Bacterial Identification and Visualization of Bacterial Biofilms Adjacent to Fracture Sites After Internal Fixation.

Objectives: The primary aims of this study were to determine if any correlation exists in cases of fracture fixation among: (1) bacterial profiles recovered from the instrumentation and adjacent tissues; (2) the type of orthopedic injury; and (3) the clinical outcome-union versus nonunion. A secondary goal was to compare culture and molecular diagnostics for identifying the bacterial species present following fracture fixation. Design: Single-institution, prospective case-control cohort study. Setting: Single level 1 trauma center. Patients: Forty-nine bony nonunion cases undergoing revision internal fixation and 45 healed fracture controls undergoing removal of hardware. Intervention: Bacterial infection was detected by standard microbial culture methods and by a pan-eubacterial domain, molecular diagnostic (MDx) assay. Confirmation of culture and MDx results was achieved with bacterial ribosomal 16S rRNA fluorescence in situ hybridization (FISH) to visualize bacterial biofilms. Main Outcome Measurements: MDx and microbial culture methods results were the primary study outcomes. Results: Ninety-four percent of the nonunion cohort and 93% of the union cohort had bacteria detected by the MDx. Seventy-eight percent of the nonunion cases and 69% of the controls were culture negative, but MDx positive. Although no significant differences in bacterial composition were observed between the cases and controls, differences were observed when cases were divided by comorbidities. Conclusion: The MDx is more sensitive than microbial culture in detecting bacterial presence. The lack of significantly different findings with regard to bacterial profile identified between the cases and controls suggests that host factors and environmental conditions are largely responsible for determining if bony union will occur. Level of Evidence: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Deletion of genes involved in the ketogluconate metabolism, Entner-Doudoroff pathway, and glucose dehydrogenase increase local and invasive virulence phenotypes in Streptococcus pneumoniae.

Streptococcus pneumoniae displays increased resistance to antibiotic therapy following biofilm formation. A genome-wide search revealed that SP 0320 and SP 0675 (respectively annotated as 5-keto-D-gluconate-5-reductase and glucose dehydrogenase) contain the highest degree of homology to CsgA of Myxococcus xanthus, a signaling factor that promotes cell aggregation and biofilm formation. Single and double SP 0320 and SP 0675 knockout mutants were created in strain BS72; however, no differences were observed in the biofilm-forming phenotypes of mutants compared to the wild type strain. Using the chinchilla model of otitis media and invasive disease, all three mutants exhibited greatly increased virulence compared to the wild type strain (increased pus formation, tympanic membrane rupture, mortality rates). The SP 0320 gene is located in an operon with SP 0317, SP 0318 and SP 0319, which we bioinformatically annotated as being part of the Entner-Doudoroff pathway. Deletion of SP 0317 also resulted in increased mortality in chinchillas; however, mutations in SP 0318 and SP 0319 did not alter the virulence of bacteria compared to the wild type strain. Complementing the SP 0317, SP 0320 and SP 0675 mutant strains reversed the virulence phenotype. We prepared recombinant SP 0317, SP 0318, SP 0320 and SP 0675 proteins and confirmed their functions. These data reveal that disruption of genes involved in the degradation of ketogluconate, the Entner-Doudoroff pathway, and glucose dehydrogenase significantly increase the virulence of bacteria in vivo; two hypothetical models involving virulence triggered by reduced in carbon-flux through the glycolytic pathways are presented.

Polymerase Chain Reaction-Electrospray-Time-of-Flight Mass Spectrometry Versus Culture for Bacterial Detection in Septic Arthritis and Osteoarthritis.

BACKGROUND: Preliminary studies have identified known bacterial pathogens in the knees of patients with osteoarthritis (OA) before arthroplasty. AIMS: The current study was designed to determine the incidence and types of bacteria present in the synovial fluid of native knee joints from adult patients with diagnoses of septic arthritis and OA. PATIENTS AND METHODS: Patients were enrolled between October 2010 and January 2013. Synovial fluid samples from the affected knee were collected and evaluated with both traditional microbial culture and polymerase chain reaction-electrospray ionization-time-of-flight mass spectrometry (molecular diagnostics [MDx]) to prospectively characterize the microbial content. Patients were grouped by diagnosis into one of two cohorts, those with clinical suspicion of septic arthritis (n = 44) and those undergoing primary arthroplasty of the knee for OA (n = 21). In all cases where discrepant culture and MDx results were obtained, we performed species-specific 16S rRNA fluorescence in situ hybridization (FISH) as a confirmatory test. RESULTS: MDx testing identified bacteria in 50% of the suspected septic arthritis cases and 29% of the arthroplasty cases, whereas culture detected bacteria in only 16% of the former and 0% of the latter group. The overall difference in detection rates for culture and MDx was very highly significant, p-value = 2.384 × 10-7. All of the culture-positive cases were typed as Staphylococcus aureus. Two of the septic arthritis cases were polymicrobial as was one of the OA cases by MDx. FISH testing of the specimens with discordant results supported the MDx findings in 91% (19/21) of the cases, including one case where culture detected S. aureus and MDx detected Streptococcus agalactiae. CONCLUSIONS: MDx were more sensitive than culture, as confirmed by FISH. FISH only identifies bacteria that are embedded or infiltrated within the tissue and is thus not susceptible to contamination. Not all suspected cases of septic arthritis contain bacteria, but a significant percent of patients with OA, and no signs of infection, have FISH-confirmed bacterial biofilms present in the knee.

Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

Comparative Genomic Analyses of the Moraxella catarrhalis Serosensitive and Seroresistant Lineages Demonstrate Their Independent Evolution.

The bacterial speciesMoraxella catarrhalishas been hypothesized as being composed of two distinct lineages (referred to as the seroresistant [SR] and serosensitive [SS]) with separate evolutionary histories based on several molecular typing methods, whereas 16S ribotyping has suggested an additional split within the SS lineage. Previously, we characterized whole-genome sequences of 12 SR-lineage isolates, which revealed a relatively small supragenome when compared with other opportunistic nasopharyngeal pathogens, suggestive of a relatively short evolutionary history. Here, we performed whole-genome sequencing on 18 strains from both ribotypes of the SS lineage, an additional SR strain, as well as four previously identified highly divergent strains based on multilocus sequence typing analyses. All 35 strains were subjected to a battery of comparative genomic analyses which clearly show that there are three lineages-the SR, SS, and the divergent. The SR and SS lineages are closely related, but distinct from each other based on three different methods of comparison: Allelic differences observed among core genes; possession of lineage-specific sets of core and distributed genes; and by an alignment of concatenated core sequences irrespective of gene annotation. All these methods show that the SS lineage has much longer interstrain branches than the SR lineage indicating that this lineage has likely been evolving either longer or faster than the SR lineage. There is evidence of extensive horizontal gene transfer (HGT) within both of these lineages, and to a lesser degree between them. In particular, we identified very high rates of HGT between these two lineages for ß-lactamase genes. The four divergent strains aresui generis, being much more distantly related to both the SR and SS groups than these other two groups are to each other. Based on average nucleotide identities, gene content, GC content, and genome size, this group could be considered as a separate taxonomic group. The SR and SS lineages, although distinct, clearly form a single species based on multiple criteria including a large common core genome, average nucleotide identity values, GC content, and genome size. Although neither of these lineages arose from within the other based on phylogenetic analyses, the question of how and when these lineages split and then subsequently reunited in the human nasopharynx is explored.

Streptococcus pneumoniae Supragenome Hybridization Arrays for Profiling of Genetic Content and Gene Expression.

There is extensive genomic diversity among Streptococcus pneumoniae isolates. Approximately half of the comprehensive set of genes in the species (the supragenome or pangenome) is present in all the isolates (core set), and the remaining is unevenly distributed among strains (distributed set). The Streptococcus pneumoniae Supragenome Hybridization (SpSGH) array provides coverage for an extensive set of genes and polymorphisms encountered within this species, capturing this genomic diversity. Further, the capture is quantitative. In this manner, the SpSGH array allows for both genomic and transcriptomic analyses of diverse S. pneumoniae isolates on a single platform. In this unit, we present the SpSGH array, and describe in detail its design and implementation for both genomic and transcriptomic analyses. The methodology can be applied to construction and modification of SpSGH array platforms, as well to other bacterial species as long as multiple whole-genome sequences are available that collectively capture the vast majority of the species supragenome.

Development and validation of an Haemophilus influenzae supragenome hybridization (SGH) array for transcriptomic analyses.

We previously carried out the design and testing of a custom-built Haemophilus influenzae supragenome hybridization (SGH) array that contains probe sequences to 2,890 gene clusters identified by whole genome sequencing of 24 strains of H. influenzae. The array was originally designed as a tool to interrogate the gene content of large numbers of clinical isolates without the need for sequencing, however, the data obtained is quantitative and is thus suitable for transcriptomic analyses. In the current study RNA was extracted from H. influenzae strain CZ4126/02 (which was not included in the design of the array) converted to cDNA, and labelled and hybridized to the SGH arrays to assess the quality and reproducibility of data obtained from these custom-designed chips to serve as a tool for transcriptomics. Three types of experimental replicates were analyzed with all showing very high degrees of correlation, thus validating both the array and the methods used for RNA profiling. A custom filtering pipeline for two-condition unpaired data using five metrics was developed to minimize variability within replicates and to maximize the identification of the most significant true transcriptional differences between two samples. These methods can be extended to transcriptional analysis of other bacterial species utilizing supragenome-based arrays.

What role do periodontal pathogens play in osteoarthritis and periprosthetic joint infections of the knee?

Through the use of polymerase chain reaction (PCR)-electron spray ionization (ESI)-time of flight (TOF)-mass spectrometry (MS), we identified multiple periodontal pathogens within joint tissues of individuals undergoing replacement arthroplasties of the knee. The most prevalent of the periodontal pathogens were Treponema denticola and Enterococcus faecalis, the latter of which is commonly associated with apical periodontitis. These findings were unique to periprosthetic joint infections (PJI) of the knee and were never observed for PJIs of other lower extremity joints (hip and ankle) or upper extremity joints (shoulder and elbow). These data were confirmed by multiple independent methodologies including fluorescent in situ hybridization (FISH) which showed the bacteria deeply penetrated inside the diseased tissues, and 454-based deep 16S rDNA sequencing. The site-specificity, the tissue investment, and the identical findings by multiple nucleic-acid-based techniques strongly suggests the presence of infecting bacteria within these diseased anatomic sites. Subsequently, as part of a control program using PCR-ESI-TOF-MS, we again detected these same periodontal pathogens in aspirates from patients with osteoarthritis who were undergoing primary arthroplasty of the knee and thus who had no history of orthopedic implants. This latter finding raises the question of whether hematogenic spread of periodontal pathogens to the knee play a primary or secondary-exacerbatory role in osteoarthritis.

There is a specific response to pH by isolates of Haemophilus influenzae and this has a direct influence on biofilm formation.

BACKGROUND: Haemophilus influenzae colonizes the nasopharynx as a commensal. Strain-specific factors allow some strains to migrate to particular anatomical niches, such as the middle ear, bronchi or blood, and induce disease by surviving within the conditions present at these sites in the body. It is established that H. influenzae colonization and in some cases survival is highly dependent on their ability to form a biofilm. Biofilm formation is a key trait in the development of chronic infection by certain isolates. This is exemplified by the contrast between the biofilm-forming strains found in middle ear infections and those isolates that survive within the blood and are rarely associated with biofilm development. RESULTS: Screening a group of H. influenzae strains revealed only slight variations in their growth across a range of pH conditions. However, some isolates responded to a pH of 8.0 by the formation of a biofilm. While the type b capsular blood isolate Eagan did not form a biofilm and grew at the same rate regardless of pH 6.8-8.0, transcriptomic analyses demonstrated that at pH 8.0 it uniquely induced a gluconate-uptake and metabolism pathway, which concurrently imports H+. A non-typeable H. influenzae, isolated from the middle ear, induced biofilm formation at pH 8.0, and at this pH it induced a series of iron acquisition genes, consistent with previous studies linking iron homeostasis to biofilm lifestyle. CONCLUSIONS: Different strains of H. influenzae cope with changes in environmental factors using strain-specific mechanisms. These pathways define the scope and mode of niche-survival for an isolate. The pH is a property that is different from the middle ear (at least pH 8.0) compared to other sites that H. influenzae can colonize and infect. The transcriptional response to increasing pH by H. influenzae varies between strains, and pH is linked to pathways that allow strains to either continue free-living growth or induction of a biofilm. We showed that a biofilm-forming isolate induced iron metabolism pathways, whereas a strain that does not form biofilm at increasing pH induced mechanisms for growth and pH homeostasis based on sugar acid transport.

Can we trust intraoperative culture results in nonunions?

OBJECTIVES: To identify the presence of bacterial biofilms in nonunions comparing molecular techniques (multiplex polymerase chain reaction and mass spectrometry, fluorescent in situ hybridization) with routine intraoperative cultures. METHODS: Thirty-four patients with nonunions were scheduled for surgery and enrolled in this ongoing prospective study. Intraoperative specimens were collected from removed implants, surrounding tissue membrane, and local soft tissue followed by standard culture analysis, Ibis's second generation molecular diagnostics (Ibis Biosystems), and bacterial 16S rRNA-based fluorescence in situ hybridization (FISH). Confocal microscopy was used to visualize the tissue specimens reacted with the FISH probes, which were chosen based on the Ibis analysis. RESULTS: Thirty-four patient encounters were analyzed. Eight were diagnosed as infected nonunions by positive intraoperative culture results. Ibis confirmed the presence of bacteria in all 8 samples. Ibis identified bacteria in a total of 30 of 34 encounters, and these data were confirmed by FISH. Twenty-two of 30 Ibis-positive samples were culture-negative. Four samples were negative by all methods of analysis. No samples were positive by culture, but negative by molecular techniques. CONCLUSIONS: Our preliminary data indicate that molecular diagnostics are more sensitive for identifying bacteria than cultures in cases of bony nonunion. This is likely because of the inability of cultures to detect biofilms and bacteria previously exposed to antibiotic therapy. LEVEL OF EVIDENCE: Diagnostic Level I. See Instructions for Authors for a complete description of levels of evidence.

Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates.

BACKGROUND: Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. RESULTS: We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. CONCLUSIONS: These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.

The microbiome of chronic rhinosinusitis: culture, molecular diagnostics and biofilm detection.

BACKGROUND: Bacteria and fungi are believed to influence mucosal inflammation in chronic rhinosinusitis (CRS). However their presence and relationship to disease is debated. This study used multiple detection methods to compare microbial diversity and microbial abundance in healthy and diseased sinonasal mucosa. The utility of contemporary detection methods is also examined. METHODS: Sinonasal mucosa was analyzed from 38 CRS and 6 controls. Bacterial and fungal analysis was performed using conventional culture, molecular diagnostics (polymerase chain reaction coupled with electrospray ionization time-of-flight mass spectrometry) and fluorescence in situ hybridization. RESULTS: Microbes were detected in all samples, including controls, and were often polymicrobial. 33 different bacterial species were detected in CRS, 5 in control patients, with frequent recovery of anaerobes. Staphylococcus aureus and Propionibacterium acnes were the most common organisms in CRS and controls, respectively. Using a model organism, FISH had a sensitivity of 78%, and a specificity of 93%. Many species were detected in both CRS and controls however, microbial abundance was associated with disease manifestation. CONCLUSIONS: This study highlights some cornerstones of microbial variations in healthy and diseased paranasal sinuses. Whilst the healthy sinus is clearly not sterile, it appears prevalence and abundance of organisms is critical in determining disease. Evidence from high-sensitivity techniques, limits the role of fungi in CRS to a small group of patients. Comparison with molecular analysis suggests that the detection threshold of FISH and culture is related to organism abundance and, furthermore, culture tends to select for rapidly growing organisms.

In vivo capsular switch in Streptococcus pneumoniae--analysis by whole genome sequencing.

Two multidrug resistant strains of Streptococcus pneumoniae - SV35-T23 (capsular type 23F) and SV36-T3 (capsular type 3) were recovered from the nasopharynx of two adult patients during an outbreak of pneumococcal disease in a New York hospital in 1996. Both strains belonged to the pandemic lineage PMEN1 but they differed strikingly in virulence when tested in the mouse model of IP infection: as few as 1000 CFU of SV36 killed all mice within 24 hours after inoculation while SV35-T23 was avirulent.Whole genome sequencing (WGS) of the two isolates was performed (i) to test if these two isolates belonging to the same clonal type and recovered from an identical epidemiological scenario only differed in their capsular genes? and (ii) to test if the vast difference in virulence between the strains was mostly - or exclusively - due to the type III capsule. WGS demonstrated extensive differences between the two isolates including over 2500 single nucleotide polymorphisms in core genes and also differences in 36 genetic determinants: 25 of which were unique to SV35-T23 and 11 unique to strain SV36-T3. Nineteen of these differences were capsular genes and 9 bacteriocin genes.Using genetic transformation in the laboratory, the capsular region of SV35-T23 was replaced by the type 3 capsular genes from SV36-T3 to generate the recombinant SV35-T3* which was as virulent as the parental strain SV36-T3* in the murine model and the type 3 capsule was the major virulence factor in the chinchilla model as well. On the other hand, a careful comparison of strains SV36-T3 and the laboratory constructed SV35-T3* in the chinchilla model suggested that some additional determinants present in SV36 but not in the laboratory recombinant may also contribute to the progression of middle ear disease. The nature of this determinants remains to be identified.

Comparative genomic analyses of 17 clinical isolates of Gardnerella vaginalis provide evidence of multiple genetically isolated clades consistent with subspeciation into genovars.

Gardnerella vaginalis is associated with a spectrum of clinical conditions, suggesting high degrees of genetic heterogeneity among stains. Seventeen G. vaginalis isolates were subjected to a battery of comparative genomic analyses to determine their level of relatedness. For each measure, the degree of difference among the G. vaginalis strains was the highest observed among 23 pathogenic bacterial species for which at least eight genomes are available. Genome sizes ranged from 1.491 to 1.716 Mb; GC contents ranged from 41.18% to 43.40%; and the core genome, consisting of only 746 genes, makes up only 51.6% of each strain's genome on average and accounts for only 27% of the species supragenome. Neighbor-grouping analyses, using both distributed gene possession data and core gene allelic data, each identified two major sets of strains, each of which is composed of two groups. Each of the four groups has its own characteristic genome size, GC ratio, and greatly expanded core gene content, making the genomic diversity of each group within the range for other bacterial species. To test whether these 4 groups corresponded to genetically isolated clades, we inferred the phylogeny of each distributed gene that was present in at least two strains and absent in at least two strains; this analysis identified frequent homologous recombination within groups but not between groups or sets. G. vaginalis appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties. Consequently, it may be appropriate to treat these four groups as separate species.

Complete genome sequence of Streptococcus pneumoniae strain ST556, a multidrug-resistant isolate from an otitis media patient.

Streptococcus pneumoniae is a major pathogen causing bacterial infection in the middle ear of humans. We previously used S. pneumoniae strain ST556, a low-passage 19F isolate from an otitis media patient, to perform a whole-genome screen for ear infection-associated genes in a chinchilla model. This report presents the complete genome sequence of ST556. The genome sequence will provide information complementary to the experimental data from our genetic study of this strain.

Differences in genotype and virulence among four multidrug-resistant Streptococcus pneumoniae isolates belonging to the PMEN1 clone.

We report on the comparative genomics and characterization of the virulence phenotypes of four S. pneumoniae strains that belong to the multidrug resistant clone PMEN1 (Spain(23F) ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinants.

Preliminary study: treatment with intramuscular interferon beta-1a results in increased levels of IL-12Rß2+ and decreased levels of IL23R+ CD4+ T - Lymphocytes in multiple sclerosis.

BACKGROUND: There are a lack of biomarkers which can be used to predict clinical outcomes for multiple sclerosis (MS) patients receiving interferon beta (IFN-ß). Thus the objective of this study was to characterize changes in CD4+ T-lymphocyte expression in an unbiased manner following initiation of intramuscular (IM) IFN-ß-1a treatment, and then to verify those findings using marker-specific assays. METHODS: Peripheral blood specimens were collected from twenty MS patients before and after treatment with intramuscular (IM) IFN-ß-1a and were used for isolation of mononuclear cells (PBMCs). mRNA expression patterns of negatively-selected CD4+ T-cells from the PBMCs were analyzed using microarray gene expression technology. IL-12 and IL-23 receptor levels on PBMC-derived CD4+ T-cells were analyzed by flow cytometry. The phosphorylation status of Stat4 was measured by performing densitometry on western blots. RESULTS: Microarray analyses demonstrated that mRNA expression of the IL-12Rß2 gene was uniformly up-regulated in response to IFN-ß-1a treatment and was associated with an increased number of IL-12Rß2+ CD4+ T-cells by flow cytometry in 4 of 6 patients. This finding was substantiated by demonstrating that Stat4 phosphorylation, a transcription factor for IL-12, was increased after treatment. Conversely, the number of IL-23R+ CD4+ T-cells was decreased following treatment. CONCLUSIONS: The IL-12 receptor shares a common subunit, the IL-12Rß2, with the IL-23 receptor. Both of these receptors have a probable role in regulating IL-17 and TH-17 cells, important mediators of inflammation in multiple sclerosis (MS). Thus, the changes in the numbers of CD4+ T-cells expressing these receptors in response to IFN-ß-1a treatment may point to an important mechanism of action for this drug, but further large scale studies are needed to confirm these preliminary observations.

Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.

Characterization of bacterial communities in venous insufficiency wounds by use of conventional culture and molecular diagnostic methods.

Microbial infections delay wound healing, but the effect of the composition of the wound microbiome on healing parameters is unknown. To better understand bacterial communities in chronic wounds, we analyzed debridement samples from lower-extremity venous insufficiency ulcers using the following: conventional anaerobic and aerobic bacterial cultures; the Ibis T5000 universal biosensor (Abbott Molecular); and 16S 454 FLX titanium series pyrosequencing (Roche). Wound debridement samples were obtained from 10 patients monitored clinically for at least 6 months, at which point 5 of the 10 sampled wounds had healed. Pyrosequencing data revealed significantly higher bacterial abundance and diversity in wounds that had not healed at 6 months. Additionally, Actinomycetales was increased in wounds that had not healed, and Pseudomonadaceae was increased in wounds that had healed by the 6-month follow-up. Baseline wound surface area, duration, or analysis by Ibis or conventional culture did not reveal significant differences between wounds that healed after 6 months and those that did not. Thus, pyrosequencing identified distinctive baseline characteristics of wounds that did not heal by the 6-month follow-up, furthering our understanding of potentially unique microbiome characteristics of chronic wounds.

Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743.

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia, we chose to investigate the ET-743 (Yondelis) biosynthetic pathway. This molecule is an approved anticancer agent obtained in low abundance (10(-4)-10(-5) % w/w) from the tunicate Ecteinascidia turbinata and is generated in suitable quantities for clinical use by a lengthy semisynthetic process. On the basis of structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium, we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anticancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogues through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.