BackgroundLung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort.ResultsOverall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas.ConclusionsThe results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1501-6) contains supplementary material, which is available to authorized users.

Resistance of clinical isolates of Candida albicans to the echinocandin drug caspofungin is slowly emerging and is linked to mutations in short conserved regions in the FKS1 gene. The most prominent changes occurred at the serine 645 position in Fks1p with substitutions of proline, tyrosine, and phenylalanine. An allele-specific real-time PCR molecular-beacon assay was developed for rapid identification of drug resistance by targeting FKS1 mutations. Mutations altering serine 645 were reliably identified in both heterozygous and homozygous states. The molecular-beacon assay was used to evaluate two large collections of spontaneous mutants from separate strains of C. albicans with resistance (MICs, >16 g/ml) to caspofungin with the goal of understanding the relationship between FKS1 mutations and echinocandin resistance. Of 85 resistant isolates recovered, all were identified with mutations in FKS1; 93% showed changes at Ser645, with 62% displaying a characteristic S645P substitution expressed as either a homozygous or a heterozygous mutation in FKS1. Two other prominent amino acid substitutions, S645Y and S645F, were found at frequencies of 22% and 8%, respectively. Three new mutations were also identified: T1922C, G1932T, and C1934G, encoding F641S, L644F, and S645C substitutions, respectively. One strain had the double amino acid substitution L644F and S645C. Allele-specific probes were combined in a multiplex assay for reliable screening of known FKS1 mutations. These data support the importance of FKS1p substitutions in echinocandin resistance and demonstrate the feasibility of applying molecular screening for routine resistance assessment.The echinocandin drugs, caspofungin, micafungin, and anidulafungin, are the first of a new class of antifungal compounds that target the fungal cell wall by blocking ␤-1,3-glucan synthase (4, 6, 9). These drugs have broad-spectrum activity against Candida and Aspergillus spp. without cross-resistance to existing agents and therefore are effective against azole-resistant yeasts and molds (1,7,15,18,19). Importantly, due to their critical effect on the cell wall, echinocandins kill yeasts (5). Use of caspofungin (CANCIDAS), the first approved echinocandin, in the clinic is expanding rapidly, and it is now widely used along with triazole drugs, such as voriconazole, for primary therapy against yeast and molds. The entry of the closely related drugs micafungin and anidulafungin will further extend the clinical scope of this highly efficacious class of drugs. As patient exposure to caspofungin grows, and as micafungin and anidulafungin augment the market, it is anticipated that the number of clinical isolates with highly elevated MICs will increase. Because the echinocandins are the first new major antifungal drug class to enter the market in decades, it is vital to assess the nature of developing resistance mechanisms to this class of drugs.Recently, we reported that specific mutations in two highly conserved regions of the Fks1p subunit of glucan synthase, a putative large po...

Gardnerella vaginalis is an important component of the human vaginal microflora. It is proposed to play a key role in the pathogenesis of bacterial vaginosis (BV), the most common vaginal condition. Here we describe the development, validation and comparative analysis of a novel molecular approach capable of G. vaginalis identification, quantification and subtyping in noncultured vaginal specimens. Using two quantitative PCR (qPCR) assays, we analysed G. vaginalis bacterial loads and clade distribution in 60 clinical vaginal-swab samples. A very high pathogen prevalence was revealed by species-specific qPCR not only among BV patients (100 %), but also in healthy women (97 %), although the G. vaginalis concentration was significantly lower in non-BV samples. G. vaginalis clades identified in vaginal specimens by subtyping multiplex qPCR, which targets four clade-specific genetic markers, had frequencies of 53 % for

Antifungal efficacies of the echinocandin drugs caspofungin, micafungin, and anidulafungin were reduced significantly in the presence of 50% human serum, which yielded nearly equivalent MICs or minimum effective concentrations against diverse Candida spp. and Aspergillus spp. Consistent with a direct drug interaction, serum decreased the sensitivity of glucan synthase to echinocandin drugs.The echinocandin drugs caspofungin, micafungin, and anidulafungin inhibit the fungal ␤-1,3-glucan synthase enzyme, which blocks the formation of glucan polymers, thereby disrupting fungal cell wall integrity (2). Animal and human studies indicate that echinocandin drugs are extensively bound to serum proteins (1,4,5,15), and serum was shown to reduce the antifungal properties of micafungin with some Candida spp. (3), yet little is known about the influence of serum on antifungal efficacy with the different echinocandin drugs.Echinocandin susceptibility in the presence or absence of 50% human serum (Sigma) was evaluated with a diverse collection of clinical isolates, laboratory strains, and reference strains of Candida spp. and Aspergillus spp., according to the guidelines in CLSI documents M27-A2 (9) and M38-A (8), respectively. Abnormal colony morphology was used to establish a minimum effective concentration (MEC) for Aspergillus spp. after 48 h of incubation at 35°C (6). Glucan synthase (GS) isolation and 50% inhibitory concentration (IC 50 ) inhibition kinetics were performed as described previously (11). A murine candidiasis model utilizing female BALB/c mice (age, 10 to 12 weeks; weight, 20 to 25 g) was used to assess the relative in vivo efficacies of echinocandin drugs (13).Serum increased caspofungin MICs an average of 2-fold, with a range of 1-to 16-fold, while it had a more pronounced effect on the other drugs, increasing the MIC an average of 16-fold with a range of 8-to 256-fold for anidulafungin and an average of 64-fold with a range of 32-to 128-fold for micafungin (Table 1). The effects of serum on MICs were assessed for other non-Candida albicans spp. The largest MIC shift for caspofungin (eightfold) was with Candida krusei, while Candida tropicalis strains showed the most significant shifts (128-fold) for both micafungin and anidulafungin. These drugs consistently showed pronounced shifts, which reflected their greater antifungal potencies in the absence of serum. These differences disappeared in the presence of 50% serum, where all three drugs showed comparable MICs.Similar serum-induced effects were observed with a collection of Aspergillus spp., where microscopically observed MECs for the three drugs shifted higher in the presence of serum. Micafungin and anidulafungin again showed the most pronounced antifungal shifts, 32-to 133-fold and 16-to 32-fold, respectively, reflecting their more active behavior in the absence of serum ( Table 2). The three drugs displayed nearly equivalent MECs in the presence of 50% serum. The reduced antifungal properties of echinocandin drugs in the presence of serum suggested...

BackgroundPan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences’ (PacBio’s) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data.ResultsAnalysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity with regard to taxonomic classification. Examination of a 250-plus species mock community demonstrated correct species-level classification of > 90% of taxa, and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes) was the predominant species throughout, but was found at distinct relative abundances by site.ConclusionsOur microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt) overcomes deficits of standard marker gene-based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0569-2) contains supplementary material, which is available to authorized users.

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.

Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidate the bacterial pathways undergoing in vivo evolutionary adaptation, we compared bacterial genomes collected over time from 13 COPD patients and identified recurrent genetic changes arising in independent bacterial lineages colonizing different patients. Besides finding changes in phase-variable genes, we found recurrent loss-of-function mutations in the ompP1 (fadL) gene. We show that loss of OmpP1/FadL function reduces this bacterium’s ability to infect cells via the hCEACAM1 epithelial receptor but also increases its resistance to bactericidal fatty acids enriched within the COPD lung, suggesting a case of antagonistic pleiotropy that restricts ΔfadL strains’ niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways.

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