Adenoid reservoir for pathogenic biofilm bacteria.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Mucin gene expression and mouse middle ear epithelium.

OBJECTIVES: To investigate the expression of recently identified human mucin genes in an in vitro model of cultured mouse middle ear epithelial cells (MMEEC). METHODS: MMEEC were established, RNA was extracted and primers were designed for RT-PCR to assess for expression of mucin genes Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc6, Muc7, Muc8, Muc9, Muc10, Muc11/12, Muc13, Muc15, Muc16, Muc17, Muc18, Muc19 and Muc20 expression. RESULTS: Mucin genes Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc9, Muc10, Muc13, Muc15, Muc16, Muc18, Muc19 and Muc20 were identified and expressed in MMEEC. The genes Muc6, Muc7, Muc8, Muc11/12 and Muc17 were not identified. CONCLUSION: Many of the mucin genes that have been recently identified in human MEE and chinchilla MEE are also expressed in MMEEC. There are differences in expression, however, which may have implications in utilizing various animal models for study of middle ear physiology and pathogenesis; specifically as it relates to mucin gene expression.

Extending the chinchilla middle ear epithelial model for mucin gene investigation.

OBJECTIVES: To investigate the expression of recently identified human mucin genes in an in vivo model of the chinchilla middle ear epithelium (CMEE). METHODS: CMEE was harvested, RNA was extracted and primers were designed for RT-PCR to assess for expression of mucin genes Muc6, Muc17 and Muc18. Further sequencing of these genes was also accomplished. RESULTS: Mucin genes Muc6, Muc17 and Muc18 was assessed and found to be identical to the expression in human and mouse MEE. CONCLUSION: This study further characterizes mucin gene expression in the CMEE and provides additional sequence data for chinchilla middle ear genes. The concordance of this gene expression data to that of both the human and mouse models further demonstrates the utility of this animal model in OM investigations.

Recombinant Enterobacter amnigenus highly toxic to Anopheles dirus mosquito larvae.

The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter amnigenus, a Gram-negative bacterium isolated from Anopheles dirus larvae gut. The toxin was placed under the regulation of various promoters in order to improve the expression level of the toxin. Amongst the recombinants obtained, E. amnigenus harboring pBS373, a plasmid which contains the toxin genes under the control of the native B. sphaericus promoter, expressed a significant amount of protein, comparable to that found in B. sphaericus 2297. In addition, this recombinant provided approximately twenty times higher toxicity against second-instar Anopheles dirus larvae when compared to B. sphaericus 2297. The procedure of obtaining this environmentally isolated bacterium from larvae gut and introducing the system for mosquito-larvicidal toxin synthesis is noteworthy. The promising result presented here provides a substantial degree of confidence for further field studies.

Efficient expression of mosquito-larvicidal proteins in a gram-negative bacterium capable of recolonization in the guts of Anopheles dirus larva.

The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae, was identified as Enterobacter amnigenus. The E. amnigenus was able to recolonize in the gut of An. dirus larva but not in those of Aedes aegypti and Culex quinquefasciatus larvae. It was able to float in water for a longer period than Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. These are desirable characteristics for a delivery vehicle of mosquito-larvicidal toxins for the control of mosquito larvae, and E. amnigenus was therefore used as a host to express the cryIVB gene of B. thuringiensis subsp. israelensis and the binary toxin genes of B. sphaericus. The recombinant E. amnigenus produced a high level of CryIVB protein, which was toxic to larvae of Ae. aegypti and An. dirus. Another E. amnigenus producing the 51-kDa protein of B. sphaericus was toxic to larvae of An. dirus and Cx. quinquefasciatus. The recombinant plasmids were stable in E. amnigenus without the presence of selective pressure for at least 23 generations. The recombinant E. amnigenus should represent a desirable biological agent for controlling mosquito larvae.

The glycogen phosphorylase-2 promoter binding protein in Dictyostelium is replication protein A.

During Dictyostelium development, glycogen degradation is a crucial event that provides glucose monomers used in the synthesis of the essential structural components for cellular differentiation. The product of the developmentally regulated glycogen phosphorylase-2 gene (gp2) catalyzes the degradation. DNA-binding proteins were found to bind to a regulatory site of the gp2 gene in a stage-dependent pattern. Gel-shift analysis of undifferentiated amoebae cell extract revealed a protein migrating at 0.40 Rf, while 17 hour differentiated cell extract produced a species migrating at 0.32 Rf. Both the 0.32 and 0.40 Rf proteins were purified and found to consist of three subunits of 18, 35 and 62 kDa (for 0.40 Rf) or 81 kDa (for 0.32 Rf). Data base searches identified the protein as the Dictyostelium homologue of replication protein A (DdRPA). Amino acid sequence analysis showed identity between the 62 and 81 kDa subunits. Incubation of cell-free extracts under appropriate conditions at low pH, resulted in conversion of the 81 kDa to the 62 kDa subunit. Northern blot analysis revealed that the levels of expression of the large subunit of DdRPA were constant throughout differentiation and the size of the mRNA was the same at all stages of development. The results raise the possibility that pH induced post-translational modifications of DdRPA are involved in events that halt cell proliferation and induce differentiation in Dictyostelium.