Cathodic Voltage-controlled Electrical Stimulation Plus Prolonged Vancomycin Reduce Bacterial Burden of a Titanium Implant-associated Infection in a Rodent Model.

BACKGROUND: Cathodic voltage-controlled electrical stimulation (CVCES) of titanium implants, either alone or combined with a short course of vancomycin, has previously been shown to reduce the bone and implant bacterial burden in a rodent model of methicillin-resistant Staphylococcus aureus (MRSA) implant-associated infection (IAI). Clinically, the goal is to achieve complete eradication of the IAI; therefore, the rationale for the present study was to evaluate the antimicrobial effects of combining CVCES with prolonged antibiotic therapy with the goal of decreasing the colony-forming units (CFUs) to undetectable levels. QUESTIONS/PURPOSES: (1) In an animal MRSA IAI model, does combining CVCES with prolonged vancomycin therapy decrease bacteria burden on the implant and surrounding bone to undetectable levels? (2) When used with prolonged vancomycin therapy, are two CVCES treatments more effective than one? (3) What are the longer term histologic effects (inflammation and granulation tissue) of CVCES on the surrounding tissue? METHODS: Twenty adult male Long-Evans rats with surgically placed shoulder titanium implants were infected with a clinical strain of MRSA (NRS70). One week after infection, the rats were randomly divided into four groups of five: (1) VANCO: only vancomycin treatment (150 mg/kg, subcutaneous, twice daily for 5 weeks); (2) VANCO + 1STIM: vancomycin treatment (same as the VANCO group) coupled with one CVCES treatment (-1.8 V for 1 hour on postoperative day [POD] 7); (3) VANCO + 2STIM: vancomycin treatment (same as the VANCO group) coupled with two CVCES treatments (-1.8 V for 1 hour on POD 7 and POD 21); or (4) CONT: no treatment. On POD 42, the implant, bone, and peripheral blood were collected for CFU enumeration and histological analysis, where we compared CFU/mL on the implants and bone among the groups. A pathologist, blinded to the experimental conditions, performed a semiquantitative analysis of inflammation and granulation tissue present in serial sections of the humeral head for animals in each experimental group. RESULTS: The VANCO + 1STIM decreased the implant bacterial burden (median = 0, range = 0-10 CFU/mL) when compared with CONT (median = 5.7 × 10(4), range = 4.0 × 10(3)-8.0 × 10(5) CFU/mL; difference of medians = -5.6 × 10(4); p < 0.001) and VANCO (median = 4.9 × 10(3), range = 9.0 × 10(2)-2.1 × 10(4) CFU/mL; difference of medians = -4.9 × 10(3); p < 0.001). The VANCO + 1STIM decreased the bone bacterial burden (median = 0, range = 0-0 CFU/mL) when compared with CONT (median = 1.3 × 10(2), range = 0-9.4 × 10(2) CFU/mL; difference of medians = -1.3 × 10(2); p < 0.001) but was not different from VANCO (median = 0, range = 0-1.3 × 10(2) CFU/mL; difference of medians = 0; p = 0.210). The VANCO + 2STIM group had implant CFU (median = 0, range = 0-8.0 × 10(1) CFU/mL) and bone CFU (median = 0, range = 0-2.0 × 10(1) CFU/mL) that were not different from the VANCO + 1STIM treatment group implant CFU (median = 0, range = 0-10 CFU/mL; difference of medians = 0; p = 0.334) and bone CFU (median = 0, range = 0-0 CFU/mL; difference of medians = 0; p = 0.473). The histological analysis showed no deleterious effects on the surrounding tissue as a result of the treatments. CONCLUSIONS: Using CVCES in combination with prolonged vancomycin resulted in decreased MRSA bacterial burden, and it may be beneficial in treating biofilm-related implant infections. CLINICAL RELEVANCE: CVCES combined with clinically relevant lengths of vancomycin therapy may be a treatment option for IAI and allow for component retention in certain clinical scenarios. However, more animal research and human trials confirming the efficacy of this approach are needed before such a clinical recommendation could be made.

Cathodic Electrical Stimulation Combined With Vancomycin Enhances Treatment of Methicillin-resistant Staphylococcus aureus Implant-associated Infections.

BACKGROUND: Effective treatments for implant-associated infections are often lacking. Cathodic voltage-controlled electrical stimulation has shown potential as a treatment of implant-associated infections of methicillin-resistant Staphylococcus aureus (MRSA). QUESTIONS/PURPOSES: The primary purpose of this study was to (1) determine if cathodic voltage-controlled electrical stimulation combined with vancomycin therapy is more effective at reducing the MRSA bacterial burden on the implant, bone, and synovial fluid in comparison to either treatment alone or no treatment controls. We also sought to (2) evaluate the histologic effects of the various treatments on the surrounding bone; and to (3) determine if the cathodic voltage-controlled electrical stimulation treatment had an effect on the mechanical properties of the titanium implant as a result of possible hydrogen embrittlement. METHODS: Thirty-two adult male Long-Evans rats (Harlan Laboratories, Indianapolis, IN, USA) with surgically placed shoulder titanium implants were infected with a clinical strain of MRSA (NRS70). One week after infection, eight animals received a treatment of cathodic voltage-controlled electrical stimulation at -1.8 V versus Ag/AgCl for 1 hour (STIM), eight received vancomycin twice daily for 1 week (VANCO), eight received the cathodic voltage-controlled electrical stimulation and vancomycin therapy combined (STIM + VANCO), and eight served as controls with no treatment (CONT). Two weeks after initial infection, the implant, bone, and synovial fluid were collected for colony-forming unit (CFU) enumeration, qualitative histological analysis by a pathologist blinded to the treatments each animal received, and implant three-point bend testing. RESULTS: The implant-associated CFU enumerated from the STIM + VANCO (mean, 3.7 × 10(3); SD, 6.3 × 10(3)) group were less than those from the CONT (mean, 1.3 × 10(6); SD, 2.8 × 10(6); 95% confidence interval [CI] of difference, -4.3 × 10(5) to -9.9 × 10(3); p < 0.001), STIM (mean, 1.4 × 10(6); SD, 2.0 × 10(6); 95% CI of difference, -2.1 × 10(6) to -1.8 × 10(3); p = 0.002), and VANCO (mean, 5.8 x 10(4); SD, 5.7 × 10(4); 95% CI of difference, -6.4 × 10(4) to -1.7 × 10(4); p < 0.001) group. The bone-associated CFU enumerated from the STIM + VANCO group (6.3 × 10(1); SD, 1.1 × 10(2)) were less than those from the CONT (mean, 2.8 × 10(5); SD, 4.8 × 10(5); 95% CI of difference, -9.4 × 10(4) to -5.0 × 10(3); p < 0.001) and STIM (mean, 2.6 × 10(4); SD, 2.5 × 10(4); 95% CI of difference, -4.1 × 10(4) to -1.6 × 10(3); p < 0.001) groups. The VANCO group (4.3 × 10(5); SD, 6.3 × 10(2)) also had lower bone-associated CFU as compared with the CONT (mean 95% CI of difference, -9.3 × 10(4) to -4.5 × 10(3); p < 0.001) and STIM (95% CI of difference, -4.0 × 10(4) to -1.5 × 10(3); p < 0.001) groups. In comparison to the synovial fluid CFU enumerated from the CONT group (mean, 3.3 × 10(4); SD, 6.0 × 10(4)), lower synovial CFU were reported for both the STIM + VANCO group (mean, 4.6 × 10(1); SD, 1.2 × 10(2); 95% CI of difference, -4.9 × 10(3) to -3.0 × 10(2); p < 0.001) and the VANCO group (mean, 6.8 × 10(1); SD, 9.2 × 10(1); 95% CI of difference, -4.9 × 10(3) to -2.8 × 10(2); p = 0.007). The histological analysis showed no discernable deleterious effects on the surrounding tissue as a result of the treatments. No brittle fracture occurred during mechanical testing and with the numbers available, no differences in implant flexural yield strength were detected between the groups. CONCLUSIONS: In this rodent model, cathodic voltage-controlled electrical stimulation combined with vancomycin is an effective treatment for titanium implant-associated infections showing greater than 99.8% reduction in bacterial burden on the implant, surrounding bone, and synovial fluid as compared with the controls and the stimulation alone groups. CLINICAL RELEVANCE: Cathodic voltage-controlled electrical stimulation combined with vancomycin may enable successful treatment of titanium orthopaedic implant-associated infections with implant retention. Future studies will focus on optimization of the stimulation parameters for complete eradication of infection and the ability to promote beneficial host tissue responses.

Moraxella catarrhalis is susceptible to antimicrobial photodynamic therapy with Photofrin.

BACKGROUND AND OBJECTIVE: Moraxella catarrhalis is a significant cause of pediatric otitis media (OM), which is the most prevalent bacterial infection in children and primary reason for antibiotic administration in this population. Moreover, biofilm formation has been implicated as a primary mechanism of chronic or recurrent OM disease. As bacterial biofilms are inherently resistant to most antibiotics and these complex structures also present a significant challenge to the immune system, there is a clear need to identify novel antimicrobial approaches to treat OM infections. In this study, we evaluated the potential efficacy of antibacterial photodynamic therapy (aPDT) with porfimer sodium (Photofrin (PF)) against planktonic as well as biofilm-associated M. catarrhalis. MATERIALS AND METHODS: The bactericidal activity of aPDT with PF was assessed against multiple recent clinical isolates of M. catarrhalis grown planktonically as well as in biofilms. The bactericidal activity of PF-aPDT was quantified by enumeration of colony forming units post-treatment. The effect of aPDT on M. catarrhalis biofilms was further investigated with scanning electron microscopy (SEM) imaging. RESULTS: aPDT with PF significantly reduced M. catarrhalis viability. Although PF-aPDT caused higher killing in planktonic grown organisms (5-6 log kill), biofilm grown bacteria also demonstrated a statistically significant reduction in viable organisms (3-4 log decrease in recoverable bacteria) following treatment as compared to saline only controls (P < 0.01). SEM studies indicated the PF-aPDT treated bacteria exhibited prominent morphological changes with visibly distorted cell membranes. CONCLUSIONS: aPDT with PF elicits significant bactericidal activity against both planktonic and biofilm-associated M. catarrhalis, suggesting this technology warrants further analysis as a potential novel antimicrobial treatment for acute or recurrent OM.

The K1 capsular polysaccharide from Acinetobacter baumannii is a potential therapeutic target via passive immunization.

The emergence of extremely resistant and panresistant Gram-negative bacilli, such as Acinetobacter baumannii, requires consideration of nonantimicrobial therapeutic approaches. The goal of this report was to evaluate the K1 capsular polysaccharide from A. baumannii as a passive immunization target. Its structure was determined by a combination of mass spectrometric and nuclear magnetic resonance (NMR) techniques. Molecular mimics that might raise the concern for autoimmune disease were not identified. Immunization of CD1 mice demonstrated that the K1 capsule is immunogenic. The monoclonal antibody (MAb) 13D6, which is directed against the K1 capsule from A. baumannii, was used to determine the seroprevalence of the K1 capsule in a collection of 100 A. baumannii strains. Thirteen percent of the A. baumannii isolates from this collection were seroreactive to MAb 13D6. Opsonization of K1-positive strains, but not K1-negative strains, with MAb 13D6 significantly increased neutrophil-mediated bactericidal activity in vitro (P < 0.05). Lastly, treatment with MAb 13D6 3 and 24 h after bacterial challenge in a rat soft tissue infection model resulted in a significant decrease in the growth/survival of a K1-positive strain compared to that of a K1-negative strain or to treatment with a vehicle control (P < 0.0001). These data support the proof of principle that the K1 capsule is a potential therapeutic target via passive immunization. Other serotypes require assessment, and pragmatic challenges exist, such as the need to serotype infecting strains and utilize serotype-specific therapy. Nonetheless, this approach may become an important therapeutic option with increasing antimicrobial resistance and a diminishing number of active antimicrobials.

Characterization of a trifunctional glucosyltransferase essential for Moraxella catarrhalis lipooligosaccharide assembly.

The human respiratory tract pathogen Moraxella catarrhalis expresses lipooligosaccharides (LOS), glycolipid surface moieties that are associated with enhanced colonization and virulence. Recent studies have delineated the major steps required for the biosynthesis and assembly of the M. catarrhalis LOS molecule. We previously demonstrated that the glucosyltransferase enzyme Lgt3 is responsible for the addition of at least one glucose (Glc) molecule, at the ß-(1-4) position, to the inner core of the LOS molecule. Our data further suggested a potential multifunctional role for Lgt3 in LOS biosynthesis. The studies reported here demonstrate that the Lgt3 enzyme possesses two glycosyltransferase domains (A1 and A2) similar to that of other bifunctional glycosyltransferase enzymes involved in surface polysaccharide biosynthesis in Escherichia coli, Pasteurella multocida and Streptococcus pyogenes. Each Lgt3 domain contains a conserved DXD motif, shown to be involved in the catalytic activity of other glycosyltransferases. To determine the function of each domain, A1 (N-terminal), A2 (C-terminal) and double A1A2 site-directed DAD to AAA mutants were constructed and the resulting LOS phenotypes of these modified strains were analyzed. Our studies indicate that the Lgt3 N-terminal A1 catalytic domain is responsible for the addition of the first ß-(1-3) Glc to the first Glc on the inner core. The C-terminal catalytic domain A2 then adds the ß-(1-4) Glc and the ß-(1-6) Glc, confirming the bifunctional nature of this domain. The results from these experiments demonstrate that Lgt3 is a novel, multifunctional transferase responsible for the addition of three Glcs with differing linkages onto the inner core of M. catarrhalis LOS.

Antimicrobial Photodynamic Therapy with Chlorin e6 Is Bactericidal against Biofilms of the Primary Human Otopathogens.

Moraxella catarrhalis, Streptococcus pneumoniae, and nontypeable Haemophilus influenzae (NTHi) are ubiquitous upper respiratory opportunistic pathogens. Together, these three microbes are the most common causative bacterial agents of pediatric otitis media (OM) and have therefore been characterized as the primary human otopathogens. OM is the most prevalent bacterial infection in children and the primary reason for antibiotic administration in this population. Moreover, biofilm formation has been confirmed as a primary mechanism of chronic and recurrent OM disease. As bacterial biofilms are inherently metabolically recalcitrant to most antibiotics and these complex structures also present a significant challenge to the immune system, there is a clear need to identify novel antimicrobial approaches to treat OM infections. In this study, we evaluated the potential efficacy of antibacterial photodynamic therapy (aPDT) with the photosensitizer chlorin e6 (Ce6) against planktonic as well as biofilm-associated M. catarrhalis, S. pneumoniae, and NTHi. Our data indicate aPDT with Ce6 elicits significant bactericidal activity against both planktonic cultures and established biofilms formed by the three major otopathogens (with an efficacy of ≥99.9% loss of viability). Notably, the implementation of a novel, dual-treatment aPDT protocol resulted in this disinfectant effect on biofilm-associated bacteria and, importantly, inhibited bacterial regrowth 24 h posttreatment. Taken together, these data suggest this novel Ce6-aPDT treatment may be a powerful and innovative therapeutic strategy to effectively treat and eradicate bacterial OM infections and, significantly, prevent the development of recurrent disease.IMPORTANCE Otitis media (OM), or middle ear disease, is the most prevalent bacterial infection in children and the primary reason for antibiotic use and surgical intervention in the pediatric population. Biofilm formation by the major bacterial otopathogens, Moraxella catarrhalis, Streptococcus pneumoniae, and nontypeable Haemophilus influenzae, has been shown to occur within the middle ears of OM patients and is a key factor in the development of recurrent disease, which may result in hearing impairment and developmental delays. Bacterial biofilms are inherently impervious to most antibiotics and present a significant challenge to the immune system. In this study, we demonstrate that antimicrobial photodynamic therapy (aPDT) using the photosensitizer chlorin e6 elicits significant bactericidal activity versus planktonic and biofilm-associated otopathogens and supports further analyses of this novel, efficacious, and promising technology as an adjunctive treatment for acute and recurrent OM.

Draft Genome Sequence of Pediatric Otitis Media Isolate Streptococcus pneumoniae Strain EF3030, Which Forms In Vitro Biofilms That Closely Mimic In Vivo Biofilms.

Here, we report the draft genome sequence of Streptococcus pneumoniae EF3030, a pediatric otitis media isolate active in biofilm assays of epithelial colonization. The final draft assembly included 2,209,198 bp; the annotation predicted 2,120 coding DNA sequences (CDSs), 4 complete rRNA operons, 58 tRNAs, 3 noncoding RNAs (ncRNAs), and 199 pseudogenes.

Serum antibody response to Moraxella catarrhalis proteins in stringently defined otitis prone children.

BACKGROUND: Moraxella catarrhalis (Mcat) is a frequent pathogen of acute otitis media (AOM) in young children. Here we prospectively assessed naturally-induced serum antibodies to four Mcat vaccine candidate proteins in stringently defined otitis prone (sOP) and non-otitis prone (NOP) children age 6-36months old following nasopharyngeal (NP) colonization, at onset of AOM and convalescence from AOM. METHODS: Serum IgG and IgM antibody against recombinant Mcat proteins, oligopeptide permease A (OppA), outer membrane protein (OMP) CD, hemagglutinin (Hag), and PilA clade 2 (PilA2), were quantitated by ELISA. RESULTS: During NP colonization by Mcat all four antigens were immunogenic in both sOP and NOP children. However, sOP children had lower antibody responses than NOP children across age 6-36months, similar to our findings for protein vaccine candidates of Streptococcus pneumoniae (Spn) and Nontypeable Haemophilus influenzae (NTHi). sOP children displayed a later and lower peak of antibody rise than NOP children for all four antigens during NP colonization of Mcat. The age-dependent increase of antibody ranked as OppA>Hag5-9>OMP CD>PilA2 in both sOP and NOP children. Lower serum antibody levels to the Mcat antigens were measured in sOP compared to NOP children at the onset of AOM. We did not find a consistent significant increase of antibody at the convalescence phase after an AOM event. CONCLUSIONS: sOP children is a highly vulnerable population that mount lower serum antibody responses to Mcat candidate vaccine proteins compared to NOP children during asymptomatic NP carriage and at onset of AOM.

Antibiotics Enhance Prevention and Eradication Efficacy of Cathodic-Voltage-Controlled Electrical Stimulation against Titanium-Associated Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa Biofilms.

Periprosthetic joint infection (PJI) develops clinically, even with antibiotic treatment, and methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are predominant causes of these infections. Due to biofilm formation, antibiotic treatment for patients with PJI can perpetuate resistance, further complicating the use of noninvasive treatments. This study evaluated cathodic-voltage-controlled electrical stimulation (CVCES) of titanium, in combination with a clinically relevant antibiotic, to synergistically prevent MRSA and P. aeruginosa PJIs by inhibiting bacterial adherence or as a treatment for eradicating established biofilms. CVCES of -1.0 V, -1.5 V, or -1.8 V (versus Ag/AgCl), with or without vancomycin for MRSA or gentamicin for P. aeruginosa, was applied to sterile titanium incubated with cultures to evaluate prevention of attachment or eradication of preestablished biofilms. Treatments were 24 h long and included open-circuit potential controls, antibiotic alone, CVCES, and CVCES plus antibiotic. Biofilm-associated and planktonic CFU were enumerated. In general, CVCES at -1.8 V alone or with antibiotic completely eradicated biofilm-associated CFU for both strains, and these parameters were also highly effective against planktonic bacteria, resulting in a >6-log reduction in MRSA and no detectable planktonic P. aeruginosa All CFU were reduced ∼3 to 5 logs from controls for prevention CVCES plus antibiotics at -1.0 V and -1.5 V against MRSA. Remarkably, there were no detectable P. aeruginosa CFU following prevention CVCES at -1.0 V or -1.5 V with gentamicin. Our results suggest that CVCES in combination with antibiotics may be an effective approach for prevention and treatment of PJI.IMPORTANCE Periprosthetic joint infections (PJIs) develop clinically in the presence of antibiotic therapies and are responsible for increased patient morbidity and rising health care costs. Many of these infections involve bacterial biofilm formation on orthopedic hardware, and it has been well established that these biofilms are refractory to most antibiotic treatments. Recent studies have focused on novel methods to prevent and eradicate infection. Cathodic-voltage-controlled electrical stimulation (CVCES) has previously been shown to be effective as a method for prevention and eradication of Gram-positive and Gram-negative infections. The present study revealed that the utility of CVCES for prevention and eradication of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa is enhanced in the presence of clinically relevant antibiotics. The synergistic effects of CVCES and antibiotics are effective in a magnitude-dependent manner. The results of this study indicate a promising alternative method to current PJI mitigation techniques.

Streptococcus pneumoniae Modulates Staphylococcus aureus Biofilm Dispersion and the Transition from Colonization to Invasive Disease.

Streptococcus pneumoniae and Staphylococcus aureus are ubiquitous upper respiratory opportunistic pathogens. Individually, these Gram-positive microbes are two of the most common causative agents of secondary bacterial pneumonia following influenza A virus infection, and they constitute a significant source of morbidity and mortality. Since the introduction of the pneumococcal conjugate vaccine, rates of cocolonization with both of these bacterial species have increased, despite the traditional view that they are antagonistic and mutually exclusive. The interactions between S. pneumoniae and S. aureus in the context of colonization and the transition to invasive disease have not been characterized. In this report, we show that S. pneumoniae and S. aureus form stable dual-species biofilms on epithelial cells in vitro When these biofilms are exposed to physiological changes associated with viral infection, S. pneumoniae disperses from the biofilm, whereas S. aureus dispersal is inhibited. These findings were supported by results of an in vivo study in which we used a novel mouse cocolonization model. In these experiments, mice cocolonized in the nares with both bacterial species were subsequently infected with influenza A virus. The coinfected mice almost exclusively developed pneumococcal pneumonia. These results indicate that despite our previous report that S. aureus disseminates into the lungs of mice stably colonized with these bacteria following influenza A virus infection, cocolonization with S. pneumoniae in vitro and in vivo inhibits S. aureus dispersal and transition to disease. This study provides novel insight into both the interactions between S. pneumoniae and S. aureus during carriage and the transition from colonization to secondary bacterial pneumonia.IMPORTANCE In this study, we demonstrate that Streptococcus pneumoniae can modulate the pathogenic potential of Staphylococcus aureus in a model of secondary bacterial pneumonia. We report that host physiological signals related to viral infection cease to elicit a dispersal response from S. aureus while in a dual-species setting with S. pneumoniae, in direct contrast to results of previous studies with each species individually. This study underscores the importance of studying polymicrobial communities and their implications in disease states.

Magnesium alloy AZ91 exhibits antimicrobial properties in vitro but not in vivo.

Magnesium alloys hold great promise for developing orthopedic implants that are biocompatible, biodegradable, and mechanically similar to bone tissue. This study evaluated the in vitro and in vivo antimicrobial properties of magnesium-9%aluminum-1%zinc (AZ91) and commercially pure titanium (cpTi) against Acinetobacter baumannii (Ab307). The in vitro results showed that as compared to cpTi, incubation with AZ91 significantly reduced both the planktonic (cpTi = 3.45e8, AZ91 = 8.97e7, p < 0.001) colony forming units (CFU) and biofilm-associated (cpTi = 3.89e8, AZ91 = 1.78e7, p = 0.01) CFU of Ab307. However, in vivo results showed no significant differences in the CFU enumerated from the cpTi and AZ91 implants following a 1-week implantation in an established rodent model of Ab307 implant associated infection (cpTi = 5.23e3, AZ91 = 2.46e3, p = 0.29). It is proposed that the in vitro results were associated with an increased pH in the bacterial culture as a result of the AZ91 corrosion process. The robust in vivo buffering capacity likely diminished this corrosion associated pH antimicrobial effect. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 221-227, 2018.

Cathodic voltage-controlled electrical stimulation of titanium for prevention of methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii biofilm infections.

Antibiotic resistance of bacterial biofilms limits available treatment methods for implant-associated orthopaedic infections. This study evaluated the effects of applying cathodic voltage-controlled electrical stimulations (CVCES) of -1.5V and -1.8V (vs. Ag/AgCl) to coupons of commercially pure titanium (cpTi) incubated in cultures of methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (A. baumannii) as a method of preventing bacterial attachment. Stimulations were applied for 2, 4, and 8h and coupon-associated and planktonic colony-forming units (CFU) were enumerated following stimulation. Compared to open circuit potential (OCP) controls, CVCES for 4h at -1.8V significantly reduced coupon-associated MRSA CFU by 99.9% (1.30×104vs. 4.45×107, p=0.047) and A. baumannii coupon-associated CFU by 99.9% (1.64×104vs. 5.93×107, p=0.001) and reduced planktonic CFU below detectable levels for both strains. CVCES at -1.8V for 8h also reduced coupon-associated and planktonic CFU below detectable levels for each strain. CVCES at -1.5V for 4 and 8h, and -1.8V for 2h did not result in clinically relevant reductions. For 4 and 8h stimulations, the current density was significantly higher for -1.8V than -1.5V, an effect directly related to the rate of water and oxygen reduction on the cpTi surface. This significantly increased the pH, a suspected influence in decreased CFU viability. The voltage-dependent electrochemical properties of cpTi likely contribute to the observed antimicrobial effects of CVCES. This study revealed that CVCES of titanium could prevent coupon-associated and planktonic CFU of Gram-positive MRSA and Gram-negative A. baumannii from reaching detectable levels in a magnitude-dependent and time-dependent manner. STATEMENT OF SIGNIFICANCE: Periprosthetic joint infection is a devastating outcome of total joint arthroplasty and has led to increased patient morbidity and rising healthcare costs. Current treatments are limited by the growing prevalence of antimicrobial resistant biofilms. Therefore, there is a growing interest in the prevention of bacterial colonization of implants. Previous work has shown that cathodic voltage-controlled electrical stimulation (CVCES) of titanium is effective both in vitro and in vivo as an antimicrobial strategy to eradicate established implant-associated biofilm infections. The present study revealed that CVCES of titanium coupons also has utility in preventing coupon-associated and planktonic colony-forming units of Gram-positive methicillin-resistant Staphylococcus aureus and Gram-negative Acinetobacter baumannii from reaching detectable levels in a magnitude-dependent and time-dependent manner.

Host Physiologic Changes Induced by Influenza A Virus Lead to Staphylococcus aureus Biofilm Dispersion and Transition from Asymptomatic Colonization to Invasive Disease.

UNLABELLED: Staphylococcus aureus is a ubiquitous opportunistic human pathogen and a major health concern worldwide, causing a wide variety of diseases from mild skin infections to systemic disease. S. aureus is a major source of severe secondary bacterial pneumonia after influenza A virus infection, which causes widespread morbidity and mortality. While the phenomenon of secondary bacterial pneumonia is well established, the mechanisms behind the transition from asymptomatic colonization to invasive staphylococcal disease following viral infection remains unknown. In this report, we have shown that S. aureus biofilms, grown on an upper respiratory epithelial substratum, disperse in response to host physiologic changes related to viral infection, such as febrile range temperatures, exogenous ATP, norepinephrine, and increased glucose. Mice that were colonized with S. aureus and subsequently exposed to these physiologic stimuli or influenza A virus coinfection developed pronounced pneumonia. This study provides novel insight into the transition from colonization to invasive disease, providing a better understanding of the events involved in the pathogenesis of secondary staphylococcal pneumonia. IMPORTANCE: In this study, we have determined that host physiologic changes related to influenza A virus infection causes S. aureus to disperse from a biofilm state. Additionally, we report that these same host physiologic changes promote S. aureus dissemination from the nasal tissue to the lungs in an animal model. Furthermore, this study identifies important aspects involved in the transition of S. aureus from asymptomatic colonization to pneumonia.

Serum antibody response to Moraxella catarrhalis proteins OMP CD, OppA, Msp22, Hag, and PilA2 after nasopharyngeal colonization and acute otitis media in children.

BACKGROUND: There is no licensed vaccine for Moraxella catarrhalis (Mcat), which is a prominent bacterium causing acute otitis media (AOM) in children and lower respiratory tract infections in adults. Nasopharyngeal (NP) colonization caused by respiratory bacteria results in natural immunization of the host. To identify Mcat antigens as vaccine candidates, we evaluated the development of naturally induced antibodies to 5 Mcat surface proteins in children 6-30 months of age during Mcat NP colonization and AOM. METHODS: Human serum IgG against the recombinant Mcat proteins, outer membrane protein (OMP) CD, oligopeptide permease (Opp)A, hemagglutinin (Hag), Moraxella surface protein (Msp)22, and PilA clade 2 (PilA2) was quantitated by using an ELISA assay. RESULTS: There were 223 Mcat NP colonization episodes documented in 111 (60%) of 184 children in the study. Thirty five Mcat AOM episodes occurred in 30 (16%) of 184 children. All 5 Mcat candidate vaccine antigens evaluated stimulated a significant rise in serum IgG levles over time from 6 to 36 months of age (P<0.001), with a rank order as follows: Msp22=OppA>OMP CD=Hag=PilA2. Children with no detectable Mcat NP colonization showed a higher serum IgG level against OppA, Hag, and Msp22 compared to those with Mcat NP colonization (P<0.05). Individual data showed that some children responded to AOM with an antibody increase to one or more of the studied Mcat proteins but some children failed to respond. CONCLUSIONS: Serum antibody to Mcat candidate vaccine proteins OMP CD, OppA, Msp22, Hag, and PilA2 increased with age in naturally immunized children age 6-30 months following Mcat NP colonization and AOM. High antibody levels against OppA, Msp22, and Hag correlated with reduced carriage. The results support further investigation of these vaccine candidates in protecting against Mcat colonization and infection.

Cathodic voltage-controlled electrical stimulation of titanium implants as treatment for methicillin-resistant Staphylococcus aureus periprosthetic infections.

Effective treatment options are often limited for implant-associated orthopedic infections. In this study we evaluated the antimicrobial effects of applying cathodic voltage-controlled electrical stimulation (CVCES) of -1.8 V (vs. Ag/AgCl) to commercially pure titanium (cpTi) substrates with preformed biofilm-like structures of methicillin-resistant Staphylococcus aureus (MRSA). The in vitro studies showed that as compared to the open circuit potential (OCP) conditions, CVCES of -1.8 V for 1 h significantly reduced the colony-forming units (CFU) of MRSA enumerated from the cpTi by 97% (1.89 × 106 vs 6.45 × 104 CFU/ml) and from the surrounding solution by 92% (6.63 × 105 vs. 5.15 × 104 CFU/ml). The in vivo studies, utilizing a rodent periprosthetic infection model, showed that as compared to the OCP conditions, CVCES at -1.8 V for 1 h significantly reduced MRSA CFUs in the bone tissue by 87% (1.15 × 105 vs. 1.48 × 104 CFU/ml) and reduced CFU on the cpTi implant by 98% (5.48 × 104 vs 1.16 × 103 CFU/ml). The stimulation was not associated with histological changes in the host tissue surrounding the implant. As compared to the OCP conditions, the -1.8 V stimulation significantly increased the interfacial capacitance (18.93 vs. 98.25 µF/cm(2)) and decreased polarization resistance (868,250 vs. 108 Ω-cm(2)) of the cpTi. The antimicrobial effects are thought to be associated with these voltage-dependent electrochemical surface properties of the cpTi.

Comparative analyses of the Moraxella catarrhalis type-IV pilus structural subunit PilA.

Moraxella catarrhalis is a Gram-negative aerobic diplococcus that is a mucosal pathogen of the upper and lower respiratory tracts in humans. In order to colonize the human host and establish an infection, M. catarrhalis must be able to effectively attach to the respiratory mucosal epithelia. Although little is known about M. catarrhalis pathogenesis, our laboratory has previously shown that expression of type IV pili (TFP) contributes to mucosal colonization. TFP are filamentous surface appendages primarily composed of a single protein subunit termed pilin, which is encoded by pilA in M. catarrhalis. These surface structures play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Our studies also indicate that unlike the pilin of the pathogenic Neisseria species, which exhibit both phase and antigenic variation, the pilin subunit of M. catarrhalis appears to be more highly conserved as there are no major pilin variants produced by a single strain and only two major PilA antigenic variants, termed clade 1 and clade 2, have been observed between strains. Moreover, we have determined that these highly conserved bacterial surface structures are expressed by all M. catarrhalis clinical isolates evaluated. Therapeutic or vaccine-based interventions that prevent or diminish nasopharyngeal colonization will likely decrease acute and recurrent M. catarrhalis infections in prone populations. Thus, our data indicate that additional studies aimed at elucidating the role of PilA in the pathogenesis and host response to M. catarrhalis infections are warranted.