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bioRxiv2023

Genome-wide Quantification of Polycistronic Transcription in Leishmania major

Leishmania major is a human-pathogenic, obligate parasite and the etiological agent of the most prevalent, cutaneous form of leishmaniasis, which is an important neglected, tropical disease with [~]1.2 Mio new infections per year. Leishmania, and the whole order Trypanosomatida, are early eukaryotes with highly diverged gene expression and regulation pathways, setting them apart from their mammalian hosts and from most other eukaryotes. Using precision run-on sequence analysis, we performed a genome-wide mapping and density analysis of RNA polymerases in isolated nuclei of the protozoan parasite Leishmania major. We map transcription initiation sites within the chromosomes and correlate them with known sites of chromatin modifications. We confirm continuous, polycistronic RNA synthesis in all RNA polymerase II-dependent gene arrays but find varying RNA polymerase activities in polycistronic transcription units (PTUs), excluding gene-specific transcription regulation, but not PTU-specific variations as possible targets of modulatory pathways. Lastly, we find evidence for transcriptional pausing of all three RNA polymerase classes, hinting at a possible mechanism of transcriptional regulation. Significance StatementLeishmania spp. are pathogens of humans and animals and cause one of the most important neglected tropical diseases. Regulation of gene expression in Leishmania but also in the related Trypanosoma is radically different from all eukaryotic model organisms, dispensing with regulated, gene-specific transcription, and relying instead on highly regulated translation. Our work sheds light on the initiation, elongation and termination of transcription, maps unidirectional, polycistronic transcription units, provides evidence for transcriptional pausing at or near starting points of RNA synthesis, and quantifies the varying transcription rates of the polycistronic transcription units. Our results will further the understanding of these important pathogens and should provide a valuable ressource for researchers in the field of eukaryotic microbiology.

microbiology↗

bioRxiv2023

pH selects for distinct N2O-reducing microbiomes in tropical soil microcosms

Nitrous oxide (N2O), a greenhouse gas with ozone destruction potential, is mitigated by the microbial reduction to dinitrogen catalyzed by N2O reductase (NosZ). Bacteria with NosZ activity have been studied at circumneutral pH but the microbiology of low pH N2O reduction has remained elusive. Acidic (pH<5) tropical forest soils were collected in the Luquillo Experimental Forest in Puerto Rico, and microcosms maintained with low (0.02mM) and high (2mM) N2O assessed N2O reduction at pH 4.5 and 7.3. All microcosms consumed N2O, but long lag times of up to 7 months were observed in microcosms with 2 mM N2O. Comparative metagenome analysis revealed that Rhodocyclaceae dominated in circumneutral microcosms under both N2O feeding regimes. In acidic microcosms, Peptococcaceae dominated in high-N2O, and Hyphomicrobiaceae in low-N2O microcosms. Seventeen metagenome-assembled genomes (MAGs) recovered from these microcosms harbored nos operons, with all eight MAGs derived from acidic microcosms carrying the clade II type nosZ, lacking nitrite reductase genes (nirS, nirK). Five of these MAGs represented novel taxa indicating an unexplored N2O-reducing diversity exists in acidic tropical soils. A survey of pH 3.5-5.7 soil metagenome datasets revealed that nosZ genes commonly occur, suggesting broad distribution of N2O reduction potential in acidic soils.

microbiology↗

bioRxiv2023

A benchmarked comparison of software packagesfor time-lapse image processing of monolayerbacterial population dynamics

Time-lapse microscopy offers a powerful approach for analysing cellular activity. In particular, this technique is valuable for assessing the behaviour of bacterial populations, which can exhibit growth and intercellular interactions in monolayer. Such time-lapse imaging typically generates large quantities of data, limiting the options for manual investigation. Several of image processing software packages have been developed to facilitate analysis. It can thus be a challenge to identify the software package best suited to a particular research goal. Here, we compare four software packages that support analysis of 2D time-lapse images of cellular populations: CellProfiler, SuperSegger-Omnipose, DeLTA, and FAST. We compare their performance against benchmarked results on time-lapse observations of E. coli populations. Performance varies across the packages, with each of the four out-performing the others in at least one aspect of the analysis. Not surprisingly, the packages that have been in development for longer showed the strongest performance. We found that deep-learning based approaches to object segmentation outperformed traditional approaches, but the opposite was true for frame-to-frame object tracking. We offer these comparisons, together with insight on usability, computational efficiency, and feature availability, as a guide to researchers seeking image processing solutions. Significance StatementTime-lapse microscopy provides a detailed window into the world of bacterial behavior. However, the vast amount of data produced by these techniques is difficult to analyze manually. We have analysed four software tools designed to process such data and compared their performance, using populations of commonly studied bacterial species as our test subjects. Our findings offer a roadmap to scientists, helping them choose the right tool for their research. This comparison bridges a gap between microbiology and computational analysis, streamlining research efforts.

microbiology↗

bioRxiv2023

Intracellular defensive symbiont is culturable and capable of transovarial, vertical transmission

Insects frequently form heritable associations with beneficial bacteria that are vertically transmitted from parent to offspring. Long term vertical transmission has repeatedly resulted in genome reduction and gene loss rendering many such bacteria incapable of independent culture. Among aphids, heritable endosymbionts often provide a wide range of context-specific benefits to their hosts. Although these associations have large impacts on host phenotypes, experimental approaches are often limited by an inability to independently cultivate these microbes. Here, we report the axenic culture of Candidatus Fukatsuia symbiotica strain WIR, a heritable bacterial endosymbiont of the pea aphid, Acyrthosiphon pisum. Whole genome sequencing revealed similar genomic features and high sequence similarity to previously described strains, suggesting the cultivation techniques used here may be applicable to Ca. F. symbiotica strains from distantly related aphids. Microinjection of the isolated strain into uninfected aphids revealed that it can reinfect developing embryos, and is maintained in subsequent generations via transovarial maternal transmission. Artificially infected aphids exhibit similar phenotypic and life history traits compared to native infections, including protective effects against an entomopathogenic Fusarium species. Overall, our results show that Ca. F. symbiotica may be a useful tool for experimentally probing the molecular mechanisms underlying heritable symbioses and antifungal defense in the pea aphid system. IMPORTANCEDiverse eukaryotic organisms form stable, symbiotic relationships with bacteria that provide benefits to their hosts. While these associations are often biologically important, they can be difficult to probe experimentally, because intimately host-associated bacteria are difficult to access within host tissues, and most cannot be cultured. This is especially true of the intracellular, maternally inherited bacteria associated with many insects, including aphids. Here, we demonstrate that a pea aphid-associated strain of the heritable endosymbiont, Candidatus Fukatsuia symbiotica, can be grown outside of its host using standard microbiology techniques, and can readily re-establish infection that is maintained across host generations. These artificial infections recapitulate the effects of native infections making this host-symbiont pair a useful experimental system. Using this system, we demonstrate that Ca. F. symbiotica infection reduces host fitness under benign conditions, but protects against a previously unreported fungal pathogen.

microbiology↗

bioRxiv2023

Quantitative measurement of microbial growth rate with Raman microspectroscopy

Rates of microbial activity and growth are fundamental to understanding environmental geochemistry and ecology. However, measuring the heterogeneity of microbial activity at the single-cell level, especially within complex populations and environmental matrices, remains a forefront challenge. Stable Isotope Probing (SIP) is a standard method for assessing microbial activity and involves measuring the incorporation of an isotopically labeled compound into microbial biomass. Here, we assess the utility of Raman microspectroscopy as a SIP technique, specifically focusing on the measurement of deuterium (2H), a tracer of microbial biomass production. We generate calibrations of microbial biomass 2H values and find that Raman microspectroscopy reliably quantifies 2H incorporation ranging between 0 and 40 at. %. Applying the results of this calibration to a SIP model, we explicitly parameterize the factors controlling microbial growth quantification, demonstrating how Raman-SIP can measure the growth of microorganisms with doubling times ranging from hours to years. Furthermore, we correlatively compare our Raman-derived measurements with those of nanoscale secondary ion mass spectrometry (nanoSIMS) to compare the relative strengths of nanoSIMS- and Raman-based SIP approaches. We find that Raman microspectroscopy is a robust, accessible methodology that can readily differentiate and quantify the growth of individual microbial cells in complex samples. ImportanceGrowth rate, the rate at which organisms grow and reproduce, is a key metric with which to evaluate microbial physiology and contributions to system-level processes. The heterogeneity of microbial growth across space, time, and populations is often difficult to capture with bulk-scale techniques. Single-cell methods hold promise for measuring the heterogeneity of microbial growth rates and responses to changing conditions in situ, without the need for cultivation of microbial isolates. In this study, we evaluated the ability of Raman microspectroscopy, a non-destructive and rapid technique, to measure the assimilation of isotopically labeled water into individual microbial cells and thereby calculate their rates of growth. We explicitly parameterize the factors controlling the quantification of microbial growth rate and compare this technique to standard methods. The framework we report allows researchers to couple single-cell and aggregate rate measurements to functional or system-level properties, a forefront challenge in microbiology.

microbiology↗

bioRxiv2023

Characterization of β-lactamase and virulence genes in Pseudomonas aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh

The emergence and spread of multidrug-resistant pathogens like Pseudomonas aeruginosa are major concerns for public health worldwide. This study aimed to investigate the prevalence of circulating P. aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh, their antibiotic susceptibility, {beta}-lactamase and virulence gene profiling using standard molecular and microbiology techniques. We collected 110 samples from five different locations, viz., BAU residential area (BAURA; n=15), BAU Healthcare Center (BAUHCC; n = 20), BAU Veterinary Teaching Hospital (BAUVTH; n=22), Poultry Market (PM; n=30) and Mymensingh Medical College Hospital (MCCH; n=23). After overnight enrichment in nutrient broth, 89 probable Pseudomonas isolates (80.90%) were screened through selective culture, gram-staining and biochemical tests. Using genus- and species-specific PCR, we confirmed 22 isolates (20.0%) as P. aeruginosa from these samples. Antibiogram profiling revealed that 100.0% P. aeruginosa isolates (n = 22) were multidrug-resistant isolates, showing resistance against Doripenem, Penicillin, Ceftazidime, Cefepime, and Imipenem. Furthermore, resistance to aztreonam was observed in 95.45% isolates. However, P. aeruginosa isolates showed a varying degree of sensitivity against Amikacin, Gentamicin, and Ciprofloxacin. The blaTEM gene was detected in 86.0% isolates, while blaCMY, blaSHV and blaOXA, were detected in 27.0%, 18.0% and 5.0% of the P. aeruginosa isolates, respectively. The algD gene was detected in 32.0% isolates, whereas lasB and exoA genes were identified in 9.0% and 5.0% P. aeruginosa isolates. However, none of the P. aeruginosa isolates harbored exoS gene. Thus, this study provides novel and important data on the resistance and virulence of P. aeruginosa currently circulating in clinical, environmental and poultry environment of Bangladesh. These data provide important insights into the emergence of {beta}-lactamase resistance in P. aeruginosa, highlighting its usefulness in the treatment and control of P. aeruginosa infections in both humans and animals.

microbiology↗

bioRxiv2023

Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal against Mycobacterium ulcerans

BackgroundBuruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a cumbersome method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against clinical M. ulcerans isolates by TKA, while comparing four methodologies: CFU/mL enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification. Methodology/Principal findingsTKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified after 1, 3, 7, 10, 14, 21 and 28 days of treatment with different methodologies. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combination were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies provided similar results with a general good correlation between CFU counting and the other methodologies. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA. Conclusions/SignificanceOur study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to the design of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment. Author summarySince 2004, when only surgery was available, Buruli ulcer (BU) treatment has improved reaching to the efficient 8-weeks all oral antibiotic course of rifampicin and clarithromycin together with wound care and, sometimes, tissue grafting and surgery. This skin neglected tropical disease caused by Mycobacterium ulcerans mainly affects people living in rural areas in under-resourced countries with limited access to health services and medicines, thus compromising patients treatment adherence. The inclusion of amoxicillin/clavulanate in BU therapy was previously described with the potential to shorten BU treatment. In this study, we confirmed the high bactericidal activity over time of rifampicin and amoxicillin/clavulanate-containing combinations against M. ulcerans, being even more effective in vitro than the antibiotics currently used to treat BU. A comparison of different methodologies that are applied in the laboratory and in the clinical environment showed good correlation between them, leading to a wide variety of biomarkers for BU research and giving the opportunity for clinical translation; the choice of the most suitable one being driven by the purpose and the context of the study. These in vitro results provide further support to the ongoing clinical trial in West Africa to evaluate if BU treatment can be shortened from 8 to 4 weeks (BLMs4BU trial: NCT05169554, PACTR202209521256638).

microbiology↗

bioRxiv2023

Pathogen-microbiome interactions and the virulence of an entomopathogenic fungus

AO_SCPLOWBSTRACTC_SCPLOWBacteria shape interactions between hosts and fungal pathogens. In some cases, bacteria associated with fungi are essential for pathogen virulence. In other systems, host associated microbiomes confer resistance against fungal pathogens. We studied an aphid-specific entomopathogenic fungus called Pandora neoaphidis in the context of both host and pathogen microbiomes. Aphids host several species of heritable bacteria, some of which confer resistance against Pandora. We first found that spores that emerged from aphids that harbored protective bacteria were less virulent against subsequent hosts and did not grow on plate media. We then used 16S amplicon sequencing to study the bacterial microbiome of fungal mycelia and spores during plate culturing and host infection. We found that the bacterial community is remarkably stable in culture despite dramatic changes in pathogen virulence. Last, we used an experimentally transformed symbiont of aphids to show that Pandora can acquire hostassociated bacteria during infection. Our results uncover new roles for bacteria in the dynamics of aphidpathogen interactions and illustrate the importance of the broader microbiological context in studies of fungal pathogenesis.

microbiology↗

bioRxiv2024

Biofilm formation and maize root-colonization of seed-endophytic Bacilli isolated from native maize landraces

Agricultural microbiology seeks to replace the use of agrochemicals with microbe-based products. Plant growth-promoting bacteria (PGPB) are often selected based on their functions in vitro, and then, their effect on plant development is tested. However, this approach neglects the study of their survival in soil, root-colonization, and the monitoring of beneficial functions in the rhizosphere. This could explain the recurrent lack of success in the transition from lab tests to field applications of natural isolates from novel habitats. In our recent studies, we found that native maize seeds from traditional agroecosystems carry endophytic bacterial communities that are dominated by members of the class Bacilli. As an approach to grasp their PGP potential, we developed protocols to test maize root-colonization of these natural isolates in 1) a short-term hydroponics assay in vitro and 2) a long-term assay in non-sterile soil pots. Our results show that in vitro biofilm formation was only partially associated to short-term colonization in vitro; furthermore, long-term root-colonization in soil pots was not correlated to the in vitro assays. This work highlights the necessity to incorporate root-colonization assays as part of the research strategies in the search for PGPB natural isolates from unexplored habitats, towards the generation of inoculants with increased success in the field.

microbiology↗

bioRxiv2024

A method for separating bacterial cultures into single cells and defined size fractions of biofilm aggregates

In vitro microbiological experiments that aim to describe differences between planktonic and biofilm aggregate populations are challenging since liquid batch cultures contain a mix of both. Here, we present a simple method for fractioning a bacterial liquid batch culture into aggregates and single cells. Stackable cell strainers with mesh sizes of 30 m and 10 m were used to filtrate 6 day old batch cultures of Pseudomonas aeruginosa to produce size fractions of 0-10m and >30m. By confocal laser scanning microscopy measurements, we show that 95.5% of the total biomass was <10 m in the "0-10m size fraction" and that 92.5% of the total biomass was >30m in the ">30m size fraction". Furthermore, the adjustment of bacterial concentration using CFU/ml was validated by quantifying the total DNA of viable bacteria in the two size fractions after DNase treatment to deplete eDNA and DNA from dead bacteria. Surprisingly, this showed that adjusting the bacterial concentration using CFU/ml was a valid method with no significant differences in total DNA from viable bacteria.

microbiology↗

bioRxiv2024

Identification, antimicrobial resistance and molecular detection of Shiga toxin-producing E. coli (STEC) isolated from raw milk of cattle in Chitwan, Nepal

Shiga toxin-producing Escherichia coli (STEC) is one of the most common foodborne pathogens leading to gastroenteritis with symptoms of mild diarrhoea to the potential life-threatening haemolytic uremic syndrome. Serotype O157:H7 has been the most predominant type worldwide but there are an increasing number of outbreaks caused by non-O157:H7 STEC serotypes. This is the first study about STEC in milk used in Nepalese communities. A total of 267 raw milk samples were taken from different sites in Chitwan District, one of the major milk-producing districts of Nepal, using multi-stage sampling. The samples underwent microbiological and molecular analysis. The overall prevalence of E. coli was found to be 29.6% (79/267) in raw cattle milk. Z3276 gene, a specific genetic marker of E. coli O157:H7 was detected only in three isolates which also carried stx genes. The overall prevalence of STEC in raw cattle milk was 11.6% (31/267) whereas the prevalence of STEC among E. coli isolates was 39.2% (31/79). The prevalence of O157:H7 STEC is 1.1% (3/267) and of non-O157:H7 STEC is 10.5% (28/267). Out of 12 antibiotics tested, the sensitivity of STEC to an antibiotic was highest with azithromycin, and levofloxacin, and the most resistant was with tetracycline, and doxycycline. The findings revealed that raw cattle milk was contaminated with STEC, posing a potential public health concern. The discovery of a greater occurrence of stx2 genes in this study highlights the possibility of more severe complications in humans. Molecular-based STEC surveillance is essential to minimize the risk of its transmission.

microbiology↗

bioRxiv2024

Limit of detection of Salmonella ser. Enteritidis using culture-based versus culture-independent diagnostic approaches

In order to prevent the spread of foodborne illnesses, the presence of pathogens in the food chain is monitored by government agencies and food producers. The culture-based methods currently employed are sensitive but time-and labour-intensive, leading to increasing interest in exploring culture-independent diagnostic tests (CIDTs) for pathogen detection. However, sensitivity and reliability of these CIDTs relative to current approaches has not been well established. To address this issue, we conducted a comparison of the limit of detection (LOD50) for Salmonella between a culture-based method and three CIDT methods: qPCR (targeting invA and stn), metabarcode (16S) sequencing, and shotgun metagenomic sequencing. Samples of chicken feed and chicken caecal contents were spiked with Salmonella serovar Enteritidis and subjected to culture-and DNA-based detection methods. To explore the impact of non-selective enrichment on LOD50, all samples underwent both immediate DNA extraction and an overnight enrichment prior to gDNA extraction. In addition to this spike-in experiment, feed and caecal samples acquired from the field were tested with culturing, qPCR, and metabarcoding. In general, LOD50 was comparable between qPCR and shotgun sequencing methods. Overnight microbiological enrichment resulted in an improvement in LOD50 with up to a three log decrease, comparable to culture-based detection. However, Salmonella reads were detected in some unspiked feed samples, suggesting false-positive detection of Salmonella. Additionally, the LOD50 in feeds was three logs lower than in caecal contents, underscoring the impact of background microbiota on Salmonella detection using all methods. IMPORTANCEThe appeal of CIDTs is increased speed with lowered cost, as well as the potential to detect multiple pathogen species in a single analysis and to monitor other areas of concern such as antimicrobial resistance genes or virulence factors. Understanding the sensitivity of CIDTs relative to current approaches will help determine the feasibility of implementing these methods in pathogen surveillance programs.

microbiology↗

bioRxiv2024

Mycobacterium ulcerans challenge strain selection for a Buruli ulcer controlled human infection model

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

microbiology↗

bioRxiv2024

Macrolactin A Is an Inhibitor of Protein Biosynthesis in Bacteria

The macrolide antibiotic, macrolactin A (McA), has been known for its antimicrobial properties since the late 1980s, but the mechanism of its antibacterial activity is still unknown. In this study, we investigated the microbiological and molecular characteristics of McA antimicrobial activity. McA effect on bacteria was found to be both bacteriostatic and bactericidal, depending on species and strains. Regarding the mechanism of action of McA, the following important results were obtained: 1) using in vivo and in vitro systems, we showed that McA is an inhibitor of protein synthesis in bacteria; 2) the concentration of McA required to inhibit protein synthesis in the E. coli cell-free model was found to be 50 times lower than the concentration required in the S. aureus cell-free model; 3) the toe-printing assay revealed that McA inhibits the first step of elongation stage of protein synthesis; 4) we identified single and multiple nucleotide polymorphisms in the gene encoding the translation elongation factor Tu (EF-Tu) by annotating the genomes of McA-resistant Bacillus pumilus McAR and its parental strain. Molecular modeling showed that the McA molecule can form non-covalent bonds with amino acids at the interface of domains 1 and 2 of EF-Tu, characterized by a relatively high docking score. Overall, our study demonstrated that McA acts as an elfamycin-like antibiotic (targeting EF-Tu), addressing a substantial gap in our understanding of the mechanism of action of macrolactin A, a representative member of macrolides.

microbiology↗

bioRxiv2024

Microbial community assembly during aging of food waste-derived hydrochar: key bacterial guilds mediate nutrient dynamics

Hydrochar aging may mitigate the adverse effects of direct application on plant growth. However, little is known about the strategies for accelerating this process and the microbiological mechanisms involved. This study explored the effects of three strategies, including the additions of straw and efficient-degrading microorganisms, on the aging processes of food waste-derived hydrochar, and the key microbial drivers. The bacterial and fungal community compositions in hydrochar were estimated at different aging periods (i.e., day 1, 7, 14 and 35) using 16S and ITS rRNA gene sequencing. The results showed that straw and microbial inoculum addition improved the temperature and shortened the aging time by [~]30%. Three bacterial guilds, mainly including Bacillus-like species, were identified that had significant correlations with the nitrogen (N) and phosphorus (P) dynamics during aging. This study demonstrates the feasibility of manipulating key microbial guilds artificially to achieve efficient harmless-treatment of hydrochar. HighlightsO_LIStraw and microbial inoculum addition reduced ageing duration by 30%. C_LIO_LIMicrobial inoculum addition increases the aging reaction temperature by 13%. C_LIO_LIBacteria, rather than fungi, play the critical role in hydrochar aging. C_LIO_LIBacillus and related species drive nitrogen and phosphorus cycles of aging. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/580608v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@b2c2c9org.highwire.dtl.DTLVardef@124ab1org.highwire.dtl.DTLVardef@e5c00borg.highwire.dtl.DTLVardef@1a31deb_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia

Defined as a pneumonia occurring after more than 48 hours of mechanical ventilation via an endotracheal tube, ventilator-associated pneumonia results from biofilm formation on the indwelling tube, seeding the patients lower airways with pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Currently there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates biofilm formation of these pathogens in a host-mimicking environment, and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilator airway mucus (SVAM) medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-SEM were employed to characterise the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication, or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved biofilm eradication of all pathogens. Our in vitro endotracheal tube (IVETT) model informs on fundamental microbiology in the ventilator-associated pneumonia context, and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants. ImportanceThe incidence of ventilator-associated pneumonia in mechanically ventilated patients is between 5-40%, increasing to 50-80% in patients suffering from coronavirus disease 2019 (COVID-19). The mortality rate of ventilator-associated pneumonia patients can reach 45%. Treatment of the endotracheal tube biofilms that cause ventilator-associated pneumonia is extremely challenging, with causative organisms able to persist in endotracheal tube biofilm despite appropriate antimicrobial treatment in 56% of ventilator-associated pneumonia patients. Flawed antimicrobial susceptibility testing often means that ventilator-associated pneumonia pathogens are insufficiently treated, resulting in patients experiencing ventilator-associated pneumonia recurrence. Here we present an in vitro endotracheal tube biofilm model that recapitulates key aspects of endotracheal tube biofilms, including dense biofilm growth and elevated antimicrobial tolerance. Thus our biofilm model can be used as a ventilated airway simulating environment, aiding the development of anti-ventilator-associated pneumonia therapies and antimicrobial endotracheal tubes that can one day improve the clinical outcomes of mechanically ventilated patients.

microbiology↗

bioRxiv2024

Identification and characterization of a skin microbiome on Caenorhabditis elegans suggests environmental microbes confer cuticle protection

In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with this microbial milieux. Here, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple skin microbiome. We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Further, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin-and gut-microbiomes. We find that some strains of bacteria, including Enterobacter sp. JUb101, are primarily found on the nematode skin, while others like Stenotrophomonas indicatrix JUb19 and Ochrobactrum vermis MYb71 are predominantly in the animals gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts. ImportanceThe genetic model organism C. elegans has recently emerged as a tool for understanding host-microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, non-pathogenic skin microbes or their function. We show that members of a modified C. elegans model microbiome, mCeMbio, can adhere to the animals cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbed C. elegans, and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wild C. elegans skin microbiomes and use C. elegans as a model for skin research.

microbiology↗

bioRxiv2024

Unraveling the Interplay between Phytochemicals and Rhizosphere Bacteria in Ficus racemosa

Plant-microbe interactions in the rhizosphere play crucial roles in plant health and ecosystem functioning. Despite extensive research, gaps persist in understanding the relationship between phytochemicals and rhizosphere bacteria, particularly in species like Ficus racemosa. This study aims to investigate this interplay by characterizing the diversity and abundance of rhizosphere bacteria in Ficus racemosa and assessing the influence of phytochemical composition on microbial community structure and function. Through a multidisciplinary approach integrating microbiology and phytochemistry, our findings contribute to a holistic understanding of rhizosphere ecology and inform strategies for optimizing plant-microbe interactions in agricultural and environmental contexts.

microbiology↗