Richard S. Berk

Identification of adenoid biofilms with middle ear pathogens in otitis-prone children utilizing SEM and FISH

OBJECTIVES Biofilms have been implicated in the development of several chronic infections. We sought to demonstrate middle ear pathogens in adenoid biofilms using scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with confocal laser scanning microscopy (CLSM). METHODS Comparative micro-anatomic investigation of adenoid mucosa using SEM and FISH with confocal scanning laser microscopic (CLSM) imaging from patients with recurrent acute otitis media (RAOM). RESULTS All otitis-prone children demonstrated biofilm surface area presence greater than 85% by SEM. FISH accompanied by CLSM imaging also demonstrated patchy biofilms All biofilms contained middle ear pathogens and were frequent in polymicrobial distributions: 4 of 6, 4 of 6 and 3 of 6 samples contained Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, respectively. CONCLUSIONS Dense adenoid biofilms may act as a reservoir for reinfection of the tubotympanum. Aspiration of planktonic middle ear pathogens existing in resistant adenoid biofilms during a viral upper respiratory tract infection may be an important event in the development of RAOM.

Analysis of adhesion, piliation, protease production and ocular infectivity of several P. aeruginosa strains

The role of bacterial piliation and protease production in Pseudomonas aeruginosa adhesion to the injured corneal epithelial surface and subsequent infectivity was examined using several bacterial strains, including three that were hyperpiliated. To initiate this study, bacteria were examined by transmission EM to confirm their piliation characteristics. The PAK strain, like pseudomonas ATCC 19660, possessed about 1-4 polar pili. The mutant PAK/PR11 lacked pili while PAK/PR1, DB2, a mutant of PAO1, and PA1244, a wild-type clinical isolate, were hyperpiliated. Ocular infectivity of these bacterial strains and mutants was examined macroscopically and histopathologically in mice and these data compared to the well-characterized ocular disease response of a murine model of infection with pseudomonas ATCC 19660. The PAK strain was infective, but less virulent than strain 19660 by both macroscopic grading and histopathological analysis of infected eyes. Infectivity of the PR11 mutant was similar to the PAK parent strain, while PR1, DB2 and 1244, all hyperpiliated, were not infective. To explore the hypothesis that hyperpiliated bacteria bound less well to cornea and thus failed to induce corneal disease, in vitro quantitative studies of bacterial adhesion were done using an ocular organ culture model. The PR1 hyperpiliated mutant bound significantly less well to cornea than the PAK parent strain, PR11 mutant or pseudomonas 19660, while DB2 and 1244 binding did not differ significantly from 19660 or PAK. Examination of protease production, another factor which may influence adhesion, revealed that only 19660 and DB2 produced detectable protease. This study provides evidence that non-piliated, non-protease producing strains such as PAK/PR11 possess alternate virulence mechanisms to facilitate binding to and infectivity of corneal tissue.

Demonstration of Nasopharyngeal and Middle Ear Mucosal Biofilms in an Animal Model of Acute Otitis Media

Objectives: We performed this study to determine the role of nasopharyngeal and middle ear (ME) biofilms in acute otitis media (AOM). Methods: Sixty female 6-month-old chinchillas, free of ME disease, were utilized. Experimental animals were inoculated with influenza A followed by Streptococcus pneumonia 7 days later. Control animals were inoculated with Sorensens phosphate buffer. Daily otoscopy and tympanometry was performed, and the animals were painlessly sacrificed on days 1, 2, 5, 8, and 14. All mucosae were harvested and prepared for scanning electron microscopy. Results: The ME inflammation, initially detected on day 2 after bacterial inoculation, peaked on day 8. Eight percent of the dually inoculated chinchillas displayed type B tympanograms, and 40% displayed type C. Otoscopic evaluation of tympanic membrane inflammation was rated from 0 to 4 (0 = normal and 4 = severe drainage and/or inflammation) according to an otoscopic grading system. Ten percent of the experimental chinchillas had a grade 2 score, 20% had grade 3, and 6.7% had grade 4. The controls demonstrated no abnormal tympanometric or otoscopic findings. Scanning electron microscopic imaging showed dense biofilms on 83% of the nasopharynges and 67% of the MEs on day 8 in the experimental animals. All animals with ME biofilms had biofilms in the nasopharynx. The controls did not demonstrate biofilm formation. Conclusions: The study parallels the natural pathogenesis of AOM in humans. The demonstration of mucosal biofilms in both the nasopharynx (58%) and the ME (47%) of animals with ME inflammation and/or infection lends further support to the importance of mucosal biofilms in the pathogenesis of AOM.

Catabolism of taurine in Pseudomonas aeruginosa.

Cell-free extracts of taurine-grown Pseudomonas aeruginosa catalyze the transamination of taurine and pyruvate resulting in the formation of L-alanine and sulfoacetaldehyde. The enzyme responsible for this activity has been partially purified in order to demonstrate its participation in a pathway of taurine degradation. Ethyl methane sulfonate treatment of Ps. aeruginosa yielded a mutant deficient in taurine transaminase and incapable of growing on taurine indicating that the enzyme is of physiological significance in this organism.

In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces phospholipase C (PLC), a heat-labile hemolysin. Histopathological analysis of PLC-treated mice revealed that the primary target organs involved in PLC-induced toxicity were the liver and kidney. Mice treated i.v. with PLC demonstrated significant tubular epithelial necrosis of the kidney with hematuria, while when given i.p. they exhibited hepatonecrosis with cellular infiltration. Splenomegaly was also a consistent finding. Results from in vitro studies indicate that PLC is toxic for mouse peritoneal cells and human leukocytes.

Biofilms and chronic otitis media: an initial exploration into the role of biofilms in the pathogenesis of chronic otitis media

PURPOSE The aim of the study was to compare the extent of biofilm infection in percentage of mucosal surface area of adenoids removed from children with otitis media with effusion (OME) vs those with recurrent acute otitis media (RAOM) and obstructive sleep apnea (OSA). MATERIALS AND METHODS Comparative microanatomical investigation of adenoid mucosa using scanning electron microscopy obtained from 30 children with OME, RAOM, and OSA was used in this study. Seventeen males and 13 females ranging in age from 9 months to 10 years were included in this study. Percentage of biofilm surface area involvement was the main measure. RESULTS Adenoids removed from patients with OME had moderately dense mature biofilms covering the mucosal surface with a mean of 27.7% of their mucosal surface covered with mature biofilms. These results were distinct from results obtained from patients diagnosed with RAOM and OSA with means of 97.6% and 0.10% of their mucosal surfaces covered with mature biofilms, respectively. These differences were statistically significant at P < .0001. CONCLUSIONS Adenoids removed from patients with OME were characterized by distinctly different percentage of biofilm mucosal surface area coverage, with significantly more biofilm presence than OSA patients but significantly less biofilm presence than RAOM patients. Although previous investigations have supported a dominant role of nasopharyngeal biofilms in RAOM pathogenesis, these results suggest nasopharyngeal biofilms may play a different role in the pathogenesis of OME and that this clinical entity may be more multifactorial in nature.

Distribution and Kinetics of the Inflammatory Cell Response to Ocular Challenge with Pseudomonas aeruginosa in Susceptible versus Resistant Mice

This study examined and characterized the distribution and kinetics of the inflammatory cell response to pseudomonas ocular challenge in susceptible C57BL/6J and resistant DBA/2J mice. Initially, in the cornea, the number of neutrophilic leukocytes (PMNs), macrophages/monocytes or lymphocytes was 2-2.5 times greater in resistant versus susceptible mice. The peak cellular response in the cornea was also greater in resistant than in susceptible animals. Further, resistant mice, when compared with susceptible mice, had a shorter duration of inflammatory cells as well as bacteria in the cornea. These data were similar for the cell infiltrate in the anterior chamber, with the exception that PMNs were initially greater in number in C57BL/6J than in DBA/2J mice. Collectively these data suggest that susceptible mice are, in general, cellularly hyporesponsive to pseudomonas ocular challenge when compared with resistant animals. This, together with persistence of inflammatory cells and bacteria in the cornea of susceptible animals, may contribute to their failure to restore corneal clarity following pseudomonas ocular challenge.

Experimental eye infections caused by Pseudomonas aeruginosa

Cells of Pseudomonas aeruginosa were titered (1.0× 104–1.0× 108 cells) to determine the minimum number required to initiate corneal infection in Swiss-Webster

Desquamation of the corneal epithelium in the immature mouse: a scanning and transmission microscopy study.

Abstract The normal immature mouse corneal epithelium as characterized by scanning electron microscopy (SEM) is composed of dark, medium and light cells covered with surface microvilli. Microvilli are more numerous on the light and medium than the dark cells. In transmission electron microscopy (TEM) light cells of SEM are electron dense. The medium and dark cells of SEM on the other hand, have pale cytoplasm with few microvilli. The process of epithelial desquamation in the above cell population was detailed sequentially. The initial step in desquamation of a surface cell is swelling and reduction in number of surface microvilli. This is followed by the appearance of dehiscences or slits in the plasma membrane of surface cells. In TEM these dehiscences are readily visible as small slits in the apical plasma membrane of cells with pale appearing cytoplasm. Next, the desquamating cell pulls away from its lateral cell borders. Finally, the cell detaches from the intermediate cell layer by rupture of desmosomes. This mechanism of desquamation would not appear to contribute to or to insure stability of the mucin component of the precorneal tear film.

Biofilm density in the pediatric nasopharynx: recurrent acute otitis media versus obstructive sleep apnea.

Objectives: We compared the biofilm surface density of adenoids removed from children with recurrent acute otitis media (RAOM) to that of adenoids removed from children with a diagnosis of obstructive sleep apnea (OSA). Methods: We performed a comparative microanatomic study of adenoid mucosa using scanning electron microscopy in patients with diagnoses of RAOM and OSA (27 female and 41 male; age range, 3 months to 15 years). Results: The adenoids removed from patients with RAOM had dense, mature biofilms covering nearly their entire mucosal surfaces. More specifically, the adenoids removed from patients with RAOM had an average of 93.53% of their mucosal surface covered, versus an average of 1.01% coverage on the adenoids removed from patients with OSA. These differences were statistically significant (p < 0.0001). Conclusions: The adenoids removed from patients with RAOM had almost their entire mucosal surface covered with biofilms, versus scant coverage for patients with OSA. Recurrent acute otitis media is notoriously resistant to antibiotic treatment, and aspirates of middle ear fluid repeatedly yield negative cultures. It is these properties that have led biofilms to become increasingly implicated in the pathogenesis of RAOM. Thus, the resistance of biofilms to antimicrobials, together with their planktonic shedding of organisms, may be an important mechanism in the development of RAOM.