There is insufficient evidence to support screening of various tick-borne diseases (TBD) related microbes alongside Borrelia in patients suffering from TBD. To evaluate the involvement of multiple microbial immune responses in patients experiencing TBD we utilized enzyme-linked immunosorbent assay. Four hundred and thirty-two human serum samples organized into seven categories followed Centers for Disease Control and Prevention two-tier Lyme disease (LD) diagnosis guidelines and Infectious Disease Society of America guidelines for post-treatment Lyme disease syndrome. All patient categories were tested for their immunoglobulin M (IgM) and G (IgG) responses against 20 microbes associated with TBD. Our findings recognize that microbial infections in patients suffering from TBDs do not follow the one microbe, one disease Germ Theory as 65% of the TBD patients produce immune responses to various microbes. We have established a causal association between TBD patients and TBD associated co-infections and essential opportunistic microbes following Bradford Hill’s criteria. This study indicated an 85% probability that a randomly selected TBD patient will respond to Borrelia and other related TBD microbes rather than to Borrelia alone. A paradigm shift is required in current healthcare policies to diagnose TBD so that patients can get tested and treated even for opportunistic infections.
In addition to oxygenic photosynthetic systems, solar radiation is utilized for energy by diverse anoxygenic photosynthetic systems. Aerobic anoxygenic phototrophic bacteria (AAPB) perform photosynthesis without producing oxygen but still live in aerobic conditions. Typically they have been reported from aquatic ecosystems, but they can also be found from polar and desert soil ecosys- tems and primary succession communities like soil crusts. Recently, AAPB have been discovered in the metagenomic data of several plant phyllospheres. By utilizing citizen science, we screened plant foliar samples from eleven different locations in Finland for AAPB by near infrared fluorescence imaging of culturable phyllosphere and endosphere bacteria. Near infrared fluorescence reports the presence of AAPB which contain Bacteriochlorophyll a molecules, embedded in Light Harvesting 1-Reaction Center complex. We found that AAPB were ubiquotous in phyllosphere communities of diverse plant species in all sampling locations. They were also consistently present in the endosphere of plant species with perennial leaves. Most of the AAPB isolated represent alphaproteobacterial genera Sphingomonas and Methylobacterium, but several isolates from genus Lichenihabitans as well as putative novel alphaproteobacterial taxa were also identified. Methylobacterial isolates were mostly detected in the phyllosphere with weak host specificity, while Sphingomonas AAPB were detected also in the endosphere of several plant species, with clear host specific taxa. We studied also the fluorescence spectral properties of several AAPBs. All the observed spectra resemble typical fluorescence spectral properties of Light Harvesting complex 1. Still, slight variation among each spectra could be obtained, revealing some physical difference among the complexes. Our results demonstrate for the first time, that AAPB are common in cold climate plant endophytic as well as epiphytic microbiomes and they build up substantial amounts of Bacteriochlorophyll a containing Light Harvesting complexes. Their putative role in plant adaptation to strong seasonality in light and temperature or tolerance of abiotic stressors remains to be investigated in future studies.
Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a simple and environmentally friendly protocol for recovering DNA from ddPCR.
The main method utilised in this study is an ELISA assay. An investigation by the University of Jyväskylä, Finland, has concluded that the patient selection and description in this Article, and in an unpublished report validating the methods used, do not justify the results presented. The Editors therefore no longer have confidence in the results and conclusions presented in this Article.
Microbial communities often respond to various challenges, such as the presence of antibiotics, as a whole. Dissecting these community-level effects into separate acting entities requires the identification of organisms that carry functional genes for the observed feature. However, unculturable microbes abound in various environments, hence making the identification challenging. Here we present a cultivation-free technique that can be utilized to link functional genes with carrying bacterial species at single-cell resolution. The developed protocol is relatively simple to use, utilizes commercially available droplet microfluidics devices, does not require toxic reagents (as compared to some previous methods), eliminates invalid signals emerging from extracellular DNA, and allows simultaneous analysis of community diversity via 16S rRNA gene sequencing. The method can be customized for any given genetic trait to accurately identify its hosting subpopulation from a heterogeneous and potentially uncultivable bacterial community.
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