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At least 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Bioprospecting Trichoderma: A Systematic Roadmap to Screen Genomes and Natural Products for Biocontrol Applications

Natural products derived from microbes are crucial innovations that would help in reaching sustainability development goals worldwide while achieving bioeconomic growth. Trichoderma species are well-studied model fungal organisms used for their biocontrol properties with great potential to alleviate the use of agrochemicals in agriculture. However, identifying and characterizing effective natural products in novel species or strains as biological control products remains a meticulous process with many known challenges to be navigated. Integration of recent advancements in various “omics” technologies, next generation biodesign, machine learning, and artificial intelligence approaches could greatly advance bioprospecting goals. Herein, we propose a roadmap for assessing the potential impact of already known or newly discovered Trichoderma species for biocontrol applications. By screening publicly available Trichoderma genome sequences, we first highlight the prevalence of putative biosynthetic gene clusters and antimicrobial peptides among genomes as an initial step toward predicting which organisms could increase the diversity of natural products. Next, we discuss high-throughput methods for screening organisms to discover and characterize natural products and how these findings impact both fundamental and applied research fields.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Dissemination of blaNDM-5 and mcr-8.1 in carbapenem-resistant Klebsiella pneumoniae and Klebsiella quasipneumoniae in an animal breeding area in Eastern China

Animal farms have become one of the most important reservoirs of carbapenem-resistant Klebsiella spp. (CRK) owing to the wide usage of veterinary antibiotics. “One Health”-studies observing animals, the environment, and humans are necessary to understand the dissemination of CRK in animal breeding areas. Based on the concept of “One-Health,” 263 samples of animal feces, wastewater, well water, and human feces from 60 livestock and poultry farms in Shandong province, China were screened for CRK. Five carbapenem-resistant Klebsiella pneumoniae (CRKP) and three carbapenem-resistant Klebsiella quasipneumoniae (CRKQ) strains were isolated from animal feces, human feces, and well water. The eight strains were characterized by antimicrobial susceptibility testing, plasmid conjugation assays, whole-genome sequencing, and bioinformatics analysis. All strains carried the carbapenemase-encoding gene bla NDM-5 , which was flanked by the same core genetic structure (IS 5 - bla NDM-5 - ble MBL - trpF - dsbD -IS 26 -IS Kox3 ) and was located on highly related conjugative IncX3 plasmids. The colistin resistance gene mcr-8.1 was carried by three CRKP and located on self-transmissible IncFII(K)/IncFIA(HI1) and IncFII(pKP91)/IncFIA(HI1) plasmids. The genetic context of mcr-8.1 consisted of IS 903 - orf - mcr-8.1-copR-baeS-dgkA - orf -IS 903 in three strains. Single nucleotide polymorphism (SNP) analysis confirmed the clonal spread of CRKP carrying- bla NDM-5 and mcr-8.1 between two human workers in the same chicken farm. Additionally, the SNP analysis showed clonal expansion of CRKP and CRKQ strains from well water in different farms, and the clonal CRKP was clonally related to isolates from animal farms and a wastewater treatment plant collected in other studies in the same province. These findings suggest that CRKP and CRKQ are capable of disseminating via horizontal gene transfer and clonal expansion and may pose a significant threat to public health unless preventative measures are taken.

Yang, Chengxia↗
DOE OSTI2022

One fold, many functions—M23 family of peptidoglycan hydrolases

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons.

Razew, Alicja↗
DOE OSTI2023

Aculeaxanthones A–E, new xanthones from the marine-derived fungus Aspergillus aculeatinus WHUF0198

Introduction Dimeric natural products are widespread in plants and microorganisms, which usually have complex structures and exhibit greater bioactivities than their corresponding monomers. In this study, we report five new dimeric tetrahydroxanthones, aculeaxanthones A−E ( 4−8 ), along with the homodimeric tetrahydroxanthone secalonic acid D ( 1 ), chrysoxanthones B and C ( 2 and 3 ), and 4−4’-secalonic acid D ( 9 ), from different fermentation batches of the title fungus. Methods A part of the culture was added to a total of 60 flasks containing 300 ml each of number II fungus liquid medium and culture 4 weeks in a static state at 28˚C. The liquid phase (18 L) and mycelia was separated from the fungal culture by filtering. A crude extract was obtained from the mycelia by ultrasound using acetone. To obtain a dry extract (18 g), the liquid phase combined with the crude extract were further extracted by EtOAc and concentrated in vacuo. The MIC of anaerobic bacteria was examined by a broth microdilution assay. To obtain MICs for aerobic bacteria, the agar dilution streak method recommended in Clinical and Laboratory Standards Institute document (CLSI) M07-A10 was used. Compounds 1−9 was tested against the Bel-7402, A-549 and HCT-116 cell lines according to MTT assay. Results and Discussion The structures of these compounds were elucidated on the base of 1D and 2D NMR and HR-ESIMS data, and the absolute configurations of the new xanthones 4−8 were determined by conformational analysis and time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations. Compounds 1–9 were tested for cytotoxicity against the Bel-7402, A549, and HCT-116 cancer cell lines. Of the dimeric tetrahydroxanthone derivatives, only compound 6 provided cytotoxicity effect against Bel-7402 cell line (IC50, 1.96 µM). Additionally, antimicrobial activity was evaluated for all dimeric tetrahydroxanthones, including four Gram-positive bacteria including Enterococcus faecium ATCC 19434, Bacillus subtilis 168, Staphylococcus aureus ATCC 25923 and MRSA USA300; four Gram-negative bacteria, including Helicobacter pylori 129, G27, as well as 26,695, and multi drug-resistant strain H. pylori 159, and one Mycobacterium M. smegmatis ATCC 607. However, only compound 1 performed activities against H. pylori G27, H. pylori 26695, H. pylori 129, H. pylori 159, S. aureus USA300, and B. subtilis 168 with MIC values of 4.0, 4.0, 2.0, 2.0, 2.0 and 1.0 μg/mL, respectively.

Wu, Jun↗
DOE OSTI2024

Genomic and phenotypic characterization of multidrug-resistant Salmonella enterica serovar Reading isolates involved in a turkey-associated foodborne outbreak

Salmonella is a global bacterial foodborne pathogen associated with a variety of contaminated food products. Poultry products are a common source of Salmonella -associated foodborne illness, and an estimated 7% of human illnesses in the United States are attributed to turkey products. From November 2017 to March 2019, the Centers for Disease Control and Prevention reported a turkey-associated outbreak of multidrug-resistant (MDR; resistant to ≥3 antimicrobial classes) Salmonella enterica serovar Reading ( S . Reading) linked to 358 human infections in 42 US states and Canada. Since S . Reading was seldom linked to human illness prior to this outbreak, the current study compared genomic sequences of S . Reading isolates prior to the outbreak (pre-outbreak) to isolates identified during the outbreak period, focusing on genes that were different between the two groups but common within a group. Following whole-genome sequence analysis of five pre-outbreak and five outbreak-associated turkey/turkey product isolates of S . Reading, 37 genes located within two distinct chromosomal regions were identified only in the pre-outbreak isolates: (1) an ~5 kb region containing four protein-coding genes including uidA which encodes beta-glucuronidase, pgdA encoding peptidoglycan deacetylase, and two hypothetical proteins and (2) an ~28 kb region comprised of 32 phage-like genes and the xerC gene, which encodes tyrosine recombinase (frequently associated with phage genes). The five outbreak isolates also had a deletional event within the cirA gene, introducing a translational frame shift and premature stop codon. The cirA gene encodes a protein with dual receptor functions: a siderophore receptor for transport of dihydroxybenzoylserine as well as a colicin Ia/b receptor. Significant differences for the identified genetic variations were also detected in 75 S . Reading human isolates. Of the 41 S . Reading isolates collected before or in 2017, 81 and 90% of the isolates contained the uidA and pgdA genes, respectively, but only 24% of the isolates collected after 2017 harbored the uidA and pgdA genes. The truncation event within the cirA gene was also significantly higher in isolates collected after 2017 (74%) compared to before or in 2017 (5%). Phenotypic analysis of the S . Reading isolates for colicin and cefiderocol sensitivities (CirA) and β-methyl-D-glucuronic acid utilization (UidA and accessory proteins) supported the genomic data. Overall, a similar genome reduction pattern was generally observed in both the turkey and human isolates of S . Reading during the outbreak period, and the genetic differences were present in genes that could potentially promote pathogen dissemination due to variation in Salmonella colonization, fitness, and/or virulence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification of novel small molecule inhibitors of twin arginine translocation (Tat) pathway and their effect on the control of Campylobacter jejuni in chickens

Introduction Control ofCampylobacterfrom farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs ofCampylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allowsCampylobacterto colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO 4 ). Methods Since mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigateCampylobacterin infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO 4 (1 mM) inC. jejuni81–176, a human isolate which is widely studied. Results Furthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO 4 for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1–T8) were selected for further studies. These selected eight Tat inhibitors cleared all testedCampylobacterstrains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO 4 in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 μg/mL for 24 h and completely cleared intracellularC. jejunicells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO 4 . Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log;p < 0.01) with minimal disruption of microbiota.In silicoanalyses predicted that T7 has better drug-like properties than T2 inhibitor and might target a key amino acid residue (glutamine 165), which is located in the hydrophobic core of TatC protein. Discussion Thus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to controlC. jejunispread on farms.

Microbiology↗
DOE OSTI2024

Impact of intense sanitization procedures on bacterial communities recovered from floor drains in pork processing plants

Pork processing plants in the United States (US) cease operations for 24–48 h every six or twelve months to perform intense sanitization (IS) using fogging, foaming, and further antimicrobial treatments to disrupt natural biofilms that may harbor pathogens and spoilage organisms. The impact such treatments have on short-term changes in environmental microorganisms is not well understood, nor is the rate at which bacterial communities return. Swab samples were collected from floor drains to provide representative environmental microorganisms at two US pork processing plants before, during, and after an IS procedure. Samples were collected from four coolers where finished carcasses were chilled and from four locations near cutting tables. Each sample was characterized by total mesophile count (TMC), total psychrophile count (TPC), and other indicator bacteria; their biofilm-forming ability, tolerance of the formed biofilm to a quaternary ammonium compound (300 ppm, QAC), and ability to protect co-inoculated Salmonella enterica. In addition, bacterial community composition was determined using shotgun metagenomic sequencing. IS procedures disrupted bacteria present but to different extents depending on the plant and the area of the plant. IS reduced TPC and TMC, by up to 1.5 Log 10 CFU only to return to pre-IS levels within 2–3 days. The impact of IS on microorganisms in coolers was varied, with reductions of 2–4 Log 10 , and required 2 to 4 weeks to return to pre-IS levels. The results near fabrication lines were mixed, with little to no significant changes at one plant, while at the other, two processing lines showed 4 to 6 Log 10 reductions. Resistance to QAC and the protection of Salmonella by the biofilms varied between plants and between areas of the plants as well. Community profiling of bacteria at the genus level showed that IS reduced species diversity and the disruption led to new community compositions that in some cases did not return to the pre-IS state even after 15 to 16 weeks. The results found here reveal the impact of using IS to disrupt the presence of pathogen or spoilage microorganisms in US pork processing facilities may not have the intended effect.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Examining the role of glycoside hydrolases in local rheology of Pseudomonas aeruginosa biofilms

Current research strategies in the treatment of biofilm infections have focused on dispersal, in which bacteria are made to vacate the extracellular polymeric substance (EPS) surrounding them and return to a planktonic state where antimicrobial treatments are more effective. Glycoside hydrolases (GHs), which cleave bonds in EPS polysaccharides, have been shown to promote dispersal in Pseudomonas aeruginosa biofilms. The dispersal mechanism is possibly due to GHs’ ability to directly release bacteria from the EPS, disrupt EPS’ ability to regulate the environment, or reduce overall mechanical stability. In this work, passive microrheology is used to examine the relevance of the last mechanism by exploring the effects of three GHs (α-amylase, cellulase, and xylanase) known to disperse P. aeruginosa on local biofilm viscoelasticity. Compared to control studies in wild-type strains, it is found that treatment with all three GHs results in statistically relatively less elastic and stiffer biofilms, indicating that changes to mechanical stability may be a factor in effective dispersal. Both cellulase and xylanase were observed to have the greatest impact in creating a less stiff and elastic biofilm; these GHs have been observed to be effective at dispersal in the published results. Each GH was further tested on biofilms grown with strains that produced EPS missing specific polysaccharide components. Cellulase specifically targeted Psl, which forms the major structural and mechanical backbone of the EPS, explaining its efficacy in dispersal. However, xylanase did not appear to exhibit any affinity to any polysaccharide within the EPS based on the microrheology results. Overall, these results suggest that the local microrheology of the biofilms is impacted by GHs and that may be one of the factors that is causing the ability of these therapeutics to enhance dispersal.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Editorial: Phenylpropanoid Systems Biology and Biotechnology

Phenylpropanoids are specialized metabolites involved in several aspects of plant growth and development and in the responses of plants to environmental stimuli. These compounds are synthesized from key intermediates of the shikimate pathway, which are structurally modified by the combined activities of lyases, transferases, ligases, reductases and oxygenases, resulting in the organ- and developmental-specific synthesis and accumulation of diverse metabolites (Vogt, 2010). The phenylpropanoid pathway provides the building blocks for lignin, suberin, and condensed tannins that play a role in structural support and mechanical strength. Lignin is a major contributor to feedstock recalcitrance and negatively affects the conversion of plant biomass into downstream products in biorefineries (Liu et al., 2021). Further, this pathway is key for the production of anthocyanins for organ pigmentation, flavonols and flavones for UV protection, various flavonoids and isoflavonoids for plant-microbe interactions, and antimicrobial phytoalexins for protection against pathogens (Deng and Lu, 2017). In addition to their biological functions in planta, phenylpropanoids are economically important metabolites. They constitute important components in the human diet, acting as nutraceutical compounds with antioxidant, chemopreventive, antimitotic, neuroprotective, cardioprotective, and anti-inflammatory activities. Several phenylpropanoids are considered high-value biochemicals employed in the production of fragrances, pharmaceuticals and biopolymers (Lin and Eudes, 2020).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Weaning transition, but not the administration of probiotic candidate Kazachstania slooffiae , shaped the gastrointestinal bacterial and fungal communities in nursery piglets

As in-feed antibiotics are phased out of swine production, producers are seeking alternatives to facilitate improvements in growth typically seen from this previously common feed additive. Kazachstania slooffiae is a prominent commensal fungus in the swine gut that peaks in relative abundance shortly after weaning and has beneficial interactions with other bacteriome members important for piglet health. In this study, piglets were supplemented with K. slooffiae to characterize responses in piglet health as well as fungal and bacterial components of the microbiome both spatially (along the entire gastrointestinal tract and feces) and temporally (before, during, and after weaning). Litters were assigned to one of four treatments: no K. slooffiae (CONT); one dose of K. slooffiae 7 days before weaning (day 14; PRE); one dose of K. slooffiae at weaning (day 21; POST); or one dose of K. slooffiae 7 days before weaning and one dose at weaning (PREPOST). The bacteriome and mycobiome were analyzed from fecal samples collected from all piglets at day 14, day 21, and day 49, and from organ samples along the gastrointestinal (GI) tract at day 21 and day 49. Blood samples were taken at day 14 and day 49 for cytokine analysis, and fecal samples were assayed for antimicrobial resistance. While some regional shifts were seen in response to K. slooffiae administration in the mycobiome of the GI tract, no remarkable changes in weight gain or health of the animals were observed, and changes were more likely due to sow and the environment. Ultimately, the combined microbiome changed most considerably following the transition from suckling to nursery diets. This work describes the mycobiome along the piglet GI tract through the weaning transition for the first time. Based on these findings, K. slooffiae administered at this concentration may not be an effective tool to hasten colonization of K. slooffiae in the piglet GI tract around the weaning transition nor support piglet growth, microbial gut health, or immunity. However, diet and environment greatly influence microbial community development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Granulibacter bethesdensis, a Pathogen from Patients with Chronic Granulomatous Disease, Produces a Penta-Acylated Hypostimulatory Glycero-D-talo-oct-2-ulosonic Acid–Lipid A Glycolipid (Ko-Lipid A)

Granulibacter bethesdensis can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact G. bethesdensis (Gb) is hypostimulatory compared to Escherichia coli, i.e., cytokine production in human blood requires 10–100 times more G. bethesdensis CFU/mL than E. coli. To better understand the pathogenicity of G. bethesdensis, we isolated its lipopolysaccharide (GbLPS) and characterized its lipid A. Unlike with typical Enterobacteriaceae, the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Manp-(1→4)-β-GlcpN3N-(1→6)-α-GlcpN-(1⇿1)-α-GlcpA tetra-saccharide substituted with five acyl chains: the amide-linked N-3′ 14:0(3-OH), N-2′ 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS that is covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance of GbLPS. In addition, the presence of Ko and only five acyl chains may explain the >10-fold lower proinflammatory potency of GbKo–lipidA compared to E. coli lipid A, as measured by cytokine induction in human blood. These unusual structural properties of the G.bethesdensis Ko–lipid A glycolipid likely contribute to immune evasion during pathogenesis and resistance to antimicrobial peptides.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

High-Throughput Microbial Community Analyses to Establish a Natural Fungal and Bacterial Consortium from Sewage Sludge Enriched with Three Pharmaceutical Compounds

Emerging and unregulated contaminants end up in soils via stabilized/composted sewage sludges, paired with possible risks associated with the development of microbial resistance to antimicrobial agents or an imbalance in the microbial communities. An enrichment experiment was performed, fortifying the sewage sludge with carbamazepine, ketoprofen and diclofenac as model compounds, with the aim to obtain strains with the capability to transform these pollutants. Culturable microorganisms were obtained at the end of the experiment. Among fungi, Cladosporium cladosporioides, Alternaria alternata and Penicillium raistrickii showed remarkable degradation rates. Population shifts in bacterial and fungal communities were also studied during the selective pressure using Illumina MiSeq. These analyses showed a predominance of Ascomycota (Dothideomycetes and Aspergillaceae) and Actinobacteria and Proteobacteria, suggesting the possibility of selecting native microorganisms to carry out bioremediation processes using tailored techniques.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular Regulatory Mechanisms Drive Emergent Pathogenetic Properties of Neisseria gonorrhoeae

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, with an estimated 87 million annual cases worldwide. N. gonorrhoeae predominantly colonizes the male and female genital tract (FGT). In the FGT, N. gonorrhoeae confronts fluctuating levels of nutrients and oxidative and non-oxidative antimicrobial defenses of the immune system, as well as the resident microbiome. One mechanism utilized by N. gonorrhoeae to adapt to this dynamic FGT niche is to modulate gene expression primarily through DNA-binding transcriptional regulators. Here, we describe the major N. gonorrhoeae transcriptional regulators, genes under their control, and how these regulatory processes lead to pathogenic properties of N. gonorrhoeae during natural infection. We also discuss the current knowledge of the structure, function, and diversity of the FGT microbiome and its influence on gonococcal survival and transcriptional responses orchestrated by its DNA-binding regulators. We conclude with recent multi-omics data and modeling tools and their application to FGT microbiome dynamics. Understanding the strategies utilized by N. gonorrhoeae to regulate gene expression and their impact on the emergent characteristics of this pathogen during infection has the potential to identify new effective strategies to both treat and prevent gonorrhea.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland—a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Whole-Genome SNP Analysis Identifies Putative Mycobacterium bovis Transmission Clusters in Livestock and Wildlife in Catalonia, Spain

The high-resolution WGS analyses of MTBC strains have provided useful insight for determining sources of infection for animal tuberculosis. In Spain, tuberculosis in livestock is caused by Mycobacterium bovis and Mycobacterium caprae, where wildlife reservoirs play an important role. We analyzed a set of 125 M. bovis isolates obtained from livestock and wildlife from Catalonia to investigate strain diversity and identify possible sources and/or causes of infection. Whole-genome SNP profiles were used for phylogenetic reconstruction and pairwise SNP distance analysis. Additionally, SNPs were investigated to identify virulence and antimicrobial resistance factors to investigate clade-specific associations. Putative transmission clusters (≤12 SNPs) were identified, and associated epidemiological metadata were used to determine possible explanatory factors for transmission. M. bovis distribution was heterogeneous, with 7 major clades and 21 putative transmission clusters. In order of importance, the explanatory factors associated were proximity and neighborhood, residual infection, livestock-wildlife interaction, shared pasture, and movement. Genes related to lipid transport and metabolism showed the highest number of SNPs. All isolates were pyrazinamide resistant, and five were additionally resistant to isoniazid, but no clade-specific associations could be determined. Our findings highlight the importance of high-resolution molecular surveillance to monitor bovine tuberculosis dynamics in a low-prevalence setting.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Performance of Graphene/Polydimethylsiloxane Surfaces against S. aureus and P. aeruginosa Single- and Dual-Species Biofilms

The increasing incidence of implant-associated infections has prompted the development of effective strategies to prevent biofilm formation on these devices. In this work, pristine graphene nanoplatelet/polydimethylsiloxane (GNP/PDMS) surfaces containing different GNP loadings (1, 2, 3, 4, and 5 wt%) were produced and evaluated on their ability to mitigate biofilm development. After GNP loading optimization, the most promising surface was tested against single- and dual-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. The antibiofilm activity of GNP/PDMS surfaces was determined by the quantification of total, viable, culturable, and viable but nonculturable (VBNC) cells, as well as by confocal laser scanning microscopy (CLSM). Results showed that 5 wt% GNP loading reduced the number of total (57%), viable (69%), culturable (55%), and VBNC cells (85%) of S. aureus biofilms compared to PDMS. A decrease of 25% in total cells and about 52% in viable, culturable, and VBNC cells was observed for P. aeruginosa biofilms. Dual-species biofilms demonstrated higher resistance to the antimicrobial activity of GNP surfaces, with lower biofilm cell reductions (of up to 29% when compared to single-species biofilms). Still, the effectiveness of these surfaces in suppressing single- and dual-species biofilm formation was confirmed by CLSM analysis, where a decrease in biofilm biovolume (83% for S. aureus biofilms and 42% for P. aeruginosa and dual-species biofilms) and thickness (on average 72%) was obtained. Overall, these results showed that pristine GNPs dispersed into the PDMS matrix were able to inhibit biofilm growth, being a starting point for the fabrication of novel surface coatings based on functionalized GNP/PDMS composites.

Oliveira, Isabel M.↗
DOE OSTI2022

Silver Nanoparticles Functionalized Nanosilica Grown over Graphene Oxide for Enhancing Antibacterial Effect

The continuous growth of multidrug-resistant bacteria due to the overuse of antibiotics and antibacterial agents poses a threat to human health. Silver nanoparticles, silica-based materials, and graphene-based materials have become potential antibacterial candidates. In this study, we developed an effective method of enhancing the antibacterial property of graphene oxide (GO) by growing nanosilica (NS) of approximately 50 nm on the graphene oxide (GO) surface. The structures and compositions of the materials were characterized through powdered X-ray diffraction (P-XRD), transmission electron microscopy (TEM), scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS), ultraviolet–visible spectroscopy (UV–VIS), dynamic light scattering (DLS), Raman spectroscopy (RM), Fourier-transform infrared spectroscopy (FTIR), Brunauer–Emmet–Teller (BET) surface area, and pore size determination. The silver nanoparticles (AgNPs) with an average diameter of 26 nm were functionalized on the nanosilica (NS) surface. The composite contained approximately 3% of silver nanoparticles. The silver nanoparticles on nanosilica supported over graphene oxide (GO/NS/AgNPs) exhibited a 7-log reduction of Escherichia coli and a 5.2-log reduction of Bacillus subtilis within one hour of exposure. Both GO/NS and GO/NS/AgNPs exhibited substantial antimicrobial effects against E. coli and B. subtilis.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Diversity in the Common Fold: Structural Insights into Class D β-Lactamases from Gram-Negative Pathogens

Class D β-lactamases (DBLs) represent a major threat to antibiotic efficacy by hydrolyzing β-lactam drugs, including last-resort carbapenems, thereby driving antimicrobial resistance in Gram-negative bacteria. The enzymes share a structurally conserved two-domain α/β architecture with seven active-site motifs and three flexible extended loops (the P-loop, Ω-loop, and newly designated B-loop) that surround the active site. While each of these loops is known to influence enzyme function, their coordinated roles have not been fully elucidated. To investigate the significance of their interplay, we compared the sequences and crystal structures of 40 DBLs from clinically relevant Gram-negative pathogens and performed molecular dynamics simulations on selected representatives. Combined structural and dynamical analyses revealed a strong correlation between B-loop architecture and carbapenemase activity in the pathogens Klebsiella and Acinetobacter, particularly regarding loop length and spatial organization. These findings emphasize the B-loop’s critical contribution, in concert with the P- and Ω-loops, in tuning active site versatility, substrate recognition, catalytic activity, and structural stability. A deeper understanding of how these motifs and loops govern DBL function may inform the development of novel antibiotics and inhibitors targeting this class of enzymes.

antibiotic resistance↗