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Results for “Microbiology”

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bioRxiv2017

Accurate Determination of Bacterial Abundances in Human Metagenomes Using Full-length 16S Sequencing Reads

DNA sequencing of PCR-amplified marker genes, especially but not limited to the 16S rRNA gene, is perhaps the most common approach for profiling microbial communities. Due to technological constraints of commonly available DNA sequencing, these approaches usually take the form of short reads sequenced from a narrow, targeted variable region, with a corresponding loss of taxonomic resolution relative to the full length marker gene. We use Pacific Biosciences single-molecule, real-time circular consensus sequencing to sequence amplicons spanning the entire length of the 16S rRNA gene. However, this sequencing technology suffers from high sequencing error rate that needs to be addressed in order to take full advantage of the longer sequence. Here, we present a method to model the sequencing error process using a generalized pair hidden Markov chain model and estimate bacterial abundances in microbial samples. We demonstrate, with simulated and real data, that our model and its associated estimation procedure are able to give accurate estimates at the species (or subspecies) level, and is more flexible than existing methods like SImple Non-Bayesian TAXonomy (SINTAX).

microbiology↗

bioRxiv2017

Pneumococcal vaccine impacts on the population genomics of non-typeable Haemophilus influenzae

Between 2008/09 and 2012/13 the molecular epidemiology of non-typeable Haemophilus influenzae (NTHi) carriage in children <5 years of age was determined; a period that included pneumococcal conjugate vaccine (PCV) 13 introduction. Significantly increased carriage in post-PCV13 years was observed and lineage-specific associations with S. pneumoniae were observed before and after PCV13 introduction. NTHi were characterised into eleven discrete, temporally stable lineages, congruent with current knowledge regarding the clonality of NTHi. This increase could not be linked to the expansion of a particular clone and demonstrates different dynamics to before PCV13 implementation during which time NTHi co-carried with vaccine serotype pneumococci.

microbiology↗

bioRxiv2017

Electron microscopy reveals unique nano forms of bacterial spores

Endospore formation under environmental stress conditions is a well-established phenomenon for members of bacterial phylum Firmicutes, among which the most well studied ones belong to genus Bacillus and Clostridium. So far, known sizes of the spores are all larger than 500 nm. Nano-forms of bacteria have been reported but the notion still remains controversial. In this study, we provide visual evidences of living nano-entities (named here as nano-spores) formed by a bacterial species Bacillus cereus under prolonged stress, which are capable of escaping though standard sterile filtration procedure. The existence of nano-forms of bacteria was initially identified in a yeast ribosome preparation. We further demonstrate the transformation of the nano-spores into mature cells upon nutrient supply. Our study not only demonstrates the ability of bacteria to get transformed into yet-unknown form in order to survive under harsh environment, but also brings to light the existence of the smallest possible form of life.

microbiology↗

bioRxiv2017

Optogenetics reprogramming of planktonic cells for biofilm formation

Single-cell behaviors play essential roles during early-stage biofilms formation. In this study, we evaluated whether biofilm formation could be guided by precisely manipulating single cells behaviors. Thus, we established an illumination method to precisely manipulate the type IV pili (TFP) mediated motility and microcolony formation of Pseudomonas aeruginosa by using a combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation and adaptive microscopy. We termed this method as Adaptive Tracking Illumination (ATI). We reported that ATI enables the precise manipulation of TFP mediated motility and microcolony formation during biofilm formation by manipulating bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) levels in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms can be controlled using ATI. Thus, the established method (i.e., ATI) can markedly promote ongoing studies of biofilms.

microbiology↗

bioRxiv2017

Automated detection of Mycobacterial growth on 96-well plates for rapid and accurate Tuberculosis drug susceptibility testing

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the minimum inhibitory concentrations (MICs) to be elucidated. The two participating laboratories each inoculated ten 96-well plates with the standard H37Rv reference strain and, after two weeks incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (> 30,000) of samples of M. tuberculosis from patients over the next few years.

microbiology↗

bioRxiv2017

Molecular Identification and Characterization of Two Rubber Dandelion Amalgaviruses

The Amalgaviridae family comprise persistent viruses that share the genome architecture of Totiviridae and gene evolutionary resemblance to Partitiviridae. Two genera have been assigned to this family, including genus Amalgavirus consisting in nine recognized species, corresponding to plant infecting viruses with dsRNA monosegmented genomes of ca. 3.4 kb. Here, we present the molecular characterization of two novel viruses detected in rubber dandelion (Taraxacum kok-saghyz). The sequenced viruses are 3,409 and 3,413 nt long, including two partially overlapping ORFs encoding a putative coat protein and an RNA-dependent RNA polymerase (RdRP). Phylogenetic insights based on the RdRP suggest them to be members of two new species within the Amalgavirus genus. Multiple independent RNAseq data suggest that the identified viruses have a dynamic distribution and low relative RNA levels in infected plants. Virus presence was not associated with any apparent symptoms on the plant hosts. We propose the names rubber dandelion latent virus 1 & 2 to the detected amalgaviruses; the first viruses to be associated to this emergent and sustainable natural rubber crop.

microbiology↗

bioRxiv2017

Timing of host feeding drives rhythms in parasite replication

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the hosts peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the hosts peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and has broad implications for applied bioscience.\n\nAuthor summaryHow cycles of asexual replication by malaria parasites are coordinated to occur in synchrony with the circadian rhythms of the host is a long-standing mystery. We reveal that rhythms associated with the time-of-day that hosts feed are responsible for the timing of rhythms in parasite development. Specifically, we altered host feeding time to phase-shift peripheral rhythms, whilst leaving rhythms driven by the central circadian oscillator in the brain unchanged. We found that parasite developmental rhythms remained synchronous but changed their phase, by 12 hours, to follow the timing of host feeding. Furthermore, our results suggest that parasites themselves schedule rhythms in their replication to coordinate with rhythms in glucose in the hosts blood, rather than have rhythms imposed upon them by, for example, host immune responses. Our findings reveal a novel relationship between hosts and parasites that if disrupted, could reduce both the severity and transmission of malaria infection.

microbiology↗

bioRxiv2017

A simple mung bean infection model for studying the virulence of Pseudomonas aeruginosa

Here we highlight the development of a simple and high throughput mung bean model to study virulence in the opportunistic pathogen Pseudomonas aeruginosa. The model is easy to setup and infection and virulence can be monitored for up to 10 days. In a first test of the model, we found that mung bean seedlings infected with PAO1 showed poor development of roots and high mortality rates compared to un-infected controls. We also found that a quorum sensing (QS) mutant was significantly less virulent when compared with the PAO1 wild type. Our work introduces a new tool for studying virulence in P. aeruginosa, that will allow for high throughput virulence studies of mutants, and for testing the in vivo efficacy of new therapies at a time when new antimicrobial drugs are desperately needed.

microbiology↗

bioRxiv2017

Light assisted antibiotics

The overuse of antibiotics is accelerating the bacterial resistance, and therefore there is a need to reduce the amount of antibiotics used for treatment. Here, we demonstrate that specific wavelengths in a narrow range around 296 nm are able to eradicate bacteria in the biofilm state more effectively, than antibiotics and the combination of irradiation and antibiotics is even better, introducing a novel concept light assisted antibiotics. The investigated wavelength range was 249 nm to 338 nm with an approximate step of 5 nm. The novel concept can significantly reduce the amount of antibiotics needed for eradicating mature bacterial biofilms. The irradiation treatment was combined with tobramycin and its efficiency was compared to combinatory antibiotic treatment and highly concentrated antibiotic monotherapy. The eradication efficacies, on mature biofilms, achieved by light assisted antibiotic and by the antibiotic monotherapy at 10-fold higher concentration, were equivalent. The present achievement could motivate the development of light assisted antibiotic treatments for treating infections.

microbiology↗

bioRxiv2017

The rust fungus Melampsora larici-populina expresses a conserved genetic program and distinct sets of secreted protein genes during infection of its two host plants, larch and poplar

Mechanims required for broad spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycle. Melampsora larici-populina infects two taxonomically unrelated plants, larch on which sexual reproduction is achieved and poplar on which clonal multiplication occurs leading to severe epidemics in plantations. High-depth RNA sequencing was applied to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia and aecia. Comparative transcriptomics of infection on poplar and larch hosts was performed using available expression data. Secreted protein was the only significantly over-represented category among differentially expressed M. larici-populina genes in basidia, pycnia and aecia compared together, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes commonly expressed on the two hosts and a fraction exhibiting a host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

microbiology↗

bioRxiv2017

ANTISEPTIC AGENTS ELICIT SHORT-TERM, PERSONALIZED AND BODY SITE-SPECIFIC SHIFTS IN RESIDENT SKIN BACTERIAL COMMUNITIES

Despite critical functions in cutaneous health and disease, it is unclear how resident skin microbial communities are altered by topical antimicrobial interventions commonly used in personal and clinical settings. Here we show that acute exposure to antiseptic treatments elicits rapid but short-term depletion of microbial community diversity and membership. Thirteen subjects were enrolled in a longitudinal treatment study to analyze the effects of topical treatments (ethanol, povidone-iodine, chlorhexidine, water) on the skin microbiome at two skin sites of disparate microenvironment: forearm and back. Treatment effects were highly dependent on personalized and body site-specific colonization signatures, which concealed community dynamics at the population level when not accounted for in this analysis. The magnitude of disruption was influenced by the identity and abundance of particular bacterial inhabitants. Lowly abundant members of the skin microbiota were more likely to be displaced, and subsequently replaced by the most abundant taxa prior to treatment. Members of the skin commensal family Propionibactericeae were particularly resilient to treatment, suggesting a distinct competitive advantage in the face of disturbance. These results provide insight into the stability and resilience of the skin microbiome, while establishing the impact of topical antiseptic treatment on skin bacterial dynamics and community ecology.

microbiology↗

bioRxiv2017

Folic acid instability and bacterial metabolism combine to impact C. elegans development and ageing

Supplementation with the synthetic oxidised folate, folic acid is used to prevent neural tube defects and other symptoms of folate deficiency. However, several unanswered questions remain over folic acid efficacy, safety and interactions with gut microbes. Prevention of a development defect caused by folate deficiency in the nematode worm Caenorhabditis elegans requires > 10 fold higher concentrations of folic acid compared to folinic acid, a reduced folate. Here we show that the major route for folic acid to restore normal development is indirect via the Escherichia coli used to feed C. elegans. This route occurs mainly via the E. coli transporter AbgT, which takes up the folic acid breakdown product para-aminobenzoate-glutamate (PABA-glu). We found that folic acid preparations, including a commercial supplement, contain 0.3- 4.0 % of this breakdown product. Previously, we have shown that inhibiting bacterial folate synthesis increases C. elegans lifespan by removing a life-shortening bacterial activity. Here, we show that folic acid restores bacterial folate synthesis and reverses this lifespan increase. It is still to be determined whether this bacterial route increases host folate levels in humans and if there are situations where increased bacterial folate synthesis has negative health complications.

microbiology↗

bioRxiv2017

The Combined activities of FtsW and MurJ are required for lipid II translocation during cell division in Escherichia coli

Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity in E. coli. However, how MurJ functions in vivo has not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localization in vivo and reveals a possible mechanism of how MurJ functions during cell division, which gives possibilities for future investigations and further antibiotics developments.

microbiology↗

bioRxiv2017

Diversity and evolution of the emerging Pandoraviridae family

With DNA genomes up to 2.5 Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting Pandoraviruses remained the most spectacular viruses since their description in 2013. Our isolation of three new strains from distant locations and environments allowed us to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses, led to the discovery of many non-coding transcripts while significantly reducing the former set of predicted protein-coding genes. We found that the Pandoraviridae exhibit an open pan genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggests that de novo gene creation is a strong component in the evolution of the giant Pandoravirus genomes.

microbiology↗

bioRxiv2017

Programmable transport of micro- and nanoparticles by Paramecium caudatum

We exploit chemo- and galvanotactic behaviour of Paramecium caudatum to design a hybrid device that allows for controlled uptake, transport and deposition of environmental micro- and nanoparticulates in an aqueous medium. Manipulation of these objects is specific, programmable and parallel. We demonstrate how device operation and output interpretation may be automated via a DIY low-cost fluorescence spectrometer, driven by a microprocessor board. The applications of the device presented range from collection and detoxification of environmental contaminants (e.g. nanoparticles), to micromixing, to natural expressions of computer logic.

microbiology↗

bioRxiv2017

Identification and characterization of siderophore producing arsenic tolerant Staphylococcus sp. TA6 and its possible involvement in arsenic geocycle

The presence of arsenic in sediments, carbonaceous rocks are geogenic, while its entry into the aquifers is mediated by several factors including microorganisms. It is well known that the microorganisms play a crucial role in the biogeochemical cycle of different elements. However, the precise role of bacteria in regulating the concentration of arsenic in Brahmaputra valley has not been investigated in detail. In this paper, we report the isolation of arsenic resistant bacterium TA6 with active arsenate reduction efficiency. The isolate was able to grow in arsenate concentration (250 mM) and arsenite (30 mM). Along with resistance to inorganic arsenic, it showed cross-tolerance to other heavy metals like Hg+2, Cd+2, Co+2, Ni+2, Cr+2. The bacterium also had a high siderophore activity (78.7 {+/-} 0.004 mol), which is positively correlated with the resistance aptitude. The biochemical test showed the TA6, a gram-positive bacterium which can hydrolyze starch and casein, produce catalase enzyme and utilizes citrate as a metabolic trait. Molecular and chemotaxonomic identification of TA6 based on 16S rRNA and FMAE analysis showed similarity with members of Staphylococcus genus with significant difference in sequence similarity and fatty acid composition. Based on 16S rRNA and FAME analysis it was identified as Staphylococcus sp. TA6. Rate of biotransformation showed bacterium could reduce ~88.2% of initial 2mM As(V) into As(III). The characterization of arsenate reductase enzyme with NADPH coupled assay showed the highest activity at pH 5.5 and temperature 50{degrees}C.

microbiology↗

bioRxiv2017

Rarefaction, alpha diversity, and statistics

Understanding the drivers of microbial diversity is a fundamental question in microbial ecology. Extensive literature discusses different methods for describing microbial diversity and documenting its effects on ecosystem function. However, it is widely believed that diversity depends on the number of reads that are sequenced. I discuss a statistical perspective on diversity, framing the diversity of an environment as an unknown parameter, and discussing the bias and variance of plug-in and rarefied estimates. I argue that by failing to account for both bias and variance, we invalidate analysis of alpha diversity. I describe the state of the statistical literature for addressing these problems, and suggest that measurement error modeling can address issues with variance, but bias corrections need to be utilized as well. I encourage microbial ecologists to avoid motivating their investigations with alpha diversity analyses that do not use valid statistical methodology.

microbiology↗

bioRxiv2017

Data Analyses and new Findings Indicate a Primordial Neurotropic Pathogen Evolved into Infectious Causes of Several CNS Neurodegenerative Diseases

The extraordinary genetic flexibility of microorganisms enables their evolution into diverse forms expressing unanticipated structures and functions. Typically, they evolve in response to selective pressures of challenging niches, enabling their evolution and survival in extreme environments wherein life forms were not thought to exist. Approaching the problem of persistent neurodegenerative CNS infections as a challenging niche for pathogen evolution led to uncovering microorganisms which expand concepts of microbial diversity. These organisms are proposed as hybrid pathogens. They express two separate sets of structures and functions: viruslike properties when intracellular, and yet also reproduce as unique prokaryotes when outside the host. Their recovery opens new opportunities to comprehend the remarkable diversity of pathogens and elucidate etiologies of unresolved CNS neurodegenerative infections. Cells infected with these agents produce virus-like particles, inclusions and cytopathic effects consistent with biopsy studies of multiple sclerosis (MS), the -synucleinopathies, and the transmissible spongiform encephalopathies (TSE) or prion diseases. The principle agents described were recovered from sheep with scrapie and are available via the Biodefense and Emerging Infections Research Resources Repository. Comparative studies with SMCA, a tick isolate inducing neurodegeneration in lab animal models, are included as supportive evidence.

microbiology↗