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bioRxiv2017

Alphaproteobacteria fix nitrogen in a Sphagnum-dominated peat bog using molybdenum-dependent nitrogenase

Microbial N2 fixation (diazotrophy) represents an important nitrogen source to oligotrophic peatland ecosystems, which are important sinks for atmospheric CO2 and susceptible to changing climate. The objectives of this study were: (i) to determine the active microbial group and type of nitrogenase mediating diazotrophy in a ombrotrophic Sphagnum-dominated peat bog (the S1 peat bog, Marcell Experimental Forest, Minnesota, USA); and (ii) to determine the effect of environmental parameters (light, O2, CO2, CH4) on potential rates of diazotrophy measured by acetylene (C2H2) reduction and 15N2 incorporation. Molecular analysis of metabolically active microbial communities suggested that diazotrophy in surface peat was primarily mediated by Alphaproteobacteria (Bradyrhizobiaceae and Beijerinckiaceae). Despite higher dissolved vanadium (V; 11 nM) than molybdenum (Mo; 3 nM) in surface peat, a combination of metagenomic, amplicon sequencing and activity measurements indicated that Mo-containing nitrogenases dominate over the V-containing form. Acetylene reduction was only detected in surface peat exposed to light, with the highest rates observed in peat collected from hollows with the highest water content. Incorporation of15N2 was suppressed 90% by O2 and 55% by C2H2, and was unaffected by CH4 and CO2 amendments. These results suggest that peatland diazotrophy is mediated by a combination of C2H2 - sensitive and C2H2 - insensitive microbes that are more active at low O2 and show similar activity at high and low CH4.\n\nImportancePrevious studies indicate that diazotrophy provides an important nitrogen source and is linked to methanotrophy in Sphagnum-dominated peatlands. However, the environmental controls and enzymatic pathways of peatland diazotrophy, as well as the metabolically active microbial populations that catalyze this process remain in question. Our findings indicate that oxygen levels and photosynthetic activity override low nutrient availability in limiting diazotrophy, and that members of the Alphaproteobacteria (Rhizobiales) catalyze this process at the bog surface using the molybdenum - based form of the nitrogenase enzyme.

microbiology↗

bioRxiv2017

Ankyrin-1 gene exhibits allelic heterogeneity in conferring protection against malaria

Allelic heterogeneity is a common phenomenon where a gene exhibit different phenotype depending on the nature of its genetic mutations. In the context of genes affecting malaria susceptibility, it allowed us to explore and understand the intricate host-parasite interactions during malaria infections. In this study, we described a gene encoding erythrocytic ankyrin-1 (Ank-1) which exhibits allelic-dependent heterogeneous phenotypes during malaria infections. We conducted an ENU mutagenesis screen on mice and identified two Ank-1 mutations, one resulted in an amino acid substitution (MRI95845), and the other a truncated Ank-1 protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection against Plasmodium chabaudi infections. Upon further examination, the Ank-1(MRI96570) mutation was found to inhibit intra-erythrocytic parasite maturation, whereas Ank-1(MW95845) caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between two Ank-1 mutations. Despite the lack of direct evidence from population studies, this data further supported the protective roles of ankyrin-1 mutations in conferring malaria protection. This study also emphasized the importance of such phenomenon to achieve a better understanding of host-parasite interactions, which could be the basis of future studies.

microbiology↗

bioRxiv2017

Transcriptomic profiling of high-density Giardia foci encysting in the murine proximal intestine

Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci that may cause localized pathology to the epithelium. Encystation is also initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression (> 10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an emerging view that parasites encyst early in localized regions in the gut, possibly as a consequence of nutrient limitation, and also impact local metabolism and physiology.

microbiology↗

bioRxiv2017

A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide

Salmonella is an intracellular pathogen that infects a wide range of hosts and can survive in macrophages. An essential mechanism uses by the macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2O2. For Tn-seq, a highly saturated Tn5 insertion library was grown in vitro under either 2.5 (H2O2L) or 3.5 mM H2O2 (H2O2H). We identified two sets of overlapping genes that are required for resistance of S. Typhimurium to H2O2L and H2O2H, and the results were validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for resistance to H2O2 included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), flagellar genes (fliBC), H2O2 scavenging enzymes, and DNA adenine methylase. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H2O2. A subset of proteins identified by Tn-seq were analyzed by targeted proteomics, and 70% of them were upregulated upon exposure to H2O2. The identified candidate genes will deepen our understanding about mechanisms of S. Typhimurium survival in macrophages, and can be exploited to develop new antimicrobial drugs.

microbiology↗

bioRxiv2017

Is the 25 year hepatitis C marathon coming to an end to declare victory?

Hepatitis C virus (HCV) which was originally recognized as posttransfusion non-A, non-B hepatitis has been a major global health problem affecting 3% of the world population. Interferon/peginterferon and ribavirin combination therapy was the backbone of chronic HCV therapy for two decades of the journey. However, the interferon based treatment success rate was around 50% with many side effects. Many chronic HCV patients with psychiatric diseases, or even cytopenias, were ineligible for HCV treatment. Now, we no longer need any injectable medicine. New direct-acting antiviral agents against HCV allowed the advance of interferon-free and ribavirin-free oral regimens with high rates of response and tolerability. The cost of the medications should not be a barrier to their access in certain parts of the world. While we are getting closer, we should still focus on preventing the spread of the disease, screening and delivering the cure globally to those in need. In the near future, development of an effective vaccine against HCV would make it possible to eradicate HCV infection worldwide completely.

microbiology↗

bioRxiv2017

Gut microbiota analysis in rats with methamphetamine-induced conditioned place preference

Methamphetamine abuse is a major public health crisis. Because accumulating evidence supports the hypothesis that the gut microbiota plays an important role in central nervous system (CNS) function, and research on the roles of the microbiome in CNS disorders holds conceivable promise for developing novel therapeutic avenues for treating CNS disorders, we sought to determine whether administration of methamphetamine leads to alterations in the intestinal microbiota. In this study, the gut microbiota profiles of rats with methamphetamine-induced conditioned place preference (CPP) were analysed through 16S rRNA gene sequencing. The faecal microbial diversity was slightly higher in the METH CPP group. The propionate-producing genus Phascolarctobacterium was attenuated in the METH CPP group, and the family Ruminococcaceae was elevated in the METH CPP group. Short chain fatty acid analysis revealed that the concentrations of propionate were decreased in the faecal matter of METH-administered rats. These findings provide direct evidence that administration of METH causes gut dysbiosis, enable a better understanding of the function of gut microbiota in the process of drug abuse, and provide a new paradigm for addiction treatment.

microbiology↗

bioRxiv2017

Preponderance of Enterovirus Species C in RD-L20B Cell Culture Negative Stool Samples from Children Diagnosed with Acute Flaccid Paralysis in Nigeria.

AbstractsRecently, a reverse transcriptase seminested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses from clinical specimen. In this study, we use the assay and its modification to screen acute flaccid paralysis (AFP) samples previously confirmed negative for enteroviruses by the RD-L20B algorithm.\n\nThirty paired stool suspensions collected in 2015 as part of the national AFP surveillance program in different states of Nigeria were analyzed in this study. The samples were previously confirmed negative for enteroviruses by the polio laboratory in accordance with the WHO recommended RD-L20B cell culture based algorithm. Two samples previously confirmed to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, and the RT-snPCR assay and its modifications. All amplicons were sequenced and enteroviruses identified using the enterovirus genotyping tool.\n\nOverall, amplicons were recovered from the two controls and 50% (15/30) of samples screened. Fourteen were successfully typed of which, 7.1% (1/14), 21.4% (3/14), 64.3% (9/14) and 7.1% (1/14) were EV-A, EV-B, EV-C and a mixture of EV-B and C (EV-C99 and E25), respectively. The two controls were identified as EV-C99 and CV-A1, both EV-Cs. The PV-2 detected had VP1 ILE143. Hence, a vaccine strain.\n\nThe results of this study showed that about 50% of enterovirus infections (including some Sabin PV2s) are being missed by the RD-L20B cell culture based algorithm. This highlights the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-Cs in Nigeria was also confirmed.

microbiology↗

bioRxiv2017

Inferring Within-Host Bottleneck Size: A Bayesian Approach

A number of approaches exist for bottleneck-size estimation with respect to within-host bacterial infections; however, some are more appropriate than others under certain circumstances. A Bayesian comparison of several approaches is made in terms of the availability of isogenic multitype bacteria (e.g., WITS), knowledge of post-bottleneck dynamics, and the suitability of dilution with monotype bacteria. The results are summarised by a guiding flowchart.\n\nA sampling approach to bottleneck-size estimation is also introduced.

microbiology↗

bioRxiv2017

An Efficient Moments-Based Inference Method for Within-Host Bacterial Infection Dynamics

Over the last ten years, isogenic tagging (IT) has revolutionised the study of bacterial infection dynamics in laboratory animal models. However, quantitative analysis of IT data has been hindered by the piecemeal development of relevant statistical models. The most promising approach relies on stochastic Markovian models of bacterial population dynamics within and among organs. Here we present an efficient numerical method to fit such stochastic dynamic models to in vivo experimental IT data. A common approach to statistical inference with stochastic dynamic models relies on producing large numbers of simulations, but this remains a slow and inefficient method for all but simple problems, especially when tracking bacteria in multiple locations simultaneously. Instead, we derive and solve the systems of ordinary differential equations for the two lower-order moments of the stochastic variables (mean, variance and covariance). For any given model structure, and assuming linear dynamic rates, we demonstrate how the model parameters can be efficiently and accurately estimated by divergence minimisation. We then apply our method to an experimental dataset and compare the estimates and goodness-of-fit to those obtained by maximum likelihood estimation. While both sets of parameter estimates had overlapping confidence regions, the new method produced lower values for the division and death rates of bacteria: these improved the goodness-of-fit at the second time point at the expense of that of the first time point. This flexible framework can easily be applied to a range of experimental systems. Its computational efficiency paves the way for model comparison and optimal experimental design.\n\nAuthor SummaryRecent advancements in technology have meant that microbiologists are producing vast amounts of experimental data. However, statistical methods by which we can analyse that data, draw informative inference, and test relevant hypotheses, are much needed. Here, we present a new, efficient inference tool for estimating parameters of stochastic models, with a particular focus on models of within-host bacterial dynamics. The method relies on matching the two lower-order moments of the experimental data (i.e., mean, variance and covariance), to the moments from the mathematical model. The method is verified, and particular choices justified, through a number of simulation studies. We then use this method to estimate models that have been previously estimated using a \"gold-standard\" maximum likelihood procedure.\n\nList of symbolsO_LIA: number of animals\nC_LIO_LIT: number of tagged strains\nC_LIO_LIn: number of organs\nC_LIO_LINi: number of bacteria in organ i\nC_LIO_LImij: migration rate from organ i to organ j\nC_LIO_LIki: killing rate in organ i\nC_LIO_LIri: replication rate in organ i\nC_LIO_LI{tau}i: observation time i\nC_LIO_LIA, B, C: matrices\nC_LIO_LI{lambda}: vector of transition rates\nC_LIO_LIB: Number of bootstrap samples\nC_LIO_LI{theta}*: MDE parameter estimate\nC_LI\n\nAbbreviationsO_LIABC: approximate Bayesian computation\nC_LIO_LIIT: isogenic tagging\nC_LIO_LILV: live vaccine\nC_LIO_LIMARE: mean absolute relative error\nC_LIO_LIMDE: minimum divergence estimate\nC_LIO_LIMLE: maximum likelihood estimate\nC_LIO_LIqPCR: quantitative polymerase chain reaction\nC_LIO_LIWITS: wildtype isogenic tagged strain\nC_LI

microbiology↗

bioRxiv2017

Tracking electron uptake from a cathode into Shewanella cells: implications for generating maintenance energy from solid substrates.

While typically investigated as a microorganism capable of extracellular electron transfer to minerals or anodes, Shewanella oneidensis MR-1 can also facilitate electron flow from a cathode to terminal electron acceptors such as fumarate or oxygen, thereby providing a model systems for a process that has significant environmental and technological implications. This work demonstrates that cathodic electrons enter the electron transport chain of S. oneidensis when oxygen is used as the terminal electron acceptor. The effect of electron transport chain inhibitors suggested that a proton gradient is generated during cathode-oxidation, consistent with the higher cellular ATP levels measured in cathode-respiring cells relative to controls. Cathode oxidation also correlated with an increase in the cellular redox (NADH/FMNH2) pool using a bioluminescent assay. Using a proton uncoupler, generation of NADH/FMNH2 under cathodic conditions was linked to reverse electron flow mediated by the proton pumping NADH oxidase Complex I. A decrease in cathodic electron uptake was observed in various mutant strains including those lacking the extracellular electron transfer components necessary for anodic current generation. While no cell growth was observed under these conditions, here we show that cathode oxidation is linked to cellular energy conservation, resulting in a quantifiable reduction in cellular decay rate. This work highlights a potential mechanism for cell survival and/or persistence in environments where growth and division are severely limited.

microbiology↗

bioRxiv2017

Cross reactivity of antibodies against microbial proteins to human tissues as basis of Crohns disease and other autoimmune diseases

BackgroundAutoimmune disease is generally a systemic inflammatory response with production of autoantibodies. In this study, we investigated the anti-microbial antibodies in circulation in cases of Crohns disease (CD), Sjogrens syndrome (SS) and other autoimmune disease and their roles in the pathogenesis of these autoimmune diseases.\n\nMaterial and methodsWestern blot was used to determine the reactivity of human plasmas from patients with CD and SS as the primary antibodies against the whole microbial extracts. The microbial proteins reactive to patients plasma were further identified and the modified sandwich ELISA assays were used to determine the blood levels of antibodies against these microbial proteins in patients with CD and SS. Antibodies against the microbial proteins are used for immunohistochemical staining of normal human tissue.\n\nResultsA group of 7 microbial proteins was identified reactive to the plasmas of patients with CD and SS including DNA-directed RNA polymerase B (RPOB), and elongation factor G (EF-G) from Staphylococcus aureus and Staphylococcus pseudintermedius (S. aureus and S. pseudintermedius), ATP synthase alpha (ATP5a) and heat shock protein 65 (Hsp65) from Mycobacterium avium subspecies paratuberculosis (MAP), elongation factor Tu (EF-Tu) and outer membrane porin C (ompC) from Escherichia Coli (E. coli). Anti-microbial antibodies can cross-react to normal human tissues. The levels of antibodies against the microbial proteins are significantly elevated in the patients with CD and SS.\n\nConclusionThe levels of antibodies against the microbial proteins are significantly elevated in CD and SS. The cross-reactivity of the anti-microbial antibodies to human tissue provides a new mechanism of pathogenesis of autoimmune diseases such as CD and SS.

microbiology↗

bioRxiv2017

cAMP-Independent Control Of Twitching Motility In Pseudomonas aeruginosa

FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through activation of the adenylate cyclase, CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimVs cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, required both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds - lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH, or expressing constitutively-active CyaB - pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV-dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimVs role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.\n\nIMPORTANCEPseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility, and are involved in surface sensing which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements - including the PilSR two-component system - to cell poles for optimal function.

microbiology↗

bioRxiv2017

Multi-Locus And Ultra-Long Amplicon Sequencing Approach To Study Microbial Diversity At Species Level Using The MinION™ Portable Nanopore Sequencer

BackgroundThe miniaturised and portable DNA sequencer MinION has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach.\n\nResultsAfter compiling a robust database by parsing and extracting the rrn bacterial region from more than 67,000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION device using R9 and R9.4 chemistries was sufficient to study two mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities.\n\nConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108) permitted to retrieve the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimising base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems.

microbiology↗

bioRxiv2017

Comprehensive mapping of HIV-1 escape from a broadly neutralizing antibody

Precisely defining how viral mutations affect HIVs sensitivity to antibodies is vital to the development and evaluation of vaccines and antibody immunotherapeutics. But despite great effort, a full map of escape mutants has not yet been delineated for even a single anti-HIV antibody. Here we describe a massively parallel experimental approach to quantify how all single amino-acid mutations to Envelope (Env) affect HIVs sensitivity to a neutralizing antibody. We applied this approach to PGT151 and identified novel sites of escape in addition to those previously defined by structural and functional studies, such as glycans at sites 611 and 637, residue 647, and sites in the fusion peptide. Evaluating the effect of each amino acid at each site lends insight into the biochemical basis of escape throughout the epitope. Thus, comprehensive mapping of HIV antibody escape gives a quantitative, mutation-level view of the ways that Env can evade neutralization.

microbiology↗

bioRxiv2017

Bacterial physiological adaptations to contrasting edaphic conditions identified using landscape scale metagenomics

Environmental factors relating to soil pH are widely known to be important in structuring soil bacterial communities, yet the relationship between taxonomic community composition and functional diversity remains to be determined. Here, we analyze geographically distributed soils spanning a wide pH gradient and assess the functional gene capacity within those communities using whole genome metagenomics. Low pH soils consistently had fewer taxa (lower alpha and gamma diversity), but only marginal reductions in functional alpha diversity and equivalent functional gamma diversity. However, coherent changes in the relative abundances of annotated genes between pH classes were identified; with functional profiles clustering according to pH independent of geography. Differences in gene abundances were found to reflect survival and nutrient acquisition strategies, with organic-rich acidic soils harboring a greater abundance of cation efflux pumps, C and N direct fixation systems and fermentation pathways indicative of anaerobiosis. Conversely, high pH soils possessed more direct transporter-mediated mechanisms for organic C and N substrate acquisition. These findings show that bacterial functional versatility may not be constrained by taxonomy, and we further identify the range of physiological adaptations required to exist in soils of varying nutrient availability and edaphic conditions.

microbiology↗

bioRxiv2017

Deciphering the combinatorial influence of diet and the microbiota on experimental colitis

Background & AimsThe complex interactions between diet and the microbiota that influence mucosal inflammation and inflammatory bowel disease are poorly understood. Experimental colitis models provide the opportunity to control and systematically perturb diet and the microbiota in parallel to quantify the contributions between multiple dietary ingredients and the microbiota on host physiology and colitis.\n\nMethodsTo examine the interplay of diet and the gut microbiota on host health and colitis, we fed over 40 different diets with varied macronutrient sources and concentrations to specific pathogen free or germ free mice either in the context of healthy, unchallenged animals or colitis models (dextran sodium sulfate (DSS) and T cell transfer).\n\nResultsDiet influenced physiology in both health and colitis across all models, with the concentration of protein and psyllium fiber having the most profound effects. Increasing dietary protein elevated gut microbial density and worsened DSS colitis severity. Depleting gut microbial density by using germ-free animals or antibiotics negated the effect of a high protein diet. Psyllium fiber influenced host physiology and attenuated colitis severity through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary protein and psyllium fiber in parallel explain most variation in gut microbial density, intestinal permeability, and DSS colitis severity, and changes in one ingredient can be offset by changes in the other.\n\nConclusionsOur results demonstrate the importance of examining complex mixtures of nutrients to understand the role of diet in intestinal inflammation.

microbiology↗

bioRxiv2017

Electrically Conductive Pili from Pilin Genes of Phylogenetically Diverse Microorganisms

The possibility that bacteria other than Geobacter species might contain genes for electrically conductive pili (e-pili) was investigated by heterologously expressing pilin genes of interest in Geobacter sulfurreducens. Strains of G. sulfurreducens producing high current densities, which are only possible with e-pili, were obtained with pilin genes from Flexistipes sinusarabici, Calditerrivibrio nitroreducens, and Desulfurivibrio alkaliphilus. The conductance of pili from these strains was comparable to native G. sulfurreducens e-pili. The e-pili derived from C. nitroreducens, and D. alkaliphilus pilin genes are the first examples of relatively long (> 100 amino acids) pilin monomers assembling into e-pili. The pilin gene from Desulfofervidus auxilii did not yield e-pili, suggesting that the hypothesis that this sulfate reducer wires itself to ANME-1 microbes with e-pili to promote anaerobic methane oxidation should be reevaluated. A high density of aromatic amino acids and a lack of substantial aromatic-free gaps along the length of long pilins may be important characteristics leading to e-pili. This study demonstrates a simple method to screen pilin genes from difficult-to-culture microorganisms for their potential to yield e-pili; reveals new potential sources for biologically based electronic materials; and suggests that a wide phylogenetic diversity of microorganisms may employ e-pili for extracellular electron exchange.

microbiology↗

bioRxiv2017

Divergent In vitro MIC Characteristics and underlying isogenic mutations in host-specialized Pseudomonas aeruginosa

Clinical isolates of Pseudomonas aeruginosa (Pa) from patients with cystic fibrosis (CF) are known to differ from those associated with infections of non-CF hosts in colony morphology, drug susceptibility patterns, and genomic hypermutability. Although Pa isolates from CF have long been recognized for their overall higher resistance rate calculated generally by reduced \"percent susceptible\", this study takes the approach to compare and contrast Etest MIC distributions between two distinct cohorts of clinical strains (n=224 from 56 CF patients and n=130 from 68 non-CF patients respectively) isolated in 2013. Logarithmic transformed MIC (logMIC) values of 11 antimicrobial agents were compared between the two groups. CF isolates tended to produce heterogeneous and widely dispersed MICs compared to non-CF isolates. By applying a test for equality of variances, we were able to confirm that the MICs generated from CF isolates against 9 out of the 11 agents were significantly more dispersed than those from non-CF (p<0.02-<0.001). Quantile-quantiles plots indicated little agreement between the two cohorts of isolates. Based on whole genome sequencing of 19 representative CF Pa isolates, divergent gain- or loss-of-function mutations in efflux and porin genes and their regulators between isogenic or intra-clonal associates were evident. Not one, not a few, but the net effect all adaptive mutational changes in the genomes of CF Pa, both shared and unshared between isogenic strains, are responsible for the divergent heteroresistance patterns. Moreover, the isogenic variations are suggestive of a bacterial syntrophic lifestyle when \"lockedo inside a host focal airway environment over prolonged periods.\n\nSignificance statementBacterial heteroresistance is associated with niche specialized organisms interacting with host species for prolonged period of time, medically characterized by \"chronic focal infections\". A prime example is found in Pseudomonas aeruginosa isogenic/non-homogeneous isolates from patient airways with cystic fibrosis. The development of pseudomonal polarizing MICs in vitro to many actively used antimicrobial agents among isogenic isolates and \"Eagle-type\" heteroresistance patterns are common and characteristic. Widespread isogenic gene lesions were evident for defects in drug transporters, DNA mismatch repair, and many other structural or cellular functions--a result of pseudomonal symbiotic response to host selection. Co-isolation of extremely susceptible and resistant isogenic Pa strains suggests intra-airway evolution of a multicellular syntrophic bacterial lifestyle, which has laboratory interpretation and clinical treatment implications.

microbiology↗