Log-phase Cultures Research Articles

Lipidomic Analysis Reveals Branched-Chain and Cyclic Fatty Acids from Angomonas deanei Grown under Different Nutritional and Physiological Conditions.

Angomonas deanei belongs to Trypanosomatidae family, a family of parasites that only infect insects. It hosts a bacterial endosymbiont in a mutualistic relationship, constituting an excellent model for studying organelle origin and cellular evolution. A lipidomic approach, which allows for a comprehensive analysis of all lipids in a biological system (lipidome), is a useful tool for identifying and measuring different expression patterns of lipid classes. The present study applied GC-MS and NMR techniques, coupled with principal component analysis (PCA), in order to perform a comparative lipidomic study of wild and aposymbiotic A. deanei grown in the presence or absence of FBS. Unusual contents of branched-chain iso C17:0 and C19:0-cis-9,10 and-11,12 fatty acids were identified in A. deanei cultures, and it was interesting to note that their content slightly decreased at the log phase culture, indicating that in the latter growth stages the cell must promote the remodeling of lipid synthesis in order to maintain the fluidity of the membrane. The combination of analytical techniques used in this work allowed for the detection and characterization of lipids and relevant contributors in a variety of A. deanei growth conditions.

A Comprehensive Approach Combining Short-Chain Polyphosphate and Bacterial Biostimulants for Effective Nutrient Solubilization and Enhanced Wheat Growth.

Phosphorus constitutes a crucial macronutrient for crop growth, yet its availability often limits food production. Efficient phosphorus management is crucial for enhancing crop yields and ensuring food security. This study aimed to enhance the efficiency of a short-chain polyphosphate (PolyP) fertilizer by integrating it with plant growth-promoting bacteria (PGPB) to improve nutrient solubilization and wheat growth. Specifically, the study investigated the effects of various bacterial strains on wheat germination and growth when used in conjunction with PolyP. To achieve this, a greenhouse experiment was conducted in which the wheat rhizosphere was amended with a short-chain PolyP fertilizer. Based on the morphological aspect, eight bacteria, designated P1 to P8, were isolated and further characterized. Plant growth-promoting traits were observed in all bacterial strains, as they presented the ability to produce Indole Acetic Acid (IAA) in significant amounts ranging from 7.5 ± 0.3 µg/mL to 44.1 ± 2 µg/mL, expressed by B. tropicus P4 and P. soyae P1, respectively. They also produced ammonia, hydrogen cyanide (HCN), and siderophores. Their effect against the plant pathogen Fusarium culmorum was also assessed, with P. reinekei P2 demonstrating the highest biocontrol activity as it presented a total inhibitory effect. Additionally, some strains exhibited the ability to solubilize/hydrolyze phosphorus, potassium, and zinc. In vivo, the initial growth potential of wheat seeds indicated that those inoculated with the isolated strains exhibited elevated germination rates and enhanced root growth. Based on their plant growth-promoting traits and performance in the germination assay, three strains were selected for producing the best results, specifically phosphorus hydrolyzation/solubilization, zinc solubilization, IAA production, HCN, and siderophores production. Wheat seeds were inoculated by drenching in a bacterial suspension containing 1010 CFU/mL of log phase culture, and an in planta bioassay was conducted in a growth chamber using three selected strains (Pseudomonas soyae P1, Pseudomonas reinekei P2, and Bacillus tropicus P4), applied either individually or with PolyP on a P-deficient soil (28 mg/kg of P Olsen). Our findings demonstrated that the combination of Pseudomonas soyae P1 and PolyP achieved the highest shoot biomass, averaging 41.99 ± 0.87 g. Notably, applying P. soyae P1 or Bacillus tropicus P4 alone yielded similar results to the use of PolyP alone. At the heading growth stage, the combination of Bacillus tropicus P4 and PolyP significantly increased the Chlorophyll Content Index (CCI) to 37.02 µmol/m2, outperforming both PolyP alone (24.07 µmol/m2) and the control (23.06 µmol/m2). This study presents an innovative approach combining short-chain PolyP with bacterial biostimulants to enhance nutrient availability and plant growth. By identifying and characterizing effective bacterial strains, it offers a sustainable alternative to conventional fertilizers.

Effect of physicochemical and microbiological factors on the development of viable but non-culturable and resuscitation states of Vibrio cholerae.

Vibrio cholerae can endure harsh environmental conditions by transitioning into viable but non-culturable (VBNC) form and resuscitate upon return of appropriate conditions. In this study, we assessed the impact of physicochemical and microbiological factors, on the development of low temperature-induced VBNC state and subsequent recovery by temperature upshift. In estuarine water, Vibrio cholerae exhibits a slower decline in culturability over a period of 77 days as compared to 10 days in fresh water. When variable cell numbers from different growth phases were used for VBNC induction, it was observed that the higher inoculum size (106-107 cfu ml-1) from the late log phase culture appears to be crucial for entering the VBNC state. Conversely, starved cells could enter the VBNC state with an initial inoculum of 104-105 cfu ml-1, followed by resuscitation as well. The addition of glucose, GlcNAc and mannitol differentially affects progression into VBNC, while the addition of tryptone, yeast extract and casamino acid facilitated early entry into the VBNC state and shortened the length of the recovery period. Altogether these findings demonstrated that the ionic strength of water, inoculum size and the availability of nutrients played distinct roles during VBNC induction and resuscitation.

Experimental Susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis (Psychodidae: Phlebotominae) to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi (Trypanosomatidae: Leishmaniinae).

The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi-Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis-parasite combinations, as well with Lu. longipalpis-uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi.

Mono and bilayer coatings of alkanethiol and silane on copper: Prevents corrosion and regulate bacterial adhesion

Self-assembling molecules (SAMs) provide a flexible system for surface modification of materials by forming films of molecular thickness. SAMs protect materials from corrosion and modify surface properties such as adhesion, adsorption and wetting due to their molecular composition and orientation. In this investigation, polished copper specimens were dip coated to form SAMs of alkanethiol and silane to study bacterial adhesion as well as for corrosion prevention. The SAM coated specimens were exposed to the log phase cultures of Pseudomonas aeruginosa (aerobic bacteria) and Desulfovibrio vulgaris (anaerobic bacteria) for 12, 24 and 48 hrs, to monitor cell adhesion and biofilm thickness by microscopy. The results showed that alkanethiols SAM promoted adhesion and biofilm formation. The biofilm thickness ranged from 10 to 38 µm for Pseudomonas and 9 – 25 µm for Desulfovibrio sp. Among the SAM coatings tested, octadecyl-trichlorosilane (ODTCS), alkane-thiol and ODTCS (bilayer SAM) showed no bacterial adhesion, however, solitary alkane-thiol SAM showed significant bacterial adhesion. The high hydrophobicity (contact angle ∼140°), antibacterial and anti-biofilm property of the ODTCS coating could have regulated bacterial adhesion. The constant climate test results showed that silane SAM prevented copper corrosion up to ∼160 days (3480 hrs), while the alkanethiol coating could protect up to 10 days (240 hrs). Confocal laser scanning microscopic images of the silane coated copper specimens showed initiation of the corrosion after 160 days of exposure, while at the end of 200 days significant copper corrosion was observed. Surface modification leads to specific biochemical interactions that regulate bacterial adhesion and prevent biofilm formation and corrosion of copper.

The effects of palladium coordination complex speciation and concentration upon the ubiquitous bacterial species Pseudomonas aeruginosa

The toxicity of three different palladium (Pd) species to Pseudomonas aeruginosa, an environmentally ubiquitous bacterial species, is reported. Palladium was added to chemically-defined minimal media as three complex ion salts, namely sodium tetrachloropalladate (Na2[PdCl4]), tetraamminepalladium(II) chloride ([Pd(NH3)4]Cl2), and potassium hexachloropalladate(IV) (K2[PdCl6]), inoculated with log-phase cultures and incubated for 24 h at 25 °C. Toxicity was tested for Pd concentrations ranging from 6.55 μg/L (0.06 μM Pd) to 250 μg/L (2.33 μM Pd). Minimum inhibitory concentrations (MICs) were determined and growth tracked via optical absorption at 600 nm. Viability and minimum bactericidal concentrations (MBCs) were measured in parallel with dilution, plating and colony forming unit (CFU) counting. MICs for all forms of Pd were 62.5 μg Pd/L, approximately 1000 times lower than previously reported values. The MBCs for PdCl42- and Pd(NH3)42+ were 62.5 μg Pd/L and 125 μg Pd/L for PdCl62-. Pd(NH3)42+ and PdCl62- culture viability at 7.8–31.3 μg Pd/L was not different from controls. However, PdCl42- culture viability was different from the other additives, with decreasing viability at sub-MBC concentrations down to 6.55 μg Pd/L. To understand the possible effect of speciation upon toxicity, the equilibrium speciation of Pd was modeled for all solutions using PHREEQC and found to be dominated by Pd(NH3)3Cl+ (65.6 % of total Pd) and Pd(NH3)42+ (34.2 % total Pd). The juxtaposition of the equilibrium calculations and the toxicity results indicates that the kinetics of ligand exchange between the palladium complexes and the growth medium could influence bacterial response.

590. Activities of Cefepime-Taniborbactam and Ceftazidime-Avibactam against Cefepime-Resistant Respiratory Gram-Negative Pathogens in a Hollow Fiber Infection Model

Abstract Background Cefepime-taniborbactam (FTB) combines cefepime (FEP), a fourth generation cephalosporin with taniborbactam, a novel inhibitor of metallo (MBL)- and serine β-lactamases (SBL). FTB 2.5g IV q8h was safe and effective in adults with complicated urinary tract infection in a Phase 3 trial (NCT03840148). FTB is also under development for hospital-acquired and ventilator-associated bacterial pneumonia (HABP/VABP). Methods An in vitro hollow fiber infection model (HFM) was used to assess resistance emergence in MBL- and/or SBL-producing Klebsiella pneumoniae (KP, n=5) and Pseudomonas aeruginosa (PA, n=3) treated with humanized exposures of FTB or ceftazidime-avibactam (CZA). Dense (≥ 7 log10 CFU/mL) log phase cultures were inoculated into HFM cartridges and treated with human equivalent doses of FEP (2g q8h), FTB (2.5g q8h), or CZA (2.5g q8h) for 4 days. KP strains collectively harbored NDM (n=2), VIM (n=1), CTX-M (n=4), SHV-ESBL (n=1), CMY (n=1), KPC (n=1), and OXA-48 (n=1). PA strains produced either VIM (n=1), CTX-M (n=1), or KPC (n=1). Pharmacokinetic profiles of FTB and CZA in HFMs, based on free drug exposures in plasma of healthy volunteers, were confirmed by LC-MS/MS. For CZA HFMs with MBL-producing strains, EDTA was added to sequester zinc to restore CZA susceptibility (MIC ≤ 8 mg/L). Viable bacteria were quantitated by serial dilution plating; subpopulations with elevated MICs (≥ 4x) were monitored on FTB- or CZA-supplemented agar. Results FEP, FTB, and CZA MICs ranged 16 to > 128 mg/L, 0.5–8 mg/L, and 2 to > 128 mg/L, respectively. FEP was inactive (n=7) or bacteriostatic (n=1, FEP MIC=16 mg/L). FTB was bactericidal (≥ 3 log kill) against all 8 strains; subpopulations with elevated FTB MICs were not detected. Against MBL producers, CZA was inactive without EDTA and was bacteriostatic (n=2) or bactericidal (n=2) only when EDTA was added to disable MBLs. Against non-MBL producers, all of which were CZA-susceptible, CZA was either bactericidal (n=1) or bacteriostatic (n=2) or allowed growth due to emergence of resistance (n=1). Conclusion Humanized exposures of FTB in a HFM were bactericidal against high inocula of MBL- and/or SBL-producing, multidrug-resistant respiratory pathogens and prevented emergence of resistance for 4 days. The results support development of FTB for HABP/VABP. Disclosures Lindsay M. Avery, PharmD, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds Lindsay M. Avery, PharmD, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds Mitchell Edwards, n/a, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds Fan Yi, PhD, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds Philip E. Sabato, PharmD, MS, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds Greg Moeck, PhD, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds Daniel C. Pevear, PhD, Venatorx Pharmaceuticals: Employee|Venatorx Pharmaceuticals: Stocks/Bonds.

Curcumin Nanodiscs Improve Solubility and Serve as Radiological Protectants against Ionizing Radiation Exposures in a Cell-Cycle Dependent Manner.

Curcumin, a natural polyphenol derived from the spice turmeric (Curcuma longa), contains antioxidant, anti-inflammatory, and anti-cancer properties. However, curcumin bioavailability is inherently low due to poor water solubility and rapid metabolism. Here, we further refined for use curcumin incorporated into “biomimetic” nanolipoprotein particles (cNLPs) consisting of a phospholipid bilayer surrounded by apolipoprotein A1 and amphipathic polymer scaffolding moieties. Our cNLP formulation improves the water solubility of curcumin over 30-fold and produces nanoparticles with ~350 µg/mL total loading capacity for downstream in vitro and in vivo applications. We found that cNLPs were well tolerated in AG05965/MRC-5 human primary lung fibroblasts compared to cultures treated with curcumin solubilized in DMSO (curDMSO). Pre-treatment with cNLPs of quiescent G0/G1-phase MRC-5 cultures improved cell survival following 137Cs gamma ray irradiations, although this finding was reversed in asynchronously cycling log-phase cell cultures. These findings may be useful for establishing cNLPs as a method to improve curcumin bioavailability for administration as a radioprotective and/or radiomitigative agent against ionizing radiation (IR) exposures in non-cycling cells or as a radiosensitizing agent for actively dividing cell populations, such as tumors.

Complete Genome Sequences of Four Strains ofErwiniatracheiphila: A Resource for Studying aBacterial Plant Pathogen with a Highly Complex Genome.

Complete Genome Sequences of Four Strains ofErwiniatracheiphila: A Resource for Studying aBacterial Plant Pathogen with a Highly Complex Genome.

Environment dependent expression of mycobacterium hormone sensitive lipases: expression pattern under ex-vivo and individual in-vitro stress conditions in M. tuberculosis H37Ra.

Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kinds of lipids. In comparison to single human Hormone Sensitive Lipase (hHSL), that is induced under nutritional stress, twelve serine hydrolases are annotated as HSL in Mycobacterium tuberculosis (mHSL). Mycobacterium is exposed to multiple stresses inside the host. Therefore, the present study was carried out to investigate if mHSL are also expressed under stress condition and if there is any correlation between various stress conditions and expression pattern of mHSL. The expression pattern of mHSL under different environmental conditions (in-vitro and ex-vivo) were studied using qRT-PCR in M. tuberculosis H37Ra strain with 16S rRNA as internal control. Out of 12, only two genes (lipU and lipY) were expressed at very low level in mid log phase culture under aerobic conditions, while 9 genes were expressed at stationary phase of growth. Ten mHSLs were expressed post-infection under ex-vivo conditions in time dependent manner. LipH and lipQ did not express at any time point under ex-vivo condition. The relative expression of most of the genes under individual stress was much higher than observed in ex-vivo conditions. The expression pattern of genes varied with change in stress condition. Different sets of mHSL genes were expressed under different individual stress conditions pointing towards the requirement of different mHSL to combat different stress conditions. Overall, most of the mHSLs have demonstrated stress dependent expression pointing towards their role in intracellular survival of mycobacteria.

Isolation and Identification of a Wastewater Siphoviridae Bacteriophage Targeting Multidrug-resistant Klebsiella pneumoniae

Background: Based on the WHO, multidrug-resistant Klebsiella pneumoniae is a priority pathogen that causes opportunistic infections and is widely spread in the environment. Phage therapy is considered a natural, safe, and very efficient alternative to treat difficult-to-treat infections. Objectives: This study aimed to isolate highly virulent, lytic bacteriophages and evaluate their efficacy for lysing multidrug-resistant K. pneumoniae. Methods: Municipal wastewater samples were collected and filtered using 0.22 µm syringe filters and cultivated with log-phase cultures of K. pneumoniae using enrichment media. After 48 h of incubation, the cultures were centrifuged, and the resultant supernatant was filtered (0.22 µm). The detection of the phage was done using the spot assay with K. pneumoniae as the host. One-step growth kinetics and bacterial reduction tests were conducted to assess the growth kinetics of the isolated phage. The stability of the isolated phage was characterized by subjecting it to various temperature and pH conditions. The chemical stability of the K. pneumoniae phage was determined by exposing it to various organic compounds. A panel of 20 bacterial strains was tested using the spot assay, as well as double agar overlying assay, to determine the host range of the isolated phage. Results: Out of 40 wastewater samples tested, only one sample was tested positive for the K. pneumoniae phage (2.5%) that was lytic against the host strain. The K. pneumoniae phage had a latent period of 15 min and a burst size of 100 virions per infected cell. It was most stable at 37°C and pH range of 6.0 to 10.0. Chemically, the K. pneumoniae phage was resistant to 10% chloroform treatment. Transmission electron micrograph indicated that the K. pneumoniae phage belonged to the order Caudovirales, family Siphoviridae, morphotype B1. Conclusions: Most of the characteristic features of the K. pneumoniae phage indicated the potential of this phage to be used in phage therapy. Hence, a comprehensive study is highly recommended to characterize the K. pneumoniae phage genome, detect its molecular interactions with the host cell, and determine its lytic activity in combination with other phages, which may lead to the efficient utilization of this phage in phage therapy against K. pneumoniae infections.

Pseudomonas aeruginosa transcriptome adaptations from colonization to biofilm infection of skin wounds

In burn patients Pseudomonas aeruginosa infection is a major cause of morbidity. Analysis of the pathogen’s gene expression as it transitions from colonization to acute and then biofilm wound infection may provide strategies for infection control. Toward this goal, we seeded log-phase P. aeruginosa (PAO1) into 3-day-old, full-thickness excision wounds (rabbit ear) and harvested the bacteria during colonization (Hrs 2 and 6), acute infection (Hr 24), and biofilm infection (Days 5 and 9) for transcriptome analysis (RNA-Seq). After 2–6 h in the wound, genes for metabolism and cell replication were down-regulated while wound-adaptation genes were up-regulated (vs. expression in log-phase culture). As the infection progressed from acute to biofilm infection, more genes became up-regulated than down-regulated, but the down-regulated genes enriched in more pathways, likely because the genes and pathways that bacteria already colonizing wounds up-regulate to establish biofilm infection are less known. Across the stages of infection, carbon-utilization pathways shifted. During acute infection, itaconate produced by myeloid cells appears to have been a carbon source because myeloid cell infiltration and the expression of the host gene, ACOD1, for itaconate production peaked coincidently with the expression of the PAO1 genes for itaconate transport and catabolism. Additionally, branched-chain amino acids are suggested to be a carbon source in acute infection and in biofilm infection. In biofilm infection, fatty acid degradation was also up-regulated. These carbon sources feed into the glyoxylate cycle that was coincidently up-regulated, suggesting it provided the precursors for P. aeruginosa to synthesize macromolecules in establishing wound infection.

Ultra-Rapid Drug Susceptibility Testing for Klebsiella pneumoniae Clinical Isolates in 60 Min by SYBR Green I/Propidium Iodide Viability Assay.

BackgroundWe aimed to optimize and validate the drug susceptibility test (DST) assay by SYBR Green I/PI (SG-PI) method using a panel of 89 Klebsiella pneumoniae clinical isolates in comparison with the conventional DST method to three most important antibiotics used for treatment of this bacterial infection, including imipenem, cefmetazole, and gentamicin.MethodsBy staining with SYBR Green I and PI dyes, green fluorescence and red fluorescence, which linearly correlated with the percentages of live and dead or membrane damaged cells, respectively, were used to produce two standard curves to calculate the relative cell membrane impermeable rates for each log and stationary phase cultures. Stationary phase K. pneumoniae cells were used in imipenem and cefmetazole SG-PI DST assay whereas log phase cells were used in the gentamicin assay. The conventional broth microdilution method was used as a gold standard for DST for comparison.ResultsData showed that after antibiotic treatment for 30–60 min, the antibiotic-resistant K. pneumoniae strains had significantly higher numbers of surviving cells than the susceptible strains at different concentrations of imipenem, cefmetazole, and gentamicin, where the average relative membrane impermeable rates were 88.5, 92.5, and 103.8% for resistant clinical strains, respectively, and 9.1, 49.3, and 71.5% for susceptible strains, respectively. Overall, the total concordances between the ultra-rapid SG-PI method and conventional minimal inhibitory concentration assay in diagnosing imipenem, cefmetazole and gentamicin resistance were high and were 96.6% (86/89), 95.4% (83/87), and 95.5% (85/89), respectively.ConclusionWe demonstrate that our novel SG-PI assay can accurately and stably detect resistance to different antibiotics in clinical isolates of K. pneumoniae in an ultra-fast manner in 60–90 min.

Engineering of the CHAPk Staphylococcal Phage Endolysin to Enhance Antibacterial Activity against Stationary-Phase Cells

Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.

Study of Viability and Effect of Liquid Formulation of Bacillus subtilis, Bbv 57 against Nematode - Fungus Disease Complex in Tomato, Lycopersicon esculentum Mill.

Viability of antagonistic Bacillus subtilis isolate, Bbv 57 was observed in nutrient broth (NB) amended with different chemicals viz., glycerol (10 mM), trehalose (10 mM) and polyvinylpyrrolidone (PVP) (2%). Among different chemical amendments tested, NB amended with glycerol (10 mM) maintained Bbv 57 population for a period of one year. Liquid formulation of Bbv 57 was prepared by adding log phase culture of the bacterium in NB amended with glycerol and further tests were carried out with this formulation. In vitro, minimum egg hatching and maximum juvenile mortality of Meloidogyne incognita and minimum mycelial growth of Fusarium oxysporum f. sp. lycopersici was observed in 5th day old formulation of Bbv 57 when compared with other day old formulations. In pot culture and field studies, maximum plant growth parameters of tomato, minimum nematode population and fungus colonies were recorded with soil drenching of liquid formulation of Bbv 57 (1000 ml/ha).

1263. Assessment of Cefepime (FEP)-Taniborbactam (TAN) Human Exposures to Suppress the Emergence of Resistance among Serine (SBL)- and Metallo-β-Lactamase (MBL)-Producing Gram-Negative Bacteria (GNB) in a Hollow Fiber Infection Model (HFIM)

BackgroundFEP-TAN (FTB) efficacy and safety are currently being evaluated in a Phase 3 trial (NCT03840148). TAN, a boronic acid-based β-lactamase inhibitor, restores susceptibility to FEP when resistance is driven by SBL or MBL (ie, NDM, VIM). This in vitro study assessed whether clinical FTB exposures suppress treatment-emergent resistance in pathogenic Enterobacterales and Pseudomonas aeruginosa.MethodsBioreactors (C2011, FiberCell) were inoculated with clinical GNB strains (N=6) using highly concentrated log phase cultures (> 108 CFU). Syringe pumps supplied humanized exposures of FEP (2 g), FTB (2 g/0.5 g), ceftazidime-avibactam (CZA, 2 g/0.5 g), each as 2 h infusions q8h, or meropenem-vaborbactam (MEV, 2 g/2 g q8h, 3 h infusion) for 7 days. Exposures were confirmed by UPLC-MS/MS for all agents. Subpopulations with elevated FTB MICs (4x) were monitored with drug-supplemented agar. CZA or MEV served as positive or negative controls for selected strains. Samples, serially removed from bioreactors, were saline-washed prior to quantitative culture to prevent drug carryover.ResultsAll strains grew rapidly in the presence of FEP (Figure 1), consistent with resistance by broth microdilution (BMD, Table 1). With the addition of TAN, there was extensive killing of the total bacterial populations by FTB, and subpopulations with elevated FTB MICs were never recovered. Like FTB against Klebsiella pneumoniae (KP) BAA-1705, CZA initially decreased the inoculum to the lower limit of detection, but unlike FTB, allowed regrowth to 3.7 log10 CFU/mL by day 7. The first dose of FTB was bactericidal against VIM+ and NDM+ KP strains while regrowth occurred prior to 8 h of MEV and CZA challenge, respectively. Notably, early failure of MEV is discordant with susceptibility by BMD (MIC= 4 µg/mL). By day 7, FTB sterilized an OXA-48+ KP strain that when challenged by MEV, grew to 9.8 log10 CFU/ml at 24 h.Figure 1. Bacterial burdens observed in the HFIM when treated with FEP alone or FEP+TAN (FTB) Table 1. Characterization of strains assessed in the HFIM; minimum inhibitory concentration (MIC) values for treatments not assessed are included in parentheses. ConclusionIn a 7-day HFIM with humanized exposures and high initial inoculums, FTB provided sustained bactericidal activity against multidrug-resistant Enterobacterales and P. aeruginosa strains harboring a diversity of β-lactamases and suppressed growth of resistant subpopulations. These data are crucial to inform understanding of the potential role for FTB in GNB infections and future clinical studies.DisclosuresLindsay M. Avery, PharmD, Venatorx Pharmaceuticals (Employee, Shareholder) Salvador Vernacchio, BS, Venatorx Pharmaceuticals (Employee, Shareholder) Lisa McLaughlin, BS, Venatorx Pharmaceuticals (Employee, Shareholder) Luigi Xerri, PhD, Venatorx Pharmaceuticals (Employee, Shareholder) Greg Moeck, PhD, Venatorx Pharmaceuticals (Employee, Shareholder) Daniel Pevear, PhD, Venatorx Pharmaceuticals (Employee, Shareholder)

Growth-associated catabolic potential of Acetoanaerobium sticklandii DSM 519 on gelatin and amino acids.

Acetoanaerobium sticklandii DSM 519 is a hyperammonia-producing anaerobe that catabolizes proteins and amino acids into organic solvents and volatile acids via the Stickland reactions. However, the specific growth rate and metabolic capability of this organism on proteins and amino acids are not yet known. Therefore, the present study was intended to evaluate its specific growth rate and metabolic potential on gelatin and amino acids in the experimental media. We carried out metabolic assay experiments to calculate its ability to utilize pure gelatin, single amino acids, and amino acid pairs at different growth phases. The results of this study show that complete assimilation of gelatin was achieved by its log-phase culture. The subsequent fermentation of amino acids was much faster than gelatin hydrolysis. The rate of gelatin degradation was associated with the growth and catabolic rates of this organism. Many amino acids were not assimilated completely for its growth and energy conservation. A log-phase culture of this organism preferably utilized l-cysteine, l-arginine, and l-serine, and released more fraction of ammonia. As shown by our analysis, the catabolic rates of these amino acids were determined by the rates of respective enzymes involved in amino acid catabolic pathways and feedback repression of ammonia. The growth kinetic data indicated that at the initial growth stage, a metabolic shift in its solventogenesis and acidogenesis phases was associated with catabolism of certain amino acids. Thus, the results of this study provide a new insight to exploit its log-phase culture as a starter for the production of biofuel components from gelatin processing industries.

Novel resveratrol derivatives have diverse effects on the survival, proliferation and senescence of primary human fibroblasts.

Resveratrol alters the cytokinetics of mammalian cell populations in a dose dependent manner. Concentrations above 25-50µM typically trigger growth arrest, senescence and/or apoptosis in multiple different cell types. In contrast, concentrations below 10µM enhance the growth of log phase cell cultures and can rescue senescence in multiple strains of human fibroblasts. To better understand the structural features that regulate these effects, a panel of 24 structurally-related resveralogues were synthesised and evaluated for their capacity to activate SIRT1, as determined by an ex-vivo SIRT1 assay, their toxicity, as measured by lactate dehydrogenase release, and their effects on replicative senescence in MRC5 human fibroblasts as measured by their effects on Ki67 immunoreactivity and senescence-associated β galactosidase activity.Minor modifications to the parent stilbene, resveratrol, significantly alter the biological activities of the molecules. Replacement of the 3,5-dihydroxy substituents with 3,5-dimethoxy groups significantly enhances SIRT1 activity, and reduces toxicity. Minimising other strong conjugative effects also reduces toxicity, but negatively impacts SIRT1 activation.At 100µM many of the compounds, including resveratrol, induce senescence in primary MRC5 cells in culture. Modifications that reduce or remove this effect match those that reduce toxicity leading to a correlation between reduction in labelling index and increase in LDH release.At 10µM, the majority of our compounds significantly enhance the growth fraction of log phase cultures of MRC5 cells, consistent with the rescue of a subpopulation of cells within the culture from senescence. SIRT1 activation is not required for rescue to occur but enhances the size of the effect.

Pseudomonas Can Survive Tailocin Killing via Persistence-Like and Heterogenous Resistance Mechanisms.

Phage tail-like bacteriocins (tailocins) are bacterially produced protein toxins that mediate competitive interactions between cocolonizing bacteria. Both theoretical and experimental research has shown there are intransitive interactions between bacteriocin-producing, bacteriocin-sensitive, and bacteriocin-resistant populations, whereby producers outcompete sensitive cells, sensitive cells outcompete resistant cells, and resistant cells outcompete producers. These so-called rock-paper-scissors dynamics explain how all three populations occupy the same environment, without one driving the others extinct. Using Pseudomonas syringae as a model, we demonstrate that otherwise sensitive cells survive bacteriocin exposure through a physiological mechanism. This mechanism allows cells to survive bacteriocin killing without acquiring resistance. We show that a significant fraction of the target cells that survive a lethal dose of tailocin did not exhibit any detectable increase in survival during a subsequent exposure. Tailocin persister cells were more prevalent in stationary- rather than log-phase cultures. Of the fraction of cells that gained detectable resistance, there was a range from complete (insensitive) to incomplete (partially sensitive) resistance. By using genomic sequencing and genetic engineering, we showed that a mutation in a hypothetical gene containing 8 to 10 transmembrane domains causes tailocin high persistence and that genes of various glycosyltransferases cause incomplete and complete tailocin resistance. Importantly, of the several classes of mutations, only those causing complete tailocin resistance compromised host fitness. This result indicates that bacteria likely utilize persistence to survive bacteriocin-mediated killing without suffering the costs associated with resistance. This research provides important insight into how bacteria can escape the trap of fitness trade-offs associated with gaining de novo tailocin resistance.IMPORTANCE Bacteriocins are bacterially produced protein toxins that are proposed as antibiotic alternatives. However, a deeper understanding of the responses of target bacteria to bacteriocin exposure is lacking. Here, we show that target cells of Pseudomonas syringae survive lethal bacteriocin exposure through both physiological persistence and genetic resistance mechanisms. Cells that are not growing rapidly rely primarily on persistence, whereas those growing rapidly are more likely to survive via resistance. We identified various mutations in lipopolysaccharide biogenesis-related regions involved in tailocin persistence and resistance. By assessing host fitness of various classes of mutants, we showed that persistence and subtle resistance are mechanisms P. syringae uses to survive competition and preserve host fitness. These results have important implications for developing bacteriocins as alternative therapeutic agents.

Detection and differential identification of typhoidal Salmonella using bacteriophages and resazurin.

This study was aimed at developing an easy to use and inexpensive biosensor for the detection of typhoidal Salmonella. The technique was designed to be used without expensive instrumentation if necessary. Bacteriophages specifically infecting three typhoidal Salmonella serovars were isolated and purified. Log-phase cultures were mixed with a high titre of a single phage (109 PFUs) in separate wells of a microtitre plate and incubated at room temperature (30°C) for 1h. After incubation, resazurin was added and the plates were incubated further for 1h. Absorbance at 570nm of each test well was measured using a commercial microplate reader and compared with that of the control well. A significant difference (p < 0.05) between the absorbance of test and control wells indicated the presence of target bacteria. With visual inspection, a delay in colour change from blue to pink was considered a positive result. The system could detect 5×104 CFUs in 120min without pre-enrichment and 10 CFUs with a pre-enrichment of 6h.