Adherence and invasion are thought to be key events in the pathogenesis of non‐typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS‐coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB::ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB::ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co‐localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti‐ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge. It is associated with an increased risk of preterm delivery, pelvic inflammatory disease, and an increased risk of acquisition of sexually transmitted infections including human immunodeficiency virus (HIV). The epidemiology of BV supports sexual transmission. However, its etiology remains unknown. At the center of the debate is whether BV is caused by a primary pathogen or a polymicrobial consortium of microorganisms that are sexually transmitted. We previously published a conceptual model hypothesizing that BV is initiated by sexual transmission of Gardnerella vaginalis. Critics of this model have iterated that G. vaginalis is found in virginal women and in sexually active women with a normal vaginal microbiota. In addition, colonization does not always lead to BV. However, recent advances in BV pathogenesis research have determined the existence of 13 different species within the genus Gardnerella. It may be that healthy women are colonized by nonpathogenic Gardnerella species, whereas virulent strains are involved in BV development. Based on our results from a recent prospective study, in addition to an extensive literature review, we present an updated conceptual model for the pathogenesis of BV that centers on the roles of virulent strains of G. vaginalis, as well as Prevotella bivia and Atopobium vaginae.

Haemophilus influenzae is a gram-negative pleiomorphic bacterium that is a common commensal/mutualist within the human airways (30). Encapsulated H. influenzae strains are overt pathogens causing invasive disease (3) and have largely been contained by a vaccine effective against the predominant capsular serotype b strains (32). In contrast, the so-called nontypeable H. influenzae (NTHi) strains lacking capsular polysaccharides remain predominant in asymptomatic carriage and localized airway infections (14, 29). These infections are mostly opportunistic in nature and include bronchiopneumonia, sinusitis, and otitis media (OM). OM is among the most common pediatric infections, causing an estimated ϳ$5 billion in costs of treatment and parents' missed work days per year (20). OM infections include chronic OM that is difficult to resolve with antibiotic therapy, and it has long been postulated that chronic OM involves the formation of bacterial biofilm communities (5, 35). In support of that hypothesis, biofilms have been visualized in tympanostomy drain tubes removed from patients with OM and on middle ear tissue from experimentally infected chinchillas (7,18,33). More recent evidence shows that NTHi and other bacterial agents are present within biofilms on tissue specimens obtained from patients with chronic and recurrent OM (13).The H. influenzae surface is covered with lipooligosaccharide (LOS) endotoxins that lack a repeating O side chain. Instead, the H. influenzae LOS features a diverse collection of LOS glycoforms that differ in the length, content, and nature of the chemical linkages found in the oligosaccharide portion. These LOS oligosaccharides include structures that are antigenically similar to host cell-surface glycolipids and may also contain the host membrane constituents sialic acid (NeuAc) and phosphorylcholine (PCho) (41). LOS confers resistance to host killing (8,9,37) and is also the primary target of the Toll-like receptor 4 pathway that mediates protection against H. influenzae in the airways (47). It has been established that NTHi strains that express NeuAc-LOS forms comprise a greater proportion of biofilm communities than of planktonic cultures, and that mutations eliminating these forms decrease biofilm formation and bacterial persistence in animal models of OM (4,12,18,43). More recently, we showed that LOS purified from biofilms has decreased potency as an inflammatory agonist, which correlated with an increase in PCho ϩ LOS forms that were present within biofilms (55). In this study, we compared the virulence

Salmonella typhimurium encounters a variety of acid stress situations during growth in host and nonhost environments. The organism can survive potentially lethal acid conditions (pH <4) if it is first able to adapt to mild or more moderate acid levels. The molecular events that occur during this adaptive process are collectively referred to as the acid tolerance response and vary depending on whether the cells are in log

Nontypeable Haemophilus influenzae (NTHi) is a leading cause of acute and chronic otitis media, which are a major public health problem worldwide. The persistence of NTHi during chronic and recurrent otitis media infections involves multicellular biofilm communities formed within the middle-ear chamber. Bacterial biofilms resist immune clearance and antibiotic therapy due in part to encasement within a polymeric matrix. In this study, the contribution of biofilms to bacterial persistence in vivo and composition of the NTHi biofilm matrix during experimental otitis media were investigated. The presence of biofilms within the chinchilla middle-ear chamber was significantly correlated with increased bacterial load in middle-ear effusions and tissue. Examination of thin sections revealed polymorphonuclear cells within a DNA lattice containing elastase and histones, which is consistent with the definition of neutrophil extracellular traps. Viable multicellular biofilm communities with biofilm phenotypes were found within the DNA lattice throughout the biofilm. Further, NTHi was resistant to both phagocytic and extracellular neutrophil killing in vitro by means of lipooligosaccharide moieties that promote biofilm formation. These data support the conclusion that NTHi subverts neutrophil extracellular traps to persist in vivo. These data also indicate that a more inclusive definition for biofilms may be warranted.

Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.

Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic ON ). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic ON . The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.Nontypeable Haemophilus influenzae (NTHI) is a commensal of the human upper airways that can cause localized opportunistic airway infections when mucociliary defenses are compromised (24). NTHI is a leading cause of otitis media with effusion (2), acute otitis media (1), chronic sinusitis (20), and pulmonary infections associated with chronic obstructive pulmonary disease (31). For many of these infections, NTHI bacteria persist within dense biofilm communities that are thought to provide resistance to host clearance (12,25,29). NTHI biofilms contain variants expressing lipooligosaccharides (LOS) that contain phosphorylcholine (PCho) and sialic acid (NeuAc) (10,19,36,41). Mutants lacking these structures are unable to establish persistent biofilms in vivo (14,19,36). The addition of PCho to LOS occurs in a phase-variable manner, and thus, NTHI populations contain discrete subpopulations of PCho-positive (PCho ϩ ) and PCho-negative (PCho Ϫ ) variants (40). PCho contributes to NTHI colonization and persistence by promoting bacterial adherence to host cells (9,32,...

Otitis media 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. Otitis media can be chronic/recurrent in nature, and a wealth of data indicates that in these cases the bacteria persist within biofilms. Epidemiological data demonstrates most cases of otitis media 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.