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bioRxiv2023

The phenotype and genotype of fermentative microbes

Fermentation is a major type of metabolism carried out by many organisms. The study of this metabolism cuts across many fields, including cell biology, animal and human health, and biofuel production. Despite this broad importance, there has been no systematic study of fermentation across many organisms. Here we explore the phenotype and genotype of fermentative prokaryotes in order to gain insight into this metabolism. We assembled a dataset containing phenotypic records of 8,350 organisms (type strains) plus 4,355 genomes and 13.6 million genes. Fermentation was widespread, being found in 30% of all organisms and across the tree of life. Fermentative organisms were more likely than non-fermentative ones to have certain phenotypic traits. Some traits (such as oxygen insensitivity) were expected, but others (such as long cells) were surprising. Fermentative organisms also had a distinct genotype, with 9,450 gene functions and 337 metabolic pathways being more common in them. In a related analysis, we identified end products (metabolites) for 1,455 organisms fermenting 100 substrates. We found 55 products were formed in nearly 300 combinations, showing fermentation is more complex than previously realized. Additionally, we built metabolic models for 406 organisms and predicted which products they form. These models did not predict all products accurately, revealing gaps in our knowledge of metabolic pathways. Our study paints a full picture of fermentation while showing there is still much to learn about this type of metabolism. The microbiology community can explore resources in this work with an interactive tool (https://github.com/thackmann/FermentationExplorer).

microbiology↗

bioRxiv2023

Soybean and cotton spermosphere soil microbiome shows dominance of soil-borne copiotrophs

The spermosphere is the transient, immediate zone of soil around imbibing and germinating seeds. It represents a habitat where there is contact between seed-associated microbes and soil microbes, but is studied less compared to other plant habitats. Previous studies on spermosphere microbiology were primarily culture-based or did not sample the spermosphere soil as initially defined in space and time. Thus, the objectives of this study were to develop an efficient strategy to collect spermosphere soils around imbibing soybean and cotton in non-sterile soil and investigate changes in microbial communities. The method employed sufficiently collected spermosphere soil as initially defined in space by constraining the soil sampled with a cork borer and confining the soil to a 12-well microtiter plate. Spermosphere prokaryote composition changed over time and depended on the crop within six hours after seeds were sown. By 12 to 18 hours, crops had unique microbial communities in spermosphere soils. Prokaryote evenness dropped following seed imbibition with the proliferation of copiotrophic soil bacteria. Due to their long history of plant growth promotion, prokaryote OTUs in Bacillus, Paenibacillus, Burkholderia, Massilia, Azospirillum, and Pseudomonas were notable genera enriched. Fungi and prokaryotes were hub taxa in cotton and soybean spermosphere networks. Additionally, the enriched taxa were not hubs in networks, suggesting other taxa besides those enriched may be important for spermosphere communities. Overall, this study advances knowledge in the assembly of the plant microbiome early in a plants life, which may have plant health implications in more mature plant growth stages.

microbiology↗

bioRxiv2023

Eelgrass (Zostera spp.) associated phytomyxids are host-specific congeneric parasites and predominant eukaryotes in the eelgrass rhizosphere on a global scale

Together with increasing environmental and anthropogenic pressures, pathogenic diseases are one of important factors contributing to the ongoing decline of seagrass meadows worldwide; yet the diversity and ecology of the microorganisms acknowledged as seagrass parasites remain critically understudied. Here we investigate phytomyxid parasites (Rhizaria: Endomyxa: Phytomyxea) of three different eelgrass (Zostera spp.) species found in the Northern hemisphere. We present molecular evidence that Plasmodiophora bicaudata, a long-recognized parasite of dwarf eelgrass taxa, is closely related to the novel phytomyxid recently discovered in root hairs of Zostera marina, and together they form a distinct clade within the order Phagomyxida, proposed here as Feldmanniella gen. nov. A full life cycle is systematically described in a phagomyxid representative for the first time, proving its conformity with the generalized phytomyxid life history, despite previous uncertainties. The presence of primary infection stages in nearly all collected eelgrass specimens, and subsequent analysis of amplicon sequences from a global Z. marina dataset, reveal phytomyxids to be ubiquitous and one of the predominant microeukaryotes associated with eelgrass roots on a global scale. Our discoveries challenge the current view of Phytomyxea as rare entities in seagrass meadows and suggest their generally low pathogenicity in natural ecosystems. Originality-Significance StatementThis study addresses a group of microbial parasites critically understudied in the marine environment. It presents complex evidence that Phytomyxea - obligate intracellular biotrophs previously considered to be rare entities in the oceans, are in fact ubiquitous endobionts of seagrasses of the genus Zostera - foundation species and important primary producers in coastal areas worldwide. Our work represents a significant contribution to the fields of aquatic microbiology and seagrass ecology and is seminal to understanding the biology of Phytomyxea outside of terrestrial ecosystems.

microbiology↗

bioRxiv2023

Unrecognized Potent Activities of Colistin Against Clinically Important mcr+ Enterobacteriaceae Revealed in Synergy with Host Immunity

Colistin (COL) is a cationic cyclic peptide that disrupts negatively-charged bacterial cell membranes and frequently serves as an antibiotic of last resort to combat multidrug-resistant Gram-negative bacterial infections. Emergence of the horizontally transferable plasmid-borne mobilized colistin resistance (mcr) determinant and its spread to Gram-negative strains harboring extended-spectrum {beta}-lactamase and carbapenemase resistance genes threatens futility of our chemotherapeutic arsenal. COL is widely regarded to have zero activity against mcr+ patients based on standard antimicrobial susceptibility testing (AST) performed in enriched bacteriological growth media; consequently, the drug is withheld from patients with mcr+ infections. However, these standard testing media poorly mimic in vivo physiology and omit host immune factors. Here we report previously unrecognized bactericidal activities of COL against mcr-1+ isolates of Escherichia coli (EC), Klebsiella pneumoniae (KP), and Salmonella enterica (SE) in standard tissue culture media containing the physiological buffer bicarbonate. Moreover, COL promoted serum complement deposition on the mcr-1+ Gram-negative bacterial surface and synergized potently with active human serum in pathogen killing. At COL concentrations readily achievable with standard dosing, the peptide antibiotic killed mcr-1+ EC, KP, and SE in freshly isolated human blood proved effective as monotherapy in a murine model of mcr-1+ EC bacteremia. Our results suggest that COL, currently ignored as a treatment option based on traditional AST, may in fact benefit patients with mcr-1+ Gram negative infections based on evaluations performed in a more physiologic context. These concepts warrant careful consideration in the clinical microbiology laboratory and for future clinical investigation of their merits in high risk patients with limited therapeutic options.

microbiology↗

bioRxiv2023

Uropathogenic Escherichia coli population structure and antimicrobial susceptibility in Norfolk, UK

SynopsisO_ST_ABSBackgroundC_ST_ABSHalf of all women have experienced a urinary tract infection (UTI) in their lifetime and this remains a persistent issue in rural counties like Norfolk, UK. In alignment globally, Uropathogenic E. coli (UPEC) are the main etiological agent for UTIs in Norfolk and are increasingly difficult to treat due to multi-drug resistance (MDR). ObjectiveWe set out to identify which clonal groups and resistance genes are disseminating in the community and hospitals in Norfolk, the first study of its kind for UPEC in this region. MethodsWe collected 217 clinical E. coli isolates causing UTIs in the community and hospital from the Clinical Microbiology laboratory at Norfolk and Norwich University Hospital. These were whole genome sequenced using the Illumina and MinION platforms for in silico multi-locus sequence typing and antibiotic resistance determinant detection. ResultsThe isolates were composed of 74 sequence types (STs); 8 lineages represented 57% of this population: ST73, ST12, ST69, ST131, ST404, ST95, ST127, and ST1193. Importantly, primary UTI screening deemed 8% of isolates to be MDR, with high rates of resistance to ampicillin (52.1%) and trimethoprim (36.2%) in hospitals. Of concern is the probable clonal expansion of MDR groups ST131 and ST1193 in hospitals and community settings with chromosomally encoded blaCTX-M-15, blaOXA-1, and aac(6)-Ib-cr5. ConclusionsThe burden of reported UTIs in Norfolk is largely caused by non-MDR isolates. The UPEC population is continually evolving, and monitoring samples with consideration of sources will help reduce burden of disease.

microbiology↗

bioRxiv2023

Sulfur disproportionating microbial communities in a dynamic, microoxic-sulfidic karst system

Biogeochemical sulfur cycling in sulfidic karst systems is largely driven by abiotic and biological sulfide oxidation, but the fate of elemental sulfur (S0) that accumulates in these systems is not well understood. The Frasassi Cave system (Italy) is intersected by a sulfidic aquifer that mixes with small quantities of oxygen-rich meteoric water, creating Proterozoic-like conditions and supporting a prolific ecosystem driven by sulfur-based chemolithoautotrophy. To better understand the cycling of S0 in this environment, we examined the geochemistry and microbiology of sediments underlying widespread sulfide-oxidizing mats dominated by Beggiatoa. Sediment populations were dominated by uncultivated relatives of sulfur cycling chemolithoautotrophs related to Sulfurovum, Halothiobacillus, Thiofaba, Thiovirga, Thiobacillus, and Desulfocapsa, as well as diverse uncultivated anaerobic heterotrophs affiliated with Bacteroidota, Anaerolineaceae, Lentimicrobiaceae, and Prolixibacteraceae. Desulfocapsa and Sulfurovum populations accounted for 12-26% of sediment 16S rRNA amplicon sequences and were closely related to isolates which carry out autotrophic S0 disproportionation in pure culture. Gibbs energy ({Delta}Gr) calculations revealed that S0 disproportionation under in situ conditions is energy yielding. Microsensor profiles through the mat-sediment interface showed that Beggiatoa mats consume dissolved sulfide and oxygen, but a net increase in acidity was only observed in the sediments below. Together, these findings suggest that disproportionation is an important sink for S0 generated by microbial sulfide oxidation in this oxygen-limited system and may contribute to the weathering of carbonate rocks and sediments in sulfur- rich environments.

microbiology↗

bioRxiv2023

RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function

The mycobacterial mutasome - minimally comprising ImuA, ImuB, and DnaE2 proteins - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, predicted to enable mutasome function via its interaction with the {beta} clamp, is a catalytically inactive member of the Y-family of DNA polymerases. Like other members of the Y family, ImuB features a recently identified amino acid motif with homology to the RecA-N-terminus (RecA-NT). In RecA, the motif mediates oligomerization of RecA monomers into RecA filaments. Given the role of ImuB in the mycobacterial mutasome, we hypothesized that the ImuB RecA-NT motif might mediate its interaction with ImuA, a RecA homolog of unknown function. To investigate this possibility, we constructed a panel of imuB alleles in which RecA-NT was removed, or mutated. Results from microbiological and biochemical assays indicate that RecA-NT is critical for the interaction of ImuB with ImuA. A region downstream of RecA-NT (ImuB-C) also appears to stabilize the ImuB-ImuA interaction, but its removal does not prevent complex formation. In contrast, replacing two key hydrophobic residues of RecA-NT, L378 and V383, is sufficient to disrupt ImuA-ImuB interaction. To our knowledge, this constitutes the first experimental evidence showing the role of the RecA-NT motif in mediating the interaction between a Y-family member and a RecA homolog.

microbiology↗

bioRxiv2023

Methylome-dependent transformation of emm1 group A streptococci

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A Streptococcus (GAS), a lethal human pathogen currently associated with an unprecedented surge of infections world-wide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this "restriction barrier" thus presents a major technical challenge. Here we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Further, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRDAG, encoded by all sequenced strains of the dominant and upsurge-associated emm1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol whereby the restriction barrier is overcome by addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRDAG strains including clinical isolates representing all emm1 lineages, and will expedite critical research interrogating the genetics of emm1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation, and the importance of defining lineage-specific mechanisms of genetic recalcitrance. ImportanceUnderstanding the mechanisms by which bacterial pathogens are able to cause disease is essential to enable the targeted development of novel therapeutics. A key experimental approach to facilitate this research is the generation of bacterial mutants, through either specific gene deletions or sequence manipulation. This process relies on the ability to transform bacteria with exogenous DNA designed to generate the desired sequence changes. Bacteria have naturally developed protective mechanisms to detect and destroy invading DNA, and these systems severely impede the genetic manipulation of many important pathogens, including the lethal human pathogen group A Streptococcus (GAS). Many GAS lineages exist, of which emm1 is dominant among clinical isolates. Based on new experimental evidence, we identify the mechanism by which transformation is impaired in the emm1 lineage and establish an improved and highly efficient transformation protocol to expedite the generation of mutants.

microbiology↗

bioRxiv2023

Volatile profiling distinguishes Streptococcus pyogenes from other respiratory streptococcal species

Sore throat is one of the most common complaints encountered in the ambulatory clinical setting. Rapid, culture-independent diagnostic techniques that do not rely on pharyngeal swabs would be highly valuable as a point-of-care strategy to guide outpatient antibiotic treatment. Despite the promise of this approach, efforts to detect volatiles during oropharyngeal infection have yet been limited. In our research study, we sought to evaluate for specific bacterial volatile organic compounds (VOC) biomarkers in isolated cultures in vitro, in order to establish proof-of-concept prior to initial clinical studies of breath biomarkers. A particular challenge for diagnosis of pharyngitis due to Streptococcus pyogenes is the likelihood that many metabolites may be shared by S. pyogenes and other related oropharyngeal colonizing bacterial species. Therefore, we evaluated whether sufficient metabolic differences are present that distinguish the volatile metabolome of Group A streptococci from other streptococcal species that also colonize the respiratory mucosa, such as S. pneumoniae and S. intermedius. In this work, we identify candidate biomarkers that distinguish S. pyogenes from other species, and establish highly produced VOCs that indicate presence of S. pyogenes in vitro, supporting future breath-based diagnostic testing for streptococcal pharyngitis. IMPORTANCEAcute pharyngitis accounts for approximately 15 million ambulatory care visits in the USA. The most common and important bacterial cause of pharyngitis is Streptococcus pyogenesis, accounting for 15% to 30% of pediatric pharyngitis. Distinguishing between bacterial and viral pharyngitis is key to management in US practice. Culture of a specimen obtained by throat swab is the standard laboratory procedure for the microbiologic confirmation of pharyngitis, however this method is time consuming which delays appropriate treatment. If left untreated, S. pyogenes pharyngitis may lead to local and distant complications. In this study, we characterized the volatile metabolomes of S. pyogenes and other related oropharyngeal colonizing bacterial species. We identify candidate biomarkers that distinguish S. pyogenes from other species and provides evidence to support future breath-based diagnostic testing for streptococcal pharyngitis.

microbiology↗

bioRxiv2023

Antimicrobial Resistance Profiles of Bacterial Contaminants from a Tertiary Hospital in Kenya: An Urgent Call for Action Against the Global Threat of Antimicrobial Resistance.

BackgroundHospital-acquired infections (HAIs) represent the most prevalent adverse event among patients in hospital settings. Contamination with pathogenic bacteria that are highly resistant in the hospital environment increases the risk of HAIs. ObjectiveThe antimicrobial resistance (AMR) patterns of hospital contaminants isolated from highly frequented surfaces in a tertiary hospital in Kenya. MethodsA total of 62 swabs were collected from selected surfaces, equipment, and health workers palms in April 2020. They were cultured and bacterial contaminants were identified using standard microbiological procedures and their AMR patterns were determined using recommended laboratory assays. ResultsOf the 62 swabs collected, 61.3% (n=38) yielded bacterial growth, from which 46 bacteria were isolated. Swab positivity varied across the departments as follows: gynecology wards (78.6%), New Born Unit (NBU) (56.2%), Pediatric ward (61.9%), and Renal Unit (45.5%). Gram negative species comprised 86.96%(n=40) while Gram positive species comprised 13.04%(n=6). Of all the 46 isolates obtained, 36.96% (n=17) were positive for the resistance markers screened. Specifically, 10.9% (n=5) showed both extended-spectrum beta-lactamases (ESBL) and carbapenem-resistant (CR) resistance, while 23.9%(n=11) were positive for ESBL production. The rest were non-resistant strains as shown by negative ESBL at 47.8% (n=22), methicillin sensitivity at 13% (n=6) and vancomycin sensitivity at 2.2% (n=1). Acinetobacter species which were most reported, had the highest resistance (36.84% (7/19). ConclusionThere was a high prevalence of contamination with resistant pathogenic bacteria species. Acinetobacter species were the most common pathogen. Interventions are needed to mitigate the problem of resistant HAI.

microbiology↗

bioRxiv2023

Plasmid-free cheater cells commonly evolve during laboratory growth

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, secretion of beta-lactamase from resistant cells, and subsequent degradation of nearby penicillin and related antibiotics, allows neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss still occurred. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated. ImportancePlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid containing cell is grown in the presence of antibiotic. Here we find that growth of plasmid-containing bacteria during laboratory conditions in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells for viability. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.

microbiology↗

bioRxiv2023

Prevalence of Erythromycin Resistant emm92-type Invasive Group A Streptococcal Infections in West Virginia, United States, 2021-2023

BackgroundIncreasing incidence of invasive group A Streptococcus (iGAS) disease has been reported in Europe and United States over the past several years. Coupled with this are observations of higher rates of resistance to non-beta lactam antimicrobials. ObjectivesThe aim of this study was to characterize iGAS and pharyngitis isolates from West Virginia (WV), a region outside of the US national active bacteria core surveillance purview, where risk factors associated with iGAS infections are prevalent. MethodsSeventy-seven invasive group A Streptococcus isolates were collected from sixty-seven unique patients at the J.W. Ruby Memorial Hospital Clinical Microbiology Laboratory in WV from 2021-23. Invasive isolates and twenty unique pharyngitis isolates were tested for clindamycin and erythromycin susceptibilities in the clinical laboratory. Patient demographic and clinical information was retrieved from patient electronic health records. Isolates were further characterized based on emm-type and detection of MLSB resistance determinants. ResultsTwenty-six (39%) isolates were of a single emm-type, emm92. All emm92 isolates were uniformly erythromycin/clindamycin resistant with inducible or constitutive MLSB resistance imparted by the plasmid-borne erm(T) gene. The majority of emm92 infections were associated with adult patients who reported intravenous drug use, whereas no pharyngitis infections were caused by an emm92 strain. Overall, fifty-one (76%) of the sixty-seven iGAS isolates were determined to carry MLSB resistance. ConclusionsIsolates of emm-type 92 predominated in this collection, were uniformly erythromycin/clindamycin resistant, and were associated with adult intravenous drug use but not with pediatric pharyngitis.

microbiology↗

bioRxiv2023

NIRis: A low-cost, versatile imaging system for NIR fluorescence detection of phototrophic cell colonies used in science and education

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350{euro} in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/543100v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@6dfcb7org.highwire.dtl.DTLVardef@ea7fbcorg.highwire.dtl.DTLVardef@1681e78org.highwire.dtl.DTLVardef@a88d51_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Genomic characterization of the C. tuberculostearicum species complex, a ubiquitous member of the human skin microbiome

Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on skin is incompletely characterized and lacks a comprehensive set of reference genomes. Our work aims to investigate the distribution of Corynebacterium species on the skin, as well as to expand the existing genome reference catalog to enable more complete characterization of skin metagenomes. We used V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity and distribution across healthy human skin. Corynebacterium tuberculostearicum is the predominant species found on human skin and we identified two distinct C. tuberculostearicum ribotypes (A & B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence. One is distributed across all body sites and the other found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five healthy individuals across seven skin sites. We generated five closed genomes of diverse C. tuberculostearicum which revealed that C. tuberculostearicum isolates are largely syntenic and carry a diversity of methylation patterns, plasmids and CRISPR/Cas systems. The pangenome of C. tuberculostearicum is open with a core genome size of 1806 genes and a pangenome size of 5451 total genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from shotgun metagenomic datasets derived from skin body sites. Finally, while the genomes from this study all fall within a C. tuberculostearicum species complex, the ribotype B isolates may constitute a new species. IMPORTANCEAmplicon sequencing data combined with isolate whole genome sequencing has expanded our understanding of Corynebacterium on the skin. Human skin is characterized by a diverse collection of Corynebacterium species but C. tuberculostearicum predominates many sites. Our work supports the emerging idea that C. tuberculostearicum is a species complex encompassing several distinct species. We produced a collection of genomes that help define this complex including a potentially new species which we are calling C. hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomics and growth in skin-like media. This isolate collection and high-quality genome resource sets the stage for developing engineered strains for both basic and translational clinical studies. Microbiomes are shaped by taxa that are both characteristic to those sites and functionally important to that community. The genus Corynebacterium is one such taxa for the human skin and nares. Foundational studies using 16S rRNA gene sequencing and shotgun metagenomics by our lab (1, 2) and others (3) have established Corynebacterium as common members of the skin microbiome. While Corynebacterium have been positively correlated with the resolution of dysbiosis associated with eczema flares (4), the importance of the Corynebacterium spp. is less defined for skin disease severity in primary immune deficient patients (5, 6). Corynebacterium spp. are predominant members of the human aerodigestive tract microbiome (nares, oral cavity and respiratory tract) (3) and participate in microbe-microbe interactions with members of nasal microbiome (7, 8). Corynebacterium have been shown to engage with the host immune system, specifically C. accolens-promoted IL23-dependent inflammation in mice on a high-fat diet (9). C. bovis and C. mastiditis have been shown to predominate the microbiome of a ADAM10-deficient mouse model (10) as well as an ADAM17-deficient mouse model of eczema (11). Finally, C. tuberculostearicum has been shown to induce inflammation in human epidermal keratinocyte cell cultures (12). These studies establish Corynebacterium spp. as key members of the skin microbiome capable of both microbe-microbe and microbe-host interactions. A critical resource for understanding the biology of Corynebacterium on the skin is a robust collection of complete reference genomes, including isolates collected from a variety of individuals and body sites. Previously published genome collections from skin- or nares-resident species include Staphylococcus epidermidis (13), Cutibacterium acnes (14) and the recent comparative analysis of Dolosigranulum pigrum (15). Of note, while emerging bioinformatic methods and pipelines are now being employed to extract nearly-complete genomes (MAGs) from metagenomic assemblies of skin samples (16), MAGs are not yet a substitute for genomes from cultured isolates to understand strain level or pangenomic diversity. In addition to functional prediction, comparative genomics is increasingly being used to augment conventional microbiological methods to define or redefine taxonomic boundaries (17, 18), as well as describe the full extent of diversity within these boundaries (19). A pangenome, which encompasses the complete set of genes present within a set of genome sequences, enables the characterization of gene-level heterogeneity within a taxonomic group. The pangenome is commonly subdivided into the core genome, referring to genes present in all strains, and the accessory or dispensable genome, referring to those present in only one or some isolates. (The accessory pangenome can be further subdivided to reflect a wider range of gene uniqueness, e.g. singletons.) Thorough characterization of taxa is limited by the availability of representative and high-quality genome assemblies. Unfortunately, with the exceptions of clinically relevant Corynebacterium spp. (e.g., C. diphtheriae, C. striatum and C. pseudotuberculosis), the genus is inadequately sequenced, with 75% of species having fewer than six genomes. This includes common skin-associated species like C. tuberculostearicum with just five unique isolate genomes, only two of which are from skin. This work seeks first to characterize the distribution of Corynebacterium across 14 skin sites from 23 healthy volunteers. The second goal of this work focuses on what we identify as the predominant skin Corynebacterium species, C. tuberculostearicum. We have sequenced 23 distinct C. tuberculostearicum strains (n=40 genomes before dereplication), a five-fold increase in the number of publicly available, unique genomes (n=5). In addition to short-read assemblies, we generated five complete genomes which, along with the type strain (DSM44922), demonstrate that C. tuberculostearicum genomes are largely syntenic and carry a number of methylation systems as well as a CRISPR/Cas system. Genes from the C. tuberculostearicum genomes in our collection fall into 5451 gene clusters comprising the species pangenome. This expanded pangenome, as compared to existing public references, improved the mapping of C. tuberculostearicum metagenomic reads from unrelated healthy volunteers. In addition, we have identified a distinct C. tuberculostearicum clade that is highly enriched on the feet that may represent a new species, tentatively designated Corynebacterium hallux.

microbiology↗

bioRxiv2023

Chronic Wound Milieu Challenges Essential Oils' Antibiofilm Activity

The treatment of infected non-healing wounds poses a significant challenge to contemporary medicine. Essential oils (EOs) are being increasingly investigated as potential antibiofilm agents for the management of biofilm-related wound infections. However, their in vitro antimicrobial activity reported in literature does not necessarily reflect the actual in vivo activity due to, among others, methodological imperfections of performed tests. In our study, we cultivated a Staphylococcus aureus biofilm in a novel IVWM (In Vitro Wound Milieu) medium designed to simulate the wound environment and compared it with biofilms cultured in the standard microbiological Tryptic Soy Broth (TSB) medium. We examined and compared critical biofilm properties such as morphology, biomass, metabolic activity, cell count, thickness, and spatial distribution of live and dead cells. Subsequently, staphylococcal biofilms and planktonic cells cultured in both media were exposed to the activity of thyme or rosemary EOs (T-EO, R-EO, respectively). We found that morphology of biofilms cultured in IVWM resembled more the morphology of biofilms visualized in the non-healing wounds than biofilms cultured in TSB. The biomass, metabolic activity, cell number, and the ratio of live to dead cells of S. aureus biofilms were all lower in IVWM compared to the TSB medium. Additionally, while EOs demonstrated overall significant anti-staphylococcal activity, their efficacy varied depending on the medium used. Generally, EOs displayed lower antimicrobial activity (against planktonic cells) in the IVWM than in the TSB medium. Interestingly, T-EO caused a higher reduction of biofilm cells in IVWM than in the TSB medium, in contrast to R-EO. Our findings suggest that EOs hold promise as agents for the treatment of biofilm-related wound infections. However, it is crucial to apply in vitro conditions that closely reflect the wound infection site to gain an accurate insight into the real-world activity of these antimicrobial/antibiofilm agents.

microbiology↗

bioRxiv2023

ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency

Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput applications. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for E. coli. Our redesigned plasmid toolkit achieved nearly 1000x higher efficiency than {lambda} Red recombination and enabled precise, stable knockouts (<134 kb) and integrations (<11 kb) of various sizes. Additionally, we constructed multi-mutants (double and triple) in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.

microbiology↗

bioRxiv2023

Restriction of the growth and biofilm formation of ESKAPE pathogens by caprine gut-derived probiotic bacteria

The accelerated rise of antimicrobial resistance (AMR) poses a significant global health risk, necessitating the exploration of alternative strategies for combating pathogenic infections. Biofilm-related infections, which are unresponsive to standard antibiotics, often require the use of higher-order antimicrobials with toxic side effects and a potential for disrupting the microbiome. Probiotic therapy, with its diverse benefits and inherent safety, is emerging as a promising approach for preventing and treating various infections and as an alternative to antibiotic therapy. In this study, we isolated novel probiotic bacteria from the gut of domestic goats (Capra hircus) and evaluated their antimicrobial and antibiofilm activities against the ESKAPE group of pathogens. We performed comprehensive microbiological, biochemical, and molecular characterizations, including analysis of the 16S-rRNA gene V1-V3 region and the 16S-23S ISR region, on 20 caprine gut-derived lactic acid bacteria (LAB). Among these, six selected LABs demonstrated substantial biofilm formation in anaerobic conditions, and exhibited robust cell surface hydrophobicity and autoaggregation properties highlighting their superior enteric colonization capability. Notably, these LAB isolates exhibited broad-spectrum growth inhibitory and anti-biofilm properties against ESKAPE pathogens. Additionally, the LAB isolates were susceptible to antibiotics listed by the European Food Safety Authority (EFSA), within the prescribed Minimum Inhibitory Concentration limits, suggesting their safety as feed additives. The remarkable probiotic characteristics exhibited by the caprine gut-derived LAB isolates in this study strongly endorse their potential as compelling alternatives to antibiotics and as direct-fed microbial (DFM) feed supplements in the livestock industry, addressing the escalating need for antibiotic-free animal products.

microbiology↗

bioRxiv2023

Amazing structural diversity of giant virus-like particles in forest soil

Large DNA viruses of the phylum Nucleocytoviricota infect diverse eukaryotic hosts from protists to humans, with profound consequences for aquatic and terrestrial ecosystems. While nucleocytoviruses are known to be highly diverse in metagenomes, knowledge of their capsid structures is restricted to a few characterized representatives. Here, we visualize giant virus-like particles (VLPs, diameter >0.2 {micro}m) directly from the environment using transmission electron microscopy. We found that Harvard Forest soils contain a higher diversity of giant VLP morphotypes than all hitherto isolated giant viruses combined. These included VLPs with icosahedral capsid symmetry, ovoid shapes similar to pandoraviruses, and bacilliform shapes that may represent novel viruses. We discovered giant icosahedral capsids with structural modifications that had not been described before including tubular appendages, modified vertices, tails, and capsids consisting of multiple layers or internal channels. Many giant VLPs were covered with fibers of varying lengths, thicknesses, densities, and terminal structures. These findings imply that giant viruses employ a much wider array of capsid structures and mechanisms to interact with their host cells than is currently known. We also found diverse tailed bacteriophages and filamentous VLPs, as well as ultra-small cells. Our study offers a first glimpse of the vast diversity of unexplored viral structures in soil and reinforces the potential of transmission electron microscopy for fundamental discoveries in environmental microbiology.

microbiology↗