Guilles first described the ability of piliated bacteria to bind to leukocytes, much has been learned about the nature of this interaction. Mannose-sensitive (MS) pili bind to specific mannose-containing receptors on the leukocyte surface. While MS pili are responsible for attachment, the relative hydrophobicity of the bacterial surface determines whether the organism is internalized. Both binding and ingestion trigger the leukocyte to respond with degranulation and enhanced oxidative activity. The response to piliated bacteria, however, is delayed as compared to bacteria opsonized with serum, which may account for the reduced bactericidal activity associated with pili-mediated phagocytosis. A number of factors appear to influence the significance of pili-mediated phagocytosis in vivo. These include natural selective pressures in the host tissue, the ability of the organism to undergo pili phase transition and the presence of serum or other host opsonic factors. Antipili antibody does not enhance leukocyte killing of MS + Escherichia coli, but does stimulate leukocyte metabolic activity. Antipili antibody may, therefore, have an adverse effect on the infectious process by promoting the extracellular release of inflammatory material from the granulocyte.

Antibacterial mechanisms of antibody to mannose-sensitive pili of Escherichia coli.

To identify mechanisms whereby antibody to mannose-sensitive pili of Escherichia coli might enhance host defenses, we evaluated the activity of antibody to pili in four antibacterial immune processes. Antiserum to pili and Fab' fragments of IgG antibody to pili inhibited the ability of homologous piliated organisms to adhere to buccal epithelial cells. However, this antiserum did not enhance intravascular clearance, complement-dependent bacteriolysis, or opsonophagocytosis. The addition of antiserum to pili to polymorphonuclear leukocytes and piliated E. coli reduced the rate of killing from 52% to 5%. The addition of complement restored the rate to 52%, but this was much less than the 99% rate achieved with polymorphonuclear leukocytes and either fresh serum or antibody to the whole bacteria. These observations suggest that the principal anti-bacterial property of antibody to mannose-sensitive pili of E. coli is inhibition of bacterial attachment. Whether the anti-opsonic effect of antibody to pili is detrimental to host defense remains to be determined.

Enhancement of mannose-mediated stimulation of human granulocytes by type 1 fimbriae aggregated with antibodies on Escherichia coli surfaces.

In the present study, we assayed protein iodination in human granulocytes after interaction of the cells with mannose-specific (MS) type 1 fimbriated (MS+) and nonfimbriated (MS-) phenotypes of Escherichia coli pretreated with various amounts of anti-E. coli and antifimbrial antibodies. The MS+ phenotype stimulated protein iodination in granulocytes and possessed potent MS activity as measured by yeast aggregometry. In contrast, the MS- phenotype lacked all these activities. MS+ pretreated with moderate concentrations of antibodies, however, showed up to a 15-fold increase in granulocyte stimulation as compared to granulocyte stimulation induced by the non-antibody-treated MS+ phenotype or by the antibody-treated MS- phenotype. This marked antibody-mediated increase in stimulation of granulocytes was (i) dependent on the antibody concentrations, (ii) markedly reduced by methyl-alpha-D-mannoside, (iii) caused by immunoglobulin G as well as by F(ab')2, (iv) caused only by antifimbrial antibodies, (v) associated with cross-linked fimbriae seen as "bundles" under an electron microscope, and (vi) mimicked by treating MS+ bacteria with a certain range of glutaraldehyde. The data taken together support the hypothesis that, although cross-linking of fimbriae reduced the density of functional MS fimbriae over the surface of antibody-treated bacteria and consequently reduced the ability of these organisms to agglutinate yeast cells, the resulting bundles of MS fimbriae were far more effective at stimulating granulocytes because, bound together, they were better equipped to aggregate the mannose-containing receptors on the granulocyte surface.

Serum and urogenital antibody responses to Escherichia coli pili in cystitis.

Antipili antibody (APA) was quantitated by enzyme-linked immunosorbent assay in the serum, urine, and vaginal secretions of 12 women with cystitis due to Escherichia coli. Pili were purified from the strain infecting each patient. The geometric mean titers of APA in serum taken at the initial visit were: immunoglobulin G (IgG), 23.7 +/- 3.9; IgA, 4.1 +/- 4.0; and IgM, 4.4 +/- 5.7. No consistent change was observed between samples obtained at 3 and 6 weeks. APA in serum from 10 control patients was assayed against a pool of pili assembled from the 12 infecting isolates. The geometric mean titers of the control sera were: IgG, 8.4 +/- 3.2; IgA, 9.6 +/- 2.4; and IgM, 0. Antipili IgG and IgM were significantly greater in the infected than in the control sera (P less than 0.05). APA was not detected in the urine or vaginal secretions from any infected or control patient. Antigenic relationships between the 12 infecting strains were investigated by indirect immune electron microscopy. All but 1 of 12 infecting strains shared antigenic determinants. Two patients had a second E. coli infection during the study. The pili of the reinfecting isolates showed partial antigenic cross-reactivity with the pili of the initial infecting bacteria. These observations indicate that although cystitis stimulates the development of serum APAs, it fails to stimulate local APA and thus may not engender any immunological barrier to subsequent recolonization.

Serological response to Escherichia coli pili in pyelonephritis.

The serological response to Escherichia coli pili was studied in 4 adult male patients with pyelonephritis and in 11 uninfected controls. Antibody to the specific pilus antigen of the infecting strain of each patient was measured with indirect immune electron microscopy. Antibody to a cross-reacting antigen of the type 1 pili of heterologous E. coli 346 was quantitated with an enzyme-linked immunosorbent assay. Few antipilus antibodies were found in the serum of control patients with either technique. All pyelonephrine patients developed an increase in specific antipilus antibodies belonging to the immunoglobulins classes G, A, and M. Antibodies to the cross-reacting type 1 pilus antigen were found in the serum of three of the four patients and were predominantly immunoglobulin G. Few or no antipilus antibodies were present in the urine of infected or control patients. These results suggest that a serological response to the pill of infecting organisms develops after pyelonephritis.

Protection against experimental pyelonephritis by antibodies to pili.

Bacterial pili have been shown to be an important virulence factor for urinary tract infections. In this report we relate the results of studies which evaluated the influence of antipili antibody on the susceptibility of rats to ascending pyelonephritis and on several antibody-mediated antibacterial mechanisms. Rats immunized with E. coli type 1 pili, and animals infected with E. coli developed antipili antibodies in their serum. Active or passive immunization of rats with pili protected the animals from ascending pyelonephritis. Antipili antibody did not mediate complement-dependent bacteriolysis, opsonophagocytosis or promote more rapid intravascular clearance of injected E. coli. Humoral immunity to pili did, however, effectively inhibit bacterial adherence to epithelial cells. These studies indicate that type 1 E. coli pili are immunogenic and that antipili antibodies afford protection from ascending pyelonephritis. They suggest further that a mechanism of protection is inhibition of bacterial adherence.

Dissociation and reassembly of Escherichia coli type 1 pili.

Escherichia coli type 1 pili, which mediate the mannose-sensitive adherence of the bacterium to eucaryotic cells, are comprised of very stable arrays of pilin protein subunits (molecular weight, approximately 17,000). Previous methods for the dissociation of pili caused their irreversible denaturation. We have found that incubation of pili in saturated guanidine hydrochloride at 37 degrees C led to their complete dissociation, as evidenced by nephelometry and electron microscopy. Gel chromatography of the dissociated pili on a Sepharose CL-6B column in the presence of saturated guanidine hydrochloride yielded a single protein peak with a molecular weight corresponding to that of pilin. Dialysis of this peak against 5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and rechromatography in the same buffer afforded a major protein peak, probably consisting of pilin dimers. About 25% of the protein in this peak bound to a mannan-sepharose column and could be eluted with methyl alpha-D-mannoside. The pilin dimer gave a single protein band upon polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (molecular weight, 16,600) or 10 M urea and penetrated completely into 7% gels in the absence of denaturants. Reassembly of the pilin dimers into pili was achieved upon dialysis against the tris(hydroxymethyl)aminomethane buffer containing 5 mM MgCl2, as observed by electron microscopy. Thus, the conditions used allow renaturation of the dissociated subunits and may aid in further studies of the structure-function relationship of pili.