Non-adherent Bacteria Research Articles

Biofilm Formation is Durably Prevented on Pre-Fabricated Antibiotic Cement Spacers Compared to Cobalt Chrome and Polyethylene

BackgroundA 2-stage revision remains the standard for managing chronic periprosthetic joint infection. Despite multiple spacer options, whether a particular one better resists biofilm formation remains unclear. Prefabricated polymethylmethacrylate (PMMA) articulating spacers containing antibiotics and a proprietary pore structure were developed to increase antibiotic elution characterized by a rapid burst phase for the initial one to two days and an extended slow-release phase for > 28 days. This in vitro study determined whether biofilm formation is prevented during the initial rapid burst phase and/or the slow-release phase. MethodsS. aureus-Xen36 was incubated in 1.5 mL of Luria-Bertani broth with PMMA discs with the proprietary pore structure either with or without gentamycin and vancomycin or with ‘Hoffman style’ positive-control discs (ultra-high molecular weight polyethylene or cobalt-chrome). Nonadherent bacteria were removed by three phosphate buffered saline rinses every 20 to 24 hours. Planktonic bacterial growth in the culture broth and biofilm formation on the discs were measured by colony forming unit (CFU) counting and resazurin reduction assays. Experiments were repeated > four times. ResultsNo detectable planktonic bacterial growth or biofilm formation occurred in cultures containing PMMA with antibiotics (≤ 15 CFUs/disc), whereas biofilms formed on PMMA without antibiotics, ultra-high molecular weight polyethylene, and cobalt-chrome (1 × 107 to 4 × 108 CFUs/disc, P < 0.0001). Biofilm formation was confirmed by a 100-fold decrease in sensitivity to vancomycin. To determine whether the antibiotic slow-release phase is sufficient to block biofilm formation, PMMA discs with antibiotics were preeluted for 14 days with multiple saline changes prior to bacterial inoculation. After antibiotic elution, still no detectable biofilms formed on PMMA discs with antibiotics (≤ 15 CFUs/disc, P < 0.0001). ConclusionsAntibiotic release during both the initial and slow-release phases prevented biofilm formation on PMMA with the proprietary pore structure. This may translate into improved infection eradication rates clinically.

Clue Cells and Pseudo Clue Cells in Different Morphotypes of Bacterial Vaginosis.

IntroductionClue cells (epithelial cells heavily covered with adherent bacteria) are an accepted clue to the diagnosis of bacterial vaginosis. However, the exact morphologic criteria of clue cells and bacterial adherence were never elaborated.Materials and MethodsWe investigated adhesive and cohesive patterns of main microbiota groups in vaginal discharge using fluorescence in situ hybridization (FISH). Samples from 500 women diagnosed with bacterial vaginosis and positive for clue cells with classic microscopy were collected from 42 gynecologic practices in Berlin and reexamined in our FISH laboratory for the spatial distribution of Bifidobacteriaceae, Gardnerella, Fannyhessea vaginae (Atopobium); low G+C (guanine+cytosine) bacteria, lactobacilli, Lactobacillus iners; Lactobacillus crispatus, Gamma-Proteobacteria; and Enterobacteriaceae, Prevotella–Bacteroides, Veillonella, and Coriobacterium groups.ResultsBacterial taxa present in vaginal smears were not accidentally assembled according to their relative abundance but were built in group-specific distribution patterns, which can be well described by two features: cohesiveness to each other and adherence to epithelial cells. Accordingly, four patterns can be distinguished: dispersed (non-adherent bacteria), dispersed adherent bacteria, cohesive (non-adherent) bacteria, and cohesive adherent bacteria. Direct cohesive adherence to the epithelial cells representing true clue cells was unique for Gardnerella species and observed only in 56% of the investigated samples. In the remaining vaginal samples, the epithelial cells were mechanically entrapped in bacterial masses, and the composition was unrelated to the epithelial cell surface, building non-adherent pseudo clue cells. The proportion of women with true clue cells in their samples from different gynecologic practices varied from 19% to 80%.DiscussionTaxon indifferent imaging is inadequate for the exact analysis of the microbial layer adjacent to the vaginal epithelial cells. Morphologically seen bacterial vaginosis is a mix of at least two different conditions: biofilm vaginosis and bacterial excess vaginosis.

Silver carboxylate-doped titanium dioxide-polydimethylsiloxane coating decreases Cutibacterium acnes adherence and biofilm formation on polyether ether ketone

BACKGROUND CONTEXTCutibacterium acnes (C. acnes) is a gram-positive facultative anaerobe found in the deep sebaceous follicles of the skin on the shoulder and back. C. acnes has been increasingly recognized as a pathogen in spinal surgical site infection (SSI) especially in the presence of instrumentation. PURPOSEThis study assesses whether a silver carboxylate-doped titanium dioxide-polydimethylsiloxane (TiO2-PDMS) coating can decrease C. acnes adherence and biofilm formation on PEEK and four other commonly used spinal implant materials, stainless steel, cobalt chromium, titanium, and titanium alloy. STUDY DESIGNWe compared the adherence of C. acnes over 24 hours between uncoated, 95:5 TiO2 to PDMS ratio with 10× silver carboxylate coating and a 100% silver carboxylate coating on each implant material, which were uniformly saw cut and sterilized. Implants were then subjected to scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). METHODSSamples were coated using 95:5 TiO2-PDMS 10× silver carboxylate, 100% silver carboxylate, or left uncoated. C. acnes was applied onto the samples and allowed to adhere for periods of 4, 8, 12, 16, or 20 hours. Nonadherent bacteria were then washed from the samples. These samples were then allowed to continue incubating for a total of 24 hours. SEM and confocal laser scanning microscope were used to visualize all samples for the presence of biofilm and quantification of C. acnes adherence at each time point. RESULTSThe 95:5 TiO2-PDMS 10× silver carboxylate coating was able to significantly decrease C. acnes adherence on PEEK after 8, 12, 16, and 20 hours of adherence. No statistical difference was found between the 95:5 TiO2-PDMS 10× silver carboxylate coating and the 100% silver carboxylate positive control. We previously observed extensive C. acnes biofilm formation on uncoated PEEK, but none on PEEK coated with either the 95:5 TiO2-PDMS 10× silver carboxylate or 100% Ag coating . Furthermore, no biofilm formation was observed on stainless steel, cobalt chromium, titanium, and titanium alloy coated with 95:5 TiO2-PDMS 10× silver carboxylate or 100% Ag coating. CONCLUSIONA 95:5 TiO2-PDMS 10× silver carboxylate coating decreases C. acnes adhesion and prevents biofilm formation on PEEK and other common orthopedic implant materials. CLINICAL SIGNIFICANCEA 95:5 TiO2-PDMS 10× silver carboxylate coating may help decrease spinal SSI due to C. acnes, especially in procedures with instrumentation.

Lactobacilli postbiotics reduce biofilm formation and alter transcription of virulence genes of Aggregatibacter actinomycetemcomitans.

Periodontitis is characterized by a dysbiotic microbial community and treatment strategies include the reestablishment of symbiosis by reducing pathogens abundance. Aggregatibacter actinomycetemcomitans (Aa) is frequently associated with rapidly progressing periodontitis. Since the oral ecosystem may be affected by metabolic end-products of bacteria, we evaluated the effect of soluble compounds released by probiotic lactobacilli, known as postbiotics, on Aa biofilm and expression of virulence-associated genes. Cell-free pH-neutralized supernatants (CFS) of Lactobacillus rhamnosus Lr32, L. rhamnosus HN001, Lactobacillus acidophilus LA5, and L. acidophilus NCFM were tested against a fimbriated clinical isolate of Aa JP2 genotype (1×107 CFU/well) on biofilm formation for 24hr, and early and mature preformed biofilms (2 and 24hr). Lactobacilli CFS partially reduced Aa viable counts and biofilms biomass, but did not affect the number of viable non-adherent bacteria, except for LA5 CFS. Furthermore, LA5 CFS and, in a lesser extent HN001 CFS, influenced Aa preformed biofilms. Lactobacilli postbiotics altered expression profile of Aa in a strain-specific fashion. Transcription of cytolethal distending toxin (cdtB) and leukotoxin (ltxA) was downregulated by CFS of LA5 and LR32 CFS. Although all probiotics produced detectable peroxide, transcription of katA was downregulated by lactobacilli CFS. Transcription of dspB was abrogated by LR32 and NCFM CFS, but increased by HN001, whereas expression of pgA was not affected by any postbiotic. Our data indicated the potential of postbiotics from lactobacilli, especially LA5, to reduce colonization levels of Aa and to modulate the expression of virulence factors implicated in evasion of host defenses.

Initial Bacterial Adhesion and Biofilm Formation on Aligner Materials.

The present study aims to assess the initial bacterial adhesion and biofilm formation on different aligner materials. A total of four different aligner materials, CA-medium (CAM), copolyester (COP), Duran (DUR), Erkodur (ERK), were tested. Stimulated human saliva was obtained from six healthy volunteers. Salivary bacteria were harvested by centrifugation, and 1 mL of the salivary suspension was injected onto each sample surface for 2 h and 3 days, respectively. The samples were then washed twice with 5 mL 0.9% NaCl solution, and non-adherent bacteria were removed. The adherent microorganisms were dislodged from the sample surfaces after ultrasonication for 4 min in 1 mL 0.9% NaCl on ice. After the incubation of the adherent salivary bacteria under both aerobic and anaerobic conditions on Columbia blood agar plates at 37 °C and 5% CO2 and in anaerobic jars overnight, several dilutions thereof were used for the determination of CFUs. This protocol was applied three times, obtaining an average of nine independent measurements for each material group. Overall, the differences between the tested aligner materials as well as between the materials and controls were not of statistical significance (p > 0.05). Regarding initial bacterial attachment and biofilm formation, the tested aligner materials are comparable to enamel and metal orthodontic brackets and can be therefore considered for clinical use. The four tested aligner materials CAM, COP, DUR, ERK showed no significant differences in initial microbial attachment and biofilm formation of aerobic and anaerobic species compared to enamel and conventional brackets.

Lipoxin A4 promotes reduction and antibiotic efficacy against Pseudomonas aeruginosa biofilm

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium commonly found in wound infections and airways of cystic fibrosis patients. P. aeruginosa readily forms biofilms which can reduce the efficacy of antibiotics used to eradicate the pathogen. We have previously shown that a Specialized Pro-resolving Mediator (SPM), Lipoxin A4 (LxA4) is a quorum sensing inhibitor which can reduce P. aeruginosa virulence. In this study, we examined the direct actions of LxA4 and RvD2 on P. aeruginosa biofilm formation and virulence gene expression. The influence of LxA4 on antibiotic efficacy and the combined effects on biofilm formation were also investigated. LxA4 and RvD2 reduced P. aeruginosa biofilm formation and virulence gene expression. LxA4 increased ciprofloxacin inhibition on biofilm formation but did not affect ciprofloxacin’s action on non-adherent bacteria. On the other hand, LxA4 increased bacterial killing action of imipenem but did not affect imipenem’s action on biofilm. We also found that LxA4 can increase ciprofloxacin’s bacterial killing ability in established biofilm. Together these results suggest that LxA4 has direct effects on P. aeruginosa biofilm formation and can increase antibiotic efficacy directly.

Early adherence and biofilm formation of Cutibacterium acnes (formerly Propionibacterium acnes) on spinal implant materials

BACKGROUNDCutibacterium acnes (C. acnes) is associated with infection following shoulder and spine surgery due to follicular pore concentrations in these anatomic regions. It has been established that it can form biofilms on surgical implant materials, which may contribute to its role in perioperative infection, but its behavior of early colonization on those materials is not yet well understood. PURPOSEThe purpose of this study was to evaluate the time to adherence and subsequent biofilm formation of C. acnes in the first 24 hours on implant materials commonly used in spinal surgery. STUDY DESIGNWe compared the colonization and behavior of C. acnes over time when applied to five commonly used spine implant materials – polyether ether ketone (PEEK), cobalt chromium (CC), stainless steel (SS), titanium, and titanium alloy. METHODSC. acnes was applied onto the samples of PEEK, CC, SS, titanium, and titanium alloy, and allowed to adhere for periods of 4, 8, 12, 16, and 20 hours. Nonadherent bacteria were then washed from the samples. These samples were then allowed to continue incubating for a total 24 hours. Scanning electron microscopy and confocal laser scanning microscopy were used to visualize all samples for the presence and quantification of C. acnes adherence at each time period. Subsequent transition to biofilm formation on these samples was assessed via scanning electron microscopy at each time period. RESULTSThe PEEK specimens exhibited the highest amount of surface biological burden in the first 24 hours compared with the other materials, which displayed little or no adherence. Rapid biofilm formation first observed at 8 hours of allowed adhesion on PEEK, whereas no significant biofilm formation was seen on the other materials during the observed time period. CONCLUSIONSAlthough C. acnes is known to have a slow proliferation rate, the results of this investigation demonstrate that it can rapidly adhere to and form a biofilm on PEEK. These data suggest that the use of PEEK implants placed during spinal surgery may facilitate early intraoperative colonization, and subsequent infection, compared with metallic implants. CLINICAL SIGNIFICANCEThe findings of this study suggest that PEEK may prove to be problematic as a choice of implant material in cases were C. acnes infection is a possibility.

2604. Changes in a Fatty Acid Kinase Associated with Daptomycin (DAP) Resistance Lead to Increased Collagen Binding and Biofilm Formation in Enterococcus faecalis

BackgroundEnterococci are a major cause of healthcare-associated infections with limited treatment options. We previously identified that mutations in dak (a gene encoding a putative fatty acid kinase), ace (a collagen adhesin) and the YxdJK stress response system are associated with DAP resistance (DAP-R) in E. faecalis (Efs) in the absence of a functional LiaFSR system. Here, we examined the role of DAK in pathogenesis by examining the ability of the mutants to produce biofilm and bind to collagen, an important protein of the extracellular matrix.MethodsPreviously, the Efs strain OG1RFΔliaR (inactive LiaFSR system, DAP susceptible) was adapted to make a DAP-R derivative (mutations in yxdK, dak, and ace), and the mutant OG1RFΔliaRΔc-dak, lacking the C-terminal domain of dak, and its complement OG1RFΔliaRΔc-dak::c-dak were constructed to study the dak mutation in isolation. Biofilm formation (BF) for the above strains was assayed after growth in tryptic soy broth with glucose in 96-well plates at 37° C for 24 hours. Bacteria were fixed with Bouin’s fixative, stained with crystal violet, and biofilm was quantitated by absorbance at 570 nm. For collagen adherence, 96-well plates were coated with 10 μg/well type I collagen, with 2% bovine serum albumin (BSA) as a control. Bacteria grown at 46° C (to induce ace expression) were added at OD 600 nm of 1.0 and allowed to bind for 2 hours. Non-adherent bacteria were removed by washing, cells were fixed, stained, and quantified as above.ResultsWhen compared with OG1RFΔliaR, the DAP-R derivative exhibited a significant increase in BF (4.6 vs. 2.4, P < 0.001). This enhanced biofilm phenotype was also seen in the OG1RFΔliaRΔc-dak mutant (6.1 vs. 2.4, P < 0.001), and reverted on complementation of the full-length dak in its native chromosomal location (2.4 vs. 2.6, p = 0.72). DAK was also found to impact adherence to collagen, with OG1RFΔliaRΔc-dak showing increased binding to collagen when compared with OG1RFΔliaR (7.9 vs. 3.4, P < 0.001), a phenotype which reverted on complementation (7.9 vs. 1.2, P < 0.001).ConclusionChanges in an enzyme involved in DAP adaptation lead to biofilm formation and adherence to extracellular matrix proteins, potentially enhancing virulence in the setting of DAP-R. Disclosures All authors: No reported disclosures.

Surface Immobilization of Nano-Silver on Polymeric Medical Devices to Prevent Bacterial Biofilm Formation.

Bacterial biofilm on medical devices is difficult to eradicate. Many have capitalized the anti-infective capability of silver ions (Ag+) by incorporating nano-silver (nAg) in a biodegradable coating, which is then laid on polymeric medical devices. However, such coating can be subjected to premature dissolution, particularly in harsh diseased tissue microenvironment, leading to rapid nAg clearance. It stands to reason that impregnating nAg directly onto the device, at the surface, is a more ideal solution. We tested this concept for a corneal prosthesis by immobilizing nAg and nano-hydroxyapatite (nHAp) on poly(methyl methacrylate), and tested its biocompatibility with human stromal cells and antimicrobial performance against biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Three different dual-functionalized substrates—high Ag (referred to as 75:25 HAp:Ag); intermediate Ag (95:5 HAp:Ag); and low Ag (99:1 HAp:Ag) were studied. The 75:25 HAp:Ag was effective in inhibiting biofilm formation, but was cytotoxic. The 95:5 HAp:Ag showed the best selectivity among the three substrates; it prevented biofilm formation of both pathogens and had excellent biocompatibility. The coating was also effective in eliminating non-adherent bacteria in the culture media. However, a 28-day incubation in artificial tear fluid revealed a ~40% reduction in Ag+ release, compared to freshly-coated substrates. The reduction affected the inhibition of S. aureus growth, but not the P. aeruginosa. Our findings suggest that Ag+ released from surface-immobilized nAg diminishes over time and becomes less effective in suppressing biofilm formation of Gram-positive bacteria, such as S. aureus. This advocates the coating, more as a protection against perioperative and early postoperative infections, and less as a long-term preventive solution.

BLEEDING UPON PROBING PATIENTS WITH LOCALIZED AGGRESSIVE PERIODONTITIS

Ponte Academic JournalJan 2018, Volume 74, Issue 1 BLEEDING UPON PROBING PATIENTS WITH LOCALIZED AGGRESSIVE PERIODONTITISAuthor(s): Gulden EresJ. Ponte - Jan 2018 - Volume 74 - Issue 1 doi: 10.21506/j.ponte.2018.1.3 Abstract:It is suggested that the affected periodontal pockets of the patients with localized aggressive periodontitis (LAgP) contain a specific flora, composed of mainly non-adherent bacteria. Most of the bacteria comprising this specific flora are gram-negative anaerobic or capnophilic, and saccharolytic rods. Porphyromonas gingivalis (P. gingivalis), which is asaccharolytic, either has not been detected in LAgP at all or has been reported in very low quantities. Contradictory results are reported on the quantities of spirochetes in LAgP lesions. Besides many studies reporting that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is one of the principal species causing LAgP, there are other studies reporting that it has not been detected in LAgP or has been detected in very low quantities. The objective of the study was to investigate the correlation between the prevalences of A. actinomycetemcomitans and of other recognized periodontopathogen species as well as spirochetes and bleeding upon probing in the regions, where the loss of attachment is observed in a group of patients with LAgP. Therefore, a study patient group was formed consisting of a total of 12 patients, comprising of 10 females and 2 males in the age range from 14 to 25, presenting for treatment to Ankara University Faculty of Dentistry, and who were diagnosed with LAgP. The patients were required not to have systemic diseases, not to have defective or overhanging restorations, and not to have a history of antibiotic use in the recent 3 months. The bleeding status of the patients, their pocket depths, and the degrees of attachment in the present pockets were identified, and then recorded. On the dental examinations, primarily, the bleeding status and the pocket depths as well as the degrees of attachment of the lesions demonstrated in the radiographies were identified and then recorded. After two weeks, by means of sterile paper cones, the samples of subgingival plaques were collected from the pockets deeper than 5 mm, which were present in the affected regions. Then, these samples were transferred to a 1 ml phosphate buffered saline (PBS) solution. Following this process, the bacterial concentrations and the proportion of the spirochetes were determined by using a phase-contrast microscope. The remaining materials were investigated by the DNA-RNA blot hybridization method. The results were analyzed statistically. The results of the analysis demonstrate that higher frequencies of spirochetes and Fusobacterium nucleatum were detected in the pockets, where bleeding developed on probing in patients with LAgP. A. actinomycetemcomitans was detected intensely in these pockets contrary to what is expected. Download full text:Check if you have access through your login credentials or your institution Username Password

Reduced ability to detect surface-related biofilm bacteria after antibiotic exposure under in vitro conditions

Background and purpose — Antibiotic treatment of patients before specimen collection reduces the ability to detect organisms by culture. We investigated the suppressive effect of antibiotics on the growth of non-adherent, planktonic, and surface-related biofilm bacteria in vitro by using sonication and microcalorimetry methods.Patients and methods — Biofilms of Staphylococcus aureus, S. epidermidis, Escherichia coli, and Propionibacterium acnes were formed on porous glass beads and exposed for 24 h to antibiotic concentrations from 1 to 1,024 times the minimal inhibitory concentration (MIC) of vancomycin, daptomycin, rifampin, flucloxacillin, or ciprofloxacin. The beads were then sonicated to dislodge biofilm, followed by culture and measurement of growth-related heat flow by microcalorimetry of the resulting sonication fluid.Results — Vancomycin did not inhibit the heat flow of staphylococci and P. acnes at concentrations ≤1,024 μg/mL, whereas flucloxacillin at >128 μg/mL inhibited S. aureus. Daptomycin inhibited heat flow of S. aureus, S. epidermidis, and P. acnes at lower concentrations (32–128 times MIC, p < 0.001). Rifampin showed inconsistent results in staphylococci due to random emergence of resistance, which was observed at concentrations ≤1,024 times MIC (i.e. 8 μg/mL). Ciprofloxacin inhibited heat flow of E. coli at ≥4 times MIC (i.e. ≥ 0.06 μg/mL).Interpretation — Whereas time-dependent antibiotics (i.e. vancomycin and flucloxacillin) showed only weak growth suppression, concentration-dependent drugs (i.e. daptomycin and ciprofloxacin) had a strong suppressive effect on bacterial growth and reduced the ability to detect planktonic and biofilm bacteria. Exposure to rifampin rapidly caused emergence of resistance. Our findings indicate that preoperative administration of antibiotics may have heterogeneous effects on the ability to detect biofilm bacteria.

Dual modes of membrane binding direct pore formation by Streptolysin O.

Effector translocation is central to the virulence of many bacterial pathogens, including Streptococcus pyogenes, which utilizes the cholesterol-dependent cytolysin Streptolysin O (SLO) to translocate the NAD(+) glycohydrolase SPN into host cells during infection. SLO's translocation activity does not require host cell membrane cholesterol or pore formation by SLO, yet SLO does form pores during infection via a cholesterol-dependent mechanism. Although cholesterol was considered the primary receptor for SLO, SLO's membrane-binding domain also encodes a putative carbohydrate-binding site, implicating a potential glycan receptor in binding and pore formation. Analysis of carbohydrate-binding site SLO mutants and carbohydrate-defective cell lines revealed that glycan recognition is involved in SLO's pore formation pathway and is an essential step when SLO is secreted by non-adherent bacteria, as occurs during lysis of erythrocytes. However, SLO also recognizes host cell membranes via a second mechanism when secreted from adherent bacteria, which requires co-secretion of SPN but not glycan binding by SLO. This SPN-mediated membrane binding of SLO correlates with SPN translocation, and requires SPN's non-enzymatic domain, which is predicted to adopt the structure of a carbohydrate-binding module. SPN-dependent membrane binding also promotes pore formation by SLO, demonstrating that pore formation can occur by distinct pathways during infection.

PTH-140 Soluble Plantain Fibre Inhibits The Epithelial Adhesion Of Diarrhoeal Pathogens Clostridium Difficile, Salmonella And Enterotoxigenic E. Coli (etec) Through Interaction With The Intestinal Epithelium

IntroductionPathogen-related diarrhoea causes significant morbidity and mortality worldwide. Our studies have previously shown that soluble dietary fibre (non-starch polysaccharides, NSP), particularly from plantain bananas, can inhibit epithelial adherence of diarrhoeal...

Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.

Pamidronate Enhances Bacterial Adhesion to Bone Hydroxyapatite. Another Puzzle in the Pathology of Bisphosphonate-Related Osteonecrosis of the Jaw?

Bacterial colonization of the denuded bone in bisphosphonate-related osteonecrosis of the jaw suggests that bisphosphonates increase bacterial adhesion and biofilm formation. This study evaluated the adhesion of gram-positive and gram-negative bacteria on hydroxyapatite coated with pamidronate, one of the most potent bisphosphonates. Twenty-five Staphylococcus aureus and 25 Pseudomonas aeruginosa strains were cultured on pamidronate-coated and uncoated hydroxyapatite discs. After incubation, nonadherent bacteria were removed by rinsing and centrifugation. Formation of a biofilm was confirmed by confocal laser 3-dimensional and scanning electron microscopy. The number of bacterial colonies was counted using quantitative cultures and mean numbers were compared using the Mann-Whitney rank sum test (statistical significance defined as P ≤ .05). The Hartree-Fock method was used for the calculation of electron interactions between hydroxyapatite ions and pamidronate. Fold increases in the number of colonies formed by S aureus and P aeruginosa in the presence of pamidronate compared with controls were 7.19 ± 4.127 and 2.87 ± 0.622, respectively. Hartree-Fock analysis showed that the reactive NH3(+) group of pamidronate may act as a steric factor, facilitating anchoring of bacteria to the hydroxyapatite surface. Alternatively, the NH3(+) group may attract bacteria by direct electrostatic interaction. Increased bacterial adhesion in the presence of bisphosphonates can promote osteomyelitis in patients with bisphosphonate-related osteonecrosis of the jaw. There may be increased infection rates when bisphosphonates are used for stabilization of prostheses in joint arthroplasty and in osteotomies and open fractures in patients treated with bisphosphonates.

Microbiota of yellow grouper (Epinephelus awoora Temminck &amp; Schlegel, 1842) fed two different diets

The rapidly growing yellow grouper industry has experienced relatively severe bacterial disease problems in China. The proliferation of pathogens in fish can be suppressed by commensal microbiota. In this background, we used nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequence analysis to investigate microbiota in the skin, gills and intestines, including adherent bacteria and non-adherent bacteria in yellow grouper fed with natural diet and complete feed. A total of 21 bacterial species were identified using phylogenetic analysis. The gamma-Proteobacteria group (81.0%, 17 species) dominated the bacterial communities in yellow grouper completely. Others belonged to Firmicutes (9.5%, two species), Actinobacteria (4.75%, one species) and Verrucomicrobia (4.75%, one species). The higher similarities (above 91%) of the DGGE band patterns in skin, gill and intestinal-non-adherent bacteria between two groups of fish indicated that existed more stable microbial communities existed in these specifically ecological niches in yellow grouper. However, considerable differences existed between two intestinal-adherent bacteria (IAB) samples; that is, compared with natural diet fed yellow grouper, higher bacterial apparent species richness and possibly less abundance existed in IAB in fish fed with complete diets, probably indicating that the community structures in IAB were affected easily and significantly by diet.

The Impact of Inoculation Parameters on the Pathogenesis of Contact Lens–Related Infectious Keratitis

Contact lens wear predisposes to Pseudomonas aeruginosa keratitis, but the mechanisms involved remain unclear. An in vivo model was used to study lens inoculation conditions enabling disease. Custom-made hydrogel contact lenses were fitted to rats after incubation in P. aeruginosa approximately 10(11) cfu/mL (3 hours) or approximately 10(3) cfu/mL (24 hours). Another group was inadvertently inoculated with a suction pen previously used with high inocula, but rinsed in ethanol and stored dry (6 months). Some corneas were tissue paper-blotted to cause fluorescein staining before lens fitting. Contralateral eyes were untreated. Twenty-four hours after disease detection, lenses were transferred to naive rats or examined by confocal microscopy before homogenization to quantify viable bacteria. After lens removal, corneas were washed to collect nonadherent bacteria and were analyzed by immunohistochemistry. All eyes challenged with unworn contaminated lenses developed keratitis after approximately 7 to 10 days. Disease delay and severity were unaffected by inoculum parameters or tissue blotting but occurred sooner with lenses transferred from infected eyes ( approximately 2 days). Worn lenses and corneal washes contained infecting bacteria. Posterior, not anterior, lens surfaces harbored P. aeruginosa biofilms that penetrated the lens matrix. Diseased corneas showed an infiltration of phagocytes and T-lymphocytes. P. aeruginosa induces keratitis in this lens-wearing model after a single inoculation. Delayed disease onset was interesting considering the greater keratitis risk during extended wear. Infection did not require the disruption of corneal barrier function before lens wear and occurred without exposure to lens care solutions. The data suggest that keratitis involves biofilm formation or other bacterial adaptations in vivo.

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro

Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces.Methods Discs of titanium and steel were incubated in the presence of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Propionibacterium acnes in Mueller Hinton broth. Non-adherent bacteria were removed by repeated rinsing of the discs. 10 parallels of each disc were subjected to 1 of 4 methods for bacterial recovery: (A) sonication of the discs, (B) scraping of the discs using surgical blades followed by streaking of the blades onto agar plates, (C) scraping of the discs followed by vortex mixing of the surgical blades, and (D) scraping of the discs followed by sonication of the surgical blades. Quantitative bacterial cultures were performed for each sampling method.Results With the exception of S. epidermidis on steel, sonication efficiently and reliably dislodged biofilm bacteria. The scraping methods employed did not detach bacteria embedded in biofilm.Interpretation Scraping of metal surfaces is not an adequate method for sampling of biofilm bacteria in vitro.

The Role of Decay-accelerating Factor as a Receptor for Helicobacter pylori and a Mediator of Gastric Inflammation

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Enteropathogenic Escherichia coli (EPEC) effector-mediated suppression of antimicrobial nitric oxide production in a small intestinal epithelial model system

In vivo studies with the mouse-specific member of the attaching and effacing (A/E) family of pathogens raised the possibility that these non-invasive enteric pathogens can specifically inhibit inducible nitric oxide synthase (iNOS) expression to prevent the production of antimicrobial nitric oxide (NO). In this study we use polarized Caco-2 cells, a model of human small intestinal epithelia, to (i) demonstrate conclusively that an A/E member, human specific enteropathogenic Escherichia coli (EPEC), can inhibit cytokine-induced iNOS expression, (ii) show that this activity is dependent on the delivery of effector molecules into host cells and (iii) investigate the mechanism of inhibition. Analysis of the level of iNOS-related mRNA, protein and NO production demonstrated that EPEC can inhibit iNOS expression at the transcriptional, by direct and indirect mechanisms, and post-transcriptional levels. This transcriptional block was linked to the failure of the iNOS-related transcriptional factor NF-kappaB, but not STAT1, to undergo phosphorylation-associated activation. A selective pressure to prevent iNOS production was evidenced by the finding that iNOS activity had a potent antimicrobial effect on adherent but not non-adherent bacteria. Moreover, given the central role NF-kappaB plays in transcribing genes associated with early host immune responses, this inhibitory mechanism presumably represents an important role in pathogenesis. Our study also provides insights into the nature of NO production in response to bacterial infection as well as the role of the locus of enterocyte effacement (LEE)-encoded effector molecules in inhibiting iNOS expression.