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.

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.

Effect of particle size of parenteral suspensions on in vitro muscle damage.

OBJECTIVE: Suspension particle size plays a key role in the release and stability of drugs for oral and parenteral formulations. However, the role of particle size in suspension formulations on tissue damage (myotoxicity) following intramuscular (IM) injection has not been systematically investigated. MATERIALS AND METHODS: Myotoxicity was assessed by the release of cumulative creatine kinase (CCK) from the isolated extensor digitorium longus (EDL) and soleus (SOL) rat muscles for selected suspensions of phenytoin, bupivicane and diazepam. Particle size effects on myotoxicity, independent of any specific drug, were also investigated using characterized non-dissolving polystyrene beads. RESULTS: Myotoxicity was quantitated by the cumulative release of creatine kinase (CCK) from these isolated muscles over 90 or 120 min. The relationship between particle size and myotoxicity was dependent upon the drug in these suspensions. Diazepam and phenytoin suspensions were found to be less myotoxic than bupivicaine. Using unmodified and carboxy modified polystyrene beads, an optimal particle size for reduced myotoxicity following IM injection ranges from approx. 500 nm to 1 µM. CONCLUSIONS: The relationship between myotoxicity of IM suspensions and particle size is dependent upon the particular drug and suspension particle size.

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.

The K1 capsular polysaccharide of Acinetobacter baumannii strain 307-0294 is a major virulence factor.

Acinetobacter baumannii is a pathogen of increasing medical importance with a propensity to be multidrug resistant, thereby making treatment challenging. Little is known of virulence traits in A. baumannii. To identify virulence factors and potential drug targets, random transposon (Tn) mutants derived from the A. baumannii strain AB307-0294 were screened to identify genes essential for growth in human ascites fluid in vitro, an inflammatory exudative fluid. These studies led to the identification of two genes that were predicted to be required for capsule polymerization and assembly. The first, ptk, encodes a putative protein tyrosine kinase (PTK), and the second, epsA, encodes a putative polysaccharide export outer membrane protein (EpsA). Monoclonal antibodies used in flow cytometric and Western analyses confirmed that these genes are required for a capsule-positive phenotype. A capsule-positive phenotype significantly optimized growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model. Importantly, the clearance of the capsule-minus mutants AB307.30 (ptk mutant, capsule minus) and AB307.45 (epsA mutant, capsule minus) was complete and durable. These data demonstrated that the K1 capsule from AB307-0294 was an important protectin. Further, these data suggested that conserved proteins, which contribute to the capsule-positive phenotype, are potential antivirulence drug targets. Therefore, the results from this study have important biologic and translational implications and, to the best of our knowledge, are the first to address the role of capsule in the pathogenesis of A. baumannii infection.

Identification and characterization of a glycosyltransferase involved in Acinetobacter baumannii lipopolysaccharide core biosynthesis.

Although Acinetobacter baumannii has emerged as a significant cause of nosocomial infections worldwide, there have been few investigations describing the factors important for A. baumannii persistence and pathogenesis. This paper describes the first reported identification of a glycosyltransferase, LpsB, involved in lipopolysaccharide (LPS) biosynthesis in A. baumannii. Mutational, structural, and complementation analyses indicated that LpsB is a core oligosaccharide glycosyl transferase. Using a genetic approach, lpsB was compared with the lpsB homologues of several A. baumannii strains. These analyses indicated that LpsB is highly conserved among A. baumannii isolates. Furthermore, we developed a monoclonal antibody, monoclonal antibody 13C11, which reacts to an LPS core epitope expressed by approximately one-third of the A. baumannii clinical isolates evaluated to date. Previous studies describing the heterogeneity of A. baumannii LPS were limited primarily to structural analyses; therefore, studies evaluating the correlation between these surface glycolipids and pathogenesis were warranted. Our data from an evaluation of LpsB mutant 307::TN17, which expresses a deeply truncated LPS glycoform consisting of only two 3-deoxy-d-manno-octulosonic acid residues and lipid A, suggest that A. baumannii LPS is important for resistance to normal human serum and confers a competitive advantage for survival in vivo. These results have important implications for the role of LPS in A. baumannii infections.

Penicillin-binding protein 7/8 contributes to the survival of Acinetobacter baumannii in vitro and in vivo.

BACKGROUND: Acinetobacter baumannii is a bacterial pathogen of increasing medical importance. Little is known about genes important for its survival in vivo. METHODS AND RESULTS: Screening of random transposon mutants of the model pathogen AB307-0294 identified the mutant AB307.27. AB307.27 contained its transposon insertion in pbpG, which encodes the putative low-molecular-mass penicillin-binding protein 7/8 (PBP-7/8). AB307.27 was significantly killed in ascites (P<.001), but its growth in Luria-Bertani broth was similar to that of its parent, AB307-0294 (P=.13). The survival of AB307.27 was significantly decreased in a rat soft-tissue infection model (P<.001) and a rat pneumonia model (P=.002), compared with AB307-0294. AB307.27 was significantly killed in 90% human serum in vitro, compared with AB307-0294 (P<.001). Electron microscopy demonstrated more coccobacillary forms of AB307.27, compared with AB307-0294, suggesting a possible modulation in the peptidoglycan, which may affect susceptibility to host defense factors. CONCLUSIONS: These findings demonstrate that PBP-7/8 contributes to the pathogenesis of A. baumannii. PBP-7/8 either directly or indirectly contributes to the resistance of AB307-0294 to complement-mediated bactericidal activity. An understanding of how PBP-7/8 contributes to serum resistance will lend insight into the role of this low-molecular-mass PBP whose function is poorly understood.