Roland R. Arnold

Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus

ABSTRACT The localization of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with Pseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc and P. aeruginosa bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined the growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a P. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibit P. aeruginosa biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.

Critical Role of Apoptotic Speck Protein Containing a Caspase Recruitment Domain (ASC) and NLRP3 in Causing Necrosis and ASC Speck Formation Induced by Porphyromonas gingivalis in Human Cells

Periodontal disease is a chronic inflammatory disorder that leads to the destruction of tooth-supporting tissue and affects 10–20 million people in the U.S. alone. The oral pathogen Porphyromonas gingivalis causes inflammatory host response leading to periodontal and other secondary inflammatory diseases. To identify molecular components that control host response to P. gingivalis in humans, roles for the NLR (NBD-LRR) protein, NLRP3 (cryopyrin, NALP3), and its adaptor apoptotic speck protein containing a C-terminal caspase recruitment domain (ASC) were studied. P. gingivalis strain A7436 induces cell death in THP1 monocytic cells and in human primary peripheral blood macrophages. This process is ASC and NLRP3 dependent and can be replicated by P. gingivalis LPS and Escherichia coli. P. gingivalis-induced cell death is caspase and IL-1 independent and exhibits morphological features consistent with necrosis including loss of membrane integrity and release of cellular content. Intriguingly, P. gingivalis-induced cell death is accompanied by the formation of ASC aggregation specks, a process not previously described during microbial infection. ASC specks are observed in P. gingivalis-infected primary human mononuclear cells and are dependent on NLRP3. This work shows that P. gingivalis causes ASC- and NLRP3-dependent necrosis, accompanied by ASC speck formation.

Lactoferrin interaction with salmonellae potentiates antibiotic susceptibility in vitro

Interaction of lactoferrin (Lf) with the cell envelope (CE) and outer membrane (OM) of Salmonella typhimurium-type strain ATCC13311 was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western-blot analyses. The peroxidase-labeled bovine Lf (BLf) and human Lf both recognized a heat-modifiable protein with an estimated molecular mass of 38 kD in the OM. Simultaneous immunoblotting with an antiporin monoclonal antibody specific for a conserved porin domain in members of enterobacteriaceae confirmed that the Lf-binding protein is a porin. Such Lf-binding porin proteins (37-39 kD range) were readily detected in nine other common Salmonella species: S. dublin, S. panama, S. rostock, S. abony, S. hartford, S, kentucky, S. pullorum, S. thompson, and S. virchow. The latter six species also demonstrated one to three weak Lf-reactive bands of low molecular weight in their CE. The antibiotic susceptibility of Salmonella in the presence of Lf was examined. A mixture containing sub-minimum inhibitory concentration (MIC) levels of Lf (MIC/4) and cefuroxime (MIC/2) inhibited the bacterial growth. Lf strongly potentiated the action of erythromycin (eightfold), whereas it increased the activity only by two-fold for ampicillin, ciprofloxacin, chloramphenicol, and rifampicin; similarly, these antibiotics also reduced the MIC of BLf by twofold in S. typhimurium. Such antimicrobial potentiation was not observed with BLf mixtures containing cefalexin, gentamycin, or polymyxin B against strain ATCC13311. BLf and cefuroxime also demonstrated potentiation of varying degrees (two to 16-fold) with nine other Salmonella species. These data established the binding of Lf to porins in salmonellae and a potentiation effect of Lf with certain antibiotics.

Synergistic Pathogenicity of Porphyromonas gingivalis and Fusobacterium nucleatum in the Mouse Subcutaneous Chamber Model

Porphyromonas gingivalis and Fusobacterium nucleatum are often coisolated from sites of infection, such as suppurative apical periodontitis. The synergistic pathogenicity of mixed infection of P. gingivalis HG 405 with F. nucleatum PK 1594 was studied in the mouse subcutaneous chamber model in groups of seven animals. The minimal dose for P. gingivalis HG 405 that was required to infect 100% of the chambers was reduced by 1,000-fold when animals were inoculated in the same chamber with 1 x 10(9)F. nucleatum PK 1594 (p < 0.001). To benefit from the presence of the fusobacteria, P. gingivalis HG 405 had to be coinoculated; inoculation in separate chambers for the same animal had no such effect (p < 0.001). Subinfective F. nucleatum inocula also benefited from the association with P. gingivalis HG 405 and uniformly established an infection when this partner was present (p < 0.001). These results suggest that the frequent and natural coexistence of P. gingivalis and F. nucleatum in diseased sites may express such a synergism in successful establishment and survival of small inocula.

Influence of Lactoferrin on Host-Microbe Interactions

The antimicrobial spectrum of lactoferrin (Lf) includes statsis, cidal, cationic, phagocytic, and colonization/decolonization effects on susceptible microorganisms. The selective interaction of Lf with microbial surface seems to play an essential role in regulating many of these events. Lf bound to specific outer membrane (OM) pore-forming proteins (porins) of various enteric bacteria. The magnitude of Lf-microbe interaction showed a direct relationship with the bacterial susceptibility to Lf. Although most bacteria expressed porins, certain strains showed resistance to Lf effects and did not demonstrate specific Lf binding. This Lf “resistance” was attributed to the shielding of porin accessibility by the carbohydrate O-antigenic chains of lipopolysaccharide (LPS). Mutants with progressive deletions in O-side-chain and core polysaccharide demonstrated increased Lf binding and progressive susceptibility to Lf effects. Lf interaction with bacterial surface affected the porin channels in the OM and potentiated the diffusion of antibiotics. Lf also affected the intestinal colonization of Escherichia coli. Lf inhibited the expression of various bacterial fimbriae and also blocked bacterial interaction with host subepithelial matrix components, such as fibronectin, fibrinogen, various collagens, and laminin. Our studies suggested that specific interaction with cell target involved the anion-binding ligands of Lf, followed by coordinate binding and oxidation of Fe2+ to Fe3+ on bacterial surface. This metal transition in the presence of suitable oxygen species seems to fulfill the necessary components of Haber-Weiss reaction and promote a targeted radical generation, with iron (Fe) serving as the Lf prosthetic group on the bacterial surface.

Antibacterial surface properties of fluoride-containing resin-based sealants.

OBJECTIVES The aim of the present study was to determine the antibacterial properties of three resin-based pit and fissure sealant products: Clinpro (3M ESPE), Embrace (Pulpdent), and UltraSeal XT plus (Ultradent). METHODS The antibacterial effects of the sealants were tested in both an agar diffusion assay and a planktonic growth inhibition assay using Streptococcus mutans and Lactobacillus acidophilus. The materials were applied to paper and enamel disks in the former and on the side walls of 96-well microtiter plates on the latter. RESULTS All materials showed either diffusible or contact antibacterial effects in the agar diffusion assays. The effect was diminished when enamel disks were used as substrate. In the planktonic growth inhibition assay, Clinpro had its effect reduced, but retained activity against both bacteria over time. L. acidophilus was more sensitive than S. mutans to UltraSeal. Embrace retained antibacterial activity against both bacteria over time. CONCLUSIONS Within the limitations of this in vitro study it can be concluded that all materials are capable of contact inhibition of L. acidophilus and S. mutans growth. Embrace has the longer lasting antibacterial activity when in solution, especially against S. mutans.

Antimicrobial activity and local release characteristics of chlorhexidine diacetate loaded within the dental copolymer matrix, ethylene vinyl acetate

In vitro results are presented for a novel oral drug-delivery system ultimately intended for treatment of oral infections in immunocompromised patients. Test samples of ethylene vinyl acetate copolymer (EVA) containing chlorhexidine diacetate (CDA) showed desirable antimicrobial properties and steady, slow release into aqueous and other media after an initial burst of drug release in the first day of liquid exposure. By washing away this initial burst, the proposed mouthguard device should be capable of sustained delivery of locally effective CDA concentrations far below systemically toxic levels. A prolonged room temperature shelf-life of at least 1 year, and effectivity against a wide range of oral bacteria and Candida species was demonstrated. Drug loaded films showed a top-to-bottom asymmetry in drug release, but good lateral homogeneity, and a linear relationship between initial CDA loading concentration (from 0.63 to 10 wt %) and days 3-14 release rates in a static aqueous environment. The EVA matrix containing CDA appears to possess many suitable properties for localized oral delivery of sustained antimicrobial activity.

Periapical inflammation and bacterial penetration after coronal inoculation of dog roots filled with RealSeal 1 or Thermafil.

INTRODUCTION The purpose of this study was to subject 2 carrier-based root filling products to a 4-month microbial challenge in a dog model with histologic markers to assess periapical inflammation and bacterial penetration of the 2 filling materials. Histologic evidence of bacterial penetration and periapical inflammation were the outcome parameters used to compare the products. METHODS Teeth were aseptically prepared and then filled with carrier-based Resilon (RealSeal 1 [RS-1], n = 25) or with carrier-based gutta-percha (Thermafil, n = 25) and were left exposed for 4 months. The first control group received a coronal seal over either RS-1 or Thermafil root fillings (n = 8). A second control group was instrumented and left completely empty (n = 8). RESULTS Histologic evidence of periapical inflammation was observed in 29% of the Thermafil group and in 9% of the RS-1 group. This difference was only significant when controlling for a possible tooth position effect on inflammation presence (P < .05). Histologic evidence of bacterial penetration was present in 9% of the RS-1 group and in 70% of the Thermafil group. The difference in penetration rates between RS-1 and Thermafil was statistically significant when controlling for any dog or tooth position effects on bacterial penetration (P < .001). Furthermore, there was a statistically significant correlation between histologic evidence of inflammation and histologic evidence of infection (P = .002). CONCLUSIONS RS-1 appeared to resist bacterial penetration more effectively than Thermafil under the conditions of this study.

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice.

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI−/−) mice. We demonstrate that murine neutrophils expressed cGKIα. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI−/− bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI−/− than in WT. Enhanced chemotaxis was also observed with cGKI−/− peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI−/− vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI−/− than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI−/− (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI−/− (∼60%) than in WT (∼90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIα down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIα on the secretagogue responses are complex, depending on the granule subtype.

In Vitro Inhibition of Bacterial Growth Using Different Dental Adhesive Systems

OBJECTIVES This study evaluated the antibacterial potential of four different adhesive systems. METHODS & MATERIALS Gluma Comfort Bond + Desensitizer, Gluma Comfort Bond, iBond and One-Up Bond F were tested against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus and Actinomyces viscosus. The inhibition of growth by calibrated preparations was quantified by the measurement of zones of inhibition on bacterial lawns. Bactericidal activity was determined as reductions in recoverable colony-forming units in bacterial suspensions exposed to test preparations. RESULTS All the preparations exhibited detectable zones of inhibition for all target bacteria through six months. When the bactericidal action was evaluated, all the materials were able to kill all the tested bacteria when tested immediately after polymerization. After one week of aging, iBond was the only material that continued to kill all of the test strains.