Cellulosilyticum ruminicola, a newly described rumen bacterium that possesses redundant fibrolytic-protein-encoding genes and degrades lignocellulose with multiple carbohydrate- borne fibrolytic enzymes.

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.

Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa.

OBJECTIVES: We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response. METHODS: Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription-polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism. RESULTS: Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM. CONCLUSIONS: Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.

Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates.

BACKGROUND: Streptococcus pneumoniae is a common respiratory pathogen and a major causative agent of respiratory infections, including otitis media (OM). Pneumococcal biofilms have been demonstrated on biopsies of the middle ear mucosa in children receiving tympanostomy tubes, supporting the hypothesis that chronic OM may involve biofilm development by pathogenic bacteria as part of the infectious process. To better understand pneumococcal biofilm formation six low-passage encapsulated nasopharyngeal isolates of S. pneumoniae were assessed over a six-eight day period in vitro. RESULTS: Multiparametric analysis divided the strains into two groups. Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin. Those with a low BFI developed less extensive biofilms and were more susceptible to azithromycin. dsDNA was present in the S. pneumoniae biofilm matrix in all strains and treatment with DNase I significantly reduced biofilm biomass. Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon. Interestingly, cpsA was downregulated in biofilms in both high and low BFI strains. CONCLUSION: All pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance. Furthermore, all strains of S. pneumoniae showed downregulation of the cpsA gene during biofilm growth compared to planktonic culture, regardless of BFI ranking, suggesting downregulation of capsule expression occurs generally during adherent growth.

Differential expression of chaperonin containing T-complex polypeptide (CCT) subunits during fetal and adult skin wound healing.

Integumentary wound healing in early fetal life is regenerative and proceeds without scar formation. Expressomic analysis of this phenomenon by differential display has previously determined that the eta subunit of the cytosolic chaperonin containing T-complex polypeptide (CCT) is downregulated in the healing fetal wound milieu. We now report that no other CCT subunit shares this distinct pattern of gene regulation as determined by limiting dilution reverse transcriptase polymerase chain reaction (RT-PCR); all seven of the remaining CCT subunits demonstrate no change in messenger RNA (mRNA) expression in healing fetal wounds compared to unwounded control tissue. The alpha subunit, however, did evidence reduced message levels in healing adult wound tissue. We herein report on the cloning and sequence of the complementary DNA (cDNA) for rabbit CCT-alpha and confirm its wound specific decrease in adult tissues through quantitative real-time RT-PCR assay. We also confirm that quantitative evaluation of CCT-alpha and CCT-zeta mRNA expression shows no change in healing fetal wounds.

Virulence phenotypes of low-passage clinical isolates of nontypeable Haemophilus influenzae assessed using the chinchilla laniger model of otitis media.

BACKGROUND: The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease. RESULTS: The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA - PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME. CONCLUSION: Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.

Codon usage comparison of novel genes in clinical isolates of Haemophilus influenzae.

A similarity statistic for codon usage was developed and used to compare novel gene sequences found in clinical isolates of Haemophilus influenzae with a reference set of 80 prokaryotic, eukaryotic and viral genomes. These analyses were performed to obtain an indication as to whether individual genes were Haemophilus-like in nature, or if they probably had more recently entered the H.influenzae gene pool via horizontal gene transfer from other species. The average and SD values were calculated for the similarity statistics from a study of the set of all genes in the H.influenzae Rd reference genome that encoded proteins of 100 amino acids or longer. Approximately 80% of Rd genes gave a statistic indicating that they were most like other Rd genes. Genes displaying codon usage statistics >1 SD above this range were either considered part of the highly expressed group of H.influenzae genes, or were considered of foreign origin. An alternative determinant for identifying genes of foreign origin was when the similarity statistics produced a value that was much closer to a non-H.influenzae reference organism than to any of the Haemophilus species contained in the reference set. Approximately 65% of the novel sequences identified in the H.influenzae clinical isolates displayed codon usages most similar to Haemophilus sp. The remaining novel sequences produced similarity statistics closer to one of the other reference genomes thereby suggesting that these sequences may have entered the H.influenzae gene pool more recently via horizontal transfer.

Construction and characterization of a highly redundant Pseudomonas aeruginosa genomic library prepared from 12 clinical isolates: application to studies of gene distribution among populations.

OBJECTIVE: To create, array, and characterize a pooled, high-coverage, genomic library composed of multiple biofilm-forming clinical strains of the opportunistic pathogen, Pseudomonas aeruginosa (PA). Twelve strains were obtained from patients with otorrhea, otitis media, and cystic fibrosis as a resource for investigating: difference in the transcriptomes of planktonic and biofilm envirovars; the size of the PA supragenome and determining the number of virulence genes available at the population level; and the distributed genome hypothesis. METHODS: High molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of approximately 1.5 kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with approximately 250,000 clones that was arrayed and subjected to quality control analyses. RESULTS: Restriction endonuclease and sequence analyses of 686 clones picked at random from the library demonstrated that >75% of the clones contained inserts larger than 0.5 kb with the desired mean insert size of 1.4 kb. Thus, this library provides better than 4.5x coverage for each of the genomes from the 12 components clinical PA isolates. Our sequencing effort ( approximately 1 million nucleotides to date) reveals that 13% of the clones present in this library are not represented in the genome of the reference P. aeruginosa strain PA01. CONCLUSIONS: Our data suggests that reliance on a single laboratory strain, such as PA01, as being representative of a pathogenic bacterial species will fail to identify many important genes, and that to obtain a complete picture of complex phenomena, including bacterial pathogenesis and the genetics of biofilm development will require characterization of the P. aeruginosa population-based supra-genome.

Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media.

CONTEXT: Chronic otitis media (OM) is a common pediatric infectious disease. Previous studies demonstrating that metabolically active bacteria exist in culture-negative pediatric middle-ear effusions and that experimental infection with Haemophilus influenzae in the chinchilla model of otitis media results in the formation of adherent mucosal biofilms suggest that chronic OM may result from a mucosal biofilm infection. OBJECTIVE: To test the hypothesis that chronic OM in humans is biofilm-related. DESIGN, SETTING, AND PATIENTS: Middle-ear mucosa (MEM) biopsy specimens were obtained from 26 children (mean age, 2.5 [range, 0.5-14] years) undergoing tympanostomy tube placement for treatment of otitis media with effusion (OME) and recurrent OM and were analyzed using microbiological culture, polymerase chain reaction (PCR)-based diagnostics, direct microscopic examination, fluorescence in situ hybridization, and immunostaining. Uninfected (control) MEM specimens were obtained from 3 children and 5 adults undergoing cochlear implantation. Patients were enrolled between February 2004 and April 2005 from a single US tertiary referral otolaryngology practice. MAIN OUTCOME MEASURES: Confocal laser scanning microscopic (CLSM) images were obtained from MEM biopsy specimens and were evaluated for biofilm morphology using generic stains and species-specific probes for H influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Effusions, when present, were evaluated by PCR and culture for evidence of pathogen-specific nucleic acid sequences and bacterial growth, respectively. RESULTS: Of the 26 children undergoing tympanostomy tube placement, 13 (50%) had OME, 20 (77%) had recurrent OM, and 7 (27%) had both diagnoses; 27 of 52 (52%) of the ears had effusions, 24 of 24 effusions were PCR-positive for at least 1 OM pathogen, and 6 (22%) of 27 effusions were culture-positive for any pathogen. Mucosal biofilms were visualized by CLSM on 46 (92%) of 50 MEM specimens from children with OME and recurrent OM using generic and pathogen-specific probes. Biofilms were not observed on 8 control MEM specimens obtained from the patients undergoing cochlear implantation. CONCLUSION: Direct detection of biofilms on MEM biopsy specimens from children with OME and recurrent OM supports the hypothesis that these chronic middle-ear disorders are biofilm-related.

Development and characterization of a pooled Haemophilus influenzae genomic library for the evaluation of gene expression changes associated with mucosal biofilm formation in otitis media.

UNLABELLED: Haemophilus influenzae is one of the most important respiratory pathogens of man. It has been etiologically associated with otitis media, otorrhea, and chronic obstructive pulmonary disease. Identification of new genomic elements will provide novel targets to fight chronic infections caused by this organism. OBJECTIVE: The new paradigm that chronic infections are caused by bacterial biofilms prompted us to study the relationship between bacterial pathogenicity, biofilm formation and bacterial communal cooperation. To do this, it is essential to determine the virulence gene sets that are involved in the above processes and whether they are present in every bacterial cell or distributed in a "communal gene-pool", the distributed genome hypothesis (DGH). We designed, constructed and characterized a highly redundant genomic DNA library comprised of the genomes of ten low passage clinical isolates of H. influenzae carrying large numbers of genes that are not present in the laboratory strains of H. influenzae. METHODS: Genomic DNA fragments of the ten clinical strains were hydro-dynamically sheared to produce a mean fragment size of 1.5-2.5 kb. The ten sheared DNAs were than pooled and used in the construction of a genomic library with 76800 clones. RESULTS: Our restriction endonuclease and sequence analyses of 800 clones demonstrate that 75% of the clones carry an insert larger than 0.5 kb. The library has an approximately 1.5 kb average insert size, and therefore, better than 4.5x redundancy for each of the genomes of the ten clinical isolates. Our sequencing effort ( approximately 1 million nucleotides to date) reveals that a high percentage of genes (75 clones, 11% of the 686 sequenced clones) present in this library are not represented in the genome of the reference strain H. influenzae Rd. CONCLUSIONS: The library, based on the above results, has a better than 4.5x coverage for each of the ten constituent genomes. On the basis of our preliminary sequencing data ( approximately 1 million nucleotides) the library lacks of highly repeated sequences, therefore, the expected genome coverage (4.5x) is not degraded. Using the prevalence of non-Rd like sequences (11%) detected during characterization of the genomic library, we estimated that the library contains DNA sequences equivalent to approximately 2 million bp, which are not represented in the reference genome of the H. influenzae Rd strain and that is greater in size than the genome of this reference strain, providing ample targets for innovative drug design.

Death and transfiguration in static Staphylococcus epidermidis cultures.

The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheet-like structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand-containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity.

The distributed genome hypothesis as a rubric for understanding evolution in situ during chronic bacterial biofilm infectious processes.

Most chronic infectious disease processes associated with bacteria are characterized by the formation of a biofilm that provides for bacterial attachment to the host tissue or the implanted medical device. The biofilm protects the bacteria from the host's adaptive immune response as well as predation by phagocytic cells. However, the most insidious aspect of biofilm biology from the host's point of view is that the biofilm provides an ideal setting for bacterial horizontal gene transfer (HGT). HGT provides for large-scale genome content changes in situ during the chronic infectious process. Obviously, for HGT processes to result in the reassortment of alleles and genes among bacterial strains, the infection must be polyclonal (polymicrobial) in nature. In this review, we marshal the evidence that all of the factors are present in biofilm infections to support HGT that results in the ongoing production of novel strains with unique combinations of genic characteristics and that the continual production of large numbers of novel, but related bacterial strains leads to persistence. This concept of an infecting population of bacteria undergoing mutagenesis to produce a 'cloud' of similar strains to confuse and overwhelm the host's immune system parallels genetic diversity strategies used by viral and parasitic pathogens.

Gene expression differences in infected and noninfected middle ear complementary DNA libraries.

OBJECTIVES: To investigate genetic differences in middle ear mucosa (MEM) with nontypeable Haemophilus influenzae (NTHi) infection. Genetic upregulation and downregulation occurs in MEM during otitis media (OM) pathogenesis. A comprehensive assessment of these genetic differences using the techniques of complementary DNA (cDNA) library creation has not been performed. DESIGN: The cDNA libraries were constructed from NTHi-infected and noninfected chinchilla MEM. Random clones were picked, sequenced bidirectionally, and submitted to the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags database, where they were assigned accession numbers. These numbers were used with the basic local alignment search tool (BLAST) to align clones against the nonredundant nucleotide database at NCBI. RESULTS: Analysis with the Web-based statistical program FatiGO identified several biological processes with significant differences in numbers of represented genes. Processes involved in immune, stress, and wound responses were more prevalent in the NTHi-infected library. S100 calcium-binding protein A9 (S100A9); secretory leukoprotease inhibitor (SLPI); beta(2)-microglobulin (B2M); ferritin, heavy-chain polypeptide 1 (FTH1); and S100 calcium-binding protein A8 (S100A8) were expressed at significantly higher levels in the NTHi-infected library. Calcium-binding proteins S100A9 and S100A8 serve as markers for inflammation and have antibacterial effects. Secretory leukoprotease inhibitor is an antibacterial protein that inhibits stimuli-induced MUC1, MUC2, and MUC5AC production. CONCLUSIONS: A number of genes demonstrate changes during the pathogenesis of OM, including SLPI, which has an impact on mucin gene expression; this expression is known to be an important regulator in OM. The techniques described herein provide a framework for future investigations to more thoroughly understand molecular changes in the middle ear, which will likely be important in developing new therapeutic and intervention strategies.

Population-level virulence factors amongst pathogenic bacteria: relation to infection outcome.

The study of population-level virulence traits among communal bacteria represents an emerging discipline in the field of bacterial pathogenesis. It has become clear over the past decade-and-a-half that bacteria exhibit many of the hallmarks of multicellular organisms when they are growing as biofilms and communicating among each other using quorum- sensing systems. Each of these population-level behaviors provides for multiple expressions of virulence that individual free-swimming bacteria do not possess. Population-level virulence traits are largely associated with chronic or persistent infections, whereas individual bacterial virulence traits are associated with acute infections. Thus, there is a natural dichotomy between acute and chronic infectious processes, which helps to explain the medical community's success in combating the former, but its utter failure in dealing with the latter. The recent recognition of multicellularity among chronic bacterial pathogens will lead the way towards new multimodality therapies.

What makes pathogens pathogenic.

Metazoans contain multiple complex microbial ecosystems in which the balance between host and microbe can be tipped from commensalism to pathogenicity. This transition is likely to depend both on the prevailing environmental conditions and on specific gene-gene interactions placed within the context of the entire ecosystem.

Strain-specific virulence phenotypes of Streptococcus pneumoniae assessed using the Chinchilla laniger model of otitis media.

BACKGROUND: Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced. METHODOLOGY/PRINCIPAL FINDINGS: For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity. CONCLUSIONS/SIGNIFICANCE: As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.

Identification of differentially expressed genes in scarless wound healing utilizing polymerase chain reaction-suppression subtractive hybridization.

Wound healing in fetal skin is well known to proceed without scarring, whereas adult (postnatal) skin wound healing is accompanied by scar formation. To identify differentially expressed genes during fetal wound (FW) healing, we have used polymerase chain reaction-suppression subtractive hybridization. This technique allows for a comparative analysis across the entire transcriptome of FW vs. unwounded fetal control tissue, including even potentially novel sequences. Our subtractive hybridization protocol identified 15 clones that are overexpressed in healing FWs, and 20 clones that are underexpressed. These include genes with both known and unknown functions. We have confirmed the differential pattern of expression for four of these candidate genes: elongation factor 1 alpha, elongation initiation factor 4e, and two transcripts thus far known only as an expressed sequence tags. With this approach, we have also identified novel genes potentially involved in scarless wound healing.

Bacterial plurality as a general mechanism driving persistence in chronic infections.

Classical methods for the study of bacterial pathogens have proven to be inadequate to inform with respect to chronic infections including those associated with arthroplasties. Modern methods of analysis have demonstrated that bacterial growth patterns, ecology, and intra-species heterogeneity are more complex than were envisioned by early microbiologists. Cultural methods were developed to study acute, epidemic infections, but it is now recognized that the phenotype associated with these diseases represents only a minor aspect of the bacterial life cycle, which consists of planktonic, attachment, biofilm, and dispersal phases. Over 99% of bacteria in natural populations are found in biofilms which contain multiple ecological niches and numerous phenotypes. Unfortunately, the effort to develop antibiotics has been directed solely at the planktonic minority (associated with systemic illness) which explains our inability to eradicate chronic infections. In this study we establish a new rubric, bacterial plurality, for the understanding of bacterial ecology and evolution with respect to chronic infection. The fundamental tenets of bacterial plurality are that the bacteria within an infecting population display multiple phenotypes and possess multiple genotypes. Phenotypic plurality is embodied in the biofilm paradigm and genotypic plurality is embodied in the concepts of the supra-genome and the distributed genome hypothesis. It is now clear that bacterial diversity provides bacterial populations, as a whole, the ability to persist in the face of a multi-faceted host response.

Molecular and imaging techniques for bacterial biofilms in joint arthroplasty infections.

Biofilm formation on surfaces is an ancient and integral strategy for bacterial survival. Billions of years of adaptation provide microbes with the ability to colonize any surface, including those used in orthopaedic surgery. Although remarkable progress has been made in the treatment of orthopaedic diseases with implanted prostheses, infection rates remain between 1% and 2%, and are higher for revision surgeries. The chronic nature of implant infections, their nonresponsiveness to antibiotics, and their frequent culture negativity can be explained by the biofilm paradigm of infectious disease. However, the role of biofilms in orthopaedic implant infections and aseptic loosening is controversial. To address these issues, we developed molecular diagnostic and confocal imaging techniques to identify and characterize biofilms associated with infected implants. We designed PCR and reverse transcription (RT)-PCR-based assays that can be used to detect bacterial infections associated with culture-negative joint effusions that distinguish between physiologically active Staphylococcus aureus and Staphylococcus epidermidis. Using clinical isolates of Pseudomonas aeruginosa, we constructed a series of reporter strains expressing colored fluorescent proteins to observe biofilms growing on 316L stainless steel and titanium orthopaedic screws. Three-dimensional structures of Pseudomonas aeruginosa and staphylococci biofilms growing on the screws were documented using confocal microscopy. The application of these tools for clinical diagnosis and biofilm research in animal and in vitro models is discussed.

Engineering approaches for the detection and control of orthopaedic biofilm infections.

Artificial joints are subject to chronic infections associated with bacterial biofilms, which only can be eradicated by the traumatic removal of the implant followed by sustained intravenous antibiotic therapy. We have adopted an engineering approach to develop electrical-current-based approaches to bacterial eradication and microelectromechanical systems that could be embedded within the implanted joint to detect the presence of bacteria and to provide in situ treatment of the infection before a biofilm can form. In the former case we will examine the combined bactericidal effects of direct and indirect electrical fields in combination with antibiotic therapy. In the latter case, bacterial detection will occur by developing a microelectromechanical-systems-based biosensor that can "eavesdrop" on bacterial quorum-sensing-based communication systems. Treatment will be effected by the release of a cocktail of pharmaceutical reagents contained within integral reservoirs associated with the implant, including a molecular jamming signal that competitively binds to the bacteria's quorum sensing receptors (which will "blind" the bacteria, preventing the production of toxins) and multiple high dose antibiotics to eradicate the planktonic bacteria. This approach is designed to take advantage of the relatively high susceptibility to antibiotics that planktonic bacteria display compared with biofilm envirovars. Here we report the development of a generic microelectromechanical systems biosensor that measures changes in internal viscosity in a base fluid triggered by a change in the external environment.

Fine mapping a gene for pediatric gastroesophageal reflux on human chromosome 13q14.

We previously mapped a gene for severe pediatric gastroesophageal reflux disease ( GERD1) to a 9-cM interval on chromosome 13q14. In this report, we present the results of DNA sequencing and allelic association analyses that were done in an attempt to clone the GERD1 gene. Using a candidate transcript approach, we screened affected individuals for mutations in all transcribed regions of all genes, putative genes, and ESTs identified within the 6.2-Mb GERD1 locus based on alignments with the GenBank cDNA databases. From a total of 50 identifiable genes and 99 EST clusters in the GERD1 locus, we identified 163 polymorphisms (143 SNPs and 20 INDELs) in 21 genes and 37 ESTs. The patterns of inheritance and/or the high population frequencies of all polymorphic alleles identified in this study argued against causative relationships between any of the alleles and the GERD phenotype. Using a subset of 51 SNPs distributed throughout the GERD1 locus, we performed case-control and family (TDT) allelic association analyses on two sets of samples. The case-control study was performed with 73 GERD cases and 93 controls, and the family study was performed using 22 small families. SNP 160 (position 38,925,329 Mb, UCSChg15 map) gave a significant P value prior to multiple test correction in both the case control and family studies, while SNP168 (at 40,442,903 Mb) showed significant association after multiple test correction in the case-control sample, but was uninformative in the family sample. The results suggest that the GERD1 gene might be located near SNP160 or SNP168.