Borreliella burgdorferi factor H-binding proteins are not required for serum resistance and infection in mammals.

The causative agent of Lyme disease (LD), Borreliella burgdorferi, binds factor H (FH) and other complement regulatory proteins to its surface. B. burgdorferi B31 (type strain) encodes five FH-binding proteins (FHBPs): CspZ, CspA, and the OspE paralogs OspEBBN38, OspEBBL39, and OspEBBP38. This study assessed potential correlations between the production of individual FHBPs, FH-binding ability, and serum resistance using a panel of infectious B. burgdorferi clonal populations recovered from dogs. FHBP production was assessed in cultivated spirochetes and by antibody responses in naturally infected humans, dogs, and eastern coyotes (wild canids). FH binding specificity and sensitivity to dog and human serum were also assessed and compared. No correlation was observed between the production of individual FHBPs and FH binding with serum resistance, and CspA was determined to not be produced in animals. Notably, one or more clones isolated from dogs lacked CspZ or the OspE proteins (a finding confirmed by genome sequence determination) and did not bind FH derived from canines. The data presented do not support a correlation between FH binding and the production of individual FHBPs with serum resistance and infectivity. In addition, the limited number and polymorphic nature of cp32s in B. burgdorferi clone DRI85A that were identified through genome sequencing suggest no strict requirement for a defined set of these replicons for infectivity. This study reveals that the immune evasion mechanisms employed by B. burgdorferi are diverse, complex, and yet to be fully defined.

Characterization of integrated hepatitis B virus DNA harboring pre-S mutations in hepatocellular carcinoma patients with ground glass hepatocytes.

Ground glass hepatocytes (GGHs) have been associated with hepatocellular carcinoma (HCC) recurrence and poor prognosis. We previously demonstrated that pre-S expression in some GGHs is resistant to current hepatitis B virus (HBV) antiviral therapies. This study aimed to investigate whether integrated HBV DNA (iDNA) is the primary HBV DNA species responsible for sustained pre-S expression in GGH after effective antiviral therapy. We characterized 10 sets of micro-dissected, formalin-fixed-paraffin-embedded, and frozen GGH, HCC, and adjacent hepatitis B surface antigen-negative stained tissues for iDNA, pre-S deletions, and the quantity of covalently closed circular DNA. Eight patients had detectable pre-S deletions, and nine had detectable iDNA. Interestingly, eight patients had integrations within the TERT and CCNE1 genes, which are known recurrent integration sites associated with HCC. Furthermore, we observed a recurrent integration in the ABCC13 gene. Additionally, we identified variations in the type and quantity of pre-S deletions within individual sets of tissues by junction-specific PacBio long-read sequencing. The data from long-read sequencing indicate that some pre-S deletions were acquired following the integration events. Our findings demonstrate that iDNA exists in GGH and can be responsible for sustained pre-S expression in GGH after effective antiviral therapy.

Long-term Sudan virus Ebola survivors maintain multiple antiviral defense mechanisms.

BACKGROUND: The critical issues of sustained memory immunity following ebolavirus disease among long-term survivors (EVD) are still unclear. METHODS: Here, we examine virus-specific immune and inflammatory responses in 12 Sudan virus (SUDV) long-term survivors from Uganda's 2000-1 Gulu outbreak, 15 years after recovery following in vitro challenge. Total RNA from isolated SUDV-stimulated and unstimulated PBMCs was extracted and analyzed. Matched serum samples were also collected to determine SUDV IgG levels and functionality. RESULTS: We detected persistent humoral (58%, 7 of 12) and cellular (33%, 4 of 12) immune responses in SUDV long-term survivors and identified critical molecular mechanisms of innate and adaptive immunity. Gene expression in immune pathways, the IFN signaling system, antiviral defense response, and activation and regulation of T- and B-cell responses were observed. SUDV long-term survivors also maintained robust virus-specific IgG antibodies capable of polyfunctional responses, including neutralizing and innate Fc effector functions. CONCLUSIONS: Data integration identified significant correlations among humoral and cellular immune responses and pinpointed a specific innate and adaptive gene expression signature associated with long-lasting immunity. This could help identify natural and vaccine correlates of protection against ebolavirus disease.

Establishment of a consensus protocol to explore the brain pathobiome in patients with mild cognitive impairment and Alzheimer's disease: Research outline and call for collaboration.

Microbial infections of the brain can lead to dementia, and for many decades microbial infections have been implicated in Alzheimer's disease (AD) pathology. However, a causal role for infection in AD remains contentious, and the lack of standardized detection methodologies has led to inconsistent detection/identification of microbes in AD brains. There is a need for a consensus methodology; the Alzheimer's Pathobiome Initiative aims to perform comparative molecular analyses of microbes in post mortem brains versus cerebrospinal fluid, blood, olfactory neuroepithelium, oral/nasopharyngeal tissue, bronchoalveolar, urinary, and gut/stool samples. Diverse extraction methodologies, polymerase chain reaction and sequencing techniques, and bioinformatic tools will be evaluated, in addition to direct microbial culture and metabolomic techniques. The goal is to provide a roadmap for detecting infectious agents in patients with mild cognitive impairment or AD. Positive findings would then prompt tailoring of antimicrobial treatments that might attenuate or remit mounting clinical deficits in a subset of patients.

The bacterial microbiota of Hunner lesion interstitial cystitis/bladder pain syndrome.

OBJECTIVE: To undertake the first comprehensive evaluation of the urinary microbiota associated with Hunner lesion (HL) interstitial cystitis/bladder pain syndrome (IC/BPS). Despite no previous identification of a distinct IC/BPS microbial urotype, HL IC/BPS, an inflammatory subtype of IC/BPS, was hypothesized most likely to be associated with a specific bacterial species or microbial pattern. PARTICIPANTS AND METHODS: The bacterial microbiota of midstream urine specimens from HL IC/BPS and age- and gender-matched IC/BPS patients without HL (non-HL IC/BPS) were examined using the pan-bacterial domain clinical-level molecular diagnostic Pacific Biosciences full-length 16S gene sequencing protocol, informatics pipeline and database. We characterized the differential presence, abundances, and diversity of species, as well as gender-specific differences between and among HL and non-HL IC/BPS patients. RESULTS: A total of 59 patients with IC/BPS were enrolled (29 HL, 30 non-HL; 43 women, 16 men) from a single centre and the microbiota in midstream urine specimens was available for comparison. The species abundance differentiation between the HL and non-HL groups (12 species) was not significantly different after Bonferroni adjustments for multiple comparisons. Similarly, the nine differentiating species noted between female HL and non-HL patients were not significantly different after similar statistical correction. However, four species abundances (out of the 10 species differences identified prior to correction) remained significantly different between male HL and non-HL subjects: Negativicoccus succinivorans, Porphyromonas somerae, Mobiluncus curtisii and Corynebacterium renale. Shannon diversity metrics showed significantly higher diversity among HL male patients than HL female patients (P = 0.045), but no significant diversity differences between HL and non-HL patients overall. CONCLUSIONS: We were not able to identify a unique pathogenic urinary microbiota that differentiates all HL from all non-HL IC/BPS. It is likely that the male-specific differences resulted from colonization/contamination remote from the bladder. We were not able to show that bacteria play an important role in patients with HL IC/BPS.

Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs.

Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.

Beyond the pan-genome: current perspectives on the functional and practical outcomes of the distributed genome hypothesis.

The principle of monoclonality with regard to bacterial infections was considered immutable prior to 30 years ago. This view, espoused by Koch for acute infections, has proven inadequate regarding chronic infections as persistence requires multiple forms of heterogeneity among the bacterial population. This understanding of bacterial plurality emerged from a synthesis of what-were-then novel technologies in molecular biology and imaging science. These technologies demonstrated that bacteria have complex life cycles, polymicrobial ecologies, and evolve in situ via the horizontal exchange of genic characters. Thus, there is an ongoing generation of diversity during infection that results in far more highly complex microbial communities than previously envisioned. This perspective is based on the fundamental tenet that the bacteria within an infecting population display genotypic diversity, including gene possession differences, which result from horizontal gene transfer mechanisms including transformation, conjugation, and transduction. This understanding is embodied in the concepts of the supragenome/pan-genome and the distributed genome hypothesis (DGH). These paradigms have fostered multiple researches in diverse areas of bacterial ecology including host-bacterial interactions covering the gamut of symbiotic relationships including mutualism, commensalism, and parasitism. With regard to the human host, within each of these symbiotic relationships all bacterial species possess attributes that contribute to colonization and persistence; those species/strains that are pathogenic also encode traits for invasion and metastases. Herein we provide an update on our understanding of bacterial plurality and discuss potential applications in diagnostics, therapeutics, and vaccinology based on perspectives provided by the DGH with regard to the evolution of pathogenicity.

Urinary fungi associated with urinary symptom severity among women with interstitial cystitis/bladder pain syndrome (IC/BPS).

PURPOSE: To correlate the presence of fungi with symptom flares, pain and urinary severity in a prospective, longitudinal study of women with IC/BPS enrolled in the MAPP Research Network. METHODS: Flare status, pelvic pain, urinary severity, and midstream urine were collected at baseline, 6 and 12 months from female IC/BPS participants with at least one flare and age-matched participants with no reported flares. Multilocus PCR coupled with electrospray ionization/mass spectrometry was used for identification of fungal species and genus. Associations between "mycobiome" (species/genus presence, relative abundance, Shannon's/Chao1 diversity indices) and current flare status, pain, urinary severity were evaluated using generalized linear mixed models, permutational multivariate analysis of variance, Wilcoxon's rank-sum test. RESULTS: The most specific analysis detected 13 fungal species from 8 genera in 504 urine samples from 202 females. A more sensitive analysis detected 43 genera. No overall differences were observed in fungal species/genus composition or diversity by flare status or pain severity. Longitudinal analyses suggested greater fungal diversity (Chao1 Mean Ratio 3.8, 95% CI 1.3-11.2, p = 0.02) and a significantly greater likelihood of detecting any fungal species (OR = 5.26, 95% CI 1.1-25.8, p = 0.04) in high vs low urinary severity participants. Individual taxa analysis showed a trend toward increased presence and relative abundance of Candida (OR = 6.63, 95% CI 0.8-58.5, p = 0.088) and Malassezia (only identified in 'high' urinary severity phenotype) for high vs low urinary symptoms. CONCLUSION: This analysis suggests the possibility that greater urinary symptom severity is associated with the urinary mycobiome urine in some females with IC/BPS.

Genome rearrangements induce biofilm formation in Escherichia coli C - an old model organism with a new application in biofilm research.

BACKGROUND: Escherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. RESULTS: Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the - 35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli. CONCLUSIONS: Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.

Specific amino acids in HIV-1 Vpr are significantly associated with differences in patient neurocognitive status.

Even in the era of combination antiretroviral therapies used to combat human immunodeficiency virus type 1 (HIV-1) infection, up to 50 % of well-suppressed HIV-1-infected patients are still diagnosed with mild neurological deficits referred to as HIV-associated neurocognitive disorders (HAND). The multifactorial nature of HAND likely involves the HIV-1 accessory protein viral protein R (Vpr) as an agent of neuropathogenesis. To investigate the effect of naturally occurring variations in Vpr on HAND in well-suppressed HIV-1-infected patients, bioinformatic analyses were used to correlate peripheral blood-derived Vpr sequences with patient neurocognitive performance, as measured by comprehensive neuropsychological assessment and the resulting Global Deficit Score (GDS). Our studies revealed unique associations between GDS and the presence of specific amino acid changes in peripheral blood-derived Vpr sequences [neuropsychological impairment Vpr (niVpr) variants]. Amino acids N41 and A55 in the Vpr sequence were associated with more pronounced neurocognitive deficits (higher GDS). In contrast, amino acids I37 and S41 were connected to measurably lower GDS. All niVpr variants were also detected in DNA isolated from HIV-1-infected brain tissues. The implication of these results is that niVpr variants alter the genesis and/or progression of HAND through differences in Vpr-mediated effects in the peripheral blood and/or the brain.

Molecular characterization of ß-lactamase genes in clinical isolates of carbapenem-resistant Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is a nosocomial pathogen which is establishing as a major cause of morbidity and mortality within the healthcare community. The success of this pathogen is largely due to its ability to rapidly gain resistance to antimicrobial therapies and its capability to persist in an abiotic environment through the production of a biofilm. Our tertiary-care hospital has showed high incidence of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. METHODS: In this study we explore both genotypic and phenotypic properties of 26 CRAB isolates: 16 isolates were collected from January 2010 to March 2011, and 10 were collected between February and May 2015. RESULTS: We determined that all 26 CRAB isolates possessed multiple ß-lactamase genes, including genes from Groups A, C, and D. Specifically, 42% of the isolates possesses the potentially plasmid-borne genes of OXA-23-like or OXA-40-like ß-lactamase. The presence of mobile gene element integron cassettes and/or integrases in 88% of the isolates suggests a possible mechanism of dissemination of antibiotic resistance genes. Additionally, the location of insertion sequence (IS) ISAba1 in promotor region of of the OXA-51-like, ADC-7, and ampC genes was confirmed. Multilocus sequence typing (MLST) demonstrated that all 26 CRAB isolates were either sequence type (ST)-229 or ST-2. Interestingly, ST-2 went from being the minority CRAB strain in the 2010-2011 isolates to the predominant strain in the 2015 isolates (from 32 to 90%). We show that the ST-2 strains have an enhanced ability to produce biofilms in comparison to the ST-229 strains, and this fact has potentially led to more successful colonization of the clinical environment over time. CONCLUSIONS: This study provides a longitudinal genetic and phenotypic survey of two CRAB sequence types, and suggests how their differing phenotypes may interact with the selective pressures of a hospital setting effecting strain dominance over a 5-year period.

Assessment of the Lower Urinary Tract Microbiota during Symptom Flare in Women with Urologic Chronic Pelvic Pain Syndrome: A MAPP Network Study.

PURPOSE: We compared culture independent assessment of microbiota of the lower urinary tract in standard culture negative female patients with urological chronic pelvic pain syndrome who reported symptom flare vs those who did not report a flare. MATERIALS AND METHODS: Initial stream (VB1) and midstream (VB2) urine specimens (233 patients with urological chronic pelvic pain syndrome) were analyzed with Ibis T-5000 Universal Biosensor system technology for comprehensive identification of microorganism species. Differences between flare and nonflare groups for presence or number of different species within a higher level group (richness) were examined by permutational multivariate analysis of variance and logistic regression. RESULTS: Overall 81 species (35 genera) were detected in VB1 and 73 (33) in VB2. Mean (SD) VB1 and VB2 species count per person was 2.6 (1.5) and 2.4 (1.5) for 86 flare cases and 2.8 (1.3) and 2.5 (1.5) for 127 nonflare cases, respectively. Overall the species composition did not significantly differ between flare and nonflare cases at any level (p=0.14 species, p=0.95 genus in VB1 and VB2, respectively) in multivariate analysis for richness. Univariate analysis, unadjusted as well as adjusted, confirmed a significantly greater prevalence of fungi (Candida and Saccharomyces) in the flare group (15.7%) compared to the nonflare group in VB2 (3.9%) (p=0.01). When adjusted for antibiotic use and menstrual phase, women who reported a flare remained more likely to have fungi present in VB2 specimens (OR 8.3, CI 1.7-39.4). CONCLUSIONS: Among women with urological chronic pelvic pain syndrome the prevalence of fungi (Candida and Saccharomyces sp.) was significantly greater in those who reported a flare compared to those who did not.

Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold standard for identification of pathogens is culture; however molecular methods for identification of microorganisms may provide a more rapid result and may be able to identify additional microorganisms that are not detected by culture. METHODS: In this study, tissue samples (n = 20) obtained after debridement of 10 patients with NSTI were analyzed by standard culture, fluorescence in situ hybridization (FISH) and multiple molecular methods. The molecular methods included analysis of microbial diversity by 1) direct 16S and D2LSU rRNA gene Microseq 2) construction of near full-length 16S rRNA gene clone libraries with subsequent Sanger sequencing for most samples, 3) the Ibis T5000 biosensor and 4) 454-based pyrosequencing. Furthermore, quantitative PCR (qPCR) was used to verify and determine the relative abundance of Streptococcus pyogenes in samples. RESULTS: For 70 % of the surgical samples it was possible to identify microorganisms by culture. Some samples did not result in growth (presumably due to administration of antimicrobial therapy prior to sampling). The molecular methods identified microorganisms in 90 % of the samples, and frequently detected additional microorganisms when compared to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods. Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also made including infection by a) Acinetobacter baumannii, b) Streptococcus pneumoniae, and c) fungi, mycoplasma and Fusobacterium necrophorum. CONCLUSION: The study emphasizes that many pathogens can be involved in NSTIs, and that no specific "NSTI causing" combination of species exists. This means that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially for fulminant life-threatening infections such as NSTI.

Search for Microorganisms in Men with Urologic Chronic Pelvic Pain Syndrome: A Culture-Independent Analysis in the MAPP Research Network.

PURPOSE: We used next-generation, state-of-the-art, culture independent methodology to survey urine microbiota of males with urologic chronic pelvic pain syndrome and control participants enrolled in the MAPP Network to investigate a possible microbial etiology. MATERIALS AND METHODS: Male patients with urologic chronic pelvic pain syndrome and matched controls were asked to provide initial, midstream and post-prostatic massage urine specimens. Specimens were analyzed with Ibis T-5000 Universal Biosensor technology to provide comprehensive identification of bacterial and select fungal species. Differences between urologic chronic pelvic pain syndrome and control study participants for the presence of species or species variation in a higher taxonomic grouping (genus) were evaluated using permutational multivariate analysis of variance and logistic regression. RESULTS: Initial and midstream urine specimens were obtained from 110 (post-prostatic massage urine in 67) participants with urologic chronic pelvic pain syndrome and 115 (post-prostatic massage urine in 62) controls. Overall 78, 73 and 54 species (42, 39 and 27 genera) were detected in initial, midstream and post-prostatic massage urine specimens, respectively. Mean (SD) initial, midstream and post-prostatic massage urine species count per person was 1.62 (1.28), 1.38 (1.36) and 1.33 (1.24) for cases, and 1.75 (1.32), 1.23 (1.15) and 1.56 (0.97) for controls, respectively. Overall species and genus composition differed significantly between participants with urologic chronic pelvic pain syndrome and controls in initial stream urine (p=0.002 species level, p=0.004 genus level), with Burkholderia cenocepacia overrepresented in urologic chronic pelvic pain syndrome. No significant differences were observed at any level in midstream or post-prostatic massage urine samples. CONCLUSIONS: Assessment of baseline culture-independent microbiological data from male subjects enrolled in the MAPP Network has identified overrepresentation of B. cenocepacia in urologic chronic pelvic pain syndrome. Future studies are planned to further evaluate microbiota associations with variable and changing urologic chronic pelvic pain syndrome symptom patterns.

Biofilm-based implant infections in orthopaedics.

The demand for joint replacement surgery is continuously increasing with rising costs for hospitals and healthcare systems. Staphylococci are the most prevalent etiological agents of orthopedic infections. After an initial adhesin-mediated implant colonization, Staphylococcus aureus and Staphylococcus epidermidis produce biofilm. Biofilm formation proceeds as a four-step process: (1) initial attachment of bacterial cells; (2) cell aggregation and accumulation in multiple cell layers; (3) biofilm maturation and (4) detachment of cells from the biofilm into a planktonic state to initiate a new cycle of biofilm formation elsewhere. The encasing of bacteria in biofilms gives rise to insuperable difficulties not only in the treatment of the infection, but also in assessing the state and the nature of the infection using traditional cultural methods. Therefore, DNA-based molecular methods have been developed to provide rapid identification of all microbial pathogens. To combat biofilm-centered implant infections, new strategies are being developed, among which anti-infective or infective-resistant materials are at the forefront. Infection-resistant materials can be based on different approaches: (i) modifying the biomaterial surface to give anti-adhesive properties, (ii) doping the material with antimicrobial substances, (iii) combining anti-adhesive and antimicrobial effects in the same coating, (iv) designing materials able to oppose biofilm formation and support bone repair.

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.

Otitis media: Interactions between host and environment, immune and inflammatory responses.

OBJECTIVE: To review and highlight progress in otitis media (OM) research in the areas of immunology, inflammation, environmental influences and host-pathogen responses from 2019 to 2023. Opportunities for innovative future research were also identified. DATA SOURCES: PubMed database of the National Library of Medicine. REVIEW METHODS: Key topics were assigned to each panel member for detailed review. Search of the literature was from June 2019 until February 2023. Draft reviews were collated, circulated, and discussed among panel members at the 22nd International Symposium on Recent Advances in Otitis Media in June 2023. The final manuscript was prepared and approved by all the panel members. CONCLUSIONS: Important advances were identified in: environmental influences that enhance OM susceptibility; polymicrobial middle ear (ME) infections; the role of adaptive immunity defects in otitis-proneness; additional genes linked to OM; leukocyte contributions to OM pathogenesis and recovery; and novel interventions in OM based on host responses to infection. Innovative areas of research included: identification of novel bacterial genes and pathways important for OM persistence, bacterial adaptations and evolution that enhance chronicity; animal and human ME gene expression, including at the single-cell level; and Sars-CoV-2 infection of the ME and Eustachian tube.

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.

Detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus colonization of healthy military personnel by traditional culture, PCR, and mass spectrometry.

BACKGROUND: Methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus colonization is associated with increased rates of infection. Rapid and reliable detection methods are needed to identify colonization of nares and extra-nare sites, particularly given recent reports of oropharynx-only colonization. Detection methods for MRSA/MSSA colonization include culture, PCR, and novel methods such as PCR/electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). METHODS: We evaluated 101 healthy military members for S. aureus colonization in the nares, oropharynx, axilla, and groin, using CHROMagar S. aureus medium and Xpert SA Nasal Complete PCR for MRSA/MSSA detection. The same subjects were screened in the nares, oropharynx, and groin using PCR/ESI-TOF-MS. RESULTS: By culture, 3 subjects were MRSA-colonized (all oropharynx) and 34 subjects were MSSA-colonized (all 4 sites). PCR detected oropharyngeal MRSA in 2 subjects, which correlated with culture findings. By PCR, 47 subjects were MSSA-colonized (all 4 sites); however, 43 axillary samples were invalid, 39 of which were associated with deodorant/anti-perspirant use (93%, p < 0.01). By PCR/ESI-TOF-MS, 4 subjects were MRSA-colonized, 2 in the nares and 2 in the oropharynx; however, neither of these correlated with positive MRSA cultures. Twenty-eight subjects had MSSA by PCR/ESI-TOF-MS, and 41 were found to have possible MRSA (S. aureus with mecA and coagulase-negative Staphylococcus (CoNS)). CONCLUSION: The overall 3% MRSA colonization rate is consistent with historical reports, but the oropharynx-only colonization supports more recent findings. In addition, the use of deodorant/anti-perspirant invalidated axillary PCR samples, limiting its utility. Defining MRSA positivity by PCR/ESI-TOF-MS is complicated by co-colonization of S. aureus with CoNS, which can also carry mecA.