Induction of inflammatory responses from THP-1 cells by cell-free filtrates from clinical isolates of Alloiococcus otitidis.

In our model system using the THP-1 monocytic cell line, whole heat-killed cells of Alloiococcus otitidis elicited several pro-inflammatory cytokines identified in ear effusions of children with otitis media (OM). Levels of these cytokines were equivalent to or greater than those elicited by a standard Gram-positive otopathogen, Streptococcus pneumoniae. The current study examined the hypothesis that extracellular material produced by A. otitidis might also contribute to the inflammatory responses in OM. Cell-free culture filtrates of recent A. otitidis isolates (n = 39) were tested for induction of pro-inflammatory cytokines from THP-1 cells primed with IFN-γ. The highest responses were from IL-8 followed by IL-1ß, and the lowest from IL-6. Filtrates from nine isolates were treated with lysozyme or proteinase K to assess the nature of the extracellular stimulants. Peptidoglycan was not a major component eliciting the responses. There was no correlation between colony type or ß-haemolysin production. Proteinase K treatment indicated extracellular proteins might induce the inflammatory responses, particularly the 70-75 ku band. Further studies on the role of the extracellular proteins of A. otitidis and cytokine responses in pathogenesis of ear infections are needed.

In vitro inflammatory responses elicited by isolates of Alloiococcus otitidis obtained from children with otitis media with effusion.

Alloiococcus otitidis is usually detected in children with otitis media (OM) by PCR as it is not often detected by routine culture. Our improved method for its isolation obtained A. otitidis from nearly 50% of 78 children with OM with effusion. The role of A. otitidis in pathogenesis of OM is unclear. This study tested two hypothesis: (1) that fresh isolates of A. otitidis would elicit pro-inflammatory cytokines from THP-1 monocytic cells equivalent to those induced by Streptococcus pneumoniae; (2) priming THP-1 cells with interferon-gamma (IFN-γ) a surrogate for virus infection, would enhance pro-inflammatory responses. Recent clinical isolates of A. otitidis, S. pneumoniae (ATCC 49619) and a blood culture isolate of S. pneumoniae (SP2) were used in the assays. Cytokines were quantified by BioRad bead assay and Luminex 200. IFN-γ priming enhanced cytokine responses. S. pneumoniae ATCC 49619 induced lower responses than SP2 for IL-1ß, IL-6, TNF-α. A. otitidis LW 27 elicited higher IL-1ß and TNF-α responses than either pneumococcal isolate. Small green colony types of A. otitidis induced higher responses than large white colony types for IL-8 and IL-1ß. The hypothesis that A. otitidis elicits cytokines observed in middle ear effusions was supported; the need to use recent clinical isolates in studies of pathogenesis was highlighted.

Isolation of Alloiococcus otitidis from Indigenous and non-Indigenous Australian children with chronic otitis media with effusion.

During the last decade Alloiococcus otitidis has been identified in specimens from patients with chronic otitis media with effusion. Whereas most of those studies employed molecular techniques, we used minor modifications of conventional microbiological methods to isolate and identify A. otitidis in samples obtained from 20/50 (40%) children referred for myringotomy. Alloiococcus otitidis was isolated from 10/22 (45%) Indigenous and 10/28 (36%) non-Indigenous children. This is the first report of isolation of A. otitidis from Australian children with chronic otitis media. All isolates were sensitive to penicillin, but 14/20 (70%) of the isolates were resistant or partially resistant to erythromycin as assessed by the E-test.

Mucoid nitrate-negative Moraxella nonliquefaciens from three patients with chronic lung disease.

Mucoid strains of Moraxella nonliquefaciens were recovered from the sputa of three indigenous Australians with chronic lung disease. These atypical strains failed to reduce nitrate, and one strain produced beta-lactamase. While the mucoid phenotype of M. nonliquefaciens has rarely been reported, the mucoid nitrate-negative biovar has never been previously reported.

A comparison of antibiotic susceptibility testing methods for cotrimoxazole with Burkholderia pseudomallei.

Melioidosis is caused by the Gram-negative soil saprophyte, Burkholderia pseudomallei and is endemic in tropical and subtropical regions of southeast Asia and northern Australia. Cotrimoxazole has been traditionally used for the therapy of melioidosis despite results indicating resistance often produced in the disc diffusion test against B. pseudomallei. This inconsistency was addressed by comparing this method with the agar dilution, MicroScan and E-test methods. The results demonstrated that by disc diffusion, 41.3% of 80 B. pseudomallei clinical isolates tested were susceptible to cotrimoxazole, whereas the MicroScan, agar dilution and the E-test demonstrated 92.5, 90 and 97.5% of the isolates to be susceptible, respectively. These results indicate that an MIC based method is required to test the susceptibility of B. pseudomallei against cotrimoxazole.