Panel 7 - Pathogenesis of otitis media - a review of the literature between 2015 and 2019.

OBJECTIVE: To perform a comprehensive review of the literature from July 2015 to June 2019 on the pathogenesis of otitis media. Bacteria, viruses and the role of the microbiome as well as the host response are discussed. Directions for future research are also suggested. DATA SOURCES: PubMed database of the National Library of Medicine. REVIEW METHODS: PubMed was searched for any papers pertaining to OM pathogenesis between July 2015 and June 2019. If in English, abstracts were assessed individually for their relevance and included in the report. Members of the panel drafted the report based on these searches and on new data presented at the 20th International Symposium on Recent Advances in Otitis Media. CONCLUSIONS: The main themes that arose in OM pathogenesis were around the need for symptomatic viral infections to develop disease. Different populations potentially having different mechanisms of pathogenesis. Novel bacterial otopathogens are emerging and need to be monitored. Animal models need to continue to be developed and used to understand disease pathogenesis. IMPLICATIONS FOR PRACTICE: The findings in the pathogenesis panel have several implications for both research and clinical practice. The most urgent areas appear to be to continue monitoring the emergence of novel otopathogens, and the need to develop prevention and preventative therapies that do not rely on antibiotics and protect against the development of the initial OM episode.

Macrolide antibiotics, bacterial populations and inflammatory airway disease.

Chronic obstructive pulmonary disease (COPD) and other inflammatory airway conditions are major causes of morbidity and mortality worldwide. Antibiotics are used to treat acute infectious exacerbations of airway disease. However, for the macrolides, a significant and growing body of evidence indicates that anti-inflammatory effects of these antibiotics, which may be independent of their antibacterial effects, are at least partially responsible for their beneficial effect. In this review, we describe current thinking on the means whereby anti-inflammatory effects of macrolides impact chronic airway disease. The current data indicate that some macrolides have immunomodulatory activity, mediated at least in part by effects on the activation of gene transcription mediated by NF-kappabeta activation that may be separable from their antimicrobial activities, and could explain their surprising efficacy in asthma and viral infections for which the role of bacteria is not established. Other, provocative work indicates that subclinical doses of macrolides may also affect signalling within and between bacterial communities, and thus impact developmental processes such as biofilm formation that are important in the establishment and persistence of chronic infections. The current data clearly suggest that activities beyond antimicrobial effects contribute significantly to the beneficial effect of macrolide therapy on inflammatory conditions.

Mycobacterium xenopi multiplies within human macrophages and enhances HIV replication in vitro.

Mycobacterium xenopi can cause opportunistic infections, particularly in persons infected with human immunodeficiency virus type 1 (HIV-1). The primary focus of this effort was to determine if M. xenopi isolates could survive and grow in human peripheral blood macrophage (MPhi), and if these isolates could promote the replication of HIV-1 in vitro. M. xenopi bacilli survived and replicated 10-fold within 48 h in human MPhi while avirulent Mycobacterium smegmatis, did not grow within the MPhi. M. xenopi bacilli when cultured with peripheral blood mononuclear cells enhanced HIV-1 replication 30- and 50-fold with the macrophage-tropic HIV-1(Ba-L) and 50- and 75-fold with T-cell-tropic strain HIV-1(LAI) by 6 days post-infection when compared to M. smegmatis. The enhanced HIV replication was associated with increased production of TNF-alpha. Partial inhibition of HIV-1 induction was observed using a neutralizing anti-TNF-alpha monoclonal antibody, pentoxifylline, and matrix metalloproteinase (MMP) inhibitor I. Similar mechanisms of pathogenesis among mycobacterial species may help elucidate better treatment approaches in HIV co-infected persons.

The predicted amino acid sequence of the Salmonella typhimurium virulence gene mviAA(+) strongly indicates that MviA is a regulator protein of a previously unknown S. typhimurium response regulator family.

The Salmonella typhimurium virulence gene mviA+ has a predicted amino acid sequence with homology to the N-terminal 112-amino-acid sequence of response regulator proteins. A previously described mutant allele (mviA), which restores virulence to avirulent LT2 strains, was shown to contain a point mutation which would be predicted to cause a single amino acid change, V-102-->G (W. H. Benjamin, Jr., J. Yother, P. Hall, and D. E. Briles, J. Exp. Med. 1,74:1073-1083, 1991). A comparison of the nucleotide sequence of mviA+ with that of the Escherichia coli and Salmonella typhi genes revealed a high degree of conservation.

Bacterial phenotypes mediated by mviA and their relationship to the mouse virulence of Salmonella typhimurium.

The focus of this study was the phenotypic characterization of Salmonella typhimurium mutants lacking the function of the response regulator mviA. The inactivation of mviA+ (mviA::kan) is shown to induce a significant change in the growth of most virulent strains, as reflected in the size of the colonies formed on agar plates. The colony phenotype observed in these strains has been designated as the small colony morphology (Scm+) phenotype. Mutants exhibiting the Scm+ phenotype are shown to be significantly attenuated for virulence in susceptible (ItyB) mice. The Scm+ phenotype therefore provides an in vitro phenotypic marker for mviA+ activity. Further examination of Scm+ mutants has revealed that they lack expression of a 55 kDa periplasmic protein which is detected in isogenic mviA+ strains. This protein has been designated mviA+ related protein A (MrpA) and was expressed in direct correlation with virulence in all S. typhimurium strains examined.

Avirulence of LT2 strains of Salmonella typhimurium results from a defective rpoS gene.

In order to identify the genetic basis for the attenuation of Salmonella typhimurium LT2 strains, experiments were performed to identify a gene(s) which restores virulence to an avirulent LT2 strain. These and further experiments confirmed that an rpoS mutation is the sole determinant of the attenuation of S. typhimurium LT2.

The mimicry of human glycolipids and glycosphingolipids by the lipooligosaccharides of pathogenic neisseria and haemophilus.

It has been known for many years that bacteria can induce autoimmune responses in humans resulting in serious disease. Recent work has shown that a number of bacteria that colonize human mucosal surfaces exclusively express antigens on their surfaces which are molecular mimics of glycosphingolipids found on human cells. These structures are important in the pathogenesis of Neisseria and Haemophilus species for both immune evasion and in the adherence and invasion of human cells. There is no evidence that colonization or infections by these bacterial species is associated with autoimmune disease.

Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium.

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- or stationary-phase growth. The acid tolerance response of logarithmically growing cells includes the participation of an alternate sigma factor, sigmaS (RpoS), commonly associated with stationary-phase physiology. Of 51 acid shock proteins (ASPs) induced during shifts to pH 4.4, 8 are clearly dependent on sigmaS for production (I. S. Lee, J. Lin, H. K. Hall, B. Bearson, and J. W. Foster, Mol. Microbiol. 17:155-167, 1995). The acid shock induction of these proteins appears to be the result of an acid shock-induced increase in the level of sigmaS itself. We have discovered that one component of a potential signal transduction system responsible for inducing rpoS expression is the product of the mouse virulence gene mviA+. MviA exhibits extensive homology to the regulatory components of certain two-component signal transduction systems (W. H. Benjamin, Jr., and P. D. Hall, abstr. B-67, p. 38, in Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993, 1993). Mutations in mviA (mviA::Km) caused the overproduction of sigmaS and sigmaS-dependent ASPs in logarithmically growing cells, as well as increases in tolerances to acid, heat, osmolarity and oxidative stresses and significant decreases in growth rate and colony size. Mutations in rpoS suppressed the mviA::Km-associated defects in growth rate, colony size, ASP production, and stress tolerance, suggesting that the effects of MviA on cell physiology occur via its control of sigmaS levels. Western blot (immunoblot) analyses of sigmaS produced from natural or arabinose-regulated promoters revealed that acid shock and MviA posttranscriptionally regulate sigmaS levels. Turnover experiments suggest that MviA regulates the stability of sigmaS protein rather than the translation of rpoS message. We propose a model in which MviA or its unknown signal transduction partner senses some consequence of acid shock, and probably other stresses, and signals the release of sigmaS from proteolysis. The increased concentration of sigmaS drives the elevated expression of the sigmaS-dependent ASPs, resulting in an increase in stress tolerance. The avirulent nature of mviA insertion mutants, therefore, appears to result from inappropriate sigmaS-dependent gene expression during pathogenesis.

Postnatal changes in selected bacterial groups of the pig colonic microflora.

The importance of the colonic microflora in health and nutrition is well known, but how they colonize and become established in the colon is not well understood. We therefore characterized the quantitative and qualitative changes of the colonic microflora during the first 120 days of postnatal development. Unlike previous studies, changes were defined for individual pigs using in situ samples collected anaerobically and aseptically from the distal colon. Although the colons were sterile at birth, they were rapidly colonized, and within 12 h bacterial densities had stabilized at 10(-9)-10(10) bacteria/g colonic content. Facultative anaerobes, notably coliforms, initially dominated the microflora, but were supplanted within 48 h after birth by obligate anaerobes, which constituted greater than 90% of the microflora thereafter. Bacteroides spp., the predominant anaerobes in the adult colon, did not markedly increase in abundance until after weaning and were still increasing by postnatal day 120. Shifts in the relative abundances of different bacterial populations throughout the first 120 days after birth confirm previous reports that the establishment of the adult colonic microflora is a gradual, sequential process, and highlight the need to focus research on anaerobic groups.

Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling.

Non-typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca2+ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell-permeant intracellular Ca2+ chelator BAPTA-AM (P = 0.022) or thapsigargin (P = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 (P < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019-stimulated IP production in a dose-dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX-sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.

Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor.

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.

Observed differences in virulence-associated phenotypes between a human clinical isolate and a veterinary isolate of Mycobacterium avium.

Mycobacterium avium, the most common opportunistic pathogen in patients with AIDS, is frequently isolated from a variety of environmental sources, but rarely can these environmental isolates be epidemiologically linked with isolates known to cause human disease. Using a number of in vitro tissue culture assays, we found significant pathogenic differences between a serotype 4 human clinical M. avium isolate and a serotype 2 veterinary isolate. Cell association of the patient strain with a human intestinal cell line was 1.7 times that of the veterinary strain. Growth of this clinical strain in human peripheral blood mononuclear cell-derived macrophages increased from 12-fold higher than that of the veterinary isolate after 2 days to 200-fold higher after 4 days. By the conclusion of each experiment, lysis of all examined host cell types and accumulation of cell debris were observed in infections with the human isolate, but monolayers remained relatively intact in the presence of the animal isolate. The two strains also differed in the ability to stimulate human immunodeficiency virus replication in coinfected host cells, with p24 antigen levels after 6 days threefold higher in the cells coinfected with the clinical strain than in those infected with the veterinary strain. If the genetic differences responsible for the phenotypes observed in these assays can be identified and characterized, it may be possible to determine which M. avium strains in the environment are potential human pathogens.

Involvement of matrix metalloproteinases in human immunodeficiency virus type 1-induced replication by clinical Mycobacterium avium isolates.

The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.