Tryptic Soy Broth Research Articles

Exploring the potential of earthworm gut bacteria for plastic degradation

The use of plastic mulch films in agriculture leads to the inevitable accumulation of plastic debris in soils. Here, we explored the potential of earthworm gut-inhabiting bacterial strains (Mycobacterium vanbaalenii (MV), Rhodococcus jostii (RJ), Streptomyces fulvissimus (SF), Bacillus simplex (BS), and Sporosarcina globispora (SG) to degrade plastic films (⌀ = 15 mm) made from commonly used polymers: low-density polyethylene film (LDPE-f), polylactic acid (PLA-f), polybutylene adipate terephthalate film (PBAT-f), and a commercial biodegradable mulch film, Bionov-B® (composed of Mater-Bi, a feedstock with PBAT, PLA and other chemical compounds). A 180-day experiment was conducted at room temperature (x̄ =19.4 °C) for different strain–plastic combinations under a low carbon media (0.1× tryptic soy broth). Results showed that the tested strain–plastic combinations did not facilitate the degradation of LDPE-f (treated with RJ and SF), PBAT-f (treated with BS and SG), and Bionov-B (treated with BS, MV, and SG). However, incubating PLA-f with SF triggered a reduction in the molecular weights and an increase in crystallinity. Therefore, we used PLA-f as model plastic to study the influence of temperature (“room temperature” & “30 °C”), carbon source (“carbon-free” & “low carbon supply”), and strain interactions (“single strains” & “strain mixtures”) on PLA degradation. SF and SF + RJ treatments significantly fostered PLA degradation under 30 °C in a low-carbon media. PLA-f did not show any degradation in carbon-free media treatments. The competition between different strains in the same system likely hindered the performance of PLA-degrading strains. A positive correlation between the final pH of culture media and PLA-f weight loss was observed, which might reflect the pH-dependent hydrolysis mechanism of PLA. Our results situate SF and its co-culture with RJ strains as possible accelerators of PLA degradation in temperatures below PLA glass transition temperature (Tg). Further studies are needed to test the bioremediation feasibility in soils.

Selenium Nanoparticle Activity against S. mutans Biofilms as a Potential Treatment Alternative for Periodontitis.

The disruption of periodontal biofilms and prevailing antimicrobial resistance issues continue to pose a great challenge to the treatment of periodontitis. Here, we report on selenium nanoparticles (SeNPs) as a treatment alternative for periodontitis by determining their antibiofilm activity against S. mutans biofilms and the potential role of particle size in disrupting biofilms. SeNPs were synthesised via a reduction reaction. Various physicochemical characterisations were conducted on the NPs, including size and shape. The microbroth dilution method was used to conduct the biofilm and antibiofilm assay against S. mutans, which was analysed by absorbance. SeNPs displayed hydrodynamic sizes as low as 46 ± 4 nm at a volume ratio of 1:5 (sodium selenite/ascorbic acid) with good monodispersity and stability. Hydrodynamic sizes of SeNPs after resuspension in tryptic soy broth supplemented with 2.5% sucrose (TSB + 2.5% suc.) and incubated at 37 °C for 24 h, ranged from 112 to 263 nm, while the zeta potential values increased to greater than -11 mV. The biofilm assay indicated that S. mutans are weakly adherent, bordering on moderately adherent biofilm producers. The minimum biofilm inhibitory concentration (MBIC) was identified at 500 µg/mL. At a 1000 µg/mL concentration, SeNPs were able to inhibit S. mutan biofilms up to 99.87 ± 2.41% at a volume ratio of 1:1. No correlation was found between antibiofilm activity and particle size; however, antibiofilm activity was proven to be concentration-dependant. SeNPs demonstrate antibiofilm activity and may be useful for further development in treating periodontitis.

Pantoea ananatis, a plant growth stimulating bacterium, and its metabolites isolated from Hydrocotyle umbellata (dollarweed)

ABSTRACT A bacterium growing on infected leaves of Hydrocotyle umbellata, commonly known as dollarweed, was isolated and identified as Pantoea ananatis. An ethyl acetate extract of tryptic soy broth (TSB) liquid culture filtrate of the bacterium was subjected to silica gel chromatography to isolate bioactive molecules. Indole was isolated as the major compound that gave a distinct, foul odor to the extract, together with phenethyl alcohol, phenol, tryptophol, N-acyl-homoserine lactone, 3-(methylthio)-1-propanol, cyclo(L-pro-L-tyr), and cyclo(dehydroAla-L-Leu). This is the first report of the isolation of cyclo(dehydroAla-L-Leu) from a Pantoea species. Even though tryptophol is an intermediate in the indoleacetic acid (IAA) pathway, we were unable to detect or isolate IAA. We investigated the effect of P. ananatis inoculum on the growth of plants. Treatment of Lemna paucicostata Hegelm plants with 4 × 109 colony forming units of P. ananatis stimulated their growth by ca. five-fold after 13 days. After 13 days of treatment, some control plants were browning, but treated plants were greener and no plants were browning. The growth of both Cucumis sativus (cucumber) and Sorghum bicolor (sorghum) plants was increased by ca. 20 to 40%, depending on the growth parameter and species, when the rhizosphere was treated with the bacterium after germination at the same concentration. Plant growth promotion by Pantoea ananatis could be due to the provision of the IAA precursor indole.

Anti-cancer activity of crude Slrp protein conjugated mesoporous silica nanoparticles in HeLa Cell Lines: An in vitro approach

Microbial based therapeutics for cancer have gained a significant interest in recent decades. The present study relies on the synthesis, analysis, and conjugation of Salmonella Leucine-rich Proteins (SlrP) with mesoporous silica nanoparticles (MSNN) to evaluate their potential anticancer activity. The SlrP proteins were effectively produced and isolated from Salmonella enterica using Tryptic Soy Broth (TSB), and the subsequent SDS-PAGE analysis verified the presence of a band at around 72 KDa. The MSN synthesis yielded particles with an average diameter of 68.05±0.87 nm and a pore diameter of 7.1 nm. In addition, we synthesized MSNMPA and then conjugated them with SlrP. Characterization studies confirmed the effective conjugation. The cytotoxicity evaluation conducted on HeLa cells revealed no substantial modification in cell viability upon treatment with MSN alone. Nevertheless, when MSNMPA/SlrP was done, it demonstrated significant cytotoxic properties, as evidenced by an IC50 value of 10 µg/mL. The results indicate that SlrP-conjugated MSN (MSNMPA/SlrP) could be utilized as promising nanocarriers for delivering anticancer proteins.

Emergence of pandrug-resistant carbapenemase-producing Enterobacterales in dogs and cats: a cross-sectional study in Egypt.

One of the most important emerging health problems is the increasing role of animals in the rapid global rise in resistance to last-resort antibiotics, such as carbapenems. However, there is limited information on the role of pet animals in harboring and spreading pandrug-resistant (PDR) carbapenemase-producing Enterobacterales (CPE), especially in Egypt. This cross-sectional study was conducted to screen for CPE in healthy and diseased pets using phenotypic and molecular methods and the NG-Test CARBA 5 immunochromatographic assay. Rectal swabs were collected from 62 dogs and 48 cats, incubated overnight in tryptic soy broth containing 10 μg of meropenem disc and subsequently cultured on MacConkey agar supplemented with meropenem (1 mg/L). Sixty-six isolates (60.6%), including 56 Klebsiella pneumoniae, seven Escherichia coli, and three K. oxytoca isolates, were confirmed to be carbapenem-resistant Enterobacterales (CRE) by the disc diffusion method, broth microdilution test, CNPt-direct, and PCR assay targeting carbapenemase genes. Forty-three (65.2%) dogs and 23 (34.8%) cats carried CPE. Of these, 35 (70.0%) were healthy (including 27 dogs and 8 cats) and 31 (52.5%) were diseased (including 16 dogs and 15 cats). bla OXA-181 was the most common gene detected (42/66, 63.6%), followed by bla IMP (40/66, 60.6%), bla OXA-48-like (29/66, 43.9%), bla KPC and bla VIM (20/66, 30.3% each), and bla NDM (17/66, 25.8%). The identified genotypes were bla KPC-2, bla IMP-1, bla VIM-1, bla NDM-1, and bla NDM-5. The CARBA 5 assay showed higher sensitivity and specificity for the detection of NDM, OXA and KPC than that for VIM and IMP genes. Antimicrobial resistance profiles of CRE isolates revealed 20 PDR, 30 extensively drug-resistant (XDR), and 16 multidrug-resistant (MDR) phenotypes. This study provides evidence of colonization with PDR CPE in dogs and cats. To manage the infection or colonization of pets in veterinary clinical settings, extended surveillance systems should be considered, and the use of critical antibiotics should be strictly controlled.

Antibacterial and Antivirulence Effects of Kale (Brassica oleracea var. sabellica) on Streptococcus intermedius

Persistent odontogenic infections caused by resistant bacterial species, such as , have consistently been associated with deep-seated infections. This in vitro study aimed to evaluate the antibacterial and antivirulence effects of kale () on . was freshly incubated in tryptic soy broth media. Three experiments per concentration of kale were conducted under aseptic conditions (i.e., disc diffusion, broth microdilution, and reverse-transcription polymerase chain reaction) to evaluate the antibacterial and antivirulence effects. The samples were then treated with 1000, 500, 250, 125, 65, 30, 15, 7, and 3 mg/mL kale; ampicillin (positive control); and tryptic soy broth (negative control). After 24-h incubation, the inhibition zone, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and relative gene expression of the virulence factor (intermedilysin []) were measured. All assays were conducted in triplicate. The findings were reported and analyzed as means ± standard deviations. The agar disc diffusion and relative gene expression were statistically analyzed using one-way analysis of variance and Tukey’s test, with the significance level set at P < 0.05. Kale showed antibacterial effects on by significantly inhibiting bacterial growth and reducing expression only at a concentration of 1000 mg/mL; it yielded an inhibition zone of 11.12 ± 1.59 mm, which was smaller than that with ampicillin. The MIC and MBC ranged from 15 to 65 mg/mL and from 500 mg/mL, respectively. Conversely, the highest concentration of kale yielded significantly less inhibition than did ampicillin. The antibacterial effects of kale may be dose-dependent. Kale can inhibit bacterial growth and suppress expression under in vitro conditions of , which is mainly involved in deep-seated odontogenic infections.

The impact of different acidic conditions and food substrates on Listeria monocytogenes biofilms development and removal using nanoencapsulated carvacrol

Listeria monocytogenes biofilms present a significant challenge in the food industry. This study explores the impact of different acidic conditions of culture media and food matrices on the development and removal of biofilms developed on stainless steel surfaces by wild-type (WT) L. monocytogenes strains as well as in two mutant derivatives, ΔsigB and ΔagrA, that have defects in the general stress response and quorum sensing, respectively. Additionally, the study investigates the efficacy of nanoencapsulated carvacrol as an antimicrobial against L. monocytogenes biofilms developed in Tryptic Soy Broth (TSB) culture media acidified to different pH conditions (3.5, 4.5, 5.5, 6.5), and in food substrates (apple juice, strained yogurt, vegetable soup, semi-skimmed milk) having the same pH levels. No biofilm formation was observed for all L. monocytogenes strains at pH levels of 3.5 and 4.5 in both culture media and food substrates. However, at pH 5.5 and 6.5, increased biofilm levels were observed in both the culture media and food substrates, with the WT strain showing significantly higher biofilm formation (3.04–6.05 log CFU cm−2) than the mutant strains (2.30–5.48 log CFU cm−2). For both applications, the nanoencapsulated carvacrol demonstrated more potent antimicrobial activity against biofilms developed at pH 5.5 with 2.23 to 3.61 log reductions, compared to 1.58–2.95 log reductions at pH 6.5, with mutants being more vulnerable in acidic environments. In food substrates, nanoencapsulated carvacrol induced lower log reductions (1.58–2.90) than the ones in TSB (2.02–3.61). These findings provide valuable insights into the impact of different acidic conditions on the development of L. monocytogenes biofilms on stainless steel surfaces and the potential application of nanoencapsulated carvacrol as a biofilm control agent in food processing environments.

Using Essential Oils to Reduce Yersinia enterocolitica in Minced Meat and in Biofilms.

Yersiniosis, one of the leading foodborne infections in the European Union, is caused by Yersinia enterocolitica. In this study, the antibacterial and antibiofilm effects of cinnamon (Cinnamomum zeylanicum Nees), clove (Syzygium aromaticum L.), oregano (Origanum vulgare L.), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.), and winter savory (Satureja montana L.) essential oils were investigated against Y. enterocolitica strains belonging to the bioserotype 4/O:3. Cinnamon essential oil showed the highest antibacterial activity, with an MIC value 0.09 µL/mL, followed by oregano and thyme essential oils, with MIC values from 0.09 to 0.18 µL/mL, and from 0.18 to 0.23 µL/mL, respectively. Thyme essential oil at 0.23 µL/g (MIC) and at 0.46 µL/g (2MIC) significantly (p < 0.05) reduced the number of Y. enterocolitica by 0.38 log CFU/g and 0.64 log CFU/g, respectively, in minced pork meat during storage at 4 °C for 4 days. The Y. enterocolitica strains formed biofilms at 15 °C and 37 °C in tryptic soy broth and Luria-Bertani broth, while no biofilms were obtained at 5 °C, and in meat broth nutrient media. Applying the minimum bactericidal concentrations of cinnamon, clove, oregano, rosemary, thyme, and winter savory essential oils on preformed biofilms led to significant reductions being observed in the range from 45.34% to 78.89%. A scanning electron microscopy assay showed the devastating impact of oregano and thyme essential oils on the morphology of Y. enterocolitica bacterial cells. In conclusion, the results of this study show that essential oils possess high anti-Yersinia and antibiofilm effects.

Investigation of Pseudomonas aeruginosa bacteria in a number of Baghdad schools and extent of their resistance to disinfectants and sterilizers

PurposeThis study was design to investigate of P. aeruginosa, an example of Gram-negative bacteria, in seven primary and secondary schools of Baghdad city, and the effects of Ethanol and Dettol of P. aeruginosa biofilm.Design/methodology/approachSeventy swabs were collected from seven primary and secondary schools of Baghdad city, Iraq, during November -December 2022. Swabs were collected from classes desk, doors handles, students hands and water taps. Standard microbiological testing methods were used on the samples for isolation and identification. The ability of bacteria to form biofilm and the effects of Ethanol and Dettol on"preformed” biofilms was examined by microtiter plate with the use of an ELISA reader.FindingsIn 70 swabs from seven primary and secondary schools, growth was observed in 33 swabs as P. aeruginosa. Primary schools were higher contaminated than secondary and water taps and door handles represented the main source of this contamination. The ability of bacteria to produce biofilm was observed in 19 (57.6%) isolates and 14 (42.4%) nonbiofilm producers. As well as, Ethanol (70%) treatment of preformed biofilms led to enhance biofilm formation and revealed significantly greater staining after 4 and 24h than Dettol (3%) compared to an untreated control (tryptic soy broth (TSB) incubation).Originality/valueStudies on P. aeruginosa in Iraqi schools are quite rare. This work is considered distinctive because it drew attention to the presence of pathogenic bacteria within primary and secondary schools, which are not considered their natural environment.

A flow cytometric assay to detect viability and persistence of Salmonella enterica subsp. enterica serotypes in nuclease-free water at 4 and 25°C.

Salmonella spp. is one of the most isolated microorganisms reported to be responsible for human foodborne diseases and death. Water constitutes a major reservoir where the Salmonella spp. can persist and go undetected when present in low numbers. In this study, we assessed the viability of 12 serotypes of Salmonella enterica subsp. enterica for 160 days in nuclease-free water at 4 and 25°C using flow cytometry and Tryptic Soy Agar (TSA) plate counts. The results show that all 12 serotypes remain viable after 160 days in distilled water using flow cytometry, whereas traditional plate counts failed to detect ten serotypes incubated at 25°C. Moreover, the findings demonstrate that 4°C constitutes a more favorable environment where Salmonella can remain viable for prolonged periods without nutrients. Under such conditions, however, Salmonella exhibits a higher susceptibility to all tested antibiotics and benzalkonium chloride (BZK). The pre-enrichment with Universal Pre-enrichment Broth (UP) and 1/10 × Tryptic Soy broth (1/10 × TSB) resuscitated all tested serotypes on TSA plates, nevertheless cell size decreased after 160 days. Furthermore, phenotype microarray (PM) analysis of S. Inverness and S. Enteritidis combined with principal component analysis (PCA) revealed an inter-individual variability in serotypes with their phenotype characteristics, and the impact of long-term storage at 4 and 25°C for 160 days in nuclease-free water. This study provides an insight to Salmonella spp. long-term survivability at different temperatures and highlights the need for powerful tools to detect this microorganism to reduce the risk of disease transmission of foodborne pathogens via nuclease-free water.

Potential Application of Bacterial Lipase from Bacillus subtilis in the Production of Biodisel

This work aims to evaluate the potential of bacterial lipase of Bacillus subtilis in biodiesel production. Assays were performed to obtain, purify, and immobilize the enzyme and compare chemical and enzymatic transesterification tests. The Tryptic Soy Broth (TSB) culture medium without supplementation showed the highest enzymatic activity and purification yields when using 60% of (NH4)2SO4. Citral was the most efficient agent for lipase immobilization. Even though the free enzymes showed a higher activity rate, the immobilized enzymes are associated with a better-quality product. In conclusion, the lipase enzyme showed positive potential in biodiesel production.

(130) Antibiotic Solutions are the Ideal Irrigation Choice for Inflatable Penile Implant Hydrophilic Surfaces

Abstract Introduction Inflatable penile prostheses (IPP) may be colonized by bacteria, which may contribute to clinical infections. Current attempts to reduce bacterial colonization include altered implant surface binding properties and mechanical irrigations with antiseptic solutions. Previous data has shown that prophylactically dipping IPPs in a solution of 2mg/mL vancomycin and 160μg/mL gentamicin (VG) reduces biofilm formation more than chlorhexidine gluconate (CHG) does. However, there is a paucity of data looking at VG as an irrigation step after a prophylactic dip. Objective To determine if VG irrigation of a previously CHG- or NS-dipped IPP decreases bacterial colonization more effectively than irrigation with normal saline. Methods Sterile 8mm diameter IPP pieces (Coloplast, Minneapolis, MN) were split into 4 groups with a minimum of 12 replicates each and submerged for 3 minutes in 3mL of normal saline (NS), 0.05% CHG in sterile water (Irrimax, Lawrenceville, GA), or VG (Sigma-Aldrich, Burlington, MA). Pieces were then rinsed in NS for five seconds, re-submerged in a separate well containing 2mL of NS or VG for 2 minutes, and finally rinsed for five seconds in a large volume of NS. After a 5 sec PBS rinse, pieces were incubated with 1 X 10^5 colony forming units per milliliter (CFU)/mL of methicillin-sensitive Staphylococcus aureus ATCC25923 in trypticase soy broth (TSB) for 48 hours. Samples were then rinsed with PBS and biofilm was removed by shaking in 0.3% Tween-20. Suspended bacteria were serially diluted, plated onto 3M PetriFilms (Maplewood, MN), and then colony counts were recorded. Mann-Whitney U tests were performed to compare outcomes, p&amp;lt;0.05 considered significant. Results Average bacterial counts with 95% CI are plotted in Figure 1. NS controls showed an average of 1.0E7 CFU/mL adherent bacteria. In this study, the introduction of VG to an NS or CHG-coated implant resulted in significant reduction in adherent bacteria; the NS-VG group showed a ~5 log decrease in adherent bacteria (3.0E1, standard deviation=3.3E4, p&amp;lt;0.01), and the CHG-VG group similarly showed a ~5 log decrease in adherent bacteria (4.4E2, standard deviation=6.9E5, p&amp;lt;0.01). Neither treatment group consistently reduced biofilm counts as effectively as the implant pieces incubated in VG as both a prophylactic dip and a rinse solution (~5 log decrease in counts, p&amp;lt;0.01). However, neither treatment group consistently reduced biofilm counts as effectively as the implant pieces incubated in VG as both a prophylactic dip and a rinse solution, also with a ~5 log decrease in adherent bacteria (1.0E2, standard deviation=2.4E2, p&amp;lt;0.01) (Figure 1). Conclusions VG is the ideal choice for both coating the implant prior to implantation and as an intraoperative irrigation solution. However, further studies are needed to compare VG with CHG as mechanical irrigants as opposed to static irrigants. Disclosure Yes, this is sponsored by industry/sponsor: Coloplast (material not financial support). Clarification: Industry funding only - investigator initiated and executed study. Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast and Boston Scientific.

(129) Prophylactic Dips and Irrigation Solutions Can Considerably Alter the Antimicrobial Efficacy of Inflatable Penile Implant Hydrophilic Surface Coatings

Abstract Introduction Inflatable penile prostheses (IPP) experience surface-associated bacterial biofilms, which may contribute to clinical infections. Current attempts to reduce bacterial colonization include antibiotic coatings, altered implant surface binding properties, and mechanical irrigations. 0.05% chlorhexidine gluconate (CHG) has been proposed as a hydrophilic surface coating and mechanical irrigation for IPP surgery. One study demonstrated reduced surface bacterial colonization on an IPP hydrophilic surface dipped and irrigated with CHG 15psi compared to normal saline. However, CHG’s efficacy has not been compared to that of an antibiotic, particularly in preventing colonization by bacteria that are commonly implicated in IPP infections. Objective To characterize the utility of 0.05% chlorhexidine or vancomycin/gentamicin (VG) as both an antibiotic dip and an intraoperative irrigation in reducing bacterial colonization on hydrophilic IPP surfaces. Methods Sterile 8mm diameter IPP pieces (Coloplast, Minneapolis, MN) underwent the following treatment protocols. 1: IPP pieces were split into three groups (10 replicates each) and submerged for 3 minutes in normal saline (NS), 0.05% CHG in sterile water (Irrimax, Lawrenceville, GA), or a solution of 2mg/mL vancomycin and 160μg/mL gentamicin (VG) (Sigma-Aldrich, Burlington, MA) 2: IPP pieces were split into six groups (&amp;gt;14 replicates each) and submerged for 3 minutes in either NS, 0.05% CHG, or VG. Pieces were then rinsed in NS for five seconds and re-submerged in a separate well containing NS, 0.05% CHG, or VG. The pieces were rinsed in NS to remove any unbound solution and then incubated with 1*10^5 CFU/mL of methicillin-sensitive Staphylococcus aureus ATCC25923 in trypticase soy broth (TSB) for 48 hours. Samples were then rinsed with PBS and adherent bacteria were removed by shaking in 0.3% Tween-20. Suspended bacteria were serially diluted, plated onto 3M PetriFilms (Maplewood, MN), and counted. Mann-Whitney U tests were performed to compare outcomes, p&amp;lt;0.05 considered significant. Results Average bacterial counts with 95% CI’s for protocols 1 and 2 are plotted in Figures 1a and 1b respectively. NS controls showed an average of 1.1E7 CFU/mL adherent bacteria. Pre-dipping the implant in 0.05% CHG inhibits bacterial growth with a nearly ~2 log decrease (4.9E5 CFU/mL, p&amp;lt;0.01) in counts. Pre-dipping with VG had greater potency, with a ~5 log decrease (3.7E1 CFU/mL. p&amp;lt;0.01). However, exposure of the previously dipped IPP surface to an antiseptic irrigation solution altered antimicrobial properties (Figure 1b). NS controls showed an average of 6.0E7 CFU/mL adherent bacteria. The introduction of NS or CHG to the VG-coated implant material resulted in a near-complete loss of antimicrobial efficacy, with a 0.5 log decrease (NS) and a slight increase (CHG) in bacterial growth (NS: 5.5E7 CFU/mL, p=0.433; CHG: 6.2E7, p=.983). VG dipping followed by VG irrigation resulted in retention of antimicrobial properties, with a similar 5 log decrease. Conclusions Dipping of the implant surface with VG was more effective than 0.05% CHG. Furthermore, the choice of irrigation solution appears to alter the efficacy of antimicrobials bound to the hydrophilic IPP surface. Surgeons should be cautious using CHG or NS as an irrigation solution, and, if utilized, should take care to limit the contact time with the antibiotic-coated implant. Disclosure Yes, this is sponsored by industry/sponsor: Coloplast (material not financial support). Clarification: Industry funding only - investigator initiated and executed study. Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast and Boston Scientific.

Using Cassava Starch Processing By-Product for Bioproduction of 1-Hydroxyphenazine: A Novel Fungicide against Fusarium oxysporum

This study aimed to develop the eco-friendly production of bioactive 1-hydroxyphenazine (HP) through fermentation using an industrial processing by-product of cassava as the main carbon/nitrogen source. Cassava starch processing by-product (CSPB) was screened as a suitable substrate for fermentation to produce HP with a high yield. Mixing CSPB with a minor amount of tryptic soy broth (TSB) at a ratio of 8/2 and with 0.05% K2HPO4 and 0.05% FeSO4 was effective in HP production by Pseudomonas aeruginosa TUN03. HP was also further scaled up through production on a bioreactor system, which achieved a higher level yield (36.5 µg/mL) in a shorter fermentation time (10 h) compared to its production in the flask (20.23 µg/mL after 3 days). In anti-fungal activity tests against various Fusarium phytopathogens, HP exhibited the most significant effect on Fusarium oxysporum F10. It could inhibit the mycelial growth of this fungus, with an inhibition rate of 68.7% and anti-spore germination activity of up to 98.4%. The results of the docking study indicate that HP effectively interacted with the protein 1TRY targeting anti-F. oxysporum, with all obtained docking parameters in the accepted range. This study supports the novel use of CSPB as the carbon/nitrogen source for P. aeruginosa fermentation to produce HP, a F. oxysporum anti-fungal agent reported here for the first time.

Abstract B043: Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer

Abstract Breast cancer is the most common cause of mortality among women worldwide. Among all subtypes, HER2-positive breast cancers are highly aggressive and have poor prognosis. Surgical resection is choice of treatment. However, surgery may not be possible if disease is widespread. Trastuzumab, a monoclonal antibody, is globally used for immunotherapy. It is a large sized molecule (141kDa) and show slow pharmacokinetics and high liver uptake. We have explored trastuzumab fragment as a radio-theranostic agent. Trastuzumab Fab was generated using Papain enzyme tagged beads, incubation time 18 to 20 h at 850 rpm in phosphate buffer. Fab were collected with ultrafiltration device, cut off 30 kDa, in phosphate buffer pH 8. Quantification of Fab was done at 280 nm. Fab characterization was done using MALDI-TOF and SDS-PAGE. To enable radiolabelling of Fab with Ga-68 and Lu-177, Fab was conjugated with bifunctional chelator, DTPA. Fab-DTPA was separated by micro bio-spin P-6 chromatography columns and the average number of DTPA/Fab were calculated. Affinity of Fab conjugated Fab-DTPA for HER2 receptors was studied using the biolayer interferometry technique (BLI). Radiolabelling of Fab with Ga-68 for PET imaging and Lu-177 for therapy was optimized. Quality control included radiochemical purity using TLC in sodium citrate (pH 5) mobile phase, pyrogenicity using PTS, and sterility by tryptic soy broth culture media. Receptor affinity and apoptosis were studied with HER2 positive SKBR-3 cells using flow cytometry assay. The SKBR-3 cell (106) were cultured in 96 well plates and incubated with a known amount of Lu-177-DTPA-Fab (0- 18.5 MBq). First in human Ga-68 Fab PET/CT and Lu-177-Fab gamma camera images were acquired after obtaining clearance from Institutional Ethics Committee and inform signed consent. Patients were recruited with histopathological proven HER2 positive breast cancer and uptake on F-18 FDG PET/CT. Ga-68-DTPA-Fab (3.5-5 mCi) and 10-15 mCi Lu-177 DTPA-Fab was injected and images were acquired at 2/3 h and up to 7 days respectively. Dominant Fab peak at 45kDa on MALD-TOF, single band on SDS-PAGE (Non-reduced or reduced) have been obtained. Two DTPA/Fab were conjugated and confirmed by MALDI-TOF. BLI data showed Kd value of trastuzumab, Fab, and conjugated DTPA-Fab as 0.2 nM, 0.57 nM, and 20 nM for HER2. High labelling efficiency (≥98%) of Lu-177-DTPA-Fab and Ga-68-DTPA-Fab was achieved. Apoptosis (%) was 1.5%- 42.5% in SKBR-3 cells treated with Lu-177-DTPA-Fab. Both formulations were found sterile and pyrogen-free. Kidney and bladder activity of Ga-68/Lu-177-DTPA-Fab demonstrate renal clearance. Blood pool activity of both tracers were high. However, it was decreased, and lesion uptake was increased with time. All the lesions on Ga-68-DTPA-Fab, same as in F-18 FDG PET/CT, were picked up accept those near heart. Good lesion retention of Lu-177-DTPA-Fab was observed showing suitability for therapy. Fab-DTPA has potential to target HER2 receptor. It could be radiolabelled with Ga-68 for PET imaging and Lu-177 for targeted therapy. Citation Format: Jaya Shukla, Yogesh Rathore, Komalpreet Kaur, Santosh Irrinky, Rajender Kumar, Amanjit Bal, Bhagwant R Mittal. Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B043.

The chronic wound milieu changes essential oils' antibiofilm activity—an in vitro and larval model study

Essential Oils (EOs) are currently being researched as potential antibiofilm agents to combat infections related to chronic wound biofilms. As documented in the literature, EOs’ in vitro antibacterial properties are often assessed using standard microbiological media and conditions that do not accurately reflect the actual environment of a chronic wound. To address this issue, In vitro Wound Milieu (IVWM) medium, which closely resembles the environment of a chronic wound, was applied for culturing S. aureus biofilms (n = 12) in this research. Biofilms cultivated in the standard Tryptic Soy Broth (TSB) medium served as a control for the experiment. Key biofilm features were analyzed and compared. Subsequently, staphylococci were exposed to the activity of thyme or rosemary EOs (T-EO and R-EO, respectively). As proof of concept, the cytotoxicity of T-EO and its antimicrobial in vivo activity were assessed using a G. mellonella larvae model. Key features of biofilm-forming cells were lower in the IVWM than in the TSB medium: biomass (up to 8 times), metabolic activity (up to 9 times), cell number (up to 100 times), and the live/dead cells ratio. Conversely, biofilm thickness was higher (up to 25%) in IVWM. These differences translated into varied responses of the biofilms to EOs exposure. The application of T-EO led to a greater reduction (up to 2 times) in 67% of biofilm-forming strains in IVWM compared to the TSB medium. Conversely, exposure to R-EO resulted in a higher reduction (up to 2.6 times) of 83% of biofilm-forming strains in TSB than in IVWM. The application of T-EO was not only non-toxic to G. mellonella larvae but also increased the survival of larvae infected with staphylococci (from 48 to 85%). Our findings suggest that EOs not only show promise as agents for treating biofilm-related wound infections but also that providing conditions reflecting the specific niche of the human body is of paramount importance in influencing the results obtained. However, before clinical application, challenges related to the methods of assessing their activity, microbial intra-species variability, and different levels of activity of various EOs should be analyzed and standardized.

Optimization of mineral nutrients to improve rhamnolipid production by Pseudomonas aeruginosa6 K‐11

AbstractBACKGROUNDRhamnolipids are biosurfactants that have their main application in bioremediation processes. They are able to increase the biodegradation and detoxification rates from industrial effluents. The north of Peru and Iquitos have been not only major oil producers but also have been responsible for oil spills. This study therefore focused on the optimization of mineral nutrients such as nitrogen, phosphorous, calcium and iron to improve rhamnolipids production by Pseudomonas aeruginosa 6 K‐11, which was isolated from oil soil in Peru.RESULTSPseudomonas aeruginosa 6 K‐11 was reactivated in Trypticase Soy Broth by scaling from 5 to 200 mL of culture medium for 8 h at each stage. The culture was centrifuged and placed in 100 mL Siegmund–Wagner medium for 36 h in a 500 mL Erlenmeyer flask. First, it was determined that sodium nitrate, monopotassium phosphate, calcium chloride and iron sulfate were the best sources of mineral nutrients. Then, by using corn oil as carbon source, the ratios obtained for carbon‐to‐nitrogen and carbon‐to‐phosphorous were 21.172 and 16.279, respectively. In addition, it was found that the optimal concentration for calcium was 0.046 g L−1 and for iron was 0.003 g L−1. The most influential variables were carbon‐to‐nitrogen and carbon‐to‐phosphorus ratio. Finally, 35.124 g L−1 rhamnolipids was produced after 168 h of fermentation with the optimal parameters.CONCLUSIONThe optimization of mineral nutrients was an important achieved goal for rhamnolipid production. In addition, this optimization can be used for modifying the culture medium to grow the biosurfactant producer strain. © 2024 Society of Chemical Industry (SCI).

Correlation between the facial skin microbiome and sensitive skin using the 2bRAD-M technique.

This study aimed to expound on the correlation between facial skin microbiome and sensitive skin (SS) using a novel sequencing technique. We applied the 2bRAD sequencing for the microbiome, which enables accurate characterization of the low-biomass microbiome at species resolution to profile facial skin microbes in SS and non-SS groups. Further, the bacterial colonies were isolated and cultured from skin surfaces to study the pro-inflammatory effect on human keratinocytes by ELISA. We accordingly identified 1142 genera and 4436 strains. In the SS group, the proportions of Actinomyces and Microbotryomycetes were significantly increased, whereas that of Acidimicrobiia was decreased. Kruskal-Wallis analysis revealed significant differences in 11 genera and 35 species, among which the proportions of Dermabacter, Chryseobacterium, Rhodotorula and Peptoniphilus A were increased in the SS group. Analysis of the top 10 genera revealed increased proportions of Cutibacterium, Corynebacterium and Staphylococcus. Moreover, the proportion of Dermabacter hominis was significantly increased by 18.9-fold in the SS group, whereas those of many Streptococcus strains were significantly decreased. Focus on the isolated bacterial colonies from skin surfaces, more yellow colonies were found in SS group when cultured in Tryptic Soy Broth medium for 48 h, and more interleukin-8 was detected on keratinocytes after yellow colonies stimulation, such as S.capitis, M.luteus. This study suggests that more SS-associated microorganisms can be identified using the 2bRAD technique even with a small sample size. Dermabacter hominis and Chryseobacterium was firstly reported with a significantly increase in SS, and the S.capitis, as well as M.luteus, but not S.aureus, may be associated with skin inflammation.

Analysis and application of volatile metabolic profiles of Escherichia coli: a preliminary GC-IMS-based study.

Nosocomial infections caused by Escherichia coli (E. coli) may pose serious risks to patients, and early identification of pathogenic bacteria and drug sensitivity results can improve patient prognosis. In this study, we clarified the composition and relative content of volatile organic compounds (VOCs) generated by E. coli in tryptic soy broth (TSB) using gas chromatography-ion mobility spectrometry (GC-IMS). We explored whether imipenem (IPM) could be utilized to differentiate between carbapenem-sensitive E. coli (CSEC) and carbapenem-resistant E. coli (CREC). The results revealed that 36 VOCs (alcohols, aldehydes, acids, esters, ketones, pyrazines, heterocyclic compounds, and unknown compounds) were detected using GC-IMS. Besides, the results indicated that changes in the relative content of VOCs as well as changes in the signal intensity of fingerprints were able to assess the growth state of bacteria during bacterial growth and help identify E. coli. Lastly, under selective pressure of IPM, volatile fingerprints of E. coli could be employed as a model to distinguish CSEC from CREC strains.

Inactivation of Salmonella Enteritidis in liquid egg yolk and egg white using bacteriophage cocktails

Salmonella Enteritidis (SE) is a significant global cause of foodborne illness, often linked to egg contamination. This study evaluated the inhibitory effects of eight bacteriophages (phages) against three SE strains isolated from poultry environments. The most effective phages were selected to formulate different phage cocktails, to enhance the efficacy and prolong inhibition. Four phage cocktails were tested at a multiplicity of infection (MOI) of 100 in tryptic soy broth (TSB), and at MOIs of 100 and 1000 in liquid egg white (EW) and egg yolk (EY) with storage at 8 °C for up to 30 days (d). The effectiveness of the phage cocktails varied significantly among bacterial strains, yet all demonstrated significant reductions compared to the positive control in liquid culture (P < 0.05). Similarly, the tested SE strains in both EW and EY showed significant reductions with phage treatments (P < 0.005), although the effectiveness was influenced by the MOI and medium composition. Treating EY proved to be more challenging, with lower magnitudes of reduction and longer treatment durations required, compared to EW. Reductions ranged from 1 to greater than 4 log CFU/mL in EW and EY after 30 d, with consistently higher reductions achieved at MOI 1000. Phage titers decreased initially, but remained stable following SE inoculation in broth and liquid eggs at 8 °C, indicating that lysis from without mechanisms may have contributed to the inhibitory effect. Notably, phages exhibited stronger attachment to SE in EW, which can be attributed to be less viscous nature of EW compared to EY. This study demonstrated that phage applications in both EW and EY effectively reduced SE counts at 8 °C, with no regrowth during long-term storage. These findings contribute to the development of biocontrol methods that enhance food safety and reduce foodborne outbreaks associated with contaminated egg products.