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At least 181 records · Page 10

Extreme Ionizing-Radiation-Resistant Bacterium

There is a growing concern that desiccation and extreme radiation-resistant, non-spore-forming microorganisms associated with spacecraft surfaces can withstand space environmental conditions and subsequent proliferation on another solar body. Such forward contamination would jeopardize future life detection or sample return technologies. The prime focus of NASA s planetary protection efforts is the development of strategies for inactivating resistance-bearing micro-organisms. Eradi cation techniques can be designed to target resistance-conferring microbial populations by first identifying and understanding their physiologic and biochemical capabilities that confers its elevated tolerance (as is being studied in Deinococcus phoenicis, as a result of this description). Furthermore, hospitals, food, and government agencies frequently use biological indicators to ensure the efficacy of a wide range of radiation-based sterilization processes. Due to their resistance to a variety of perturbations, the nonspore forming D. phoenicis may be a more appropriate biological indicator than those currently in use. The high flux of cosmic rays during space travel and onto the unshielded surface of Mars poses a significant hazard to the survival of microbial life. Thus, radiation-resistant microorganisms are of particular concern that can survive extreme radiation, desiccation, and low temperatures experienced during space travel. Spore-forming bacteria, a common inhabitant of spacecraft assembly facilities, are known to tolerate these extreme conditions. Since the Viking era, spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Members of the non-sporeforming bacterial community such as Deinococcus radiodurans can survive acute exposures to ionizing radiation (5 kGy), ultraviolet light (1 kJ/m2), and desiccation (years). These resistive phenotypes of Deinococcus enhance the potential for transfer, and subsequent proliferation, on another solar body such as Mars and Europa. These organisms are more likely to escape planetary protection assays, which only take into account presence of spores. Hence, presences of extreme radiation-resistant Deinococcus in the cleanroom facility where spacecraft are assembled pose a serious risk for integrity of life-detection missions. The microorganism described herein was isolated from the surfaces of the cleanroom facility in which the Phoenix Lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. This bacterium exhibits very low 16SrRNA similarity with any other environmental isolate reported to date. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Deinococcus and represents a novel species. The name Deinococcus phoenicis was proposed after the Phoenix spacecraft, which was undergoing assembly, testing, and launch operations in the spacecraft assembly facility at the time of isolation. D. phoenicis cells exhibited higher resistance to ionizing radiation (cobalt-60; 14 kGy) than the cells of the D. radiodurans (5 kGy). Thus, it is in the best interest of NASA to thoroughly characterize this organism, which will further assess in determining the potential for forward contamination. Upon the completion of genetic and physiological characteristics of D. phoenicis, it will be added to a planetary protection database to be able to further model and predict the probability of forward contamination.

Vaishampayan, Parag A.↗

Diazotrophic Bacteria and Their Mechanisms to Interact and Benefit Cereals

Plant-growth-promoting bacteria (PGPB) stimulate plant growth through diverse mechanisms. In addition to biological nitrogen fixation, diazotrophic PGPB can improve nutrient uptake efficiency from the soil, produce and release phytohormones to the host, and confer resistance against pathogens. The genetic determinants that drive the success of biological nitrogen fixation in nonlegume plants are understudied. These determinants include recognition and signaling pathways, bacterial colonization, and genotype specificity between host and bacteria. This review presents recent discoveries of how nitrogen-fixing PGPB interact with cereals and promote plant growth. We suggest adopting an experimental model system, such as the Setaria–diazotrophic bacteria association, as a reliable way to better understand the associated mechanisms and, ultimately, increase the use of PGPB inoculants for sustainable agriculture. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Biochemistry & Molecular Biology↗

Understanding the dynamic interactions of root-knot nematodes and their host: role of plant growth promoting bacteria and abiotic factors

Root-knot nematodes (Meloidogyne spp., RKN) are among the most destructive endoparasitic nematodes worldwide, often leading to a reduction of crop growth and yield. Insights into the dynamics of host-RKN interactions, especially in varied biotic and abiotic environments, could be pivotal in devising novel RKN mitigation measures. Plant growth-promoting bacteria (PGPB) involves different plant growth-enhancing activities such as biofertilization, pathogen suppression, and induction of systemic resistance. We summarized the up-to-date knowledge on the role of PGPB and abiotic factors such as soil pH, texture, structure, moisture, etc. in modulating RKN-host interactions. RKN are directly or indirectly affected by different PGPB, abiotic factors interplay in the interactions, and host responses to RKN infection. We highlighted the tripartite (host-RKN-PGPB) phenomenon with respect to (i) PGPB direct and indirect effect on RKN-host interactions; (ii) host influence in the selection and enrichment of PGPB in the rhizosphere; (iii) how soil microbes enhance RKN parasitism; (iv) influence of host in RKN-PGPB interactions, and (v) the role of abiotic factors in modulating the tripartite interactions. Furthermore, we discussed how different agricultural practices alter the interactions. Finally, we emphasized the importance of incorporating the knowledge of tripartite interactions in the integrated RKN management strategies.

59 BASIC BIOLOGICAL SCIENCES↗

Extreme Ionizing-Radiation-Resistant Bacterium

There is a growing concern that desiccation and extreme radiation-resistant, non-spore-forming microorganisms associated with spacecraft surfaces can withstand space environmental conditions and subsequent proliferation on another solar body. Such forward contamination would jeopardize future life detection or sample return technologies. The prime focus of NASA s planetary protection efforts is the development of strategies for inactivating resistance-bearing microorganisms. Eradification techniques can be designed to target resistance-conferring microbial populations by first identifying and understanding their physiologic and biochemical capabilities that confers its elevated tolerance (as is being studied in Deinococcus phoenicis, as a result of this description). Furthermore, hospitals, food, and government agencies frequently use biological indicators to ensure the efficacy of a wide range of radiation- based sterilization processes. Due to their resistance to a variety of perturbations, the non-spore forming D. phoenicis may be a more appropriate biological indicator than those currently in use. The high flux of cosmic rays during space travel and onto the unshielded surface of Mars poses a significant hazard to the survival of microbial life. Thus, radiation-resistant microorganisms are of particular concern that can survive extreme radiation, desiccation, and low temperatures experienced during space travel. Spore-forming bacteria, a common inhabitant of spacecraft assembly facilities, are known to tolerate these extreme conditions. Since the Viking era, spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Members of the non-spore-forming bacterial community such as Deinococcus radiodurans can survive acute exposures to ionizing radiation (5 kGy), ultraviolet light (1 kJ/sq m), and desiccation (years). These resistive phenotypes of Deinococcus enhance the potential for transfer, and subsequent proliferation, on another solar body such as Mars and Europa. These organisms are more likely to escape planetary protection assays, which only take into account presence of spores. Hence, presences of extreme radiation-resistant Deinococcus in the cleanroom facility where spacecraft are assembled pose a serious risk for integrity of life-detection missions. The microorganism described herein was isolated from the surfaces of the cleanroom facility in which the Phoenix Lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. This bacterium exhibits very low 16SrRNA similarity with any other environmental isolate reported to date. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Deinococcus and represents a novel species. The name Deinococcus phoenicis was proposed after the Phoenix spacecraft, which was undergoing assembly, testing, and launch operations in the spacecraft assembly facility at the time of isolation. D. phoenicis cells exhibited higher resistance to ionizing radiation (cobalt-60; 14 kGy) than the cells of the D. radiodurans (5 kGy). Thus, it is in the best interest of NASA to thoroughly characterize this organism, which will further assess in determining the potential for forward contamination. Upon the completion of genetic and physiological characteristics of D. phoenicis, it will be added to a planetary protection database to be able to further model and predict the probability of forward contamination.

Vaishampayan, Parag A.↗

Systematic discovery of pseudomonad genetic factors involved in sensitivity to tailocins

Tailocins are bactericidal protein complexes produced by a wide variety of bacteria that kill closely related strains and may play a role in microbial community structure. Thanks to their high specificity, tailocins have been proposed as precision antibacterial agents for therapeutic applications. Compared to tailed phages, with whom they share an evolutionary and morphological relationship, bacterially produced tailocins kill their host upon production but producing strains display resistance to self-intoxication. Though lipopolysaccharide (LPS) has been shown to act as a receptor for tailocins, the breadth of factors involved in tailocin sensitivity, and the mechanisms behind resistance to self-intoxication, remain unclear. Here, we employed genome-wide screens in four non-model pseudomonads to identify mutants with altered fitness in the presence of tailocins produced by closely related pseudomonads. Our mutant screens identified O-antigen composition and display as most important in defining sensitivity to our tailocins. In addition, the screens suggest LPS thinning as a mechanism by which resistant strains can become more sensitive to tailocins. Furthermore, we validate many of these novel findings, and extend these observations of tailocin sensitivity to 130 genome-sequenced pseudomonads. This work offers insights into tailocin–bacteria interactions, informing the potential use of tailocins in microbiome manipulation and antibacterial therapy.

59 BASIC BIOLOGICAL SCIENCES↗

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB

ABSTRACT Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

59 BASIC BIOLOGICAL SCIENCES↗

Prevalence of erythromycin-resistant emm92 -type invasive group A streptococcal infections among injection drug users in West Virginia, United States, 2021–23

Abstract Background Increasing incidence of invasive group A Streptococcus (iGAS) disease has been reported in Europe and the USA over the past several years. Coupled with this are observations of higher rates of resistance to erythromycin and clindamycin. Objectives To characterize iGAS and pharyngitis isolates from West Virginia (WV), a US state outside of the national Active Bacteria Core surveillance purview, where risk factors associated with iGAS infections are prevalent. Methods Seventy-seven invasive group A Streptococcus isolates were collected from 67 unique patients at the J.W. Ruby Memorial Hospital Clinical Microbiology Laboratory in WV from 2021 to 2023. Invasive isolates and 20 unique pharyngitis isolates were tested for clindamycin and erythromycin susceptibility in the clinical laboratory. Patient demographic and clinical information was retrieved from patient electronic health records. Isolates were further characterized based on emm subtype and detection of MLSB resistance determinants. Results Twenty-six (39%) isolates were of a single emm92 type. All emm92 isolates were uniformly erythromycin/clindamycin resistant with inducible or constitutive MLSB resistance imparted by the plasmid-borne erm(T) gene. The majority of emm92 infections were associated with adult patients who reported IV drug use, whereas no pharyngitis infections were caused by an emm92 strain. Overall, 51 (76%) of the 67 iGAS isolates were determined to carry MLSB resistance. Conclusions Isolates of emm92 type (clonal subtype emm92.0) were associated with iGAS infections in adult IV drug users, but not with paediatric pharyngitis, and were uniformly resistant to erythromycin and clindamycin.

Powell, Lillie M.↗

Self-Assembling and Pore-Forming Peptoids as Antimicrobial Biomaterials

Bacterial infections have been a serious threat to mankind throughout history. Natural antimicrobial peptides (AMPs) and their membrane-disruption mechanism have generated an immense interest in the design and development of synthetic mimetics that could overcome the intrinsic drawbacks of AMPs, such as their susceptibility to proteolytic degradation. Herein, by exploiting the self-assembly and pore-forming capabilities of sequence-defined peptoids, we discovered a new family of low molecular weight peptoid antibiotics that exhibit excellent broad-spectrum activity and high selectivity toward a panel of clinically significant Gram-positive and Gram-negative bacterial strains, including vancomycin-resistant E. faecalis (VREF), methicillin-resistant S. aureus (MRSA), methicillin-resistant S. epidermidis (MRSE), E. coli, P. aeruginosa, and K. pneumoniae. Tuning peptoid sidechain chemistry and structure enabled us to tune the efficacy of antimicrobial activity. Mechanistic studies using Transmission Electron Microscopy (TEM), bacterial membrane depolarization and lysis, and time-kill kinetics assays along with molecular dynamics simulations reveal that these peptoids kill both Gram-positive and Gram-negative bacteria through a membrane-disruption mechanism. In conclusion, these robust and biocompatible peptoid-based antibiotics can provide a valuable tool for combating the emerging drug resistance.

59 BASIC BIOLOGICAL SCIENCES↗

Disinfection susceptibility of waterborne pseudomonads and Legionellae under simulated space vehicle conditions

The sensitivity of waterborne bacteria from iodinated systems to iodine is examined with particular attention to the recovery of the organisms. The use of iodine as a disinfectant for space-vehicle water is described, and references are made to studies of iodine sensitivity and the relationship between growth rate and iodine sensitivity. Growth following iodination is discussed, and bacterial responses to nutrient restriction are examined for both P aeruginosa and Legionella pneumophila. The low level of organic nutrients in spacecraft water allows the selection for bacteria that are less sensitive to halogens. The formation of biofilms within the water-treatment system enhances bacterial resistance to iodine, and in the case of high-quality water it is shown that sublethal doses of iodine can stimulate bacterial growth. Water treatment should therefore be based on antecedent growth conditions, nutrient limitation, biofilm formation, and ambient selective pressures.

Mcfeters, Gordon A.↗

N-terminal domain swapping: A new paradigm for spermidine/spermine N -acetyltransferase (SSAT) protein structures?

Enterococcus faecalis is a multi-drug-resistant human pathogen that is found in a variety of environments and is challenging to treat. Under stress conditions, some bacteria regulate intracellular polyamine concentrations via polyamine acetyltransferases to reduce their toxicity. The E. faecalis genome encodes two polyamine acetyltransferases: PmvE and BltD. Both of these proteins belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily. It is unclear why there are two enzymes with similar substrate specificities in this organism. To better understand the structure/function relationship of the E. faecalis BltD enzyme, we determined its crystal structure and performed additional assays to explore its oligomeric state and enzymatic activity. The goal was to determine whether there were structural or catalytic differences between this enzyme and other polyamine acetyltransferases that could explain this redundancy and be exploited for future development of targeted inhibitors for this important human pathogen. We found the BltD enzyme was structurally unique due to its N-terminal domain swapped dimer. However, this enzyme adopts a catalytically active monomer rather than dimer in solution. This indicates the crystal structure we obtained may represent a state that forms at high protein and salt concentrations and at low pH used during crystallization. The BltD dimer found in the crystal may represent a unique view of how an inhibitory peptide or molecule could be designed to occupy its active site. Additionally, this structure shows the extensive flexibility of the N-terminal portion of the E. faecalis BltD enzyme.

59 BASIC BIOLOGICAL SCIENCES↗

Cryo-electron microscopy structure of a zinc uptake ABC transporter

Zn2+ is an essential micronutrient required for many biological processes. Microorganisms employ high-affinity Zn2+ ABC transporters to take up zinc from the environment when it is scarce, yet the mechanism of zinc uptake and its regulation remain poorly understood. Here, we report the cryo-electron microscopy structure of the Zn2+ ABC transporter complex ZnuB-ZnuC from Escherichia coli. It contains two ZnuB transport and two ZnuC regulatory subunits. The ZnuB homodimer is in an outward-facing, closed conformation with a large hydrophilic cavity at the dimer interface. Each ZnuC subunit contains an N-terminal nucleotide-binding domain and a C-terminal zinc-sensing domain (ZSD). Zn2+ binding to the ZSD locks the transporter in a closed state, whereas under low-Zn2+ conditions, the disorder of ZSD permits ATP-driven zinc uptake. Zn2+ ABC transporters are ubiquitously utilized by pathogenic bacteria to compete with hosts for Zn2+. These findings reveal potential therapeutic targets for disrupting Zn2+ homeostasis in antibiotic-resistant pathogens.

59 BASIC BIOLOGICAL SCIENCES↗

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↗

C6 Hydroxymethyl-Substituted Carbapenem MA-1-206 Inhibits the Major Acinetobacter baumannii Carbapenemase OXA-23 by Impeding Deacylation

Acinetobacter baumannii has become a major nosocomial pathogen, as it is often multidrug-resistant, which results in infections characterized by high mortality rates. The bacterium achieves high levels of resistance to β-lactam antibiotics by producing β-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of b-lactam antibiotics have been the drugs of choice for treating A. baumannii infections. However, their effectiveness has been significantly diminished due to the pathogen’s production of carbapenem-hydrolyzing class D β-lactamases (CHDLs); thus, new antibiotics and inhibitors of these enzymes are urgently needed. Here, we describe a new carbapenem antibiotic, MA-1-206, in which the canonical C6 hydroxyethyl group has been replaced with hydroxymethyl. The antimicrobial susceptibility studies presented here demonstrated that this compound is more potent than meropenem and imipenem against A. baumannii producing OXA-23, the most prevalent CHDL of this pathogen, and also against strains producing the CHDL OXA-24/40 and the class B metallo-β-lactamase VIM-2. Our kinetic and mass spectrometry studies revealed that this drug is a reversible inhibitor of OXA-23, where inhibition takes place through a branched pathway. X-ray crystallographic studies, molecular docking, and molecular dynamics simulations of the OXA-23-MA-1-206 complex show that the C6 hydroxymethyl group forms a hydrogen bond with the carboxylated catalytic lysine of OXA-23, effectively preventing deacylation. These results provide a promising strategy for designing a new generation of CHDL-resistant carbapenems to restore their efficacy against deadly A. baumannii infections. Carbapenem antibiotics are the drugs of choice for treatment of deadly infections caused by Gram-negative bacteria. However, their efficacy is severely compromised by the wide spread of carbapenem-hydrolyzing class D β-lactamases (CHDLs). The importance of this research is the discovery that substitution of the canonical hydroxyethyl group of carbapenems by a hydroxymethyl significantly enhances stability against inactivation by the major CHDL of Acinetobacter baumannii, OXA-23. These results provide a novel strategy for designing next-generation, carbapenemase-stable carbapenems to fight multidrug-resistant infections caused by Gram-negative pathogens

59 BASIC BIOLOGICAL SCIENCES↗

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Hagen, Live↗

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↗

Molecular dynamics simulations of the secondary-binding site in disaccharide-modified glycopeptide antibiotics

Oritavancin is a semisynthetic glycopeptide antibiotic used to treat severe infections by multidrug-resistant Gram-positive pathogens. Oritavancin is known to be a thousand times more potent than vancomycin against Gram-positive bacteria due to the additional interactions with bacterial peptidoglycan (PG) facilitated by a secondary-binding site. The presence of this secondary-binding site is evident in desleucyl-oritavancin, an Edman degradation product of oritavancin, still retaining its potency against Gram-positive bacteria, whereas desleucyl-vancomycin is devoid of any antimicrobial activities. Herein, using explicit solvent molecular dynamics (MD) simulations, steered MD simulations, and umbrella sampling, we show evidence of a secondary-binding site mediated by the disaccharide-modified hydrophobic sidechain of oritavancin interactions with the pentaglycyl-bridge segment of the PG. The interactions were characterized through comparison to the interaction of PG with chloroeremomycin, vancomycin, and the desleucyl analogs of the glycopeptides. Our results show that the enhanced binding of oritavancin to PG over the binding of the other complexes studied is due to an increase in the hydrophobic effect, electrostatic and van der Waals interactions, and not the average number of hydrogen bonds. Our ranking of the binding interactions of the biomolecular complexes directly correlates with the order based on their experimental minimum inhibitory concentrations. The results of our simulations provide insight into the modification of glycopeptides to increase their antimicrobial activities or the design of novel antibiotics against pathogenic Gram-positive bacteria.

60 APPLIED LIFE SCIENCES↗

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

NASA Discipline Number 04-10↗

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

NASA Discipline Plant Biology↗