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bioRxiv2024

Dual quorum-sensing control of purine biosynthesis drives pathogenic fitness of Enterococcus faecalis

Enterococcus faecalis is a resident of the human gut, though upon translocation to the blood or body tissues, it can be pathogenic. Here we discover and characterize two peptide-based quorum-sensing systems that transcriptionally modulate de novo purine biosynthesis in E. faecalis. Using a comparative genomic analysis, we find that most enterococcal species do not encode this system; E. moraviensis, E. haemoperoxidus and E. caccae, three species that are closely related to E. faecalis, encode one of the two systems, and only E. faecalis encodes both systems. We show that these systems are important for the intracellular survival of E. faecalis within macrophages and for the fitness of E. faecalis in a murine wound infection model. Taken together, we combine comparative genomics, microbiological, bacterial genetics, transcriptomics, targeted proteomics and animal model experiments to describe a paired quorum sensing mechanism that directly influences central metabolism and impacts the pathogenicity of E. faecalis.

microbiology↗

bioRxiv2024

Semi-Automatic Segmentation of Pseudomonas koreensis and Escherichia coli for Bacterial Growth Characterization

Bacterial characterization is a crucial discipline within microbiology. Given the manual and labor-intensive nature of this task, our aim is to introduce a semi-automatic segmentation method that enhances efficiency while preserving the rich details of bacterial colonies. We propose using the k-means clusterization algorithm to analyze and segment images of bacterial cultures, specifically those of Pseudomonas koreensis and Escherichia coli. Unlike existing methods that focus primarily on colony counting, our approach emphasizes morphological characterization. In some bacterial cultures, colonies are not well-defined, making manual counting or other automated counting methods unfeasible; i.e. the bacterial growth area is not easily identifiable, thus precise growth tracking is not feasible. Our method enables bacterial growth characterization even in these cases. Our computer vision system identifies and quantifies the diverse morphologies within P. koreensis and E. coli cultures, determining their relative occupancy in an image. Our approach provides valuable insights into the composition, growth patterns, and developmental stages of bacterial colonies, designed to assist both novice and expert microbiologists in bacterial analysis.

microbiology↗

bioRxiv2024

Pseudomonas aeruginosa population dynamics in a vancomycin-induced murine model of gastrointestinal carriage

Pseudomonas aeruginosa is a common nosocomial pathogen and a major cause of morbidity and mortality in hospitalized patients. Multiple reports highlight that P. aeruginosa gastrointestinal colonization may precede systemic infections by this pathogen. Gaining a deeper insight into the dynamics of P. aeruginosa gastrointestinal carriage is an essential step in managing gastrointestinal colonization and could contribute to preventing bacterial transmission and progression to systemic infection. Here, we present a clinically relevant mouse model relying on parenteral vancomycin pretreatment and a single orogastric gavage of a controlled dose of P. aeruginosa. Robust carriage was observed with multiple clinical isolates, and carriage persisted for up to 60 days. Histological and microbiological examination of mice indicated that this model indeed represented carriage and not infection. We then used a barcoded P. aeruginosa library along with the sequence tag-based analysis of microbial populations (STAMPR) analytic pipeline to quantify bacterial population dynamics and bottlenecks during the establishment of the gastrointestinal carriage. Analysis indicated that most of the P. aeruginosa population was rapidly eliminated in the stomach, but the few bacteria that moved to the small intestine and the caecum expanded significantly. Hence, the stomach constitutes a significant barrier against gastrointestinal carriage of P. aeruginosa, which may have clinical implications for hospitalized patients. IMPORTANCEWhile P. aeruginosa is rarely part of the normal human microbiome, carriage of the bacterium is quite frequent in hospitalized patients and residents of long-term care facilities. P. aeruginosa carriage is a precursor to infection. Options for treating infections caused by difficult-to-treat P. aeruginosa strains are dwindling, underscoring the urgency to better understand and impede pre-infection stages, such as colonization. Here, we use vancomycin-treated mice to model antibiotic-treated patients who become colonized with P. aeruginosa in their gastrointestinal tracts. We identify the stomach as a major barrier to the establishment of gastrointestinal carriage. These findings suggest that efforts to prevent gastrointestinal colonization should focus not only on judicious use of antibiotics but also on investigation into how the stomach eliminates orally ingested P. aeruginosa.

microbiology↗

bioRxiv2024

Activation and Evasion of the FEAR Pathway by RNA Viruses

We recently identified the FACT-ETS-1 Antiviral Response (FEAR) pathway as an interferon-independent innate immune response that restricts DNA virus replication and is countered by poxvirus-encoded A51R proteins (Rex et al., 2024, Nature Microbiology). The human FEAR pathway is mediated by the FACT complex, consisting of hSpt16 and SSRP1 subunits, that remodels chromatin to activate expression of the antiviral transcription factor, ETS-1. To counter this pathway, poxvirus A51R proteins tether SUMOylated hSpt16 subunits to microtubules to prevent ETS-1 expression. While these observations indicate a role for the FEAR pathway in DNA virus restriction, it was unclear if RNA viruses interact with this pathway. Here, we show that RNA viruses are also restricted by the FEAR pathway, yet encode mechanisms distinct from poxviruses to counter this response. We show vesicular stomatitis virus (VSV), a rhabdovirus, utilizes its matrix (M) protein to promote proteasome-dependent degradation of SUMOylated hSpt16 and to block ETS-1 nuclear import. Strains encoding mutant M proteins that cannot antagonize the FEAR pathway exhibit replication defects in human cells that can be rescued by hSpt16 or ETS-1 depletion. Moreover, FACT inhibitor treatment enhanced the replication of oncolytic VSV strains encoding defective M proteins in restrictive cancer cells, suggesting FEAR pathway inhibition may improve oncolytic virotherapy. Strikingly, we provide evidence that the inability of VSV M to degrade SUMOylated Spt16 in lepidopteran insect cells results in abortive infection, suggesting VSV-Spt16 interactions influence virus host range. Lastly, we show that human and murine paramyxovirus target SUMOylated Spt16 proteins for degradation in human and murine cells utilizing a conserved N-terminal motif in their accessory "C" proteins. Collectively, our study illustrates that DNA and RNA viruses have independently evolved diverse mechanisms to antagonize SUMOylated host Spt16 proteins, underscoring the physiological importance of the FEAR pathway to antiviral immunity.

microbiology↗

bioRxiv2024

Metagenomic insights into an enigmatic gammaproteobacterium that is important for carbon cycling in cave ecosystems worldwide

Caves are windows into the subsurface through which we can directly evaluate the microbiological processes responsible for rock weathering and biogeochemical cycling in more expansive areas of Earths subsurface. However, many cave microbial communities are dominated by microorganisms from uncultivated groups with unknown genomic capabilities that cannot be resolved from marker gene surveys alone. An example of this is a genus of Gammaproteobacteria known as "wb1-P19", which are ubiquitous and abundant in rRNA gene surveys from limestone and volcanic caves around the world. We recovered a nearly complete metagenome-assembled genome (MAG) representing a population of wb1-P19 from Lehman Caves in Great Basin National Park, Nevada, USA, and used it to identify additional MAGs representing this group from the Frasassi Caves in Italy and in publicly available databases. Although members of the wb1-P19 group have often been thought to be autotrophs that oxidize inorganic nitrogen compounds, we show that wb1-P19 are actually obligate or facultative methanotrophs capable of aerobic and anaerobic growth. Based on genomic classification, wb1-P19 are members of upland soil cluster {gamma} (USC{gamma}; part of the newly proposed order "Candidatus Methyloligotrophales"), and are likely important for methane consumption and carbon cycling in caves and other subterranean ecosystems globally.

microbiology↗

bioRxiv2024

Clostridium scindens: an endocrine keystone species in the mammalian gut

Clostridium scindens is a keystone human gut microbial taxonomic group that, while low in abundance, has a disproportionate effect on bile acid and steroid metabolism in the mammalian gut. Numerous studies indicate that the two most studied strains of C. scindens (i.e., ATCC 35704 and VPI 12708) are important for a myriad of physiological processes in the host. We focus on both historical and current microbiological and molecular biology work on the Hylemon-Bjorkhem pathway and the steroid-17,20-desmolase pathway that were first discovered in C. scindens. Our most recent analysis now calls into question whether strains currently defined as C. scindens represent two separate taxonomic groups. Future directions include developing genetic tools to further explore the physiological role bile acid and steroid metabolism by strains of C. scindens, and the causal role of these pathways in host physiology and disease.

microbiology↗

bioRxiv2024

Sodium Chloride Promotes the Growth of Bacterial Soil Isolate and Antimicrobial Activity of Polymixin B

The decomposition of organic matter in the soil, soil salinity, and soil acidity are influenced by soil microbiology, which also controls the recycling and processing of nutrients in the soil. These factors affect soil fertility and ecological stability. This manuscript is focused on the isolation of seven gram-negative bacteria found in the agricultural soil of The Neotia University campus, West Bengal, India. These isolates have been found to be mesophilic based on their study of their growth profiles, which revealed that under the same incubation conditions, the isolates displayed rising development patterns between 24 and 120 hours. The presence of sodium and potassium chloride modulates the growth and development of bacterial strains (DNI1, DNI2, DNI3, DNI4, DNI5, DNI6, and DNI7) during laboratory set up. The sodium and potassium chloride composition of culture media that effectively regulates the development of bacterial isolates has been determined using the combinational method of salt treatment. The ideal physical factor required for the growth and development of microorganisms has been demonstrated by the pH and temperature titration during this study. The powerful antibiotics known as polymyxin B, which are nonribosomal lipopeptides produced by Paenibacillus polymyxa, are especially effective against Gram-negative bacteria. Because multidrug-resistant Gram-negative bacteria have few other choices for treating infections, the use of polymyxins in clinical settings has increased despite their toxicity in the past. This study offers an update on the most current findings about the bioactivity of soil isolates and their significant relationships to temperature, pH, salt, and polymixin B sensitivity. The toxicity impact may be reduced by enhancing polymixin Bs antibacterial activity with salt treatment in clinical research.

microbiology↗

bioRxiv2024

Tracing active bugs in microbial communities by BONCAT and click chemistry-based enrichment of newly synthesised proteins

A comprehensive understanding of microbial community dynamics is fundamental to the advancement of environmental microbiology, human health, and biotechnology. Metaproteomics, i.e. the analysis of all proteins in a microbial community, provides insights into these complex systems. Microbial adaptation and activity depend to an important extent on newly synthesized proteins (nP), however, the distinction between nP and bulk proteins is challenging. The application of bioorthogonal non-canonical amino acid tagging (BONCAT) with click chemistry has demonstrated efficacy in the enrichment of nP in pure cultures. However, the transfer of this technique to microbial communities has proven challenging and has therefore not been used on microbial communities before. To address this, a new workflow with efficient and specific nP enrichment was developed using a laboratory-scale mixture of labelled E. coli and unlabelled yeast. This workflow was successfully applied to an anaerobic microbial community with initially low BONCAT efficiency. A substrate shift from glucose to ethanol selectively enriched nP with minimal background. The identification of bifunctional alcohol dehydrogenase and a syntrophic interaction between an ethanol-utilizing bacterium and two methanogens (hydrogenotrophic and acetoclastic) demonstrates the potential of metaproteomics targeting nP to trace microbial activity in complex microbial communities.

microbiology↗

bioRxiv2024

Experimental capture of genomic islands defines a widespread class of genetic element capable of non-autonomous transfer

Bacteria acquire new genes by horizontal gene transfer (HGT), typically mediated by mobile genetic elements (MGEs). While plasmids, bacteriophages and certain integrative and conjugative elements (ICEs) are well characterized, the broader diversity of MGEs remains poorly understood. Here, we propagated the bacterium Pseudomonas fluorescens SBW25 in the presence of filtrate obtained from garden compost communities. Genome sequencing of derived colonies revealed acquisition of three different mobile elements, each integrated immediately downstream of tmRNA, flanked by direct repeats, and encoding a tyrosine integrase (intY) and putative phage defense systems. Absent are genes with recognized roles in autonomous transfer. Interrogation of DNA sequence databases showed that similar elements are widespread in the genus Pseudomonas and beyond, with Vibrio Pathogenicity Island-1 (VPI-1) from V. cholerae as a notable example. Bioinformatic analyses demonstrate frequent horizontal transfer among diverse hosts. Detailed analysis of a single element, I55, showed that it is transferred between cells by a jumbo phage, and confers a fitness benefit via a type II restriction-modification system. Significance StatementThe impact of horizontal gene transfer on the evolution of bacteria outpaces that driven by spontaneous mutation, but knowledge of the range of mediators, the genes mobilized, and the mechanisms of movement have largely depended on inferences stemming from bioinformatics. Here we describe a real-time evolution experiment in which a single focal strain propagated with filtrate from a complex microbial community captured genetic elements carrying a diverse cargo of genes whose mobility was previously uncharacterized. The elements represent a widespread class of mobile DNA dependent only on a tyrosine integrase targeting a highly conserved genomic site. Genetic analysis of one element shows that it confers a significant fitness advantage via defense against bacteriophages, and hijacks a jumbo bacteriophage for intercellular transfer. Our findings reveal new insights into the hidden diversity and dynamics of MGEs in natural environments. ClassificationBiological Sciences / Microbiology

microbiology↗

bioRxiv2024

A comprehensive comparative analysis of bacterial communities within cryoconite holes of Arctic and Antarctic glaciers as a response to global warming changes

Cryoconite holes, water-filled ponds with sediment at the bottom, play a crucial role for glacial microbiological ecosystems. While they have been widely studied in various regions of the world (e.g., the Arctic, Antarctic, Alps, Himalayas, Central Andes), the novelty of the analyses presented here lies in their deeper exploration of metagenomic data. This study provides a comparative analysis of environmental factors influencing microbial biodiversity, with an additional focus on explaining the effects of pollution on bacterial communities.

microbiology↗

bioRxiv2024

Wild type domestication: loss of intrinsic metabolic traits concealed by culture in rich media

BackgroundBacteria are typically isolated on rich media to maximise isolation success, removing them from their native evolutionary context. This eliminates selection pressures, enabling otherwise deleterious genomic events to accumulate. Here we present a cautionary tale of these quiet mutations which can persist unnoticed in bacterial culture lines. MethodsWe used a combination of microbiological culture (standard and minimal media conditions), whole genome sequencing and metabolic modelling to investigate putative Klebsiella pneumoniae L-histidine auxotrophs. Additionally, we used genome-scale metabolic modelling to predict auxotrophies among completed public genomes (n=2,637). ResultsTwo sub-populations were identified within a K. pneumoniae frozen stock, differing in their ability to grow in the absence of L-histidine. These sub-populations were the same strain, separated by eight single nucleotide variants and an insertion sequence-mediated deletion of the L-histidine biosynthetic operon. The His- sub-population remained undetected for >10 years despite its in inclusion in independent laboratory experiments. Genome-scale metabolic models predicted 0.8% public genomes contained [≥]1 auxotrophy, with purine/pyrimidine biosynthesis and amino acid metabolism most frequently implicated. DiscussionWe provide a definitive example of the role of standard rich media culture conditions in obscuring biologically relevant mutations i.e. nutrient auxotrophies, and estimate the prevalence of such auxotrophies using public genome collections. While the prevalence is low, it is not insignificant given the thousands of K. pneumoniae that are isolated for global surveillance and research studies each year. Our data serve as a pertinent reminder that rich-media culturing can cause unnoticed wild type domestication.

microbiology↗

bioRxiv2024

GenomicGapID: Leveraging Spatial Distribution of Conserved Genomic Sites for Broad-Spectrum Microbial Identification

Bacterial detection and identification methods can be broadly classified as either untargeted with expansive taxonomic coverage or targeted with narrow taxonomic focus. Untargeted approaches, such as culture and sequencing, are often time-consuming and/or costly, whereas targeted methods, such as PCR, can offer faster and more cost-effective results but require a priori knowledge of the likely pathogen to select the appropriate assay. GenomicGapID, a novel approach that leverages the spatial distribution of conserved genetic regions across microbial genomes, represents a significant advancement in the field of microbial identification. This technique has the potential to provide the taxonomic breadth of culture and sequencing, while maintaining the speed, simplicity, and cost-effectiveness of PCR. By leveraging the conservation and relative positioning of highly conserved coding regions across different species, GenomicGapID enables the development of universal primer sets that amplify the non-conserved gaps between these regions. This creates a unique electrophoretic signature that facilitates rapid and accurate target agnostic microbial identification. In this study, we apply the principles of GenomicGapID to the critical task of identifying clinical pathogens. We focus on expanding the coverage of a previously developed universal bacterial identification system, which initially targeted the 16s-23s internal transcribed spacer (ITS) region and was capable of discerning 45 pathogens. To enhance this system, we assembled a comprehensive database of 189 clinically relevant bacterial species. We then identified conserved primer binding sites that produce unique amplicon size signatures for each species. While we found that the use of amplicon size signatures alone would require an impractical number of universal primer sets, we demonstrate that this challenge can be effectively mitigated through concurrent melt analysis. Ultimately, we show that just three universal primer sets, guided by the GenomicGapID framework, are sufficient to cover 189 clinical bacterial pathogens, representing a majority of microbes identified in positive cultures in a clinical microbiology setting, with experimental validation of a subset of these pathogens. This study not only enhances the existing universal bacterial identification system but also establishes GenomicGapID as a versatile and powerful tool in microbial diagnostics and beyond with the potential to open new areas of investigation in genomics, with significant implications for molecular biology, clinical practice, and public health.

microbiology↗

bioRxiv2024

Impact of media brand on cefiderocol disk diffusion results

IntroductionCefiderocol is a siderophore cephalosporin that utilizes iron transport systems to cross cell membranes. This unique strategy complicates antimicrobial susceptibility testing (AST) due to variable iron content in media. While guidance for using iron-depleted media exists for broth microdilution (BMD), disk diffusion (DD) with commercial media is a common AST method in the clinical laboratory. We investigated cefiderocol DD result variability using multiple Mueller-Hinton agar (MHA) brands. MethodsDD results using Remel (Thermo Fisher Scientific, San Diego, CA), Hardy (Hardy Diagnostics, Springboro, OH), and BBL (Becton Dickenson, East Rutherford, NJ) MHA were compared to BMD using iron-depleted cation-adjusted Mueller-Hinton broth. BMD reproducibility, BMD trailing endpoints, and DD intra- and inter-brand variability in zones of inhibition were investigated. ResultsForty-seven multidrug-resistant clinical isolates and three Antibiotic Resistance bank isolates composed of Pseudomonas aeruginosa, carbapenemase (CP-) and non-carbapenemase-producing (non-CP-) carbapenem-resistant Enterobacterales (CRE), Acinetobacter baumannii complex, Stenotrophomonas maltophilia, and Burkholderia cepacia complex were tested. Categorical agreement (CA) [≥]90% was only demonstrated using CLSI breakpoints with BBL agar. Intra- and inter-brand variability in DD were greatest for P. aeruginosa and CRE, with 30% (6/20) and 16.7% (3/18) exhibiting discrepant AST interpretations, respectively. Isolates not susceptible to cefiderocol via BMD were commonly associated with AST interpretation errors and lower CA. ConclusionsUsing commercial MHA for DD resulted in frequent AST interpretation discrepancies, particularly for isolates that were not susceptible to cefiderocol by BMD. Methods and quality control may need to be revisited to ensure the reliability of DD for cefiderocol AST. IMPORTANCE STATEMENTThe novel mechanism of action of cefiderocol overcomes a variety of resistance mechanisms associated with gram-negative bacteria and positions the agent as an attractive option for infections involving multidrug-resistant pathogens. The availability of accurate, timely antimicrobial susceptibility testing methods for cefiderocol in clinical microbiology laboratories is critical as cefiderocol-resistant isolates have been described and may contribute to treatment failure. Iron-depleted broth microdilution testing may not be feasible for many clinical laboratories. While disk diffusion is an appealing, practical method to implement, our data demonstrate reproducibility issues across agar brands, most notably for organisms that do not test susceptible to cefiderocol via broth microdilution. Discrepant errors and misclassifications of resistant isolates as susceptible, and susceptible isolates as resistant, may mislead clinicians and compromise treatment efficacy. More work is needed to standardize practical yet reproducible methods for cefiderocol antimicrobial susceptibility testing.

microbiology↗

bioRxiv2024

Phytochemical characterization and antimicrobial properties of Azadirachtin indica against selected bacterial human pathogens

The study was conducted to assess the phytochemical and antibacterial potentials of Azadirechtin indica against Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa using microbiological techniques. Ethanol and water were used to extract the active components from the Azadirechtin indica leaves. Salmonella typhi showed range of inhibition zone from 11mm to 16mm at 50% to 90% concentrations of the plant ethanolic extract. Staphylococcus aureus had a range of inhibition zone from 10mm to 13mm at 50% to 90 % concentrations of the plant ethanolic extract and Pseudomonas aeruginosa had a range of inhibition zone from 10mm to 12mm at 50% to 90% concentrations of the plant ethanolic extract. Ethanol extract showed greater antibacterial activity against the test organisms when compared with the aqueous extract. Preliminary screening of phytochemicals in the leaf extract of Azadirechtin indica revealed the presence of alkaloids (7.376mg/100g), saponins (12.041mg/100g), flavonoids (2.641mg/100g), tannins (3.431mg/100g) and cyannogenic glycosides (0.952 mg/100g). This result provides scientific support for Azadirechtin indica to be further investigated and used as drug.

microbiology↗

bioRxiv2024

Promiscuous and unbiased recombination underlies the sequence-discrete species of the SAR11 lineage in the deep ocean

Surveys of microbial communities (metagenomics) or isolate genomes have revealed sequence-discrete species. That is, members of the same species usually show >95% Average Nucleotide Identity (ANI) of shared genes among themselves vs. <83% ANI to members of other species while genome pairs showing between 83-95% ANI are comparatively rare. In these surveys, aquatic bacteria of the ubiquitous SAR11 clade (Class Alphaproteobacteria), which play a major role in carbon cycling, are an outlier and often do not exhibit discrete species boundaries, suggesting the potential for alternate modes of genetic differentiation. To explore evolution in SAR11, we analyzed high-quality, single-cell amplified genomes (SAGs) and companion metagenomes from an oxygen minimum zone (OMZ) in the Eastern Tropical Pacific Ocean, where the SAR11 make up [~]20% of the total microbial community. Our results show that SAR11 do form several sequence-discrete species, but their ANI range of discreteness is shifted to lower identities between 86-91%, with intra-species ANI ranging between 91-100%. Measuring recent gene exchange among these genomes based on a newly developed methodology revealed higher frequency of homologous recombination within compared to between species that affects sequence evolution at least twice as much as diversifying point mutation across the genome. Recombination in SAR-11 appears to be more promiscuous compared to other prokaryotic species and has facilitated the spreading of adaptive mutations within the species, further promoting the high intra-species diversity observed. These results implicate rampant, genome-wide homologous recombination as the mechanism that underlies the evolution of SAR11 into discrete species. SignificanceDistinguishing "species" is a pressing issue in microbiology, partly because the mechanisms that create and maintain clusters of genetically similar microbes are sparsely documented for natural populations. By leveraging high quality single-cell genomic data and a novel method for assessing homologous recombination, we show that rampant homologous recombination maintains species-level clusters of genomes for the most abundant order of marine bacteria, suggesting that these genomes may be evolving sexually to a much greater extent than previously thought. Therefore, our results identify a mechanism explaining the evolution of species in a major microbial group and have implications for understanding microbial diversity and the species concept more broadly.

microbiology↗

bioRxiv2024

Resource landscape shapes the composition and stability of the human vaginal microbiota

The vaginal microbiota is associated with the health of women and newborns alike. Despite its comparatively simple composition relative to other human microbiota systems, the mechanisms underpinning the dynamics and stability of vaginal microbial communities remain elusive. A crucial, yet so far underexplored, aspect of vaginal microbiota ecology is the role played by nutritional resources. Glycogen and its derivatives, produced by vaginal epithelia, are accessible to all bacterial constituents of the microbiota. Concurrently, free sialic acid and fucose offer supplementary nutritional resources to bacterial strains capable of cleaving them from glycans, which are structurally integral to mucus. Notably, bacteria adept at sialic acid exploitation are often correlated with adverse clinical outcomes and are frequently implicated in bacterial vaginosis (BV). In this study, we introduce a novel mathematical model tailored to human vaginal microbiota dynamics to explore the interactions between bacteria and their respective nutritional landscape. Our resource-based model examines the impact of the relative availability of glycogen derivatives (accessible to all bacterial species) and sialic acid (exclusive to some BV-associated bacteria) on the composition of the vaginal microbiota. Our findings elucidate that the success of BV-associated bacteria is intricately linked to their exclusive access to specific nutritional resources. This private access fortifies communities dominated by BV-associated bacteria, rendering them resilient to compositional transitions. We empirically corroborate our model prediction with longitudinal clinical data on microbiota composition and previously unpublished metabolomic profiles obtained from a North American cohort. The insights gleaned from this study shed light on potential pathways for BV prevention and treatment. Significance statementThe vaginal microbiota has a notable impact on womens health at various stages of life, namely puberty, infection protection, sexual health, fertility, pregnancy, and menopausal changes. At present, most non-anti-microbial products developed to mitigate adverse vaginal symptoms emphasise competitive interactions through acids (boric or lactic acid) or probiotics as a means to "rebalance" microbiota communities. Despite recent advances in profiling the composition of vaginal microbiota communities, there remains a major gap in our mechanistic understanding of how to maintain or reinstate a resilient Lactobacillus-dominated microbiota that improves vaginal health and outcomes. This study explores the role of nutritional resources in the vaginal microbiota by introducing a mathematical model that analyses how access to specific nutrients like glycogen derivatives and sialic acid affects the balance of bacterial vaginosis (BV) and non-BV-associated bacteria. Our findings, supported by original cohort-derived microbiological and metabolomics data, demonstrate that exclusive access to these nutrients is linked to the dominance and resilience of BV-associated bacteria, providing new insights for BV prevention and treatment.

microbiology↗

bioRxiv2024

Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers

Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 102 CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results redout (Fluorescence vs FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.

microbiology↗

bioRxiv2024

Intraspecific Diversity of Staphylococcus aureus Populations Isolated from Cystic Fibrosis Respiratory Infections

Chronic bacterial infections are often polymicrobial, comprising multiple bacterial species or variants of the same species. Because chronic infections may last for decades, they have the potential to generate high levels of intraspecific variation through within-host diversification over time, and the potential for superinfections to occur through the introduction of multiple pathogen populations to the ongoing infection. Traditional methods for identifying infective agents generally involve isolating one single colony from a given sample, usually after selecting for a specific pathogen or antibiotic resistance profile. Isolating a recognized virulent or difficult to treat pathogen is an important part of informing clinical treatment and correlative research; however, these reductive methods alone, do not provide researchers or healthcare providers with the potentially important perspective on the true pathogen population structure and dynamics over time. To begin to address this limitation, in this study, we compare findings on Staphylococcus aureus single colonies versus and pools of colonies taken from fresh sputum samples from three patients with cystic fibrosis to isolates collected from the same sputum samples and processed by the clinical microbiology laboratory. Phenotypic and genotypic analysis of isolated S. aureus populations revealed coexisting lineages in two of three sputum samples as well as population structures that were not reflected in the single colony isolates. Altogether, our observations presented here demonstrate that clinically relevant diversity can be missed with standard sampling methods when assessing chronic infections. More broadly, this work outlines the potential impact that comprehensive population-level sampling may have for both research efforts and more effective treatment practices. Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article.

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