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bioRxiv2022

Rapid detection of Staphylococcus aureus and Streptococcus pneumoniae by real-time analysis of volatile metabolites

Rapid detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment. In this study, we aimed to evaluate the technical feasibility of Secondary Electro-Spray Ionization-High Resolution Mass Spectrometry (SESI-HRMS) as a diagnostic tool for rapid detection of bacterial infections and compare its performance with the current standard of diagnostics. We compared the time required to confirm growth of the pathogenic bacteria Staphylococcus aureus and Streptococcus pneumoniae by conventional detection by culture and MAL-DI-TOF vs. detection of specific volatile organic compounds (VOCs) produced by these human pathobionts. SESI-HRMS could consistently detect VOCs produced by S. aureus or S. pneumoniae on blood agar plates within minutes, allowing to positively identify bacteria within hours. Unique S. aureus and S. pneumoniae features were detected already at bacterial densities as low as [~]103 colony forming units. Rich mass spectral fingerprints allowed for the distinction of these two bacteria on a species and even strain level. To give an incentive towards clinical application of this technology, further analyzed 17 clinical samples previously diagnosed by conventional methods. We predominantly obtained a separation of samples which showed growth (i.e. presence of living bacteria) compared to samples with no bacterial growth (i.e. presence of dead bacteria). We conclude that SESI-HRMS allows rapid identification of unique bacterial features. Further development of real-time analysis of clinical samples by SESI-HRMS will shorten the time required for microbiological diagnosis with a high level of confidence and sensitivity and should help to improve patients tailored treatment. IMPORTANCEA timely identification of a pathogenic bacteria causing the infection is of pivotal importance for the initiation of an adequate antimicrobial therapy. In this regard, different technologies have been developed with the aim to achieve a highly reliable, specific, and overall fast identification of pathogenic bacteria. However, conventional diagnostic techniques still require long preprocessing times (hours to days) to acquire enough biological material for an accurate identification of the pathogen. Therefore, in this work, we aimed to further shorten the detection time of current gold standards for microbiological diagnostics by providing a system capable of a fast, sensitive and specific discrimination of different pathogenic bacteria. This system relies on the real-time mass spectrometric detection of volatile organic compounds (VOCs) produced by a given organism during its growth, potentially leading to a significant shortening of the time required to obtain a positive reliable diagnostic.

microbiology↗

bioRxiv2022

Within-host genotypic and phenotypic diversity of contemporaneous carbapenem-resistant Klebsiella pneumoniae from blood cultures of patients with bacteremia

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are major pathogens globally. It is unknown whether bloodstream infections (BSIs) by CRKP and other bacteria are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous CRKP from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole genome sequences of 10 strains from single colonies from CRKP-positive blood cultures in each of 6 patients (Illumina HiSeq). All strains were sequence type (ST)-258 K. pneumoniae that were unique by core genome single nucleotide polymorphism phylogeny, antibiotic resistance and virulence genes, capsular polysaccharide (CPS) gene mutations, and/or plasmid loss. Strains from each of 3 patients that differed in antibiotic resistance, virulence and/or CPS gene content underwent long-read sequencing for genome completion (Oxford Nanopore), and were tested for phenotypes in vitro and pathogenicity during mouse BSIs. Genetically distinct strains within individual patients exhibited significant differences in carbapenem, beta-lactam/beta-lactamase inhibitor and other antibiotic responses, CPS production, mucoviscosity, and susceptibility to serum killing. In 2 patients, strains differed significantly in their ability to infect organs and cause mortality in mice. In conclusion, we identified genotypic and phenotypic variant ST258 K. pneumoniae strains from blood cultures of individual patients, which were not detected by the clinical laboratory at time of BSI diagnosis. The data support a new paradigm of CRKP population diversity during BSIs. If validated for other BSIs, within-host bacterial diversity may have profound implications for medical, microbiology laboratory and infection prevention practices, and for understanding emergence of antibiotic resistance and pathogenesis. IMPORTANCEIn processing positive microbiologic cultures, standard clinical laboratory practice is to test a single bacterial strain from each morphologically distinct colony. We performed comprehensive whole genome sequence analyses on 10 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from positive blood cultures from each of 6 patients. Our findings that all strains were genetically unique and that genetic variants manifested differences in phenotypes like antibiotic responsiveness and virulence suggest that CRKP bloodstream infections may be commonly caused by mixed bacterial populations. Results raise questions about laboratory protocols and treatment decisions that are directed against a single strain. The observation that pan-genome analyses revealed inter-strain differences that were not evident by studying core genomes has important implications for investigating nosocomial outbreaks and transmission. Data also suggest a model of pathogenesis of CRKP infections, in which environmental pressures in vivo may select for outgrowth of variants that manifest antibiotic resistance, tolerance or specific virulence attributes.

microbiology↗

bioRxiv2022

A comprehensive study of the microbiome and resistome of chicken waste from intensive farms

The application of chicken waste to farmland could be detrimental to public health. It may contribute to the dissemination of antibiotic resistance genes (ARG) and antibiotic-resistant bacteria (ARB) from feces and their subsequent entry to the food chain. The present study analyzes the metagenome and resistome of chicken manure and litter obtained from a commercial chicken farm in Poland. ARB were isolated, identified and screened for antibiogram fingerprints using standard microbiological and molecular methods. The physicochemical properties of the chicken waste were also determined. ARG, integrons, and mobile genetic elements (MGE) in chicken waste were analyzed by high-throughput SmartChip qPCR. The results confirm the presence of many ARGs, probably located in MGE, which can be transferred to other bacteria. Potentially pathogenic or opportunistic microorganisms and phytopathogens were isolated. More than 50% of the isolated strains were classified as multi-drug resistant, and the remainder were resistant to at least one antibiotic class; these pose a real risk of entering groundwater and contaminating the surrounding environment. Our results indicate that while chicken manure can be sufficient sources of the nutrients essential for plant growth, its microbiological aspects make this material highly dangerous to the environment.

microbiology↗

bioRxiv2022

Energy dissipation in early detection of cellular responses to metabolic challenges

Metabolic alterations have been recognized to underly the etiology of many diseases. Herein, cellular energy dissipation was evaluated as a novel non-specific global biomarker of metabolic alterations. Energy dissipation, measured as heat by microcalorimetry, was maximal during Saccharomyces cerevisiae adaptation to growth conditions before fast proliferation took place. This response was further augmented by 95 % in media where nutrient assimilation was more difficult, and by 133 % under sub-optimal non-carbon nutrient levels. In this last case, the increase in energy dissipation (1) reflected changes in amino acid and glycolytic metabolism and (2) anticipated changes in the growth curve significantly later observed by traditional microbiological measurements. It was, therefore, an early marker of adaptive responses that compensated for sub-optimal nutrient levels and maintained phenotypic stability. Compensatory responses buffer systems against perturbations and delay the onset of diseases. Microcalorimetry can, therefore, provide a biomarker development platform for early disease-diagnosis. HIGHLIGHTSO_LIEnergy dissipation measurements detect cell responses to metabolic challenges. C_LIO_LIThe detection by microcalorimetry occurs considerably earlier than by traditional microbiological measurements. C_LIO_LISub-optimal non-carbon nutrient levels impact energy dissipation long before cell proliferation. C_LIO_LIEnergy dissipation is highly sensitive to increased nutrient assimilation difficulty. C_LI

microbiology↗

bioRxiv2022

Microbial diversity and community dynamics in an active, high CO2 subsurface rift ecosystem

The Eger Rift subsurface is characterized by frequent seismic activity and consistently high CO2 concentrations, making it a unique deep biosphere ecosystem and a suitable site to study the interactions between volcanism, tectonics, and microbiological activity. Pulses of geogenic H2 during earthquakes may provide substrates for methanogenic and chemolithotrophic processes, but very little is currently known about the role of subsurface microorganisms and their cellular processes in this type of environment. To assess the impact of geologic activity on microbial life, we analyzed the geological, geochemical, and microbiological composition of rock and sediment samples from a 240 m deep drill core, running across six lithostratigraphic zones. In addition, we evaluated diversity as well as metabolic attributes of bacterial and archaeal communities. Our investigation revealed a distinct low biomass community, with a surprisingly diverse Archaea population, providing strong support that methanogenic archaea reside in the Eger subsurface. Geochemical analysis revealed sulfate and sodium concentrations as high as 1000 mg L-1 in sediment samples from a depth between 50 and 100 m and in weathered rock samples collected below 200 m. Most microbial signatures could be assigned to common soil and water bacteria, which together with the occurrence of freshwater Cyanobacteria at specific depths, emphasize the heterogenous, groundwater movement driven nature of this terrestrial subsurface environment. Although not as frequently and abundantly as initially expected, our investigations also found evidence for anaerobic, autotrophic, and acidophilic communities in Eger Rift sediments, as sulfur cycling taxa like Thiohalophilus and Desulfosporosinus were specifically enriched at depths below 100 m. The detection of methanogenic, halophilic, and ammonia oxidizing archaeal populations demonstrate that the unique features of the Eger Rift subsurface environment provide the foundation for diverse types of microbial life, including the microbial utilization of geologically derived CO2 and when available H2, as a primary energy source.

microbiology↗

bioRxiv2023

Amine recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor

Bacteria contain many different receptor families that sense different signals permitting an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Due to a significant sequence divergence, the signal recognized by sensor domains is only poorly reflected in overall sequence identity. Biogenic amines are of central physiological relevance for microorganisms and serve for example as substrates for aerobic and anaerobic growth, neurotransmitters or osmoprotectants. Based on protein structural information and sequence analysis, we report here the identification of a sequence motif that is specific for amine-sensing dCache sensor domains (dCache_1AM). These domains were identified in more than 13,000 proteins from 8,000 bacterial and archaeal species. dCache_1AM containing receptors were identified in all major receptor families including sensor kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases. The screening of compound libraries and microcalorimetric titrations of selected dCache_1AM domains confirmed their capacity to specifically bind amines. Mutants in the amine binding motif or domains that contain a single mismatch in the binding motif, had either no or a largely reduced affinity for amines, illustrating the specificity of this motif. We demonstrate that the dCache_1AM domain has evolved from the universal amino acid sensing domain, providing novel insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and thus holds a strong promise to address an important bottleneck in microbiology: the identification of signals that stimulate numerous receptors.

microbiology↗

bioRxiv2023

The heme oxygenase-1 metalloporphyrin inhibitor stannsoporfin enhances the bactericidal activity of a novel regimen for multidrug-resistant tuberculosis in a murine model

Multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) poses significant challenges to global tuberculosis (TB) control efforts. Host-directed therapies (HDT) offer a novel approach for TB treatment by enhancing immune-mediated clearance of Mtb. Prior preclinical studies found that inhibition of heme oxygenase-1 (HO-1), an enzyme involved in heme metabolism, with tin-protoporphyrin IX (SnPP) significantly reduced mouse lung bacillary burden when co-administered with the first-line antitubercular regimen. Here we evaluated the adjunctive HDT activity of a novel HO-1 inhibitor, stannsoporfin (SnMP), in combination with a novel MDR-TB regimen comprising a next-generation diarylquinoline, TBAJ-876 (S), pretomanid (Pa), and a new oxazolidinone, TBI-223 (O) (collectively, SPaO) in Mtb-infected BALB/c mice. After 4 weeks of treatment, SPaO + SnMP 5 mg/kg reduced mean lung bacillary burden by an additional 0.69 log10 (P=0.01) relative to SPaO alone. As early as 2 weeks post-treatment initiation, SnMP adjunctive therapy differentially altered the expression of pro-inflammatory cytokine genes, and CD38, a marker of M1 macrophages. Next, we evaluated the sterilizing potential of SnMP adjunctive therapy in a mouse model of microbiological relapse. After 6 weeks of treatment, SPaO + SnMP 10 mg/kg reduced lung bacterial burdens to 0.71 {+/-} 0.23 log10 CFU, a 0.78 log-fold greater decrease in lung CFU compared to SpaO alone (P=0.005). However, adjunctive SnMP did not reduce microbiological relapse rates after 5 or 6 weeks of treatment. SnMP was well tolerated and did not significantly alter gross or histological lung pathology. SnMP is a promising HDT candidate requiring further study in combination with regimens for drug-resistant TB.

microbiology↗

bioRxiv2023

Frass fertilizers from mass-reared insects: species variation, heat treatment effects, and implications for soil application

Insect farming has gained popularity as a resource-efficient and eco-friendly method of managing organic wastes by converting them into high-quality protein, fat, and frass. Insect frass is a powerful organic fertilizer, enriching the soil with essential plant nutrients and enhancing plant defense mechanisms through chitin stimulation. Given the importance of frass commercialization for many insect farmers and the use of increasingly diverse organic wastes as insect feedstock, the need for legal guidelines to enable clean production practices has emerged. The recent introduction of a legal definition for frass and heat treatment requirements by the EU commission marks a significant step towards standardizing its quality. However, frass composition is influenced by numerous factors, and little is known about the processes shaping its nutritional profiles and contributing to its maturation. Here, we analyzed the physicochemical, plant-nutritional, and microbiological properties of black soldier fly, yellow mealworm, and Jamaican field cricket frass from mass-rearing operations and assessed the impact of hygienizing heat treatment. Frass properties varied significantly across insect species, revealing concentrations of plant available nutrients reaching as high as 7000 {micro}g NH4+, 150 {micro}g NO2-NO3--N, and 20 mg P per g of total solids. Heat treatment affected microbial activity by reducing basal respiration and microbial biomass carbon, but also reducing viable counts of pathogenic E. coli and Salmonella sp. In terms of microbiome composition, alpha diversity showed no significant differences between fresh and heat-treated frass samples within each insect species, but significant distinctions were observed across the three insect species. The soil application of frass reactivated and boosted soil microbial activity, suggesting no long-term detrimental effects on microorganisms. These results further highlight the potential of insect frass as nutrient rich organic fertilizer, with promising benefits for soil health and nutrient cycling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/559882v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1935756org.highwire.dtl.DTLVardef@4af4d6org.highwire.dtl.DTLVardef@1104318org.highwire.dtl.DTLVardef@a4e568_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Physicochemical and microbiological assessment of frass from black soldier fly (BSF), yellow mealworm (YMW), and Jamaican field cricket (JFC) and the impact of heat treatment. TS = total solids, VS = volatile solids, TDS = total dissolved solids, BR = basal respiration, Cmic = microbial biomass carbon, MQ = metabolic quotient. n.s.: p > 0.05, *: p <= 0.05, **: p <= 0.01, ***: p <= 0.001, ****: p <= 0.0001, o.o.R. = out of range, b.d.l. = below detection limit. C_FIG

microbiology↗

bioRxiv2023

Microbial ecology of coastal northern Gulf of Mexico waters

Estuarine and coastal ecosystems are of high economic and ecological importance, owing to their diverse communities and the disproportionate role they play in carbon cycling, particularly in carbon sequestration. Organisms inhabiting these environments must overcome strong natural fluctuations in salinity, nutrients, and turbidity as well as numerous climate change-induced disturbances such as land loss, sea level rise, and, in some locations, increasingly severe tropical cyclones that threaten to disrupt future ecosystem health. The northern Gulf of Mexico (nGoM) along the Louisiana coast contains dozens of estuaries, including the Mississippi-Atchafalaya River outflow, which dramatically influence the region due to their vast upstream watershed. Nevertheless, the microbiology of these estuaries and surrounding coastal environments have received little attention. To improve our understanding of microbial ecology in the understudied coastal nGoM, we conducted a 16S rRNA gene amplicon survey at eight sites and multiple timepoints along the Louisiana coast and one inland swamp spanning freshwater to high brackish salinities, totaling 47 duplicated Sterivex (0.2-2.7 {micro}m) and prefilter (> 2.7 {micro}m) samples. We cataloged over 13,000 ASVs from common freshwater and marine clades such as SAR11 (Alphaproteobacteria), Synechococcus (Cyanobacteria), and acI and Candidatus Actinomarina (Actinobacteria). We observed preferences for freshwater or marine habitats in many organisms and also characterized a group of taxa with specialized distributions across brackish water sites, supporting the hypothesis of an endogenous brackish-water community. Additionally, we observed brackish-water preferences for several aquatic clades typically considered marine or freshwater taxa, such as SAR11 subclade II, SAR324, and the acI Actinobacteria. The data presented here expand the geographic coverage of microbial ecology in estuarine communities, help delineate the autochthonous and allochthonous members of these environments, and provide critical aquatic microbiological baseline data for coastal and estuarine sites in the nGoM.

microbiology↗

bioRxiv2024

Long-term imaging of three-dimensional hyphal development using the ePetri dish

Imaging three-dimensional microbial development and behavior over extended periods is crucial for advancing microbiological studies. Here, we introduce an upgraded ePetri dish system specifically designed for extended microbial culturing and 3D imaging, addressing the limitations of existing methods. Our approach includes a sealed growth chamber to enable long-term culturing, and a multi-step reconstruction algorithm that integrates 3D deconvolution, image filtering, ridge, and skeleton detection for detailed visualization of the hyphal network. The system effectively monitored the development of Aspergillus brasiliensis hyphae over a seven-day period, demonstrating the growth mediums stability within the chamber. The systems 3D imaging capability was validated in a volume of 5.5 mm x 4 mm x 0.5 mm, revealing a radial growth pattern of fungal hyphae. Additionally, we show that the system can identify potential filter failures that are undetectable with 2D imaging. With these capabilities, the upgraded ePetri dish represents a significant advancement in long-term 3D microbial imaging, promising new insights into microbial development and behavior across various microbiological research areas.

microbiology↗

bioRxiv2024

Extreme diversity of phage amplification rates and phage-antibiotic interactions revealed by PHORCE

Growth rate plays a fundamental role in microbiology and serves as an important proxy for fitness in evolution. While high-throughput measurements of bacterial growth rates are easily performed in any microbiology lab, similar methods are lacking for bacteriophages. This gap hinders systematic comparisons of important phage phenotypes, such as their amplification rate in bacterial populations and their bactericidal effect, across different phages and environmental conditions. Here, we show that the amplification rate of lytic phages can be quantified by analyzing bacterial population growth and collapse dynamics under phage predation using a parsimonious mathematical model - an approach termed PHORCE (Phage-Host Observation for Rate estimation from Collapse Events). We found that the resulting phage amplification rate captures the bactericidal effect independent of initial phage and bacterial population sizes and across different growth conditions. Using high-throughput PHORCE, we found that the amplification rates of Escherichia coli phages vary widely by more than three orders of magnitude. Furthermore, our approach suggests that phage-antibiotic interactions are predominantly determined by the antibiotic, and not by the phage. In particular, the ribosome-inhibiting antibiotic doxycycline generally showed antagonism with phage amplification, whereas the DNA-damaging antibiotic nitrofurantoin was synergistic. This framework provides a means to quantitatively characterize phage phenotypes and may facilitate future high-throughput phage screens for antibacterial applications.

microbiology↗

bioRxiv2024

Subsurface Microbial Colonization at Mineral-Filled Veins in 2-Billion-Year-Old Igneous Rock from the Bushveld Complex, South Africa

Recent advances in subsurface microbiology have demonstrated the habitability of multi-million-year-old igneous rocks, despite the scarce energy supply from rockwater interactions. Given the minimal evolution coupled with exceedingly slow metabolic rates in subsurface ecosystems, spatiotemporally stable igneous rocks can sustain microbes over geological time scales. This study investigated 2-billion-year-old igneous rock in the Bushveld Complex, South Africa, where ultradeep drilling is being executed by the International Continental Scientific Drilling Program (ICDP). New procedures were successfully developed to simultaneously detect indigenous and contaminant microbial cells in a drill core sample. Precision rock sectioning coupled with infrared, fluorescence and electron microscopy imaging of the rock section with submicron resolution revealed microbial colonization in veins filled with smectite. The entry and exit of microbial cells in the veins are severely limited by tight packing with smectite, the formation of which supplies energy sources for long-term habitability. Further microbiological characterization of drilled rock cores from the Bushveld Complex will expand the understanding of microbial evolution in deep igneous rocks over 2 billion years.

microbiology↗

bioRxiv2024

Antibiotic resistance detection and concomitant species identification of ESKAPE pathogens by proteomics

Antimicrobial resistance (AMR) is an increasing challenge for therapy of bacterial infections. Currently, patient treatment is guided by antimicrobial susceptibility testing (AST) using phenotypic assays and species identification by MALDI-ToF biotyping. Bacterial phenotype prediction using omics technologies could offer several advantages over current diagnostic methods. It would allow species identification and AST to be combined in a single measurement, it would eliminate the need for secondary cultivation and could enable the prediction of phenotypes beyond AMR, such as virulence. In this study, the potential of proteomics for clinical microbiology was evaluated in an analysis of 126 clinical isolates covering 16 species including all ESKAPE genera and 29 of the most common AMR determinants. For this purpose, a flexible workflow was developed, which enables to report the AMR phenotype and the species of primary cultures within 2h. Proteomics provided high specificity (99.9%) and sensitivity (94.4 %) for AMR detection, while allowing species identification from very large sequence databases with high accuracy. The results show, that proteomics is well suited for phenotyping clinical bacterial isolates and has the potential to become a valuable diagnostic tool for clinical microbiology in the future.

microbiology↗

bioRxiv2024

Non-Optical, Label-free Electrical Capacitance Imaging of Microorganisms

Many fundamental insights into microbiology have come from imaging, which is typically synonymous with optical techniques. However, the sample preparation needed for many optical microscopy methods such as labeling, fixing, or genetic modification, limits the range of species and environments we can investigate. Here we demonstrate the use of electrical capacitance measurements as a non-optical method for imaging live microbial samples. In electrical capacitance imaging (ECI), samples are positioned in contact with a semiconductor sensor array, and localized capacitance measurements are made across the array. From these measurements, we generate textured images of a variety of microbial colonies. We determine that capacitance is correlated with local sample thickness by comparing ECI data to 3D confocal scans. We further illustrate with ECI that a difference in capacitance signal allows microbial species to be spatially distinguished in co-culture conditions. In order to highlight the versatility of our system, we capture the cross-sectional development of floating pellicle biofilms in a liquid culture at millimeter length scales during weeks-long time-lapse experiments. These novel results establish a new low-cost and portable platform which can be used for spatially and temporally resolved experiments in diverse environments with a wide variety of microbial species. IMPORTANCEMicrobes live in diverse environments, and occupy biological roles across many timescales. Investigating the full scope of microbial activity requires imaging systems appropriate to each context. Though optical microscopy is powerful, the use of light, lenses, and other hardware limits where it can be applied. At the same time, existing non-optical imaging methods are frequently destructive to samples and require extensive equipment. In this paper we present a non-optical imaging system that is small, cheap, requires no sample labeling, and is compatible with a variety of microbial species. Our system uses semiconductor chips to measure the inherent material properties of a sample with spatial sensitivity, producing images of microbes contrasted against their environment and each other. Our technique captures label-free, micrometer-resolution images with a pocket-sized device, enabling microbiological imaging experiments in new environments with new species.

microbiology↗

bioRxiv2024

A Comprehensive Bibliometric Investigation on Antimicrobials from Fungal Origins with a Biotechnological Perspective

Interest in research related to fungi exploration past few years, given its relevance to bioactive compounds production as these metabolites are a promising source of bacteriostatic or fungistatic compounds. Therefore, their use represents a significant alternative to traditional antibiotics by minimizing the risks related to microbial resistance. In this context, this work aimed to: assess the volume of annual publications on the subject and identify key players, analyze the collaboration network among researchers, and check the patents filed on this topic. For this purpose, the Bibliometrix R-package was used, as well as scientific metadata from the Web of Science and Scopus databases (n=506). In total, 256 sources, authors (n=2,526), keywords (n=1,812), and references (n=19,315) were analyzed from 1989 to 2023. The academic debate on the subject has been promoted by India (29%), United Kingdom (UK) (7%), China (6%) and the United States of America (USA) (6%). The authors identified as the most cited were Liu J (n=142), followed by Jesu Arockiaraj (n=106). There is a knowledge predominance of publications focusing on the life science disciplines. The most prolific institutions were the National Research Center (n=20) and the University of Pittsburgh (n=13). The most cited journals were the World Journal of Microbiology & Biotechnology (n=719) and Applied Microbiology and Biotechnology (n=661). Finally, the United States Patent and Trademark Office represented 85% of the patents filed on the subject (n=28,303). Together, these results can direct researchers and Biotech industries in their search for the most important sources related to antimicrobial biotechnology.

microbiology↗

bioRxiv2024

A method for authenticating the fidelity of Cryptococcus neoformans knockout collections

Gene knockout strain collections are important tools for discovery in microbiology. Cryptococcus neoformans is the only human pathogenic fungus with an available genome-wide deletion collection, and this resource is widely used by the research community. We uncovered mix-ups in the assembly of the commercially available C. neoformans deletion collection of [~]6,000 unique strains acquired by our lab. While pursuing the characterization of a gene-of-interest, the corresponding deletion strain from the C. neoformans KO collection displayed several interesting phenotypes associated with virulence. However, RNAseq analysis identified transcripts for the putative knockout gene, and the absence of transcripts for a different knockout strain found in the same plate position in an earlier partial knockout collection, raising the possibility that plates from one collection were substituted for the other. This was supported by determining the size of the nourseothricin (NAT)-resistance cassette used to generate the two separate knockout libraries and was confirmed by RNAseq and genome sequencing. Here we report that our KN99 collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. ImportanceGene knockout strain collections are important tools for discovery in microbiology. Cryptococcus neoformans is the only human pathogenic fungus with an available genome-wide deletion collection, and this resource is widely used by the research community. Here we report that our KN99 collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. Above all, this article serves as a reminder to 2015 library collection strains before undertaking large phenotyping experiments.

microbiology↗

bioRxiv2025

A mechanistic understanding of the effect of Staphylococcus aureus VraS histidine kinase single point mutation on antibiotic resistance

Bacterial genomic mutations in Staphylococcus aureus (S. aureus) have been detected in isolated resistant clinical strains, yet their mechanistic effect on the development of antimicrobial resistance remains unclear. The resistance-associated regulatory systems acquire adaptive mutations under stress conditions that may lead to a gain of function effect and contribute to the resistance phenotype. Here, we investigate the effect of a single-point mutation (T331I) in VraS histidine kinase, part of the VraSR two-component system in S. aureus. VraSR senses and responds to environmental stress signals by upregulating gene expression for cell wall synthesis. A combination of enzyme kinetics, microbiological, and transcriptomic analysis revealed the mechanistic effect of the mutation on VraS and S. aureus. Michaelis Mentens kinetics show that the VraS mutation caused an increase in the autophosphorylation rate of VraS and enhanced its catalytic efficiency. The introduction of the mutation through recombineering coupled with CRISPR-Cas9 counterselection to the Newman strain wild-type (WT) genome doubled the minimum inhibitory concentration of three cell wall-targeting antibiotics. The mutation caused an enhanced S. aureus growth rate at sub-lethal doses of the antibiotics, confirming the causative effect of mutation on bacterial persistence. Transcriptomic analysis showed a genome-wide alteration in gene expression levels and protein-protein interaction network of the mutant compared to the WT strain after exposure to vancomycin. The results suggest that vraS mutation causes several mechanistic changes at the protein and cellular levels that favor bacterial survival under antibiotic stress and cause the mutation-harboring strains to become the dominant population during infection. ImportanceRising antimicrobial resistance (AMR) is a global health problem. Mutations in the two- component system have been linked to drug- resistance in Staphylococcus aureus, yet the exact mechanism through which these mutations work is understudied. We investigated the T331I mutation in the vraS gene linked to sensing and responding to cell wall stress. The mutation caused changes at the protein level by increasing the catalytic efficiency of VraS kinase activity. Introducing the mutation to the genome of an S. aureus strain resulted in changes in the phenotypic antibiotic susceptibility, growth kinetics, and genome-wide transcriptomic alterations. By a combination of enzyme kinetics, microbiological, and transcriptomic approaches, we highlight how small genetic changes can significantly impact bacterial physiology and survival under antibiotic stress. Understanding the mechanistic basis of antibiotic resistance is crucial to guide the development of novel therapeutic agents to combat AMR.

microbiology↗

bioRxiv2025

Diversity and single-cell activity of endolithic microbes in sediment-hosted carbonate nodules within and below the sulfate-methane transition zone

Authigenic carbonate concretions ( nodules) precipitate in marine seep sediments as a result of anaerobic oxidation of methane (AOM). These rocks host active endolithic microbial communities and persist as important methane sinks. Still, how these communities and their activity differ from those in adjacent seep sediments, particularly as a function of proximity to the sulfate-methane transition zone (SMTZ), remains understudied. Here, we sampled sediments and nodules within and below the SMTZ (0-57 cm deep) at four active deep-sea seep areas in Santa Monica Basin, CA. Measurements of high nodule porosities (43-51%) coupled to strong similarities between sediment and nodule 16S rRNA-based community profiles, including below the SMTZ, suggest continued perfusion and exchange between buried nodules and surrounding sediment. Shared, depth-dependent transitions in methanotrophic taxa (ANME-1, ANME-2, ANME-3) and methanogenic taxa (Methanofastidiosales) below the SMTZ were also consistent with trends in porewater methane and sulfate concentrations, porewater DIC, and nodule {delta}13C values -- underscoring the impact of different geochemical conditions on community structure and suggestive of under-characterized physiological plasticity in ANME-1. Laboratory-based BONCAT incubations of nodules within the SMTZ over [~]14-weeks revealed active sulfide production and translationally active endolithic microorganisms. However, cells from parallel nodule incubations recovered below the SMTZ showed weak-to-negligible BONCAT-based activity despite similar cell abundances -- suggestive of low activity on shorter timescales or dormancy. Together, these data challenge the interpretation of passively recorded microbiological signatures in seep sediment-hosted carbonate nodules and expand our understanding of how these endolithic communities may be actively shaped by past and present conditions. ImportanceThis study advances earlier investigations of microbial communities in buried seep carbonate nodules by integrating microbiological profiles of nodules and sediments, sediment geochemistry, single-cell activity measurements, and nodule mineral, geochemical, and physical characteristics within and below the sulfate-methane transition zone in deep-sea methane seeps. This approach allows us to view how nodule-hosted, endolithic microbial communities change relative to their surrounding sediments across multiple geochemical contexts and better understand how formation history and environmental conditions might affect community identity and metabolic function. Results indicate that the seep nodule and surrounding sediment communities are closely linked across diverse geochemical conditions. This connectivity between sediments and carbonate nodules is distinct from that observed in exhumed seep carbonates, with implications regarding how microbial community composition within these nodules are interpreted, suggesting that instead of a passive recorder of the communities at the time of formation, these nodules appear to retain diverse, metabolically viable communities.

microbiology↗