Jutta M. Loeffler

Phage Lytic Enzyme Cpl-1 as a Novel Antimicrobial for Pneumococcal Bacteremia

ABSTRACT Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are badly needed. We report the use of Cpl-1, the lytic enzyme of a pneumococcal bacteriophage, as an intravenous therapy for pneumococcal bacteremia in a mouse model. A 2,000-μg dose of Cpl-1 reduced pneumococcal titers from a median of log10 4.70 CFU/ml to undetectable levels (<log10 2.00 CFU/ml) within 15 min. This dose given 1 h after intravenous infection led to 100% survival at 48 h, compared to the 20% survival of buffer-treated controls. In advanced bacteremia, treatment with two doses at 5 and 10 h still resulted in significantly longer survival (P < 0.0001) and a hazard ratio of 0.29 (95% confidence interval, 0.04 to 0.35). The enzyme is immunogenic, but the treatment efficacy was not significantly diminished after previous intravenous exposure of mice and hyperimmune rabbit serum did not neutralize the activity. Cpl-1 is also very effective as a topical nasal treatment against colonization by S. pneumoniae. In vitro, the enzyme is active against many serotypes of S. pneumoniae, independent of their penicillin resistance, and it is very specific for this species. Bacteriophage enzymes are unusual but extremely effective antimicrobials and represent a new weapon against infections with resistant bacteria.

A dominant complement fixation pathway for pneumococcal polysaccharides initiated by SIGN-R1 interacting with C1q

The intricate system of serum complement proteins provides resistance to infection. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial binding C3 fragments recognized by leukocytes. The spleen and C3 provide resistance against blood-borne S. pneumoniae infection. To better understand the mechanisms involved, we studied SIGN-R1, a lectin that captures microbial polysaccharides in spleen. Surprisingly, conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-R1(+) spleen macrophages, and formation of C3 ligands. We found that SIGN-R1 directly bound the complement C1 subcomponent, C1q, and assembled a C3 convertase, but without the traditional requirement for either antibody or factor B. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway.

The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen

SIGN-R1, a recently discovered C-type lectin expressed at high levels on macrophages within the marginal zone of the spleen, mediates the uptake of dextran polysaccharides by these phagocytes. We now find that encapsulated Streptococcus pneumoniae are rapidly cleared by these macrophages from the bloodstream, and that capture also takes place when different cell lines express SIGN-R1 after transfection. To assess the role of the capsular polysaccharide of S. pneumoniae (CPS) in the interaction of SIGN-R1 with pneumococci, we first studied binding and uptake of serotype 14 CPS in transfected cells. Binding was observed and was of a much higher avidity (3,000-fold) for CPS 14 than dextran. The CPSs from four different serotypes were also cleared by marginal zone macrophages in vivo. To establish a role for SIGN-R1 in this uptake, we selectively down-regulated expression of the lectin by pretreatment of the mice with SIGN-R1 antibodies, including a newly generated hamster monoclonal called 22D1. For several days after this transient knockout, the marginal zone macrophages were unable to take up either CPSs or dextrans. Therefore, marginal zone macrophages in mice have a receptor that interacts with capsular pneumococcal polysaccharides, setting the stage for further studies of the functional consequences of this interaction.

Dexamethasone Aggravates Hippocampal Apoptosis and Learning Deficiency in Pneumococcal Meningitis in Infant Rats

In an infant rat model of pneumococcal meningitis the effect of dexamethasone on neuronal injury in the hippocampus and on learning disability after recovery from the disease was examined. Treatment with dexamethasone or vehicle was started 18 h after infection, concomitant with antibiotics. Neuronal apoptosis in the hippocampal dentate gyrus 34 h after infection was significantly aggravated by dexamethasone treatment compared with vehicle controls (p = 0.02). Three weeks after acute pneumococcal meningitis, learning capacity of animals was assessed in the Morris water maze. The results showed a significantly impaired learning performance of infected animals treated with dexamethasone compared with vehicle controls (p = 0.01). Dexamethasone had no effect on hippocampal injury or learning in uninfected controls. Thus, dexamethasone as adjuvant therapy increased hippocampal cell injury and reduced learning capacity in this model of pneumococcal meningitis in infant rats.

Therapeutic Effects of Bacteriophage Cpl-1 Lysin against Streptococcus pneumoniae Endocarditis in Rats

ABSTRACT Cpl-1, a pneumococcal phage lytic enzyme, was tested in rats with experimental endocarditis due to Streptococcus pneumoniae WB4. High-dose regimen Cpl-1 eliminated pneumococci from blood within 30 min and decreased bacterial titers in vegetations (>4 log10 CFU/g) within 2 h. Rapid bacterial lysis induced by Cpl-1 treatment increased cytokine secretion noticeably.

Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains.

ABSTRACT Pal and Cpl-1, two purified bacteriophage lytic enzymes, were tested for their in vitro activity, alone and in combination, against several serotypes of Streptococcus pneumoniae, including penicillin-resistant strains. The enzymes demonstrated synergism in their ability to cleave the bacterial peptidoglycan and thus may be more efficient for the prevention and elimination of pneumococcal colonization.

Novel Strategy to Prevent Otitis Media Caused by Colonizing Streptococcus pneumoniae

In early childhood, 70%–83% of children experience at least one episode of acute otitis media (AOM) [1,2]. Streptococcus pneumoniae is the most common bacterial agent identified as the causative agent of these infections, although there is increasing evidence that a variety of respiratory viruses play a prominent role in the development and pathogenesis of AOM [1]. Even though the heptavalent pneumococcal conjugate vaccine appears to be making an impact on the incidence of this disease among children in the United States, AOM (with more than 24 million diagnoses annually) remains the leading reason for physician visits and antibiotic prescriptions among preschool-aged children [2,3]. Frequent use of antibiotics for AOM has led to a vicious cycle of diminishing returns: increased exposure has led to increasing drug resistance, which in turn makes the infections more difficult to treat, necessitating new drugs and more treatment. Recently, purified bacteriophage (phage) cell wall hydrolases, or lysins, have shown promise as novel anti-infectives due to their ability to eradicate nasal carriage of gram-positive pathogens, particularly S. pneumoniae [4,5]. These highly active enzymes are produced by phages to disrupt the bacterial cell wall for the release of progeny phage. Here, we show that the Cpl-1 lysin, which is specific for S. pneumoniae [6], prevents AOM in a novel mouse model that mimics the natural pathogenesis of this common infection. Current animal models for AOM have critical limitations. Modeling AOM in mice requires invasive and artificial procedures to establish infection, and sacrifice of the animals to determine outcomes. Larger animals such as chinchillas and ferrets may develop infection by more natural routes, but use of these models is limited by their size and complexity [7,8]. Ideally, we wished to develop a non-invasive mouse model that was permissive of natural infection. We engineered a piliated strain of S. pneumoniae, known to efficiently colonize mucosal surfaces (a type 19F strain obtained from B. Henriques-Normark, ST16219F) [9], to express luciferase [10]. Groups of five mice maintained in a BL2 facility were infected intranasally with 1 × 105 or 1 × 106 colony-forming units (CFU) of this bioluminescent strain under light anesthesia with 2.5% inhaled isoflurane using an established infection model approved by the St. Jude Childrens Research Hospital animal care and use committee [11]. Animals were followed daily for development of infection for two weeks and thrice weekly for another four weeks. Within 72 hours of pneumococcal infection, 100% of mice (10/10) were visibly colonized with bacteria in the anterior portion of their nose, and 70% (7/10) had developed AOM. These infections of the middle ear all resolved by bioluminescent imaging within 48 hours, and no mice had evidence of AOM six days after challenge or later. Nasal colonization persisted for a median of 27 days (range 17–34 days). Around half of all children are colonized with S. pneumoniae [12]. Alteration of eustachian tube function or disruption of mucosal surfaces through viral infection allows colonizing bacteria to ascend into the middle ear, triggering AOM [1]. To model this phenomenon, we infected mice that had been stably colonized by pneumococcus with influenza virus and followed them for development of AOM. Although all mice had been colonized prior to infection with virus, 63% of virus-infected mice (19/30) developed AOM compared to 0% (0/10) of mice mock-infected with phosphate buffered saline (PBS) (Figure 1). Twenty-one of thirty mice in the virus group had experienced AOM after introduction of the bacteria in the first 72 hours post colonization (with resolution before viral challenge), while eight of ten mice in the PBS control group had experienced AOM with resolution (unpublished data). Both de novo and recurrent infections were seen in the virus-infected mice, with no correlation to whether they had previously had AOM. This is the first mouse model of AOM in which infection develops in a manner analogous to that observed in children. Figure 1 Visualization of Bioluminescent Bacteria Inside Live, Anesthetized Animals Shows the Induction of Otitis Media Using this novel and powerful model, we sought to test our hypothesis that reduction or elimination of colonizing pneumococci with purified Cpl-1 lysin [6] would prevent the development of AOM. Prior to infection with influenza virus, mice colonized with pneumococcus for seven days were treated twice four hours apart with either 1,000 ug of Cpl-1 intranasally or enzyme buffer (mock treatment). At the time of the second treatment, nine of ten animals (90%) had cleared the pneumococcus from the nose, compared to zero of ten (0%) treated with enzyme buffer. The Cpl-1 lysin was 100% effective in preventing AOM, as no animal treated with lysin developed a secondary bacterial infection following influenza infection, while eight of ten (80%) mice that were mock-treated developed AOM (Figure 2). In the one mouse in which colonization persisted despite lysin treatment, the amount of bacteria present decreased dramatically (78% reduction in flux of light through the nose). In addition, no mice treated with Cpl-1 and observed for the duration of these experiments developed toxicity or illness attributable to the enzyme as determined by clinical observation, weight loss, and histopathology (unpublished data). Figure 2 Treatment with Lysin Eliminates Colonization and Prevents the Development of Otitis Media This novel model of naturally developing AOM will be useful in studies of prevention and treatment of this important and common infection. Our data on the use of lysin in the model suggest that a strategy of decolonizing children of S. pneumoniae may prevent many cases of AOM, particularly those with chronic AOM. Importantly, these data also suggest that elimination or even reduction of resident pathogenic bacteria can prevent secondary bacterial complications of influenza. In support of this view, a recent study of a pediatric pneumococcal vaccine in South Africa showed a 31% decrease in the incidence of virus-associated pneumonia compared to controls [13]. Secondary bacterial infections account for much of the morbidity and approximately 25% of all deaths during seasonal epidemics of influenza, as well as 50%–95% of deaths during pandemics of influenza [14]. It is very likely that a reduction in pneumococcal colonization in susceptible populations such as infants and the elderly during a pandemic or annual influenza outbreak could result in a concomitant reduction in morbidity and mortality. With worldwide concerns over a potentially incipient pandemic with highly pathogenic influenza viruses of the H5N1 subtype, further study of novel therapeutics such as phage-derived lysins to prevent these infections is warranted.

The Free Radical Scavenger α-Phenyl-Tert-Butyl Nitrone Aggravates Hippocampal Apoptosis and Learning Deficits in Experimental Pneumococcal Meningitis

The effect of adjuvant therapy with the radical scavenger alpha-phenyl-tert-butyl nitrone (PBN; 100 mg/kg given intraperitoneally every 8 h for 5 days) on brain injury and learning function was evaluated in an infant rat model of pneumococcal meningitis. Meningitis led to cortical necrotic injury (median, 3.97% [range, 0%-38.9%] of the cortex), which was reduced to a median of 0% (range, 0%-30.9%) of the cortex (P<.001) by PBN. However, neuronal apoptosis in the hippocampal dentate gyrus was increased by PBN, compared with that by saline (median score, 1.15 [range, 0.04-1.73] vs. 0.31 [range, 0-0.92]; P<.001). Learning function 3 weeks after cured infection, as assessed by the Morris water maze, was decreased, compared with that in uninfected control animals (P<.001). Parallel to the increase in hippocampal apoptosis, PBN further impaired learning in infected animals, compared with that in saline-treated animals (P<.02). These results contrast with those of an earlier study, in which PBN reduced cortical and hippocampal neuronal injury in group B streptococcal meningitis. Thus, in pneumococcal meningitis, antioxidant therapy with PBN aggravates hippocampal injury and learning deficits.

Phage Lytic Enzyme Cpl-1 for Antibacterial Therapy in Experimental Pneumococcal Meningitis

Treatment of bacterial meningitis caused by Streptococcus pneumoniae is increasingly difficult, because of emerging resistance to antibiotics. Recombinant Cpl-1, a phage lysin specific for S. pneumoniae, was evaluated for antimicrobial therapy in experimental pneumococcal meningitis using infant Wistar rats. A single intracisternal injection (20 mg/kg) of Cpl-1 resulted in a rapid (within 30 min) decrease in pneumococci in cerebrospinal fluid (CSF) by 3 orders of magnitude lasting for 2 h. Intraperitoneal administration of Cpl-1 (200 mg/kg) led to an antibacterial effect in CSF of 2 orders of magnitude for 3 h. Cpl-1 may hold promise as an alternative treatment option in pneumococcal meningitis.

Synergistic Killing of Streptococcus pneumoniae with the Bacteriophage Lytic Enzyme Cpl-1 and Penicillin or Gentamicin Depends on the Level of Penicillin Resistance

ABSTRACT A combination of Cpl-1, a bacteriophage lytic enzyme, and penicillin, gentamicin, levofloxacin, or azithromycin was tested against Streptococcus pneumoniae strains with various susceptibilities to penicillin. Activities of Cpl-1 and gentamicin were increasingly synergistic with a decreasing penicillin MIC, while Cpl-1 and penicillin showed synergy against an extremely penicillin-resistant strain.