Distinct cutaneous bacterial assemblages in a sampling of South American Amerindians and US residents

The human skin harbors complex bacterial communities. Prior studies showing high inter-individual variation focused on subjects from developed countries. We therefore compared cutaneous bacterial communities of Amerindians in the Venezuelan Amazon with subjects in the United States. Forearm skin specimens were studied from healthy Amerindians in Platanillal village in Amazonas State, and from healthy persons in New York and Colorado. All skin sampling used similar swab/buffer techniques. Multiplexed V2-targeted 16S rRNA gene pyrosequencing yielded high quality sequences from 112 samples. The results show 20 phyla, with three (Proteobacteria, Firmicutes, Actinobacteria) predominating. US residents and Venezuelan Amerindians had significantly different forearm skin bacterial community compositions, with United States dominated by Propionibacterium. Among the Amerindians, there was a deep split based on bacterial community membership, with 30 and 42 samples, respectively, falling into each of the two groups, not associated with age, gender, or body mass index. One Amerindian group had diversity similar to the United States, but was dominated by Staphylococcus rather than Propionibacterium. The other Amerindian group was significantly more diverse and even than the US or the other Amerindian group, and featured a broad range of Proteobacteria. The results provide evidence that ethnicity, lifestyle and/or geography are associated with the structure of human cutaneous bacterial communities.     

An Investigation of Freezing of Supercooled Water on Anti-Freeze Protein Modified Surfaces

This work investigates how functionalization of aluminium surfaces with natural type III Anti-Freeze Protein (AFP) affects the mechanism of heterogeneous ice nucleation. First the bulk ice nucleation properties of distilled water and aqueous solution of AFP were evaluated by differential scanning calorimetry. Then the modified surface was characterized by Secondary Ions Mass Spectroscopy (SIMS), Fourier Transform InfraRed (FTIR) spectroscopy and contact angle measurement. Freezing experiments were then conducted in which water droplets underwent a slow controlled cooling. This study shows that compared to uncoated aluminium, the anti-freeze proteins functionalized surfaces exhibit a higher and narrower range of freezing temperature. It was found that these proteins that keep living organisms from freezing in cold environment act in the opposite way once immobilized on surfaces by promoting ice nucleation. Some suggestions regarding the mechanism of action of the observed phenomena were proposed based on the Classical Nucleation Theory (CNT).     

A Branched Biosynthetic Pathway Is Involved in Production of Roquefortine and Related Compounds in Penicillium chrysogenum

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.     

A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum,Zeamine-Related Antibiotics

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.     

Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial,Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community''s bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.     

Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda

Background

WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low–cost, qualitative viral–failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda.

Methods

We conducted a cross–sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV–1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VL_ref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml).

Results

496 paired VFA and VL_ref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%–87.1%), 93% specificity (95% CI: 89.7%–96.4%), 89.3% accuracy (95% CI: 85%–92%) and an agreement kappa = 0.72 as compared to the VL_ref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively.

Conclusions

VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second–line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV–1 treatment in RLS.