Acquisition of oral microbes and associated systemic responses of newborn nonhuman primates.

The acquisition and development of the complex oral microbiome remain ill defined. While selected species of oral bacteria have been examined in relation to their initial colonization in neonates, a more detailed understanding of the dynamics of the microbiome has been developed only in adults. The current investigation used a nonhuman primate model to document the kinetics of colonization of the oral cavities of newborns and infants by a range of oral commensals and pathogens. Differences in colonization were evaluated in newborns from mothers who were maintained on an oral hygiene regimen pre- and postparturition with those displaying naturally acquired gingivitis/periodontitis. The results demonstrate distinct profiles of acquisition of selected oral bacteria, with the transmission of targeted pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, being passed on primarily from mothers with gingivitis/periodontitis. This colonization resulted in defined patterns of systemic antibody responses in the infants. The significant relative risk measures for infection with the pathogens, as well as the relationship of oral infection and blood serum antibody levels, were consistent with those of the newborns from mothers with gingivitis/periodontitis. These findings indicate that the early acquisition of potentially pathogenic oral bacterial species might impact the development of mucosal responses in the gingiva and may provide an enhanced risk for the development of periodontitis later in life.

Periodontitis in pregnant baboons: systemic inflammation and adaptive immune responses and pregnancy outcomes in a baboon model.

BACKGROUND/OBJECTIVES: Chronic periodontal infections have been suggested to contribute to the risk of adverse pregnancy outcomes. MATERIAL AND METHODS: This study describes the relationship of patterns of systemic inflammatory mediators and IgG antibody to 20 oral bacteria in pregnant female baboons (Papio anubis) coupled with clinical features of ligature-induced periodontitis, as risk indicators for adverse pregnancy outcomes. Animals showing a preterm delivery and/or low birth weight newborns, as well as those pregnancies resulting in spontaneous abortion, stillbirth, or fetal demise were tabulated as adverse pregnancy outcomes. RESULTS: A significantly greater frequency of the periodontitis group neonates had a low birth weight (18.1%; p = 0.008) and decreased gestational age (9.8%). Spontaneous abortion/stillbirth/fetal demise were increased in the periodontitis (8.7%) versus the control group (3.8%) (p = 0.054). The baseline oral clinical presentation of the experimental animals did not relate to the adverse pregnancy outcomes. Animals with the greatest extent/severity of periodontitis progression during the initial ½ of gestation (ie. to mid-pregnancy) had the greatest risk for adverse pregnancy outcomes. Baseline biological parameters indicating historical responses of the animals to periodontal challenge demonstrated individual variation in selected mediators, some of which became more differential during ligature-induced periodontitis. The relationship of clinical parameters to systemic inflammatory responses was consistent with a temporal contribution to adverse pregnancy outcomes in a subset of the animals. CONCLUSIONS: These results support a link between periodontitis and adverse pregnancy outcomes in the baboons and provide a prospective experimental model for delineating the biologic parameters that contribute to a causal relationship between chronic oral infections and birth events.

Systemic inflammatory responses in progressing periodontitis during pregnancy in a baboon model.

This study tested the hypothesis that pregnant female baboons exhibit increased levels of various inflammatory mediators in serum resulting from ligature-induced periodontitis, and that these profiles would relate to periodontal disease severity/extent in the animals. The animals were sampled at baseline (B), mid-pregnancy (MP; two quadrants ligated) and at delivery (D; four quadrants ligated). All baboons developed increased plaque, gingival inflammation and bleeding, pocket depths and attachment loss following placement of the ligatures. By MP, both prostaglandin E(2) (PGE(2)) and bactericidal permeability inducing factor (BPI) were greater than baseline, while increased levels of interleukin (IL)-6 occurred in the experimental animals by the time of delivery. IL-8, MCP-1 and LBP all decreased from baseline through the ligation phase of the study. Stratification of the animals by baseline clinical presentation demonstrated that PGE(2), LBP, IL-8 and MCP-1 levels were altered throughout the ligation interval, irrespective of baseline clinical values. IL-6, IL-8 and LBP were significantly lower in the subset of animals that demonstrated the least clinical response to ligation, indicative of progressing periodontal disease. PGE(2), macrophage chemotactic protein (MCP)-1, regulated upon activation, normal T cell expressed and secreted (RANTES) and LBP were decreased in the most diseased subset of animals at delivery. Systemic antibody responses to Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Campylobacter rectus were associated most frequently with variations in inflammatory mediator levels. These results provide a profile of systemic inflammatory mediators during ligature-induced periodontitis in pregnant baboons. The relationship of the oral clinical parameters to systemic inflammatory responses is consistent with a contribution to adverse pregnancy outcomes in a subset of the animals.

Periodontitis in pregnancy: clinical and serum antibody observations from a baboon model of ligature-induced disease.

BACKGROUND: Chronic oral infections that elicit host responses leading to periodontal disease are linked with various sequelae of systemic diseases. This report provides seminal information on the clinical and adaptive immunologic characteristics of a baboon model of ligature-induced periodontitis during pregnancy. METHODS: Female Papio anubis were evaluated for periodontal health at baseline. Ligatures were tied around selected teeth to initiate oral inflammation and periodontitis. Then the animals were bred. At midpregnancy ( approximately 90 days), a clinical evaluation was performed, and additional ligatures were tied on teeth in the contralateral quadrants to maintain progressing periodontitis throughout pregnancy. A final clinical evaluation was done for all experimental teeth after delivery, and ligatures were removed. Serum was collected at all sampling intervals for the determination of antibody levels to a group of 20 oral bacteria. Unligated animals served as controls. RESULTS: At baseline, 16% of animals exhibited minimal plaque and gingival inflammation without periodontal disease. The remaining baboons demonstrated varying levels of inflammation/bleeding, and approximately 20% of the population had periodontal pocketing (>3 mm). Ligated animals expressed increased levels of inflammation and increased probing depths and clinical attachment loss (AL) and could be stratified into multiple subsets postligation based upon changes in clinical parameters at midpregnancy and at delivery. Baboons were categorized into disease susceptibility groups (periodontal disease susceptibility 1 through 4) that described the extent/severity of induced disease during pregnancy. Control animals showed minimal periodontal changes during gestation. Significant differences in serum antibody to multiple oral bacteria were found in animals presenting with periodontitis at baseline and during the 6 months of ligature-induced disease. A significant correlation to antibody to P. gingivalis, which was sustained throughout ligation and pregnancy, was observed with disease presentation. CONCLUSIONS: The clinical presentation at baseline, reflecting the natural history of oral disease in these animals, suggests individual variation that is reflected in the characteristics of the adaptive immune responses to oral bacteria. The variability in the response to ligation with resulting periodontal disease provides a model to document prospectively the relationship between oral and systemic health outcomes.

In vitro environmental regulation of Porphyromonas gingivalis growth and virulence.

Porphyromonas gingivalis appears to be a major contributor to periodontal disease, especially soft tissue destruction, which is reflected by the ability to cause invasive, spreading lesions, and tissue inflammation in a murine abscess model. This study investigated the role of hemin on the regulation of growth and virulence of P. gingivalis strains. P. gingivalis strains W50, A7A1-28, 3079, 381, W50/BEI, and NG4B19 were grown in broth and on blood agar plates. P. gingivalis cells grown under iron-depleted conditions for multiple passages showed significantly decreased lesion size in mice, in contrast to cells grown under iron-normal (5 microg/ml) and iron-elevated conditions. Statistically significant (P < 0.01) decreases in gingipain enzyme activity were found among the strains grown under iron-depleted conditions. P. gingivalis grown in the presence of blood induced significantly different lesion type, lesion size, lesion onset, and mortality. Elevated hemin resulted in increased cell-associated iron in P. gingivalis, which increased the capacity of the microorganism to survive at times of iron deprivation. These results indicate that hemin or iron availability regulates multiple aspects related to P. gingivalis virulence, including growth, survival, gingipain levels, and iron accumulation.

Vancomycin-resistant enterococci colonization in patients at seven hemodialysis centers.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are increasing in prevalence at many institutions, and are often reported in dialysis patients. We studied the prevalence of and risk factors for VRE at seven outpatient hemodialysis centers (three in Baltimore, MD, USA, and four in Richmond, VA, USA). METHODS: Rectal or stool cultures were performed on consenting hemodialysis patients during December 1997 to April 1998. Consenting patients were recultured during May to July 1998 (median 120 days later). Clinical and laboratory data and functional status (1 to 10 scale: 1, normal function; 9, home attendant, not totally disabled; 10, disabled, living at home) were recorded. RESULTS: Of 478 cultures performed, 20 (4.2%) were positive for VRE. Among the seven centers, the prevalence of VRE-positive cultures varied from 1.0 to 7.9%. Independently significant risk factors for a VRE-positive culture were a functional score of 9 to 10 (odds ratio 6.9, P < 0.001), antimicrobial receipt within 90 days before culture (odds ratio 6.1, P < 0.001), and a history of injection drug use (odds ratio 5.4, P = 0.004). CONCLUSIONS: VRE-colonized patients were present at all seven participating centers, suggesting that careful infection-control precautions should be used at all centers to limit transmission. In agreement with previous studies, VRE colonization was more frequent in patients who had received antimicrobial agents recently, underscoring the importance of judicious antimicrobial use in limiting selection for this potential pathogen.

Periodontal diseases: to protect or not to protect is the question?

For decades, investigations have identified local and systemic humoral immune responses to microorganisms comprising the supra- and subgingival biofilms in the oral cavity. Inflammation and tissue destruction in the periodontium are accompanied by alterations in the quantity, quality, and specificity of antibody. The conundrum in this scenario is the existence of a substantial plasma cell infiltrate at sites of periodontal lesions and a seemingly robust antibody response in the oral cavity and the serum, apparently coincident with progressing disease. Consequently, much effort has been expended to elucidate the critical characteristics of protective humoral responses and to develop strategies for enhancing these unique features. We and others have conducted studies attempting to distinguish disease susceptibility associated with: i) variations in response levels significantly increased to some species with disease, minimal response to others; ii) functional comparisons of antibody subclass differences, genetic regulation, and maturation of responses; iii) microbial and antigenic specificity of the antibody focus on specific pathogens and identification of selected antigens as targets for immunoprotection; and, iv) kinetics of responses during disease and therapeutic interventions linking immune changes with infection and as a measure of treatment success. This report summarizes varied research designs and results, to provide a profile of antibody in health, gingivitis, and periodontitis. These profiles may be used to provide a framework focusing on the humoral response to commensal microorganisms and likely pathogens, as they emerge in the biofilm--etiologic for or in response to disease processes. Models for antibody as a diagnostic adjunct and for predicting protective antibody responses are suggested. These concepts are likely relevant for considering vaccine approaches to periodontitis.

Cloning and expression of two novel hemin binding protein genes from Treponema denticola.

Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed, T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of the hbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in several T. denticola ATCC strains and clinical isolates, but not in Treponema pectinovorum, Treponema socranskii, or Escherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, called hbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. The hbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

Serratia liquefaciens bloodstream infections from contamination of epoetin alfa at a hemodialysis center.

BACKGROUND: In a one month period, 10 Serratia liquefaciens bloodstream infections and 6 pyrogenic reactions occurred in outpatients at a hemodialysis center. METHODS: We performed a cohort study of all hemodialysis sessions on days that staff members reported S. liquefaciens bloodstream infections or pyrogenic reactions. We reviewed procedures and cultured samples of water, medications, soaps, and hand lotions and swabs from the hands of personnel. RESULTS: We analyzed 208 sessions involving 48 patients. In 12 sessions, patients had S. liquefaciens bloodstream infections, and in 8, patients had pyrogenic reactions without bloodstream infection. Sessions with infections or reactions were associated with higher median doses of epoetin alfa than the 188 other sessions (6500 vs. 4000 U, P=0.03) and were more common during afternoon or evening shifts than morning shifts (P=0.03). Sessions with infections or reactions were associated with doses of epoetin alfa of more than 4000 U (multivariate odds ratio, 4.0; 95 percent confidence interval, 1.3 to 12.3). A review of procedures revealed that preservative-free, single-use vials of epoetin alfa were punctured multiple times, and residual epoetin alfa from multiple vials was pooled and administered to patients. S. liquefaciens was isolated from pooled epoetin alfa, empty vials of epoetin alfa that had been pooled, antibacterial soap, and hand lotion. All the isolates were identical by pulsed-field gel electrophoresis. After the practice of pooling epoetin alfa was discontinued and the contaminated soap and lotion were replaced, no further S. liquefaciens bloodstream infections or pyrogenic reactions occurred at this hemodialysis facility. CONCLUSIONS: Puncturing single-use vials multiple times and pooling preservative-free epoetin alfa caused this outbreak of bloodstream infections in a hemodialysis unit. To prevent similar outbreaks, medical personnel should follow the manufacturer's guidelines for the use of preservative-free medications.

Evaluation of a reporting system for bacterial contamination of blood components in the United States.

BACKGROUND: The transfusion of blood components contaminated with bacteria may have serious clinical consequences, but few data are available on the incidence of these events. A national effort to assess the frequency of blood component bacterial contamination associated with transfusion reaction (the BaCon Study) was initiated to better estimate their occurrence. STUDY DESIGN AND METHODS: Standard reporting criteria, data collection forms, and a standardized reporting protocol were developed in collaboration with the American Red Cross, AABB, and the Department of Defense. Episodes reported to the BaCon Study were compared with those reported to the FDA's national reporting systems to estimate the extent to which all serious reactions associated with bacterial contamination were captured. RESULTS: During the first 2 years, 38 episodes meeting study criteria were reported; 21 were laboratory-confirmed. The estimated proportion of episodes reported to the BaCon Study (i.e., completeness of coverage) was lower than that reported to the FDA during the same period (0.33 vs. 0.68), but the positive predictive value was higher (0.66 vs.0.28). CONCLUSION: Despite the complexity of obtaining reports from a large number of United States hospitals and transfusion centers, the feasibility and usefulness of the BaCon Study were shown. This study was the only national study in the United States to monitor adverse clinical events associated with bacterial contamination of blood components. By building on hospital-based reporting of transfusion-related adverse events, the BaCon Study serves as a model for the study of other complications associated with blood and blood components.

Transfusion-transmitted bacterial infection in the United States, 1998 through 2000.

BACKGROUND: Bacterial contamination of blood components can result in transfusion-transmitted infection, but the risk is not established. STUDY DESIGN AND METHODS: Suspected cases of transfusion-transmitted bacteremia were reported to the CDC by participating blood collection facilities and transfusion services affiliated with the American Red Cross, AABB, or Department of Defense blood programs from 1998 through 2000. A case was defined as any transfusion reaction meeting clinical criteria in which the same organism species was cultured from a blood component and from recipient blood, with the organism pair confirmed as identical by molecular typing. RESULTS: There were 34 cases and 9 deaths. The rate of transfusion-transmitted bacteremia (in events/million units) was 9.98 for single-donor platelets, 10.64 for pooled platelets, and 0.21 for RBC units; for fatal reactions, the rates were 1.94, 2.22, and 0.13, respectively. Patients at greatest risk for death received components containing gram-negative organisms (OR, 7.5; 95% CI, 1.3-64.2; p = 0.009). CONCLUSION: Bacterial contamination of blood is an important cause of transfusion-transmitted infection; infection risk from platelet transfusion is higher compared with that from RBCs, and, overall, the risk of infection from bacterial contamination now may exceed that from viral agents. Recipients of components containing gram-negative organisms are at highest risk for transfusion-related death. The results of this study may help direct efforts to improve transfusion-related patient safety.

Transfusion-related sepsis due to Serratia liquefaciens in the United States.

BACKGROUND: Severe, often fatal, transfusion reactions due to bacterial contamination of blood components continue to occur. Serratia liquefaciens, an unusual human pathogen, is a recently recognized potential cause of transfusion-related sepsis. CASE REPORTS: Five episodes of transfusion-related sepsis and endotoxic shock due to S. liquefaciens were reported to the CDC from July 1992 through January 1999. One episode has been described. The remaining four, all fatal, are described here: three associated with RBC transfusion and one associated with transfusion of platelets. In each instance, the source of contamination could not be found. The implicated units tended to be older (mean RBC age 28 days), and visual discoloration was noted in each RBC unit, although usually in retrospect. CONCLUSION: S. liquefaciens is an increasingly recognized cause of transfusion-related sepsis and is associated with a high mortality rate. S. liquefaciens can contaminate both RBCs and platelets, but the mechanism(s) of contamination remain unknown. Increased attention to pretransfusion visual inspection may avert the transfusion of some S. liquefaciens-contaminated RBC units. However, more sensitive rapid diagnostic tests are needed to further reduce the risk of transfusion-related sepsis and endotoxic shock.

Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5'-phosphate-dependent protein acting as a haemolytic enzyme.

Cystalysin is a C(beta)-S(gamma) lyase from the oral pathogen Treponema denticola catabolyzing L-cysteine to produce pyruvate, ammonia and H(2)S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5'-phosphate (PLP)-dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 A resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S-containing amino acid substrates yielding H(2)S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin-L-aminoethoxyvinylglycine (AVG) complex revealed a 'dead end' ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin-AVG complex may provide the chemical basis for rational drug design.

Effects of 0.12% chlorhexidine gluconate on experimental gingivitis in non-human primates: clinical and microbiological alterations.

OBJECTIVE: This study examined the efficacy of 0.12% chlorhexidine gluconate (Peridex) to reduce gingival inflammation in the absence of mechanical hygiene and its effect on the oral microbial ecology in a non-human primate (NhP) model of gingivitis. DESIGN: Twelve NhP were stratified based on existing inflammation into two groups of six NhP per group. Oral hygiene was performed on both groups so as to reach a level of gingival health (BOP < or = 0.3) at the conclusion of the hygiene phase. One group received 30 ml of 0.12% chlorhexidine gluconate twice daily 7 days/week, and a second group received 30 ml of placebo (distilled water colored to match the active) using the same regimen for 10 weeks. MEASUREMENT OUTCOMES: Clinical parameters including plaque (PLI), pocket depth (PD), attachment level (AL), and bleeding on probing (BOP) were evaluated at 2-week intervals. Subgingival plaque samples were collected by paper point at 2-week intervals and cultured for predominant cultivable bacteria. RESULTS: By week 2, there was a difference in BOP between the groups, which reached statistical significance by week 4. This difference in BOP was maintained throughout the course of the study. Chlorhexidine gluconate (0.12%) had no significant effect on PLI, PD, or AL; although PD was greater in the placebo group after week 2 and throughout the study. Microbiologically, at week 4, the treatment group had a reduction in total bacterial counts, as well as Gram positive bacteria, and total black pigmented bacteria, compared to the placebo group. However, only the differences in Actinomyces spp. reached significance. Interestingly, when both groups received only one treatment/day on the weekends (i.e., day 6 and 7), an associated loss of statistically significant differences between the two groups was observed. Additional experiments dosing the non-human primates once daily, 5 days/week yielded no significant differences in clinical parameters, including bleeding, when compared with the placebo group. CONCLUSION: Non-human primates provided a model system of gingivitis for testing antimicrobial agent effects on the subgingival ecology and accompanying inflammatory responses. Chlorhexidine gluconate (0.12%), even in the absence of mechanical hygiene, was effective in inhibiting clinical signs of inflammation, associated with alterations in the subgingival microbial ecology, most notably Actinomyces spp.

Gingival crevicular fluid inflammatory mediators and bacteriology of gingivitis in nonhuman primates related to susceptibility to periodontitis.

The hypothesis to be tested was that the microbiota and resulting local host inflammatory response characteristics in oral conditions of high levels of chronic gingival inflammation increases susceptibility to progressing periodontitis. This study used cynomolgus monkeys, Macaca fascicularis (nonhuman primates), with high and low levels of long-standing gingival inflammation to define the profiles of gingival crevicular fluid mediators, cytokines and immunoglobulins; describe the subgingival microbiota; and evaluate their susceptibility to ligature-induced periodontitis. Sixteen nonhuman primates were stratified into two groups (HI, LO) based upon Bleeding Index as a measure of the natural level of inflammation (HI = 1.26 +/- 0.45; LO = 0.22 +/- 0.16). The host mediator levels, subgingival microbiota, and clinical characteristics of the LO and HI groups were compared after 30 days of oral hygiene, during a 30 day experimental gingivitis (7, 14, and 30 days), and during periodontitis (30, 60, and 90 days). The results demonstrated that nonhuman primates with high levels of long-standing gingival inflammation when compared to those nonhuman primates with low inflammation show: 1) different inflammatory mediator profiles in gingival crevicular fluid (particularly for immunoglobulin A (IgA) and IgG levels), 2) a different quantitative and qualitative subgingival microbiota; and 3) a similar progression of periodontitis. Thus, while variations in host inflammatory responses to local factors exist in the nonhuman primates, an extensive subgingival challenge (such as ligation) may negate these individual differences.

Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts.

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.

Isolation and chemical analysis of a lipopolysaccharide from the outer membrane of the oral anaerobic spirochete Treponema pectinovorum.

Isolation of a putative lipopolysaccharide from the surface of the oral treponeme, Treponema pectinovorum, revealed it to contain larger amounts of 3-deoxy-D-manno-octulosonic acid compared with other oral Treponema species. This molecule was isolated from the outer membrane of T. pectinovorum and had chemical characteristics of a putative lipopolysaccharide. The yield of lipopolysaccharide was between 0.6% and to 1.1% of the bacterial dry weight. The purified molecule was resistant to the action of proteinases and consisted of both sugars and lipids. 3-Deoxy-D-manno-octulosonic acid and hexoses accounted for 6.1-8.7% and 17.6-20.2%, respectively of the dry weight. Carbohydrate compositional analysis revealed the presence of glucose, galactose, 2-acetamido-2-deoxy-glucose, rhamnose and 6-deoxy-talose in the molar ratio of 1.00:0.96:0.19:0.88:0.98, respectively. No heptose was detected. The fatty acid analysis determined the presence of straight chain, C13:00, C14:00, C15:00 and C17:00 acids, as well as branched chain, C13:00, C14:00 and two species of C15:00, acids. Electrophoretic analysis indicated that the lipopolysaccharide was present as two major species.

Hemoxidation and binding of the 46-kDa cystalysin of Treponema denticola leads to a cysteine-dependent hemolysis of human erythrocytes.

Cystalysin, a 46-kDa protein isolated from the cytosol of Treponema denticola, was capable of both cysteine dependent hemoxidation and hemolysis of human and sheep red blood cells. The activities were characteristic of a cysteine desulfhydrase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting analysis of the interaction of cystalysin with the red blood cells revealed an interaction of the protein with the red blood cell membrane. Substrates for the enzyme (including L-cysteine and beta-chloroalanine) enhanced the interaction, which occurred with both whole red blood cells as well as with isolated and purified red blood cell ghosts. SDS-PAGE and western immunoblotting employing anti-hemoglobin serum revealed that, during the hemoxidative events, the hemoglobin molecule associated with the red blood cell membrane, forming putative Heinz bodies. Spectrophotometric analysis of the hemoxidative events (cystalysin + cysteine + red blood cells) revealed a chemical modification of the native hemoglobin to sulfhemoglobin and methemoglobin. Hemoxidation also resulted in the degradation of both the red blood cell alpha- and beta-spectrin. The results presented suggest that the interaction of cystalysin with the red blood cell membrane results in the chemical oxidation of the hemoglobin molecule as well as an alteration in the red blood cell membrane itself.

A multistate nosocomial outbreak of Ralstonia pickettii colonization associated with an intrinsically contaminated respiratory care solution.

From 1 February through 30 April 1998, 4 hospitals reported a total of 34 patients colonized with Ralstonia pickettii. All but 1 had been exposed to 0.9% saline solution manufactured by 1 company (Modudose; Kendall, Mainsfield, MA), which was used during endotracheal suctioning. Culture of saline solution from previously unopened vials yielded R. pickettii. All available product and patient isolates were genotypically related by pulsed-field gel electrophoresis (PFGE) analysis. The contaminated saline solution was manufactured at the same plant that had been associated with a similar outbreak in 1983. The 1983 and 1998 R. pickettii isolates were unrelated, as determined by PFGE. In both 1983 and 1998, a 0. 2-microm cartridge filter was used for terminal sterilization. The detection of R. pickettii should alert hospital personnel to the possibility of product contamination. In this outbreak, prompt notification of public health agencies resulted in rapid notification of other health care providers, which likely prevented additional outbreaks.