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bioRxiv2018

Validation of a 14-drug microtitre plate that includes delamanid and bedaquiline for susceptibility testing of M. tuberculosis

UKMYC5 is a 96-well microtitre plate designed by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to enable the measurement of minimum inhibitory concentrations (MICs) of 14 different anti-TB compounds for >30,000 clinical tuberculosis isolates. Unlike the MYCOTB plate, on which UKMYC5 is based, the plate included two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents using a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers using three methods (mirrored-box, microscope and Vizion Digital viewing system) after 7, 10, 14 and 21 days incubation. In addition, all EQA strains were whole-genome sequenced and phenotypically characterized by 7H10/7H11 agar proportion method (APM) and MGIT960. We conclude that the UKMYC5 plate is optimally read using the Vizion system after 14 days incubation, achieving an inter-reader agreement of 97.9% and intra- and inter-laboratory reproducibilities of 95.6% and 93.1%, respectively. The mirrored-box had similar reproducibilities. Strains classified as resistant by APM, MGIT960 or the presence of mutations known to confer resistance consistently record elevated MICs compared with those strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain which the APM measured MICs close the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds due to specific genetic variation.

microbiology↗

bioRxiv2018

The smallest intestine (TSI) - a low volume in vitro model of the small intestine with increased throughput

AimsTo develop a low volume in vitro model with increased throughput simulating the human small intestine, incorporating the presence of ileal microbiota, to study microbial behaviour in the small intestine.\n\nMethods and ResultsThe Smallest Intestine in vitro model (TSI) was developed. During the simulated passage of the small intestine bile is absorbed by dialysis and pH adjusted to physiologically relevant level. A consortium of seven representative bacterial members of the ileum microbiota is included in the ileal stage of the model. It was found that the model can be applied for the testing of survival of probiotic bacteria in upper intestine. Moreover, it was found, that the presence of the ileal microbiota significantly impacted probiotic survival.\n\nConclusionsTSI allows testing a substantial number of samples, at low cost and short time, and is thus suitable as an in vitro screening platform. Multiples of five reactors can be added to increase the number of biological and technical replicates.\n\nSignificance and Impact of the StudyTSI serves as an alternative for costly and low throughput in vitro models currently existing on the market. Above that, significance of small intestine bacteria presence on survival of probiotic bacteria in the upper intestine was proved.

microbiology↗

bioRxiv2018

Oscillating bacterial expression states generate herd immunity to viral infection

Hypermutable loci are widespread in bacteria as mechanisms for rapid generation of phenotypic diversity, enabling individual populations to survive fluctuating, often antagonistic, selection pressures. As observed for adaptive immunity, hypermutation may facilitate survival of multiple, spatially-separated bacterial populations. We developed an oscillating prey assay to examine bacteriophage (phage) spread through populations of Haemophilus influenzae whose phage receptor gene, lic2A, is switched ON and OFF by mutations in a hypermutable tetranucleotide repeat tract. Phage extinction was frequently observed when the proportion of phage-resistant sub-populations exceeded 34%. In silico modelling indicated that phage extinction was interdependent on phage loss during transfer between populations and the frequency of resistant populations. In a fixed-area oscillating prey assay, heterogeneity in phage resistance was observed to generate vast differences in phage densities across multiple bacterial populations resulting in protective quarantining of some populations from phage attack. We conclude that phase-variable hypermutable loci produce bacterial herd immunity with resistant intermediary-populations acting as a barricade to reduce the viral load faced by phage-sensitive sub-populations. This paradigm of meta-population protection is applicable to evolution of hypermutable loci in multiple bacteria-phage and host-pathogen interactions.\n\nImportanceHerd immunity is a survival strategy wherein populations are protected against invading pathogens by resistant individuals within the population acting as a barrier to spread of the infectious agent. Although, this concept is normally only applied to higher eukaryotes, prokaryotic organisms also face invasion by infectious agents, such as bacterial viruses, bacteriophage (phage). Here we use novel experimental approaches and mathematical modelling, to show that bacteria exhibit a form of herd immunity through stochastically generated resistant variants acting as barricades to phage predation of sensitive cells. With hypermutable loci found in many prokaryotic systems, this phenomenon may be widely applicable to phage-bacteria interactions and could even impact phage-driven evolution in bacteria.

microbiology↗

bioRxiv2018

Yeast transformation efficiency is enhanced by TORC1- and eisosome-dependent signalling

Transformation of bakers yeast (Saccharomyces cerevisiae) plays a key role in several experimental techniques, yet the molecular mechanisms underpinning transformation are still unclear. Addition of amino acids to the growth and transformation medium increases transformation efficiency. Here, we show that target of rapamycin complex 1 (TORC1) activated by amino acids enhances transformation via ubiquitin-mediated endocytosis. We created mutants of the TORC1 pathway, -arrestins, and eisosome-related genes. Our results demonstrate that the TORC1-Npr1-Art1/Rsp5 pathway regulates yeast transformation. Based on our previous study, activation of this pathway results in a 13-fold increase in transformation efficiency, or greater. Additionally, we suggest DNA is taken up by domains at the membrane compartment of Can1 (MCC) in the plasma membrane formed by eisosomes. Yeast studies on transformation could be used as a platform to understand the mechanism of DNA uptake in mammalian systems, which is clinically relevant to optimise gene therapy.

microbiology↗

bioRxiv2018

Galleria mellonella as an Insect Model for P. destructans, the Cause of White-Nose Syndrome in Bats

Pseudogymnoascus destructans is the fungal pathogen responsible for White-nose Syndrome (WNS), a disease that has killed millions of bats in North America over the last decade. A major obstacle to research on P. destructans has been the lack of a tractable infection model for monitoring virulence. Here, we establish a high-throughput model of infection using larvae of Galleria mellonella, an invertebrate used to study host-pathogen interactions for a wide range of microbial species. We demonstrate that P. destructans can kill G. mellonella larvae in an inoculum-dependent manner when infected larvae are housed at 13{degrees}C or 18{degrees}C. Larval killing is an active process, as heat-killed P. destructans spores caused significantly decreased levels of larval death compared to live spores. We also show that fungal spores that were germinated prior to inoculation were able to kill larvae 3-4 times faster than non-germinated spores. Lastly, we identified chemical inhibitors of P. destructans and used G. mellonella to evaluate these inhibitors for their ability to reduce virulence. We demonstrate that two chemicals, trifluoperazine and amphotericin B, can effectively block larval killing by P. destructans and thereby establish that this infection model can be used to screen biocontrol agents against this fungal pathogen.

microbiology↗

bioRxiv2018

Commensal-Pathogen Competition Impacts Host Viability

While the structure and regulatory networks that govern the activity of the type-six secretion system (T6SS) of Vibrio cholerae are becoming increasingly clear, we know less about the role of the T6SS in disease. Under laboratory conditions, V. cholerae uses the T6SS to outcompete many Gram-negative species, including other V. cholerae strains and human commensal bacteria. However, the role of these interactions has not been resolved in an in-vivo setting. We used the Drosophila melanogaster model of cholera to define the contribution of the T6SS to V. cholerae pathogenesis. Here, we demonstrate that interactions between the T6SS and host commensals impact pathogenesis. Inactivation of the T6SS, or removal of commensal bacteria attenuates disease severity. Re-introduction of the Gram-negative commensal bacterium Acetobacter pasteurianus into a germ-free host is sufficient to restore T6SS-dependent pathogenesis. Together, our data demonstrate that the T6SS acts on commensal bacteria to promote the pathogenesis of V. cholerae.

microbiology↗

bioRxiv2018

Septal Secretion of Protein A in Staphylococcus aureus Requires SecA and Lipoteichoic Acid Synthesis

Surface proteins of Staphylococcus aureus are secreted across septal membranes for assembly into the bacterial cross-wall. This localized secretion requires the YSIRK/GXXS motif signal peptide, however the mechanisms supporting precursor trafficking are not known. We show here that the signal peptide of staphylococcal protein A (SpA) is cleaved at the YSIRK/GXXS motif. A signal peptide mutant defective for cleavage can be crosslinked to SecA, SecDF and LtaS. SecA depletion blocks precursor targeting to septal membranes, whereas deletion of secDF diminishes SpA secretion into the cross-wall. Depletion of LtaS blocks lipoteichoic acid synthesis and promotes precursor trafficking to peripheral membranes. We propose a model whereby SecA directs SpA precursors to lipoteichoic acid-rich septal membranes for YSIRK/GXXS motif cleavage and secretion into the cross-wall.

microbiology↗

bioRxiv2018

The Streptococcus pneumoniae Competence-Induced BriC Peptide Promotes Nasopharyngeal Colonization and Impacts Biofilm Development

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

microbiology↗

bioRxiv2018

Data-mining of Antibiotic Resistance Genes Provides Insight into the Community Structure of Ocean Microbiome

BackgroundAntibiotics have been spread widely in environments, asserting profound effects on environmental microbes as well as antibiotic resistance genes (ARGs) within these microbes. Therefore, investigating the associations between ARGs and bacterial communities become an important issue for environment protection. Ocean microbiomes are potentially large ARG reservoirs, but the marine ARG distribution and its associations with bacterial communities remain unclear.\n\nMethodswe have utilized the big-data mining techniques on ocean microbiome data to analysis the marine ARGs and bacterial distribution on a global scale, and applied comprehensive statistical analysis to unveil the associations between ARG contents, ocean microbial community structures, and environmental factors by reanalyzing 132 metagenomic samples from the Tara Oceans project.\n\nResultsWe identified in total 1,926 unique ARGs and found that: firstly, ARGs are more abundant and diverse in the mesopelagic zone than other water layers. Additionally, ARG-enriched genera are closely connected in co-occurrence network. We also found that ARG-enriched genera are often more abundant than their ARG-less neighbors. Furthermore, we found that samples from the Mediterranean that is surrounded by human activities often contain more ARGs.\n\nConclusionOur research for investigating the marine ARG distribution and revealing the association between ARG and bacterial communities provide a deeper insight into the marine bacterial communities. We found that ARG-enriched genera were often more abundant than their ARG-less neighbors in the same environment, indicating that genera enriched with ARGs might possess an advantage over others in the competition for survival in the oceanic microbial communities.

microbiology↗

bioRxiv2018

SUCCESSIVE BLOODMEALS ENHANCE VIRUS DISSEMINATION WITHIN MOSQUITOES AND INCREASE TRANSMISSION POTENTIAL

SUMMARY PARAGRAPHThe recent Zika virus (ZIKV) and chikungunya virus (CHIKV) epidemics highlight the explosive nature of arthropod-borne (arbo) viruses transmitted by Aedes aegypti mosquitoes1,2. Vector competence and the extrinsic incubation period (EIP) are two key entomological parameters used to assess the public health risk posed by arboviruses3. These are typically measured empirically by offering mosquitoes an infectious bloodmeal and temporally sampling mosquitoes to determine infection and transmission status. This approach has been used for the better part of a century; however, it does not accurately capture the biology and behavior of many mosquito vectors which refeed frequently (every 2-3 days)4. Here we demonstrate that administration of a second non-infectious bloodmeal significantly shortens the EIP of ZIKV-infected Ae. aegypti by enhancing virus escape from the mosquito midgut. Similarly, a second bloodmeal increased the competence of this species for dengue virus and CHIKV. This effect was also observed for ZIKV in Aedes albopictus, suggesting that this species might be a more important vector than once thought and that this phenomenon may be common among other virus-vector pairings. Modeling of these findings reveals that a shortened EIP would result in a significant increase in the basic reproductive number, R0. This increase helps explain how Ae.aegypti can sustain an explosive epidemic like ZIKV despite its relatively poor vector competence in single-feed laboratory trials. Together, these data demonstrate a direct and unrecognized link between mosquito feeding behavior, EIP, and vector competence.

microbiology↗

bioRxiv2018

Direct Imaging of the circular chromosome of a live bacterium

New assays for quantitative imaging1-6 and sequencing7-11 have yielded great progress towards understanding the organizational principles of chromosomes. Yet, even for the well-studied model bacterium Escherichia coli, many basic questions remain unresolved regarding chromosomal (sub-)structure2,11, its mechanics1,2,12 and dynamics13,14, and the link between structure and function1,15,16. Here we resolve the spatial organization of the circular chromosome of bacteria by directly imaging the chromosome in live E. coli cells with a broadened cell shape. The chromosome was observed to exhibit a torus topology with a 4.2 m toroidal length and 0.4 m bundle thickness. On average, the DNA density along the chromosome shows dense right and left arms that branch from a lower-density origin of replication, and are connected at the terminus of replication by an ultrathin flexible string of DNA. At the single-cell level, the DNA density along the torus is found to be strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. We show that prominent domain boundaries at the terminus and origin of replication are induced by MatP proteins, while weaker transient domain boundaries are facilitated by the global transcription regulators HU and Fis. These findings provide an architectural basis for the understanding of the spatial organization of bacterial genomes.

microbiology↗

bioRxiv2018

Insights into circovirus host range from the genomic fossil record.

A diverse range of DNA sequences derived from circoviruses (family Circoviridae) have been identified in samples obtained from humans and domestic animals, often in association with pathological conditions. In the majority of cases, however, little is known about the natural biology of the viruses from which these sequences are derived. Endogenous circoviral elements (CVe) are DNA sequences derived from circoviruses that occur in animal genomes and provide a useful source of information about circovirus-host relationships. In this study we screened genome assemblies of 675 animal species and identified numerous circovirus-related sequences, including the first examples of CVe derived from cycloviruses. We confirmed the presence of these CVe in the germline of the elongate twig ant (Pseudomyrmex gracilis), thereby establishing that cycloviruses infect insects. We examined the evolutionary relationships between CVe and contemporary circoviruses, showing that CVe from ants and mites group relatively closely with cycloviruses in phylogenies. Furthermore, the relatively random interspersal of CVe from insect genomes with cyclovirus sequences recovered from vertebrate samples, suggested that contamination might be an important consideration in studies reporting these viruses. Our study demonstrates how endogenous viral sequences can inform metagenomics-based virus discovery. In addition, it raises doubts about the role of cycloviruses as pathogens of humans and other vertebrates.

microbiology↗

bioRxiv2018

The Viral protein Corona Directs Viral Pathogenesis and Amyloid Aggregation

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. Additionally, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta peptide (A{beta}42), a major constituent of amyloid plaques in Alzheimers disease, in-vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

microbiology↗

bioRxiv2018

Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors

Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes depending on the cell or tissue type. The virus Spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abl kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titers and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which can make experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titers by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying host cell pathways required for coronavirus infection. This will provide insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.

microbiology↗

bioRxiv2018

A genomic island of Streptomyces coelicolor harbors the self-contained regulon of an ECF sigma factor

Streptomycetes constitute the largest genus of actinobacteria, living predominantly in soil and decaying vegetation. The bacteria are widely known for their filamentous morphologies and their capacity to synthesize antibiotics and other biologically active molecules. More than a decade ago, we and others identified 22 genomic islands that Streptomyces coelicolor M145 possesses and other Streptomyces strains lack. One of these genomic islands, Genomic Island (GI) 6, encodes an extracytoplasmic function (ECF) sigma factor that we were characterizing in separate work. Here we report that artificial induction of the ECF sigma factor, which is encoded by SCO3450, causes the transcription of approximately one-fourth of GI 6, or ~26 mostly contiguous genes, to increase. More than half of the regulon encodes putative enzymes involved in small molecule metabolism. A putative haloacid dehalogenase is present. Genes encoding two putative anti-sigma factors flank SCO3450, the three genes residing within the regulon. Our data suggest that the ECF sigma factor and its regulon are a self-contained transcriptional unit that can be transferred by horizontal gene transfer. To our knowledge, only one other example has been identified of an ECF sigma factor and its contiguous regulon appearing to be transferrable by horizontal gene transfer [18,19]. Because the regulon appears not to be induced by the 44 growth conditions recently examined by Byung-Kwan Cho and colleagues [20], if it confers fitness to S. coelicolor, the regulon likely does so in as-yet unknown situations. Those situations might range from scavenging to detoxification to even communication within microbial communities.\n\nIMPORTANCEStreptomyces bacteria grow as hyphae that colonize soil and differentiate into spores when nutrients become scarce. In their terrestrial habitats, the bacteria encounter diverse conditions. Presumably so that the bacteria can cope with those conditions, the chromosomes of streptomycetes are highly dynamic, varying greatly in structure not only between species but also between closely related strains of a single species. The bacteria also have large numbers of extracytoplasmic function (ECF) sigma factors, which undoubtedly help the microorganisms respond to the plethora of challenges coming from the environment. This work illustrates these two threads of Streptomyces biology dovetailing: Genetic adaptability through horizontal gene transfer seems to have enabled Streptomyces coelicolor to acquire a self-contained transcriptional unit that consists of an ECF sigma factor and its regulon. The suggested facile movement of the regulon between microbial hosts indicates the value of the metabolism of small molecules possibly mediated by the regulon.

microbiology↗

bioRxiv2018

Single Cell Observations Show Persister Cells Wake Based on Ribosome Content

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising naturally at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through seven sets of experiments), we used microscopy of single cells to determine that persisters have low levels of antibiotic-corrupting proteins and that their resuscitation is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found for persister cell resuscitation: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division, and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

microbiology↗

bioRxiv2018

Mixing is required for uniform reconstitution of filter-dried protein antigens in a single-injection vaccine formulation

Ambient temperature filter dried vaccine formulations have been proposed to simultaneously achieve thermostability and offer a ready-to-use immunisation device that combines reconstitution and injection. Vaccine concentration should be uniform at the point of injection, but the uniformity following direct reconstitution of filter-dried vaccines has not been reported. We present here a study of vaccine mixing and release following dissolution of filter-dried model protein and toxoid antigens within a single syringe, filter and needle unit. Release was better for filters made from glass than cellulose. Without additional mixing, uniformity was poor and only 41% of input protein was released from protein filter-dried onto glass fibre. In contrast, adding a simple glass bead and mixing by inversion, 100% release antigen solution was achieved, with uniform concentration at exit from the needle throughout a simulated injection. Adsorption onto alum adjuvant had no detectable effect on vaccine dissolution and mixing. The uniformity and yield of low doses of diphtheria and tetanus toxoid was also improved by mixing, albeit with a lower yield of 60-68%. We conclude that uniformity and mixing should be studied to ensure safety and efficacy of directly reconstituted filter-dried vaccine formulations.

microbiology↗

bioRxiv2018

Impact of DNA polymerase choice on assessment of bacterial communities by a Legionella genus-specific next-generation sequencing approach

The library preparation step is a major source of bias in NGS-based studies. Several PCR-related factors might negatively influence the application of NGS tools in environmental studies and diagnostics. Among the most understudied factors are DNA polymerases. In our study, we evaluated the effect of DNA polymerase type on the characterisation of bacterial communities, more precisely Legionella, using a genus-specific NGS approach. The assay with proof-reading high fidelity KAPA HiFi showed better amplification yield than the one with widely used non-proofreading HotStarTaq. Legionella community richness metrics were significantly overestimated with HotStarTaq. However, the choice of DNA polymerase did not significantly change the community profiling and composition. These results substantiate the use of proof-reading high fidelity DNA polymerases in NGS assays and highlight the need of considering the impact of different DNA polymerases in comparative studies and future guidelines for NGS-based diagnostic tools.

microbiology↗