Effects of squamous cell carcinoma and smoking status on oropharyngeal and laryngeal microbial communities.

BACKGROUND: Still, little is known about microbial dysbiosis in oropharyngeal and laryngeal tissue as risk factor for development of local squamous cell carcinoma. The site-specific microbiota at these regions in healthy and cancer tissue and their modulation by environmental factors need to be defined. METHODS: The local microbiota of cancer tissue and healthy controls was profiled by 16S rRNA gene amplicon sequencing and statistical analysis using 111 oropharyngeal and 72 laryngeal intraoperative swabs. RESULTS: Oropharynx and larynx harbor distinct microbial communities. Clear effects of both smoking and cancer were seen in the oropharynx whereas effects in the larynx were minor. CONCLUSION: The distinct microbial communities at larynx and oropharynx partially explain why the effects of cancer and smoking were distinct at those sites. Thus, the use of microbiota supposed to mirror community changes in another target location should be avoided and more studies on the actual cancerous environment are necessary.

Gender-specific changes of the gut microbiome correlate with tumor development in murine models of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a dismal outcome. To improve understanding of sequential microbiome changes during PDAC development we analyzed mouse models of pancreatic carcinogenesis (KC mice recapitulating pre-invasive PanIN formation, as well as KPC mice recapitulating invasive PDAC) during early tumor development and subsequent tumor progression. Diversity and community composition were analyzed depending on genotype, age, and gender. Both mouse models demonstrated concordant abundance changes of several genera influenced by one or more of the investigated factors. Abundance was significantly impacted by gender, highlighting the need to further elucidate the impact of gender differences. The findings underline the importance of the microbiome in PDAC development and indicate that microbiological screening of patients at risk and targeting the microbiome in PDAC development may be feasible in future.

The biliary microbiome in ischaemic-type biliary lesions can be shaped by stenting but is resilient to antibiotic treatment.

This study aims to characterize the biliary microbiome as neglected factor in patients with ischaemic-type biliary lesions (ITBL) after liver transplantation. Therefore, the V1-V2 region of the 16S rRNA gene was sequenced in 175 bile samples. Samples from patients with anastomotic strictures (AS) served as controls. Multivariate analysis and in silico metagenomics were applied cross-sectionally and longitudinally. The microbial community differed significantly between ITBL and AS in terms of alpha and beta diversity. Both, antibiotic treatment and stenting were associated independently with differences in the microbial community structure. In contrast to AS, in ITBL stenting was associated with pronounced differences in the biliary microbiome, whereas no differences associated with antibiotic treatment could be observed in ITBL contrasting the pronounced differences found in AS. Bacterial pathways involved in the production of antibacterial metabolites were increased in ITBL with antibiotic treatment. After liver transplantation, the biliary tract harbours a complex microbial community with significant differences between ITBL and AS. Fundamental changes in the microbial community in ITBL can be achieved with biliary stenting. However, the effect of antibiotic treatment in ITBL was minimal. Therefore, antibiotics should be administered wisely in order to reduce emerging resistance of the biliary microbiome towards external antibiotics.

Eradication of Chronic HCV Infection: Improvement of Dysbiosis Only in Patients Without Liver Cirrhosis.

BACKGROUND AND AIMS: It is well accepted that liver diseases and their outcomes are associated with intestinal microbiota, but causality is difficult to establish. The intestinal microbiota are altered in patients with hepatitis C. As chronic HCV infection can now be cured in almost all patients, it is an ideal model to study the influence of liver disease on the microbiota. APPROACH AND RESULTS: We aimed to prospectively analyze the changes in the gut microbiome in patients who received direct-acting antivirals (DAA) and achieved sustained virological response (SVR). Amplicon sequencing of the V1-V2 region in the 16S ribosomal RNA gene was performed in stool samples of patients with chronic hepatitis C. Patients in the treatment group received DAA (n = 65), whereas in the control group, no DAA were given (n = 33). Only patients achieving SVR were included. The alpha diversity increased numerically but not significantly from baseline to SVR at week 24 or 48 (SVR24/48; 2.784 ± 0.248 vs. 2.846 ± 0.224; P = 0.057). When stratifying for the presence of liver cirrhosis, a significant increase in diversity was only seen in patients without cirrhosis. Differences in the microbial community structure induced by the achievement of SVR were only observed in patients without liver cirrhosis. In patients with liver cirrhosis and in the control group, no significant differences were observed. CONCLUSIONS: In conclusion, the achievement of SVR24/48 in patients with chronic HCV was associated with changes in the intestinal microbiota. However, these changes were only seen in patients without liver cirrhosis. A major role of liver remodeling on the intestinal microbiota is indicated by the dynamics of the intestinal microbial community structure depending on the stage of fibrosis in patients resolving chronic hepatitis C.

The impact of proton pump inhibitors on the intestinal microbiota in chronic hepatitis C patients.

Objectives: Proton pump inhibitors (PPI), a class of drugs commonly used, are known to be associated with changes in the intestinal microbiota. Published studies were done in heterogeneous cohorts which could hamper conclusions drawn as effects of diseases were not taken into consideration. We aimed to elucidate differences in the intestinal microbiota being associated to the use of PPI in a cohort study of patients with chronic hepatitis C. Material and Methods: The 16S rDNA gene was analyzed in stool samples of patients with and without PPI use. Patients with concomitant medication influencing the microbiota were excluded. Results were compared with the clinical course of hepatitis C patients with decompensated liver cirrhosis. Results: No differences in alpha diversity could be observed, while the microbial community structure differed significantly, especially in patients with liver cirrhosis. The relative abundance of Streptococcus spp., Enterobacter spp. and Haemophilus spp. was significantly increased in patients with PPI use irrespectively of the stage of liver disease. Finally, in patients with decompensated liver cirrhosis due to chronic HCV infection only in these using PPI bacterial phylotypes were isolated. Conclusions: PPI use was associated with significant alterations in the microbial community in patients with chronic hepatitis C, which were even pronounced in patients with liver cirrhosis. In patients with decompensated liver cirrhosis due to chronic HCV infection, the use of PPI may promote infections either directly or indirectly through changes in the microbial community structure. Future studies should further investigate long-term impact on the microbiota and the clinical outcome.

Intestinal microbiota in patients with chronic hepatitis C with and without cirrhosis compared with healthy controls.

BACKGROUND & AIMS: The importance of the intestinal microbiota for the onset and clinical course of many diseases, including liver diseases like non-alcoholic steatohepatitis and cirrhosis, is increasingly recognized. However, the role of intestinal microbiota in chronic hepatitis C virus (HCV) infection remains unclear. METHODS: In a cross-sectional approach, the intestinal microbiota of 95 patients chronically infected with HCV (n=57 without cirrhosis [NO-CIR]; n=38 with cirrhosis [CIR]) and 50 healthy controls (HC) without documented liver diseases was analysed. RESULTS: Alpha diversity, measured by number of phylotypes (S) and Shannon diversity index (H'), decreased significantly from HC to NO-CIR to CIR. S and H' correlated negatively with liver elastography. Analysis of similarities revealed highly statistically significant differences in the microbial communities between HC, NO-CIR and CIR (R=.090; P<1.0×10-6 ). Stratifying for HCV genotypes even increased the differences. In addition, we observed distinct patterns in the relative abundance of genera being either positive or negative correlated with diseases status. CONCLUSIONS: This study shows that not only the stage of liver disease but also HCV infection is associated with a reduced alpha diversity and different microbial community patterns. These differences might be caused by direct interactions between HCV and the microbiota or indirect interactions facilitated by the immune system.

Bacterioplankton Biogeography of the Atlantic Ocean: A Case Study of the Distance-Decay Relationship.

In order to determine the influence of geographical distance, depth, and Longhurstian province on bacterial community composition and compare it with the composition of photosynthetic micro-eukaryote communities, 382 samples from a depth-resolved latitudinal transect (51°S-47°N) from the epipelagic zone of the Atlantic ocean were analyzed by Illumina amplicon sequencing. In the upper 100 m of the ocean, community similarity decreased toward the equator for 6000 km, but subsequently increased again, reaching similarity values of 40-60% for samples that were separated by ~12,000 km, resulting in a U-shaped distance-decay curve. We conclude that adaptation to local conditions can override the linear distance-decay relationship in the upper epipelagial of the Atlantic Ocean which is apparently not restrained by barriers to dispersal, since the same taxa were shared between the most distant communities. The six Longhurstian provinces covered by the transect were comprised of distinct microbial communities; ~30% of variation in community composition could be explained by province. Bacterial communities belonging to the deeper layer of the epipelagic zone (140-200 m) lacked a distance-decay relationship altogether and showed little provincialism. Interestingly, those biogeographical patterns were consistently found for bacteria from three different size fractions of the plankton with different taxonomic composition, indicating conserved underlying mechanisms. Analysis of the chloroplast 16S rRNA gene sequences revealed that phytoplankton composition was strongly correlated with both free-living and particle associated bacterial community composition (R between 0.51 and 0.62, p < 0.002). The data show that biogeographical patterns commonly found in macroecology do not hold for marine bacterioplankton, most likely because dispersal and evolution occur at drastically different rates in bacteria.

Co-occurrence Analysis of Microbial Taxa in the Atlantic Ocean Reveals High Connectivity in the Free-Living Bacterioplankton.

We determined the taxonomic composition of the bacterioplankton of the epipelagic zone of the Atlantic Ocean along a latitudinal transect (51°S-47°N) using Illumina sequencing of the V5-V6 region of the 16S rRNA gene and inferred co-occurrence networks. Bacterioplankon community composition was distinct for Longhurstian provinces and water depth. Free-living microbial communities (between 0.22 and 3 µm) were dominated by highly abundant and ubiquitous taxa with streamlined genomes (e.g., SAR11, SAR86, OM1, Prochlorococcus) and could clearly be separated from particle-associated communities which were dominated by Bacteroidetes, Planktomycetes, Verrucomicrobia, and Roseobacters. From a total of 369 different communities we then inferred co-occurrence networks for each size fraction and depth layer of the plankton between bacteria and between bacteria and phototrophic micro-eukaryotes. The inferred networks showed a reduction of edges in the deepest layer of the photic zone. Networks comprised of free-living bacteria had a larger amount of connections per OTU when compared to the particle associated communities throughout the water column. Negative correlations accounted for roughly one third of the total edges in the free-living communities at all depths, while they decreased with depth in the particle associated communities where they amounted for roughly 10% of the total in the last part of the epipelagic zone. Co-occurrence networks of bacteria with phototrophic micro-eukaryotes were not taxon-specific, and dominated by mutual exclusion (~60%). The data show a high degree of specialization to micro-environments in the water column and highlight the importance of interdependencies particularly between free-living bacteria in the upper layers of the epipelagic zone.

Exploring the bacterial assemblages along the human nasal passage.

The human nasal passage, from the anterior nares through the nasal vestibule to the nasal cavities, is an important habitat for opportunistic pathogens and commensals alike. This work sampled four different anatomical regions within the human nasal passage across a large cohort of individuals (n = 79) comprising individuals suffering from chronic nasal inflammation clinically known as chronic rhinosinusitis (CRS) and individuals not suffering from inflammation (CRS-free). While individuals had their own unique bacterial fingerprint that was consistent across the anatomical regions, these bacterial fingerprints formed into distinct delineated groups comprising core bacterial members, which were consistent across all four swabbed anatomical regions irrespective of health status. The most significant observed pattern was the difference between the global bacterial profiles of swabbed and tissue biopsy samples from the same individuals, being also consistent across different anatomical regions. Importantly, no statistically significant differences could be observed concerning the global bacterial communities, any of the bacterial species or the range of diversity indices used to compare between CRS and CRS-free individuals, and between two CRS phenotypes (without nasal polyps and with nasal polyps). Thus, the role of bacteria in the pathogenesis of sinusitis remains uncertain.

Frequent detection of HCV RNA and HCVcoreAg in stool of patients with chronic hepatitis C.

BACKGROUND AND OBJECTIVE: HCV is transmitted mainly by parenteral routes. However, unprotected anal intercourse has also been identified as a risk factor for HCV infection. HCV RNA can be detected in blood, saliva, and bile, but the presence of HCV in stool has not been investigated yet. STUDY DESIGN: Therefore, stool samples of 98 patients were collected prospectively. Specific HCV primers were used to identify samples positive for HCV RNA. HCV RNA-positive samples were tested for HCVcoreAg with the Architect HCVAg assay (Abbott). Presence of occult blood was investigated by the hemoCARE guajak test. Viral stability and infectivity of recombinant HCV particles was investigated in vitro by incubation of genotype 2a chimeric virus Jc1 with bile and stool suspensions. RESULTS: HCV RNA could be detected in 68 out of 98 stool samples from patients with chronic hepatitis C and 16 samples also tested positive for HCVcoreAg. Presence of HCV RNA in stool was more frequent in male than in female and in patients with low platelet counts but was not associated with the detection of occult blood. Stool suspensions and to a lesser extent bile reduced the in vitro infectivity of genotype 2a chimeric Jc1 virus even though infection of Huh7 cells was not completely abrogated. CONCLUSIONS: In summary, this study shows for the first time that HCV can frequently be detected in stool samples of chronically infected patients irrespective of occult bleeding. We suggest that stool can be a potential source for HCV infection and thus unprotected anal intercourse should be avoided.

Low diversity of planktonic bacteria in the tropical ocean.

The diversity of macro-organisms increases towards the equator, with almost no exceptions. It is the most conserved biogeographical pattern on earth and is thought to be related to the increase of temperature and productivity in the tropics. The extent and orientation of a latitudinal gradient of marine bacterioplankton diversity is controversial. Here we studied the euphotic zone of the Atlantic Ocean based on a transect covering ~12.000 km from 51°S to 47 °N. Water samples were collected at 26 stations at five depths between 20 and 200 m and sequentially filtered through 8 µm, 3 µm and 0,22 µm filters, resulting in a total of 359 samples. Illumina sequencing of the V5-V6 region of the 16S rRNA gene revealed a clear biogeographic pattern with a double inverted latitudinal gradient. Diversity was higher in mid-latitudinal regions of the Atlantic Ocean and decreased towards the equator. This pattern was conserved for bacteria from all three planktonic size fractions. Diversity showed a non-linear relationship with temperature and was negatively correlated with bacterial cell numbers in the upper depth layers (<100 m). The latitudinal gradients of marine bacterial diversity and the mechanisms that govern them are distinct from those found in macro-organisms.

The microbial community structure of the cotton rat nose.

The cotton rat nose is commonly used as a model for Staphylococcus aureus colonization, as it is both physiologically and anatomically comparable to the human nares and can be easily colonized by this organism. However, while the colonization of the human anterior nares has been extensively studied, the microbial community structure of cotton rat noses has not been reported so far. We describe here the microbial community structure of the cotton rat (Sigmodon hispidus) nose through next-generation sequencing of 16S rRNA gene amplicons covering the V1-V2 region and the analysis of nearly full length 16S rRNA genes of the major phylotypes. Roughly half of the microbial community was composed of two undescribed species of the genus Campylobacter, with phylotypes belonging to the genera Catonella, Acholeplasma, Streptobacillus and Capnocytophaga constituting the predominant community members. Thus, the nasal community of the cotton rat is uniquely composed of several novel bacterial species and may not reflect the complex interactions that occur in human anterior nares. Mammalian airway microbiota may, however, be a rich source of hitherto unknown microbes.

High-resolution taxonomic profiling of the subgingival microbiome for biomarker discovery and periodontitis diagnosis.

The oral microbiome plays a key role for caries, periodontitis, and systemic diseases. A method for rapid, high-resolution, robust taxonomic profiling of subgingival bacterial communities for early detection of periodontitis biomarkers would therefore be a useful tool for individualized medicine. Here, we used Illumina sequencing of the V1-V2 and V5-V6 hypervariable regions of the 16S rRNA gene. A sample stratification pipeline was developed in a pilot study of 19 individuals, 9 of whom had been diagnosed with chronic periodontitis. Five hundred twenty-three operational taxonomic units (OTUs) were obtained from the V1-V2 region and 432 from the V5-V6 region. Key periodontal pathogens like Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia could be identified at the species level with both primer sets. Principal coordinate analysis identified two outliers that were consistently independent of the hypervariable region and method of DNA extraction used. The linear discriminant analysis (LDA) effect size algorithm (LEfSe) identified 80 OTU-level biomarkers of periodontitis and 17 of health. Health- and periodontitis-related clusters of OTUs were identified using a connectivity analysis, and the results confirmed previous studies with several thousands of samples. A machine learning algorithm was developed which was trained on all but one sample and then predicted the diagnosis of the left-out sample (jackknife method). Using a combination of the 10 best biomarkers, 15 of 17 samples were correctly diagnosed. Training the algorithm on time-resolved community profiles might provide a highly sensitive tool to detect the onset of periodontitis.

Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/ß hydrolase family.

Several members of the C-C MCP (meta-cleavage product) hydrolase family demonstrate an unusual ability to hydrolyse esters as well as the MCPs (including those from mono- and bi-cyclic aromatics). Although the molecular mechanisms responsible for such substrate promiscuity are starting to emerge, the full understanding of these complex enzymes is far from complete. In the present paper, we describe six distinct α/ß hydrolases identified through genomic approaches, four of which demonstrate the unprecedented characteristic of activity towards a broad spectrum of substrates, including p-nitrophenyl, halogenated, fatty acyl, aryl, glycerol, cinnamoyl and carbohydrate esters, lactones, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate and 2-hydroxy-6-oxohepta-2,4-dienoate. Using structural analysis and site-directed mutagenesis we have identified the three residues (Ser32, Val130 and Trp144) that determine the unusual substrate specificity of one of these proteins, CCSP0084. The results may open up new research avenues into comparative catalytic models, structural and mechanistic studies, and biotechnological applications of MCP hydrolases.

Degradation of 2,3-dihydroxybenzoate by a novel meta-cleavage pathway.

2,3-Dihydroxybenzoate is the precursor in the biosynthesis of several siderophores and an important plant secondary metabolite that, in bacteria, can be degraded via meta-cleavage of the aromatic ring. The dhb cluster of Pseudomonas reinekei MT1 encodes a chimeric meta-cleavage pathway involved in the catabolism of 2,3-dihydroxybenzoate. While the first two enzymes, DhbA and DhbB, are phylogenetically related to those involved in 2,3-dihydroxy-p-cumate degradation, the subsequent steps are catalyzed by enzymes related to those involved in catechol degradation (DhbCDEFGH). Characterization of kinetic properties of DhbA extradiol dioxygenase identified 2,3-dihydroxybenzoate as the preferred substrate. Deletion of the encoding gene impedes growth of P. reinekei MT1 on 2,3-dihydroxybenzoate. DhbA catalyzes 3,4-dioxygenation with 2-hydroxy-3-carboxymuconate as the product, which is then decarboxylated by DhbB to 2-hydroxymuconic semialdehyde. This compound is then subject to dehydrogenation and further degraded to citrate cycle intermediates. Transcriptional analysis revealed genes of the dhB gene cluster to be highly expressed during growth with 2,3-dihydroxybenzoate, whereas a downstream-localized gene encoding 2-hydroxymuconic semialdehyde hydrolase, dispensable for 2,3-dihydroxybenzoate metabolism but crucial for 2,3-dihydroxy-p-cumate degradation, was only marginally expressed. This is the first report describing a gene cluster encoding enzymes for the degradation of 2,3-dihydroxybenzoate.

A poke into the diversity and associations within human anterior nare microbial communities.

The anterior nares are the major reservoir for Staphylococcus aureus in humans, where nasal carriage has a crucial function as a source for invasive infections. Despite various investigations on aerobic community members based on traditional cultivation methods, little is known on the overall microbial composition and complex in situ interactions, but such knowledge is highly warranted for effective S. aureus control strategies. As assessed using advanced culture-independent approaches, this study provides a comprehensive survey of the anterior nare bacterial community of 40 individuals. Previously undiscovered co-colonization patterns and natural variations in species composition were revealed and provide insights for future intervention strategies for the control of health-care- and community-associated S. aureus infections.

A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate.

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.

Difference in kinetic behaviour of catechol 2,3-dioxygenase variants from a polluted environment.

In a previous environmental survey of a polluted area, the authors identified two catechol 2,3-dioxygenase (C23O) sequences predominant in environmental bacterial isolates mineralizing benzene and/or toluene and also in soil DNA extracts. In the present study, using information of stable operon arrangement and conserved gene sequences, the complete C23O ORFs of these two variants were cloned, sequenced and overexpressed. The variants differ in six nucleotide positions, and the putative protein sequences differ only by a single amino acid, Tyr or His, at position 218. Even though the three-dimensional model does not suggest a significant influence of such an amino acid substitution on enzyme function, the Tyr218 variant differed significantly from the His218 variant in lower turnover number and in lower apparent K(m) for catecholic substrates. These results are evidence of the importance for enzyme function of amino acids not directly influencing active site structure and prove the utility of recovering polymorphisms evolved and selected for special functions in natural ecosystems.

Importance of different tfd genes for degradation of chloroaromatics by Ralstonia eutropha JMP134.

The tfdC(I)D(I)E(I)F(I,) and tfdD(II)C(II)E(II)F(II) gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd(I)-encoded enzymes was usually higher than that of tfd(II)-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD(II)-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.