Nicole R. Luke-Marshall

The K1 Capsular Polysaccharide from Acinetobacter baumannii Is a Potential Therapeutic Target via Passive Immunization

ABSTRACT 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.

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.

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

ABSTRACT 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. 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.

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 β-(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 β-(1-3) Glc to the first Glc on the inner core. The C-terminal catalytic domain A2 then adds the β-(1-4) Glc and the β-(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.

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.

Moraxella catarrhalis is susceptible to antimicrobial photodynamic therapy with Photofrin

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.

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.

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

ABSTRACT 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. 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.