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bioRxiv2016

Recent Outbreaks of Shigellosis in California Caused by Two Distinct Populations of Shigella sonnei With Increased Virulence or Fluoroquinolone Resistance

Shigella sonnei has caused unusually large outbreaks of shigellosis in California in 2014 - 2015. Preliminary data indicated the involvement of two distinct yet related bacterial populations, one from San Diego and San Joaquin (SD/SJ) and one from the San Francisco (SF) Bay area. Whole genome sequencing of sixty-eight outbreak and archival isolates of S. sonnei was performed to investigate the microbiological factors related to these outbreaks. Both SD/SJ and SF populations, as well as almost all of the archival S. sonnei isolates belonged to sequence type 152 (ST152). Genome-wide SNP analysis clustered the majority of California (CA) isolates to an earlier described global Lineage III, which has persisted in CA since 1986. Isolates in the SD/SJ population had a novel Shiga-toxin (STX)-encoding lambdoid bacteriophage, most closely related to that found in an Escherichia coli O104:H4 strain responsible for a large outbreak. However, the STX genes (stxla and stxlb) from this novel phage had sequences most similar to the phages from S. flexneri and S. dysenteriae. The isolates in the SF population yielded evidence of fluoroquinolone resistance acquired via the accumulation of point mutations in gyrA and parC genes. Thus, the CA S. sonnei lineage continues to evolve by the acquisition of increased virulence and antibiotic resistance, and enhanced monitoring is advocated for its early detection in future outbreaks.

Microbiology↗

bioRxiv2016

Extreme metagenomics using nanopore DNA sequencing: a field report from Svalbard, 78 N

In the field of observation, chance favours only the prepared mind (Pasteur). Impressive developments in genomics have led microbiology to its third \"Golden Age\". However, conventional metagenomics strategies necessitate retrograde transfer of samples from extreme or remote environments for later analysis, rendering the powerful insights gained retrospective in nature, striking a contrast with Pasteurs dictum. Here we implement highly portable USB-based nanopore DNA sequencing platforms coupled with field-adapted environmental DNA extraction, rapid sequence library generation and off-line analyses of shotgun metagenome and 16S ribosomal RNA gene amplicon profiles to characterize microbiota dwelling within cryoconite holes upon Svalbard glaciers, the Greenland Ice Sheet and the Austrian Alps. We show in-field nanopore sequencing of metagenomes captures taxonomic composition of supraglacial microbiota, while 16S rRNA Furthermore, comparison of nanopore data with prior 16S rRNA gene V1-V3 pyrosequencing from the same samples, demonstrates strong correlations between profiles obtained from nanopore sequencing and laboratory based sequencing approaches. gene amplicon sequencing resolves bacterial community responses to habitat changes. Finally, we demonstrate the fidelity and sensitivity of in-field sequencing by analysis of mock communities using field protocols. Ultimately, in-field sequencing potentiated by nanopore devices raises the prospect of enhanced agility in exploring Earths most remote microbiomes.

Microbiology↗

bioRxiv2016

16S rRNA Gene Sequencing as a Clinical Diagnostic Aid for Gastrointestinal-related Conditions

Accurate detection of the microorganisms underlying gut dysbiosis in the patient is critical to initiate the appropriate treatment. However, most clinical microbiology techniques used to detect gut bacteria were developed over a century ago and rely on culture-based approaches that are often laborious, unreliable, and subjective. Further, culturing does not scale well for multiple targets and detects only a minority of the microorganisms in the human gastrointestinal tract. Here we present a clinical test for gut microorganisms based on targeted sequencing of the prokaryotic 16S rRNA gene. We tested 46 clinical prokaryotic targets in the human gut, 28 of which can be identified by a bioinformatics pipeline that includes sequence analysis and taxonomic annotation. Using microbiome samples from a cohort of 897 healthy individuals, we established a reference range defining clinically relevant relative levels for each of the 28 targets. Our assay accurately quantified all 28 targets and correctly reflected 38/38 verification samples of real and synthetic stool material containing known pathogens. Thus, we have established a new test to interrogate microbiome composition and diversity, which will improve patient diagnosis, treatment and monitoring. More broadly, our test will facilitate epidemiological studies of the microbiome as it relates to overall human health and disease.

microbiology↗

bioRxiv2017

Identifying the abundant and active microorganisms common to full scale anaerobic digesters

Anaerobic digestion is widely applied to treat organic waste at wastewater treatment plants. Characterisation of the underlying microbiology represents a source of information to develop strategies for improved operation. To this end, we investigated the microbial community composition of thirty-two full-scale digesters over a six-year period using 16S rRNA gene amplicon sequencing. Sampling of the sludge fed into these systems revealed that several of the most abundant populations were likely inactive and immigrating with the influent. This observation indicates that a failure to consider immigration will interfere with correlation analysis and give an inaccurate picture of the active microbial community. Furthermore, several abundant OTUs could not be classified to genus level with commonly applied taxonomies, making inference of their function unreliable. As such, the existing MiDAS taxonomy was updated to include these abundant phylotypes. The communities of individual plants surveyed were remarkably similar - with only 300 OTUs representing 80% of the total reads across all plants, and 15% of these identified as likely inactive immigrating microbes. By identifying the abundant and active taxa in anaerobic digestion, this study paves the way for targeted characterisation of the process important organisms towards an in-depth understanding of the microbial ecology of these biotechnologically important systems.

microbiology↗

bioRxiv2017

Validation and Implementation of CLIA-Compliant Whole Genome Sequencing (WGS) in Public Health Laboratory

BackgroundPublic health microbiology laboratories (PHL) are at the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards.\n\nObjectives1) Establish the performance specifications of WGS applications used in PHL to conform with CLIA (Clinical Laboratory Improvements Act) guidelines for laboratory developed tests (LDT), 2) Develop quality assurance (QA) and quality control (QC) measures, 3) Establish reporting language for end users with or without WGS expertise, 4) Create a validation set of microorganisms to be used for future validations of WGS platforms and multi-laboratory comparisons and, 5) Create modular templates for the validation of different sequencing platforms.\n\nMethodsMiSeq Sequencer and Illumina chemistry (Illumina, Inc.) were used to generate genomes for 34 bacterial isolates with genome sizes from 1.8 to 4.7 Mb and wide range of GC content (32.1%-66.1%). A customized CLCbio Genomics Workbench - shell script bioinformatics pipeline was used for the data analysis.\n\nResultsWe developed a validation panel comprising ten Enterobacteriaceae isolates, five gram-positive cocci, five gram-negative non-fermenting species, nine Mycobacterium tuberculosis, and five miscellaneous bacteria; the set represented typical workflow in the PHL. The accuracy of MiSeq platform for individual base calling was >99.9% with similar results shown for reproducibility/repeatability of genome-wide base calling. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from MLST and genotyping tests were 100%. A test report format was developed for the end users with and without WGS knowledge.\n\nConclusionWGS was validated for routine use in PHL according to CLIA guidelines for LDTs. The validation panel, sequencing analytics, and raw sequences will be available for future multi-laboratory comparisons of WGS in PHL. Additionally, the WGS performance specifications and modular validation template are likely to be adaptable for the validation of other platforms and reagents kits.

microbiology↗

bioRxiv2017

Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing

Culture of multiple periprosthetic tissue samples is the current gold-standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through sonication fluid culture of explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI.\n\nWe compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopaedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent samples.\n\nCompared to sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69(88%,95%CI 77-94%) (derivation samples 35/38[92%,79-98%]; validation 26/31[84%,66-95%]), and genus-level sensitivity was 64/69(93%,84-98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97(88%,79-93%) (derivation 43/50[86%,73-94%], validation 42/47[89%,77-96%]). High levels of human DNA contamination were seen despite use of laboratory methods to remove it. Rigorous laboratory good practice was required to prevent bacterial DNA contamination.\n\nWe demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

microbiology↗

bioRxiv2017

Lysinibacillus fusiformis M5 induces increased complexity in Bacillus subtilis 168 colony biofilms via hypoxanthine

In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, or systems biology amongst others. The sociomicrobiology of multispecies biofilms, however, is still poorly understood. Here, we report a screening system that allowed us to identify soil bacteria, which induce architectural changes in biofilm colonies when cocultured with B. subtilis. We identified the soil bacterium Lysinibacillus fusiformis M5 as inducer of wrinkle-formation in B. subtilis colonies mediated by a diffusible signaling molecule. This compound was isolated by bioassay-guided chromatographic fractionation. The elicitor was identified to be the purine hypoxanthine using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. We show that the induction of wrinkle formation by hypoxanthine is not dependent on signal recognition by the histidine kinases KinA, KinB, KinC, and KinD, which are generally involved in phosphorylation of the master regulator Spo0A. Likewise, we show that hypoxanthine signaling does not induce the expression of biofilm-matrix related operons epsA-O and tasA-sipW-tapA. Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation of B. subtilis biofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression, but rather caused by the excess of hypoxanthine within B. subtilis cells, which may lead to cell stress and death.\n\nIMPORTANCEBiofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be favorable, for instance in crop protection. In nature, biofilms are commonly found as multispecies communities displaying complex social behaviors and characteristics. The study of interspecies interactions will thus lead to a better understanding and use of biofilms as they occur outside laboratory conditions. Here, we present a screening method suitable for the identification of multispecies interactions, and showcase L. fusiformis as a soil bacterium that is able to live alongside B. subtilis and modify the architecture of its biofilms.

microbiology↗

bioRxiv2017

Response Of Microbial Community Function To Fluctuating Geochemical Conditions Within A Legacy Radioactive Waste Trench Environment

During the 1960s, small quantities of radioactive materials were co-disposed with chemical waste at the Little Forest Legacy Site (Sydney, Australia) in three-metre-deep, unlined trenches. Chemical and microbial analyses, including functional and taxonomic information derived from shotgun metagenomics, were collected across a six-week period immediately after a prolonged rainfall event to assess how changing water levels impact upon the microbial ecology and contaminant mobility. Collectively, results demonstrated that oxygen-laden rainwater rapidly altered the redox balance in the trench water, strongly impacting microbial functioning as well as the radiochemistry. Two contaminants of concern, plutonium and americium, were shown to transition from solid-iron-associated species immediately after the initial rainwater pulse, to progressively more soluble moieties as reducing conditions were enhanced. Functional metagenomics revealed the potentially important role that the taxonomically-diverse microbial community played in this transition. In particular, aerobes dominated in the first day followed by an increase of facultative anaerobes/denitrifiers at day four. Towards the mid-end of the sampling period, the functional and taxonomic profiles depicted an anaerobic community distinguished by a higher representation of dissimilatory sulfate reduction and methanogenesis pathways. Our results have important implications to similar near-surface environmental systems in which redox cycling occurs.\n\nImportanceThe role of chemical and microbiological factors in mediating the biogeochemistry of groundwaters from trenches used to dispose of radioactive materials during the 1960s is examined in this study. Specifically, chemical and microbial analyses, including functional and taxonomic information derived from shotgun metagenomics, were collected across a six-week period immediately after a prolonged rainfall event to assess how changing water levels influence microbial ecology and contaminant mobility.\n\nResults demonstrate that oxygen-laden rainwater rapidly altered the redox balance in the trench water, strongly impacting microbial functioning as well as the radiochemistry. Two contaminants of concern, plutonium and americium, were shown to transition from solidiron-associated species immediately after the initial rainwater pulse, to progressively more soluble moieties as reducing conditions were enhanced. Functional metagenomics revealed the important role that the taxonomically-diverse microbial community played in this transition. Our results have important implications to similar near-surface environmental systems in which redox cycling occurs.

microbiology↗

bioRxiv2017

Microbial Metagenome Of Urinary Tract Infection

Urine culture and microscopy techniques are used to profile the bacterial species present in urinary tract infections. To gain insight into the urinary flora in infection and health, we analyzed clinical laboratory features and the microbial metagenome of 121 clean-catch urine samples. 16S rDNA gene signatures were successfully obtained for 116 participants, while whole genome shotgun sequencing data was successfully generated for samples from 49 participants. Analysis of these datasets supports the definition of the patterns of infection and colonization/contamination. Although 16S rDNA sequencing was more sensitive, whole genome shotgun sequencing allowed for a more comprehensive and unbiased representation of the microbial flora, including eukarya and viral pathogens, and of bacterial virulence factors. Urine samples positive by whole genome shotgun sequencing contained a plethora of bacterial (median 41 genera/sample), eukarya (median 2 species/sample) and viral sequences (median 3 viruses/sample). Genomic analyses revealed cases of infection with potential pathogens (e.g., Alloscardovia sp, Actinotignum sp, Ureaplasma sp) that are often missed during routine urine culture due to species specific growth requirements. We also observed gender differences in the microbial metagenome. While conventional microbiological methods are inadequate to identify a large diversity of microbial species that are present in urine, genomic approaches appear to comprehensively and quantitatively describe the urinary microbiome.

microbiology↗

bioRxiv2017

Salmonella enterica Serovar Typhimurium ST313 Responsible For Gastroenteritis In The UK Are Genetically Distinct From Isolates Causing Bloodstream Infections In Africa

The ST313 sequence type of Salmonella enterica serovar Typhimurium causes invasive non-typhoidal salmonellosis amongst immunocompromised people in sub-Saharan Africa (sSA). Previously, two distinct phylogenetic lineages of ST313 have been described which have rarely been found outside sSA. Following the introduction of routine whole genome sequencing of Salmonella enterica by Public Health England in 2014, we have discovered that 2.7% (79/2888) of S. Typhimurium from patients in England and Wales are ST313. Of these isolates, 59/72 originated from stool and 13/72 were from extra-intestinal sites. The isolation of ST313 from extra-intestinal sites was significantly associated with travel to Africa (OR 12 [95% CI: 3,53]). Phylogenetic analysis revealed previously unsampled diversity of ST313, and distinguished UK-linked isolates causing gastroenteritis from African-associated isolates causing invasive disease. Bayesian evolutionary investigation suggested that the two African lineages diverged from their most recent common ancestors independently, circa 1796 and 1903. The majority of genome degradation of African ST313 lineage 2 is conserved in the UK ST313 lineages and only 10/44 pseudogenes were lineage 2-specific. The African lineages carried a characteristic prophage and antibiotic resistance gene repertoire, suggesting a strong selection pressure for these horizontally-acquired genetic elements in the sSA setting. We identified an ST313 isolate associated with travel to Kenya that carried a chromosomally-located blaCTX-M-15, demonstrating the continual evolution of this sequence type in Africa in response to selection pressure exerted by antibiotic usage.\n\nThe S. Typhimurium ST313 sequence type has been primarily associated with invasive disease in Africa. Here, we highlight the power of routine whole-genome-sequencing by public health agencies to make epidemiologically-significant deductions that would be missed by conventional microbiological methods. The discovery of ST313 isolates responsible for gastroenteritis in the UK reveals new diversity in this important sequence type. We speculate that the niche specialization of sub-Saharan African ST313 lineages is driven in part by the acquisition of accessory genome elements.

microbiology↗

bioRxiv2017

Correlated expression of archaeal ammonia oxidation machinery across disparate environmental and culture conditions

Thaumarchaea are ubiquitous in marine habitats where they participate in carbon and nitrogen cycling. Although metatranscriptomes suggest thaumarchaea are active microbes in marine waters, we understand little about how thaumarchaeal gene expression patterns relate to substrate utilization and activity. Here, we report the global transcriptional response of the marine ammonia-oxidizing thaumarchaeon Candidatus Nitrosopelagicus brevis str. CN25 to ammonia limitation using RNA-Seq. We further describe the genome and transcriptome of Ca. N. brevis str. U25, a new strain capable of urea utilization. Ammonia limitation in CN25 resulted in reduced expression of transcripts coding for ammonia oxidation proteins, and increased expression of a gene coding an Hsp20-like chaperone. Despite significantly different transcript abundances across treatments, two ammonia monooxygenase subunits (amoAB), a nitrite reductase (nirK), and both ammonium transporter genes were always among the most abundant transcripts, regardless of growth state. Ca. N. brevis str. U25 cells expressed a urea transporter 139-fold more than the urease catalytic subunit ureC. Gene co-expression networks derived from culture transcriptomes and ten thaumarchaea-enriched metatranscriptomes revealed a high degree of correlated gene expression across disparate environmental conditions and identified a module of genes, including amoABC and nirK, that we hypothesize to represent the core ammonia oxidation machinery.\n\nOriginality-Significance StatementDiscovering gene function in fastidious or uncultivated lineages remains one of the biggest challenges in environmental microbiology. Here, we use an approach that combines controlled laboratory experiments with in situ transcript abundance data from the environment to identify genes that share similar transcription patterns in marine ammonia-oxidizing thaumarchaea. These findings demonstrate how transcriptomes from microbial cultures can be used together with complex environmental samples to identify suites of co-expressed genes that are otherwise enigmatic and provide new insights into the mechanism of ammonia oxidation. Our results add to the growing body of literature showing that relatively small changes in transcript abundance are linked to large changes in growth in organisms with reduced genomes, suggesting they have limited capacity for metabolic regulation or that they rely on mechanisms other than transcriptional regulation to deal with a fluctuating environment.

microbiology↗

bioRxiv2017

Chemotaxis allows bacteria to overcome host-generated reactive oxygen species that constrain gland colonization

The epithelial layer of the gastrointestinal tract contains invaginations, called glands or crypts, which are colonized by symbiotic and pathogenic microorganisms and may function as designated niches for certain species. Factors that control gland colonization are poorly understood, but bacterial chemotaxis aids occupation of these sites. We report here that a Helicobacter pylori cytoplasmic chemoreceptor, TlpD, is required for gland colonization in the stomach. tlpD mutants demonstrate gland colonization defects characterized by a reduction in the percent of glands colonized, but not in number of bacteria per gland. Consistent with TlpDs reported role in reactive oxygen species (ROS) avoidance, tlpD mutants showed hallmarks of exposure to large amounts of ROS. To assess the role of host-generated ROS in TlpD-dependent gland colonization, we utilized mice that lack either the ability to generate epithelial hydrogen peroxide or immune cell superoxide. tlpD gland colonization defects were rescued to wild-type H. pylori levels in both of these mutants. These results suggest that multiple types of innate immune generated ROS production limit gland colonization and that bacteria have evolved specific mechanisms to migrate through this gauntlet to establish in the glands.\n\nClassification: Biological sciences; microbiology\n\nSignificance statementMicrobial colonization of the gastrointestinal tract occurs at distinct sites within the tissue including glandular structures found in the stomach and intestine. Multiple lines of evidence suggest that glands supply niches that promote chronic microbial colonization, a process that is critical for symbiotic and pathogenic bacteria to maintain themselves. In this report, we show that host-produced reactive oxygen species (ROS) constrain gland colonization by the gastric pathogen Helicobacter pylori. A bacterial cytoplasmic chemoreceptor, TlpD, allows H. pylori to avoid ROS and enhances H. pyloris ability to colonize a broad swath of glands. We propose that hosts limit gland access and spread by producing ROS, and bacteria counter with chemotactic responses that allow navigation through this gauntlet.

microbiology↗

bioRxiv2017

Antimicrobial resistant Klebsiella pneumoniae carriage and infection in specialized geriatric care wards linked to acquisition in the referring hospital

BackgroundKlebsiella pneumoniae is a leading cause of extended-spectrum beta-lactamase (ESBL) producing hospital-associated infections, for which elderly patients are at increased risk.\n\nMethodsWe conducted a 1-year prospective cohort study, in which a third of patients admitted to two geriatric wards in a specialized hospital were recruited and screened for carriage of K. pneumoniae by microbiological culture. Clinical isolates were monitored via the hospital laboratory. Colonizing and clinical isolates were subjected to whole genome sequencing and antimicrobial susceptibility testing.\n\nResultsK. pneumoniae throat carriage prevalence was 4.1%, rectal carriage 10.8% and ESBL carriage 1.7%. K. pneumoniae infection incidence was 1.2%. The isolates were diverse, and most patients were colonized or infected with a unique phylogenetic lineage, with no evidence of transmission in the wards. ESBL strains carried blaCTX-M-15 and belonged to clones associated with hospital-acquired ESBL infections in other countries (ST29, ST323, ST340).\n\nOne also carried the carbapenemase blaIMP-26. Genomic and epidemiological data provided evidence that ESBL strains were acquired in the referring hospital. Nanopore sequencing also identified strain-to-strain transmission of a blaCTX-M-15 FIBK/FIIK plasmid in the referring hospital.\n\nConclusionsThe data suggest the major source of K. pneumoniae was the patients own gut microbiome, but ESBL strains were acquired in the referring hospital. This highlights the importance of the wider hospital network to understanding K. pneumoniae risk and infection control. Rectal screening for ESBL organisms upon admission to geriatric wards could help inform patient management and infection control in such facilities.\n\nSummaryPatients own gut microbiota were the major source of K. pneumoniae, but extended-spectrum beta-lactamase strains were acquired in the referring hospital. This highlights the potential for rectal screening, and the importance of the wider hospital network, for local risk management.

microbiology↗

bioRxiv2017

Outbreak of invasive wound mucormycosis in a burn unit due to multiple strains of Mucor circinelloides f. circinelloides resolved by whole genome sequencing

Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole genome sequencing (WGS) analysis.\n\nWe analyzed M. circinelloides isolates from patients in our burn unit (BU1) together with non-outbreak isolates from burn unit 2 (BU2, Paris area) and from France over a two-year period (2013-2015). For each isolate, WGS and a de novo genome assembly was performed from read data extracted from the aligned contig sequences of the reference genome (1006PhL).\n\nA total of 21 isolates were sequenced including 14 isolates from six BU1 patients. Phylogenetic classification showed that the clinical isolates clustered in four highly divergent clades. Clade1 contained at least one of the strains from the six epidemiologically-linked BU1 patients. The clinical isolates seemed specific to each patient. Two patients were infected with more than two strains from different clades suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients shared one strain in BU1, suggesting direct transmission or contamination with the same environmental source.\n\nWGS coupled with precise epidemiological data and analysis of several isolates per patients revealed in our study a complex situation with both potential cross-transmission and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.\n\nImportanceInvasive wound mucormycosis (IWM) is a severe infection due to the environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used Whole Genome Sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient-specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission between the patients. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on whole genome sequencing and careful sampling.

microbiology↗

bioRxiv2018

A metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities

Measurements of the carbon stable isotope ratio ({delta}13C) are widely used in biology to address major questions regarding food sources and metabolic pathways used by organisms. Measurement of these so called stable carbon isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals have led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide limited taxonomic resolution, such as with the use of lipid biomarkers, or are limited in throughput, such as NanoSIMS imaging of single cells.\n\nHere we present \"direct Protein-SIF\" and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate {delta}13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we show that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis showed that direct Protein-SIF confirms previous physiological hypotheses and can provide unexpected new insights into the symbionts metabolism. This confirms the usefulness of this new approach to accurately determine {delta}13C values for different species in microbial community samples.\n\nSignificanceTo understand the roles that microorganisms play in diverse environments such as the open ocean and the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression. These methods often only provide indirect evidence for which substrates are used by a microorganism in a community. The direct Protein-SIF method that we developed allows linking microbial species in communities to the environmental carbon sources they consume by determining their stable carbon isotope signature. Direct Protein-SIF also allows assessing which carbon fixation pathway is used by autotrophic microorganisms that directly assimilate CO2.

microbiology↗

bioRxiv2018

Enterococcus faecalis AHG0090 is a genetically tractable bacterium and produces a secreted peptidic bioactive that suppresses NF-kB activation in human gut epithelial cells

Enterococcus faecalis is an early coloniser of the human infant gut and contributes to the development of intestinal immunity. To better understand the functional capacity of E. faecalis we constructed a broad host range RP4 mobilisable vector, pEHR513112, that confers chloramphenicol resistance and used a metaparental mating approach to isolate E. faecalis AHG0090 from a faecal sample collected from a healthy human infant. We demonstrated that E. faecalis AHG0090 is genetically tractable and could be manipulated using traditional molecular microbiology approaches. E. faecalis AHG0090 was comparable to the gold-standard anti-inflammatory bacterium Faecalibacterium prausnitzii A2-165 in its ability to suppress cytokine mediated NF-[Kcy]B activation in human gut derived LS174T goblet cell-like and Caco-2 enterocyte-like cell lines. E. faecalis AHG0090 and F. prausnitzii A2-165 produced secreted low molecular weight NF-[Kcy]B suppressive peptidic bioactives. Both bioactives were sensitive to heat and proteinase K treatments although the E. faecalis AHG0090 bioactive was more resilient to both forms of treatment. As expected, E. faecalis AHG0090 suppressed IL-1{beta} induced NF-[Kcy]B-p65 subunit nuclear translocation and expression of the NF-[Kcy]B regulated genes IL-6, IL-8 and CXCL-10. Finally, we determined that E. faecalis AHG0090 is distantly related to other commensal strains and likely encodes niche factors that support effective colonisation of the infant gut.

microbiology↗

bioRxiv2018

Experimentally Induced Metamorphosis in Axolotl (Ambystoma mexicanum) Under Constant Diet Restructures Microbiota Accompanied by Reduced Limb Regenerative Capacity

Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite life-long neoteny in nature and in captive-bred colonies, metamorphosis of these animals can be experimentally induced by administering Thyroid hormones (THs). However, biological consequences of this experimental procedure, such as host microbiota response and implications for regenerative capacity, remain largely unknown. Here, we systematically compared host bacterial microbiota associated with skin, stomach, gut tissues and fecal samples based on 16S rRNA gene sequences, along with limb regenerative capacity, between neotenic and metamorphic Axolotls. Our results show that distinct bacterial communities inhabit individual organs of Axolotl and undergo substantial restructuring through metamorphosis. Drastic restructuring was observed for skin microbiota, highlighted by a major transition from Firmicutes-enriched to Proteobacteria-enriched relative abundance and precipitously decreased diversity. Remarkably, shifts in microbiota was accompanied by a steep reduction in limb regenerative capacity. Fecal microbiota of neotenic and metamorphic Axolotl shared relatively higher similarity, suggesting that diet continues to shape microbiota despite fundamental transformations in the host digestive organs. The results provide novel insights into microbiological and regenerative aspects of Axolotl metamorphosis and will establish a baseline for future in-depth studies.

microbiology↗

bioRxiv2018

Core Genome Multi Locus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

The use of whole genome sequencing (WGS) using next generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through Single Nucleotide Polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome Multilocus Sequence Typing (cgMLST) has not been explored so far. The current study developed an allele-based method cgMLST and compared its performance to the genome-wide SNP approach and the traditional MLVA on a defined sample collection. The dataset comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 epidemiologically unrelated human and animal isolates collected in Italy. The cgMLST scheme generated in this study contained 2,687 targets of the B. melitensis 16M reference genome (75.4% of the complete genome). We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be [≤]4 loci in the cgMLST and [≤]10 in WGS SNP analysis. CgMLST and SNP analysis provided much higher phylogenetic distance resolution than MLVA, particularly for strains belonging to the same lineage thus allowing diverse and unrelated genotypes to be identified with greater confidence. The application of this cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

microbiology↗