The effects of antimicrobial peptides WAM-1 and LL-37 on multidrug-resistant Acinetobacter baumannii.

Increasing multidrug resistance (MDR) in Acinetobacter baumannii warrants therapeutic alternatives, and the bactericidal nature of antimicrobial peptides (AMPs) offers a possible approach. In this study, we examined the interaction of cathelicidin AMPs WAM-1, a marsupial AMP, and LL-37, a human AMP, with A. baumannii clinical isolates. We characterized the antibiotic resistance of the isolates, the bacteriostatic and bactericidal effects of these AMPs, synergistic activity with antibiotics, and their effects on biofilm formation and dispersal. All clinical isolates were resistant to commonly prescribed antibiotics, with four of seven isolates showing MDR. WAM-1 and LL-37 showed variable activity in clinical isolates, with WAM-1 having a stronger bacteriostatic effect than LL-37 and showing rapid bactericidal activity against clinical isolates. Furthermore, synergistic bactericidal activity was observed with WAM-1 and commonly prescribed antibiotics. Both peptides were able to inhibit biofilm formation in all clinical isolates at some concentrations, and WAM-1 dispersed mature biofilm in most isolates. LL-37 was unable to disperse mature biofilms in any strains. Further studies must be done to elucidate the true value of these alternative treatments, but these results suggest that MDR A. baumannii's susceptibility to AMPs may result in innovative therapeutics to prevent or treat these infections.

Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media.

Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease.

Influenza A virus alters pneumococcal nasal colonization and middle ear infection independently of phase variation.

Streptococcus pneumoniae (pneumococcus) is both a widespread nasal colonizer and a leading cause of otitis media, one of the most common diseases of childhood. Pneumococcal phase variation influences both colonization and disease and thus has been linked to the bacteria's transition from colonizer to otopathogen. Further contributing to this transition, coinfection with influenza A virus has been strongly associated epidemiologically with the dissemination of pneumococci from the nasopharynx to the middle ear. Using a mouse infection model, we demonstrated that coinfection with influenza virus and pneumococci enhanced both colonization and inflammatory responses within the nasopharynx and middle ear chamber. Coinfection studies were also performed using pneumococcal populations enriched for opaque or transparent phase variants. As shown previously, opaque variants were less able to colonize the nasopharynx. In vitro, this phase also demonstrated diminished biofilm viability and epithelial adherence. However, coinfection with influenza virus ameliorated this colonization defect in vivo. Further, viral coinfection ultimately induced a similar magnitude of middle ear infection by both phase variants. These data indicate that despite inherent differences in colonization, the influenza A virus exacerbation of experimental middle ear infection is independent of the pneumococcal phase. These findings provide new insights into the synergistic link between pneumococcus and influenza virus in the context of otitis media.

Residence of Streptococcus pneumoniae and Moraxella catarrhalis within polymicrobial biofilm promotes antibiotic resistance and bacterial persistence in vivo.

Otitis media (OM) is an extremely common pediatric ailment caused by opportunists that reside within the nasopharynx. Inflammation within the upper airway can promote ascension of these opportunists into the middle ear chamber. OM can be chronic/recurrent in nature, and a wealth of data indicates that in these cases, the bacteria persist within biofilms. Epidemiological data demonstrate that most cases of OM are polymicrobial, which may have significant impact on antibiotic resistance. In this study, we used in vitro biofilm assays and rodent infection models to examine the impact of polymicrobial infection with Moraxella catarrhalis and Streptococcus pneumoniae (pneumococcus) on biofilm resistance to antibiotic treatment and persistence in vivo. Consistent with prior work, M. catarrhalis conferred beta-lactamase-dependent passive protection from beta-lactam killing to pneumococci within polymicrobial biofilms. Moreover, pneumococci increased resistance of M. catarrhalis to macrolide killing in polymicrobial biofilms. However, pneumococci increased colonization in vivo by M. catarrhalis in a quorum signal-dependent manner. We also found that co-infection with M. catarrhalis affects middle ear ascension of pneumococci in both mice and chinchillas. Therefore, we conclude that residence of M. catarrhalis and pneumococci within the same biofilm community significantly impacts resistance to antibiotic treatment and bacterial persistence in vivo.

Passive immunization with Pneumovax® 23 and pneumolysin in combination with vancomycin for pneumococcal endophthalmitis.

BACKGROUND: Capsule and pneumolysin (PLY) are two major virulence factors of Streptococcus pneumoniae. S. pneumoniae is one of the leading causes of bacterial endophthalmitis. The aim of this study is to determine whether passive immunization with the 23-valent pneumococcal polysaccharide vaccine (Pneumovax® 23; PPSV23) or PLY protects against pneumococcal endophthalmitis. METHODS: New Zealand white rabbits were passively immunized with antiserum to PLY, PPSV23, a mixture of PPSV23/PLY, or PBS (mock). Vitreous was infected with a clinical strain of S. pneumoniae. In a separate group of experiments, vancomycin was injected 4 hours post-infection (PI) for each passively immunized group. Severity of infection, bacterial recovery, myeloperoxidase (MPO) activity and percent loss of retinal function were determined. RESULTS: Passive immunization with each antiserum significantly lowered clinical severity compared to mock immunization (PPSV23 = 9.19, PPSV23/PLY = 10.45, PLY = 8.71, Mock = 16.83; P = 0.0467). A significantly higher bacterial load was recovered from the vitreous of PLY passively immunized rabbits 24 hours PI (7.87 log10 CFU) compared to controls (7.10 log10 CFU; P = 0.0134). Retinas from immunized rabbits were more intact. Vitreous of PLY (2.88 MPO untis/mL) and PPSV23/PLY (2.17) passively immunized rabbits had less MPO activity compared to controls (5.64; P = 0.0480), and both passive immunizations (PLY = 31.34% loss of retinal function, PPSV23/PLY = 27.44%) helped to significantly preserve retinal function compared to controls (64.58%; P = 0.0323). When vancomycin was administered 4 hours PI, all eyes were sterile at 24 hours PI. A significantly lower clinical severity was observed for rabbits administered the combination immunization (5.29) or PPSV23 (5.29) with vancomycin treatment compared to controls (17.68; P = 0.0469). CONCLUSIONS: Passive immunization with antisera to these antigens is effective in reducing clinical severity of pneumococcal endophthalmitis in rabbits. Addition of vancomycin to immunization is effective at eliminating the bacteria.

Serine protease PKF of Acinetobacter baumannii results in serum resistance and suppression of biofilm formation.

Acinetobacter baumannii is an important nosocomial pathogen. Infections are often preceded by intubation or catheter use, promoting the formation of biofilm, and some strains are able to cause severe cases of bacteremia because of their ability to resist killing by complement. We identified a secreted serine protease, termed "PKF," that provided resistance to complement killing and suppressed biofilm formation. Serum resistance was abrogated in A. baumannii treated with protease inhibitors, as well as in a PKF-negative mutant. Serum resistance could be restored by recombinant PKF, which was shown to reduce the complement activity of normal human serum by almost 50%. PKF was shown to inhibit biofilm formation, because the PKF-negative mutant and wild-type A. baumannii treated with protease inhibitors produced biofilm that could be inhibited by addition of recombinant PKF. Our data indicate that PKF is required for serum resistance and that it suppresses biofilm formation in A. baumannii.

Moxifloxacin and cholesterol combined treatment of pneumococcal keratitis.

PURPOSE: Compare the efficacy of treatment of pneumococcal keratitis with cholesterol, moxifloxacin, or a mixture of the two (moxifloxacin/cholesterol). MATERIALS AND METHODS: New Zealand white rabbits were injected intrastromally with 10(6) colony-forming units (CFU) of a clinical keratitis strain of Streptococcus pneumoniae. Eyes were examined before and after treatment of topical drops every 2 hr from 25 to 47 hr post-infection (PI). Corneas were harvested to quantitate bacterial CFU, and myeloperoxidase (MPO) activity was measured at 48 hr PI. Eyes were extracted for histology. Minimal inhibitory concentrations (MICs) were determined for each compound. RESULTS: Eyes treated with moxifloxacin/cholesterol had a significantly lower mean slit lamp examination (SLE) score than eyes treated with phosphate-buffered saline (PBS), moxifloxacin alone, or cholesterol alone (P ≤ 0.02). A significantly lower log(10) CFU was recovered from corneas treated with moxifloxacin/cholesterol and moxifloxacin alone as compared to corneas of eyes treated with PBS or cholesterol alone (P < 0.01). At 48 hr PI, significantly lower MPO activity was quantitated from eyes treated with moxifloxacin/cholesterol as compared to eyes treated with cholesterol or moxifloxacin alone (P ≤ 0.046). Eyes treated with moxifloxacin/cholesterol had fewer immune cells and less corneal destruction than eyes from all other treatment groups. The MIC for moxifloxacin alone was 0.125 µg/mL, and cholesterol alone was unable to inhibit growth at any of the concentrations tested. The MIC for moxifloxacin when combined with 1% cholesterol was 0.0625 µg/mL. CONCLUSIONS: Treatment with a mixture of moxifloxacin and cholesterol significantly lowers the severity of infection caused by pneumococcal keratitis as compared to treatment with moxifloxacin alone, cholesterol alone, or PBS. This treatment mixture eradicates the bacteria in the cornea, unlike treatment with PBS or cholesterol alone. Using cholesterol with moxifloxacin as a treatment for bacterial keratitis could help lower the clinical severity of the infection.

Serum resistance and biofilm formation in clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii has few known virulence factors and yet causes a variety of opportunistic infections. Many gram-negative bacteria are directly killed by complement, but we hypothesized that A. baumannii would be resistant to serum killing. A serum bactericidal assay assessed the resistance of seven A. baumannii isolates to serum killing, and C2-deficient serum was used to examine its activation of the alternative pathway. Flow cytometry was utilized to determine whether complement regulator factor H (FH) was bound by A. baumannii, and to assay C3 deposition on cells. A microtiter biofilm assay compared biofilm production among isolates. Of seven isolates, four were serum sensitive and three were serum resistant. The C2-deficient serum demonstrated that A. baumannii can activate the alternative pathway. None of the isolates bound FH. Serum-resistant strains accumulated little C3 when exposed to human serum, while sensitive strains had a high amount of surface C3 deposition. Biofilm production varied extensively among strains. Most serum-resistant isolates formed a substantial amount of biofilm, while sensitive isolates produced negligible amounts of biofilm. Our data indicate that some strains of A. baumannii are resistant to serum killing and produce biofilms and by understanding the resistance mechanisms used by this bacterium, we can further elucidate its complex pathogenicity.

A comparison of pneumolysin activity and concentration in vitro and in vivo in a rabbit endophthalmitis model.

The purpose of this study was to determine whether the in vitro activity and concentration of Streptococcus pneumoniae pneumolysin correlated to the pathogenesis of S. pneumoniae endophthalmitis. Five S. pneumoniae clinical endophthalmitis strains were grown in media to similar optical densities (OD), and extracellular milieu was tested for pneumolysin activity by hemolysis of rabbit red blood cells. Pneumolysin concentration was determined using a sandwich ELISA. Rabbit vitreous was injected with 10(2) colony-forming units (CFU) of 1 of 2 different strains with low hemolytic activity (n = 10 and 12 for strains 4 and 5, respectively) or 1 of 3 different strains with high hemolytic activity (n = 12 per strain). Pathogenesis of endophthalmitis infection was graded by slit lamp examination (SLE) at 24 hours post-infection. Bacteria were recovered from infected vitreous and quantitated. The SLE scores of eyes infected with strains having high hemolytic activity were significantly higher than the scores of those infected with strains having low hemolytic activity (P < 0.05). Pneumolysin concentration in vitro, however, did not correlate with hemolysis or severity of endophthalmitis. Bacterial concentrations from the vitreous infected with 4 of the strains were not significantly different (P > 0.05). These data suggest that pneumolysin hemolytic activity in vitro directly correlates to the pathogenesis of S. pneumoniae endophthalmitis. The protein concentration of pneumolysin, however, is not a reliable indicator of pneumolysin activity.