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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Chicken Production and Human Clinical Escherichia coli Isolates Differ in Their Carriage of Antimicrobial Resistance and Virulence Factors

Contamination of food animal products by Escherichia coli is a leading cause of foodborne disease outbreaks, hospitalizations, and deaths in humans. Chicken is the most consumed meat both in the United States and across the globe according to the U.S. Department of Agriculture. Although E. coli is a ubiquitous commensal bacterium of the guts of humans and animals, its ability to acquire antimicrobial resistance (AMR) genes and virulence factors (VFs) can lead to the emergence of pathogenic strains that are resistant to critically important antibiotics. Thus, it is important to identify the genetic factors that contribute to the virulence and AMR of E. coli. In this study, we performed in-depth genomic evaluation of AMR genes and VFs of E. coli genomes available through the National Antimicrobial Resistance Monitoring System GenomeTrackr database. Our objective was to determine the genetic relatedness of chicken production isolates and human clinical isolates. To achieve this aim, we first developed a massively parallel analytical pipeline (Reads2Resistome) to accurately characterize the resistome of each E. coli genome, including the AMR genes and VFs harbored. We used random forests and hierarchical clustering to show that AMR genes and VFs are sufficient to classify isolates into different pathogenic phylogroups and host origin. We found that the presence of key type III secretion system and AMR genes differentiated human clinical isolates from chicken production isolates. These results further improve our understanding of the interconnected role AMR genes and VFs play in shaping the evolution of pathogenic E. coli strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Efficacy of Cathelicidin-Mimetic Antimicrobial Peptoids against Staphylococcus aureus

Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly-N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (<30 min) killing of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at low micromolar concentrations without having apparent cytotoxic side effects in vivo. Additionally, these novel peptoids show excellent efficacy against biofilm formation and detachment for both MSSA and MRSA. In comparison, conventional antibiotics were unable to detach or prevent formation of biofilms. One cationic 12mer, Peptoid 1, shows great promise, as it could prevent formation of and detach biofilms at concentrations as low as 1.6 μM. The use of a bioluminescent S. aureus murine incision wound model demonstrated clearance of infection in peptoid-treated mice within 8 days, conveying another advantage these peptoids have over conventional antibiotics. These results provide clear evidence of the potential for antimicrobial peptoids for the treatment of S. aureus wound infections.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Functional Biomimetic Polymers with Antimicrobial Activity

At PNNL, we have developed a completely new class of synthetic biomimetic sequence-defined polymers based on triazine chemistry, which we call TZPs. PNNL's new polymers offer the opportunity to achieve similar functions to natural macromolecules; specifically we designed and synthesized a diversity of test molecules for antimicrobial activity. Antimicrobial activity against a variety of wild type and drug resistant pathogens was found.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

In silico analyses of diversity and dissemination of antimicrobial resistance genes and mobile genetics elements, for plasmids of enteric pathogens

The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Hydroxyapatite/TiO 2 Nanomaterial with Defined Microstructural and Good Antimicrobial Properties

Due to the growing number of people infected with the new coronavirus globally, which weakens immunity, there has been an increase in bacterial infections. Hence, knowledge about simple and low-cost synthesis methods of materials with good structural and antimicrobial properties is of great importance. A material obtained through the combination of a nanoscale hydroxyapatite material (with good biocompatibility) and titanium dioxide (with good degradation properties of organic molecules) can absorb and decompose bacteria. In this investigation, three different synthesis routes used to prepare hydroxyapatite/titanium dioxide nanomaterials are examined. The morphology and semiquantitative chemical composition are characterized by scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDX). The obtained materials’ phase and structural characterization are determined using the X-ray powder diffraction method (XRD). The crystallite sizes of the obtained materials are in the range of 8 nm to 15 nm. Based on XRD peak positions, the hexagonal hydroxyapatite phases are formed in all samples along with TiO 2 anatase and rutile phases. According to SEM and TEM analyses, the morphology of the prepared samples differs depending on the synthesis route. The EDX analysis confirmed the presence of Ti, Ca, P, and O in the obtained materials. The IR spectroscopy verified the vibration bands characteristic for HAp and titanium. The investigated materials show excellent antimicrobial and photocatalytic properties.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Cecropin P1 antimicrobial peptide modulates differential expression of immune relevant genes in rainbow trout ( Oncorhynchus mykiss ) gill cell line, RTgill-W1

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 μM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides

The use of non-standard toxicity models is a hurdle in the early development of antimicrobial peptides towards clinical applications. Herein we report an extensive in vitro and in vivo toxicity study of a library of 24 peptide-based antimicrobials with narrow spectrum activity towards veterinary pathogens. The haemolytic activity of the compounds was evaluated against four different species and the relative sensitivity against the compounds was highest for canine erythrocytes, intermediate for rat and human cells and lowest for bovine cells. Selected peptides were additionally evaluated against HeLa, HaCaT and HepG2 cells which showed increased stability towards the peptides. Therapeutic indexes of 50–500 suggest significant cellular selectivity in comparison to bacterial cells. Three peptides were administered to rats in intravenous acute dose toxicity studies up to 2– 8 × MIC. None of the injected compounds induced any systemic toxic effects in vivo at the concentrations employed illustrating that the correlation between the different assays is not obvious. This work sheds light on the in vitro and in vivo toxicity of this class of promising compounds and provides insights into the relationship between the different toxicity models often employed in different manners to evaluate the toxicity of novel bioactive compounds in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Halogenation as a tool to tune antimicrobial activity of peptoids

Antimicrobial peptides have attracted considerable interest as potential new class of antibiotics against multi-drug resistant bacteria. However, their therapeutic potential is limited, in part due to susceptibility towards enzymatic degradation and low bioavailability. Peptoids (oligomers of N-substituted glycines) demonstrate proteolytic stability and better bioavailability than corresponding peptides while in many cases retaining antibacterial activity. In this study, we synthesized a library of 36 peptoids containing fluorine, chlorine, bromine and iodine atoms, which vary by length and level of halogen substitution in position 4 of the phenyl rings. As we observed a clear correlation between halogenation of an inactive model peptoid and its increased antimicrobial activity, we designed chlorinated and brominated analogues of a known peptoid and its shorter counterpart. Short brominated analogues displayed up to 32-fold increase of the activity against S. aureus and 16- to 64-fold against E. coli and P. aeruginosa alongside reduced cytotoxicity. The biological effect of halogens seems to be linked to the relative hydrophobicity and self-assembly properties of the compounds. By small angle X-ray scattering (SAXS) we have demontrated how the self-assembled structures are dependent on the size of the halogen, degree of substitution and length of the peptoid, and correlated these features to their activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Antimicrobial properties of nanostructured surfaces – demonstrating the need for a standard testing methodology

Bioinspired nanostructured materials that exhibit antimicrobial properties are being synthesized and tested at increasing rates for use in healthcare, manufacturing processes, and diagnostics. Although progress has been made in improving and understanding their bactericidal activity, arguably, the biggest problem currently in the field is the lack of a standard testing methodology that allows for optimal characterization and better comparison of emerging nanostructures. In this work, we examine two forms of nanostructured silicon that vary in their ability to kill certain bacterial species due to different physical mechanisms and derive guidelines for the comparative testing. We perform a comprehensive evaluation of methodologies used extensively in the field (e.g., colony counting and live dead analysis) and the novel application of high-throughput flow cytometry. The data reveal how the techniques are complementary but not always directly equivalent or correlative. Therefore, comparison of results obtained using different methodologies on different materials can be grossly misleading. We report significant variations in bactericidal efficiencies depending on experimental environments (medium type, etc.) and methodologies employed. In addition, we demonstrate how cytometry is yet another powerful complementary tool that can aid the mechanistic understanding of antimicrobial activities of rough surfaces. Besides standardization for comparison, ultimately, evaluation methods need to consider anticipated applications. Then and only then can the true potential (or limitation) of a novel material be determined for its suitability for advancement in a particular field of use.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2021

Nanocomplex made up of antimicrobial metallo-supramolecules and model biomembranes – characterization and enhanced fluorescence

Antimicrobial pentatopic 2,2':6',2"-terpyridines that form 3-D supramolecular hexagonal prisms with Cd 2+ through coordination driven self-assembly can be entrapped by lipid discoidal bicelles, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) lipid, forming a well-defined nanocomplex. Structural characterization performed by very small angle neutron scattering, small angle X-ray scattering and transmission electron microscopy suggests that the hexagonal prisms are preferably located at the rim of bicellar discs with the hexagonal face in parallel with the bilayers, instead of face-to-face stacking. Such a configuration reduces the π–π interaction and consequently enhances the fluorescence emission. Since novel supramolecules were reported to have antibiotic functions, in this work we provide insight into the interactions of antimicrobial supermolecules with lipid membranes, leading to potential theranostic applications.

36 MATERIALS SCIENCE↗
DOE OSTI2020

amPEPpy 1.0: a portable and accurate antimicrobial peptide prediction tool

Antimicrobial peptides (AMPs) are promising alternative antimicrobial agents. Currently, however, portable, user-friendly and efficient methods for predicting AMP sequences from genome-scale data are not readily available. In this work, we present amPEPpy, an open-source, multi-threaded command-line application for predicting AMP sequences using a random forest classifier.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Antimicrobial activity of peptoids against Metallo-β-lactamase-producing Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa , and other WHO priority pathogens, including Candida auris

Abstract Aims The World Health Organization has identified ESKAPE bacteria and Candida auris as priority pathogens, emphasizing an urgent need for novel antimicrobials to combat them. This study aimed to explore the therapeutic potential of antimicrobial peptidomimetics, specifically peptoids with sequence-specific N-substituted glycines, against ESKAPEE pathogens, including metallo-β-lactamase (MBL) producers, as well as C. auris strains. Methods and results This study evaluated activity of the peptoids against the multidrug-resistant priority pathogens. The peptoid TM8 (with an N-decyl alkyl chain) demonstrated a geometric mean minimum inhibitory concentration (MIC) of 7.8 μg ml−1 against MBL-producing bacteria, and 5.5 μg ml−1 against C. auris. TM8 showed synergy with ciprofloxacin, enhancing its effectiveness 4-fold against NDM-1-producing Klebsiella pneumoniae. No antagonism was seen when TM8 was used with either conventional antibiotics or antifungals. Peptoids that had therapeutic indices below 3 were generally more hydrophobic, due to either alkyl chains or bromine. Scanning electron microscopy and live-dead staining assay on peptoid-treated C. auris confirmed morphological changes and killing activity, respectively. Furthermore, the peptoid could effectively inhibit biofilm formation by C. auris. Conclusion Peptoids demonstrated antibacterial activity against ESKAPEE, particularly against MBL-producing Gram-negative bacteria. Additionally, they exhibited antifungal and anti-biofilm activities against C. auris strains.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Designing Antifouling and Antimicrobial Interfaces: Structural Characterization using CryoEM, Automated Microscopy, and AI Image Segmentation

The design of functionalized surfaces for interactions with biological systems is critical across sectors such as healthcare, energy, and agriculture. Tailoring materials for specific applications, such as antifouling and antimicrobial surfaces, demands a comprehensive understanding of topology and chemistry across multiple length and time scales on both biological and materials systems. This work presents the development and characterization of nanostructured surfaces with controlled topographies and chemistries that enhance bacterial membrane disruption, reduce biofilm formation, and improve antimicrobial and antifouling capabilities. Two specific use cases will be presented - the use of cellulose nanocrystals (CNCs) for bacterial growth inhibition and the development of antifouling surfaces to prevent protein and bacterial adsorption [1-4]. By leveraging large language models (LLMs) for image segmentation and training [5], we enable automated analysis of terabyte-scale cryogenic electron microscopy (cryoEM) datasets. This analysis provides statistical insights into the biotic/abiotic interface and facilitates automated electron microscopy experiments to mitigate time and dose. The integration of cryogenic electron tomography (cryoET) and cryogenic focused ion beam (cryoFIB) milling enables high-resolution, near-native-state imaging and 3D reconstructions of bio/material interfaces [6]. Orthogonal characterization techniques and computational modeling further enhances our understanding, offering a robust platform for the design and optimization of next-generation functional surfaces [7].

Williams, Alexis [ORNL] (ORCID:0000000252835822)↗
DOE OSTI2021

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. Here we provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Transcriptomics reveal a mechanism of niche defense: two beneficial root endophytes deploy an antimicrobial GH18-CBM5 chitinase to protect their hosts

Effector secretion is crucial for root endophytes to establish and protect their ecological niche. We used time-resolved transcriptomics to monitor effector gene expression dynamics in two closely related Sebacinales, Serendipita indica and Serendipita vermifera, during symbiosis with three plant species, competition with the phytopathogenic fungus Bipolaris sorokiniana, and cooperation with root-associated bacteria. We observed increased effector gene expression in response to biotic interactions, particularly with plants, indicating their importance in host colonization. Some effectors responded to both plants and microbes, suggesting dual roles in intermicrobial competition and plant–microbe interactions. A subset of putative antimicrobial effectors, including a GH18-CBM5 chitinase, was induced exclusively by microbes. Functional analyses of this chitinase revealed its antimicrobial and plant-protective properties. We conclude that dynamic effector gene expression underpins the ability of Sebacinales to thrive in diverse ecological niches with a single fungal chitinase contributing substantially to niche defense.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes bla NDM-5 (1.10%, 2/181), mcr -1 (1.10%, 2/181), tet (X4) (1.10%, 2/181), and cfr (6.08%, 11/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified ast A (37.02%, 67/181), hly A (1.66%, 3/181), hly B (1.66%, 3/181) and hly D (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.

Zhou, Wei↗
DOE OSTI2021

Atomic Force Microscopy to Characterize Antimicrobial Peptide-Induced Defects in Model Supported Lipid Bilayers

Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such collateral hemolytic damage hinders the potential therapeutic applicability of the AMP as an anti-microbial. Elucidating the mechanisms underlying the AMP–membrane interactions is challenging due to the variations in the chemical and structural features of the AMPs, the complex compositional variations of cell membranes and the inadequacy of any single experimental technique to comprehensively probe them. (1) Background: Atomic Force Microscopy (AFM) imaging can be used in combination with other techniques to help understand how AMPs alter the orientation and structural organization of the molecules within cell membranes exposed to AMPs. The structure, size, net charge, hydrophobicity and amphipathicity of the AMPs affect how they interact with cell membranes of differing lipid compositions. (2) Methods: Our study examined two different types of AMPs, a 20-amino acid, neutral, α-helical (amphipathic) peptide, alamethicin, and a 13-amino acid, non-α-helical cationic peptide, indolicidin (which intramolecularly folds, creating a hydrophobic core), for their interactions with supported lipid bilayers (SLBs). Robust SLB model membranes on quartz supports, incorporating predominantly anionic lipids representative of bacterial cells, are currently not available and remain to be developed. Therefore, the SLBs of zwitterionic egg phosphatidylcholine (PC), which represents the composition of a mammalian cell membrane, was utilized as the model membrane. This also allows for a comparison with the results obtained from the Quartz Crystal Microbalance with Dissipation (QCM-D) experiments conducted for these peptides interacting with the same zwitterionic SLBs. Further, in the case of alamethicin, because of its neutrality, the lipid charge may be less relevant for understanding its membrane interactions. (3) Results: Using AFM imaging and roughness analysis, we found that alamethicin produced large, unstable defects in the membrane at 5 µM concentrations, and completely removed the bilayer at 10 µM. Indolicidin produced smaller holes in the bilayer at 5 and 10 µM, although they were able to fill in over time. The root-mean-square (RMS) roughness values for the images showed that the surface roughness caused by visible defects peaked after peptide injection and gradually decreased over time. (4) Conclusions: AFM is useful for helping to uncover the dynamic interactions between different AMPs and cell membranes, which can facilitate the selection and design of more efficient AMPs for use in therapeutics and antimicrobial applications.

Swana, Kathleen W.↗
DOE OSTI2024

Genetic Complexity of CC5 Staphylococcus aureus Isolates Associated with Sternal Bursitis in Chickens: Antimicrobial Resistance, Virulence, Plasmids, and Biofilm Formation

Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of Staphylococcus aureus isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 S. aureus isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems.

Silva, Vanessa (ORCID:0000000194068433)↗