Culture Growth Research Articles

Performance of Gram Stain Characteristics for Predicting Culture Growth in Lower Respiratory Specimens Across Multiple Institutions

Abstract Data from Gram stains performed on clinical specimens can guide clinical decision-making and downstream laboratory workflows. However, the extent to which Gram stain results predict gold-standard culture results, and the generalizability of this performance across sites, warrants additional exploration. We sought to discover which Gram stain features would be associated with clinically significant culture growth (“growth”). To do so, 574 remnant specimens from the lower respiratory tract collected from four institutions were analyzed. A standardized Gram stain and culture protocol was performed across all study sites. Final reference organism identification was assigned using MALDI-ToF MS, and clinically significant growth was defined as any organism reported to the medical record. Gram stains with mixed microorganisms detected were considered concordant if ≥2 organisms of different Gram stain appearances were identified in culture. Growth was observed in 53% of specimens, including 25% of specimens in which Gram stain demonstrated no organisms. The 95% confidence intervals (CI) of the site-wise growth rates were 30-42%, 42-52%, 51-67%, and 60-77% (p < 0.001 by Chi-squared). By Gram stain morphology, 53-69% of specimens with Gram-positive cocci grew any clinically significant bacteria, while 31-49% grew a concordant Gram-positive cocci. 65-85% of specimens with Gram-negative bacilli grew any clinically significant bacteria, while 59-74% grew a concordant Gram-negative bacilli. 63-83% of specimens with mixed microorganisms grew 2 or more clinically relevant bacteria of differing Gram stain morphologies. Specimens from bronchoalveolar lavages (BALs) were less likely to have growth (30-42%) than endotracheal aspirates (66-77%). Polymorphonuclear leukocyte (PMNs) abundance was associated with culture growth, with Gram stains showing no, rare, few, moderate, or abundant PMNs demonstrating growth 28, 44, 52, 60, and 67% of the time (p < 0.001). Specimens with None and few squamous epithelial cells on Gram stain demonstrated growth 38-52% and 59-68% of the time (p < 0.001). No significant difference in growth was observed for red blood cells. Organism abundance on Gram stain was significantly associated with culture growth, with growth rate CIs being 20-32% for no organisms, 44-62% for rare, 58-77% for few, 70-93% for moderate, and 81-95% for abundant organisms seen. The extent to which these associations could guide the development of decision rules or machine learning models that demonstrate acceptable negative predictive value for pathogen growth to alleviate the operational burden of low-utility cultures is an exciting future direction to explore.

Host cell-specific metabolism of linoleic acid controls Toxoplasma gondii growth in cell culture.

The obligate intracellular parasite Toxoplasma gondii can infect and replicate in any warm-blooded cell tested to date, but much of our knowledge about T. gondii cell biology comes from just one host cell type: human foreskin fibroblasts (HFFs). To expand our knowledge of host-parasite lipid interactions, we studied T. gondii in intestinal epithelial cells, the first site of host-parasite contact following oral infection and the exclusive site of parasite sexual development in feline hosts. We found that highly metabolic Caco-2 cells are permissive to T. gondii growth even when treated with high levels of linoleic acid (LA), a polyunsaturated fatty acid (PUFA) that kills parasites in HFFs. Caco-2 cells appear to sequester LA away from the parasite, preventing membrane disruptions and lipotoxicity that characterize LA-induced parasite death in HFFs. Our work is an important step toward understanding host-parasite interactions in feline intestinal epithelial cells, an understudied but important cell type in the T. gondii life cycle.

Inhibition of OXPHOS induces metabolic rewiring and reduces hypoxia in murine tumor models

IntroductionTumor hypoxia is a feature of many solid malignancies and is known to cause radio resistance. In recent years it has become clear that hypoxic tumor regions also foster an immunosuppressive phenotype and are involved in immunotherapy resistance. It has been proposed that reducing the tumors’ oxygen consumption will result in an increased oxygen concentration in the tissue and improve radio- and immunotherapy efficacy. The aim of this study is to investigate the metabolic rewiring of cancer cells by pharmacological attenuation of oxidative phosphorylation (OXPHOS) and subsequently reduce tumor hypoxia. Material and methodsThe metabolic effects of three OXPHOS inhibitors IACS-010759, atovaquone and metformin were explored by measuring oxygen consumption rate, extra cellular acidification rate, and [18F]FDG uptake in 2D and 3D cell culture. Tumor cell growth in 2D cell culture and hypoxia in 3D cell culture were analyzed by live cell imaging. Tumor hypoxia and [18F]FDG uptake in vivo following treatment with IACS-010759 was determined by immunohistochemistry and ex vivo biodistribution respectively. ResultsIn vitro experiments show that tumor cell metabolism is heterogeneous between different models. Upon OXPHOS inhibition, metabolism shifts from oxygen consumption through OXPHOS towards glycolysis, indicated by increased acidification and glucose uptake. Inhibition of OXPHOS by IACS-010759 treatment reduced diffusion limited tumor hypoxia in both 3D cell culture and in vivo. Although immune cell presence was lower in hypoxic areas compared with normoxic areas, it is not altered following short term OXPHOS inhibition. DiscussionThese results show that inhibition of OXPHOS causes a metabolic shift from OXPHOS towards increased glycolysis in 2D and 3D cell culture. Moreover, inhibition of OXPHOS reduces diffusion limited hypoxia in 3D cell culture and murine tumor models. Reduced hypoxia by OXPHOS inhibition might enhance therapy efficacy in future studies. However, caution is warranted as systemic metabolic rewiring can cause adverse effects.

Impact of pre-exposure stress on the growth and viability of Lactobacillus acidophilus in regular, buriti pulp and orange byproduct fermented milk products

The loss of probiotics viability in yogurts and fermented milk is a significant challenge in producing yogurt and fermented milk. Thus, pre-exposure of probiotics to stress conditions can be a viable alternative to increase the probiotic viability. Moreover, the use of fruit pulp and agro-industrial residues in these products has demonstrated promising results in promoting growth and improving the viability of probiotics. Thus, this study aimed to evaluate the effects of pre-exposure to acid, oxidative and osmotic stress on the growth and viability of Lactobacillus acidophilus in yogurts and naturally fermented milk containing buriti (Mauritia flexuosa Mart.) pulp or orange byproduct. L. acidophilus was individually pre-exposed to acid, oxidative, and osmotic stress and used in the production of yogurts and fermented milk to determine both the acidification profile and growth of the cultures. Furthermore, during cold storage, the post-acidification profiles and viability of microbial cultures added to the yogurts and fermented milk were monitored. Results showed that pre-exposure to stress conditions influenced the growth parameters as the growth rate (μ) and lag phase (λ) of L. acidophilus and the starter cultures of S. thermophilus and L. delbrueckii subsp. bulgaricus. Moreover, an increase in the viability of L. acidophilus - pre-exposed to acid stress - was observed on the 21st day of storage of natural yogurts containing orange byproduct compared with non-stressful conditions. This study reports new data on the growth of probiotic cultures pre-exposed to stress conditions in products added of pulps and agro-industrial residues, which have not yet been shown in the literature.

Characterization of the dynamic viscosity of cell cultures and its effect on mixing performance in a spinner flask bioreactor

Computational fluid dynamics (CFD) models have been developed to simulate cell culturing bioreactors but assume water-like viscosity properties due to significant data gaps. This study characterized the dynamic viscosity of HEK-293 cell cultures and evaluated its effect on mixing performance in a spinner flask bioreactor. Viscosity measurements indicated that the cell culture media, media with microcarriers, and cell cultures presented shear thinning behaviors within the measured shear rate range of 1–100 s−1. The viscosity also increased with the microcarrier concentrations and growth of cell culture. The CFD model, incorporating dynamic viscosity data, showed that shear stress and Kolmogorov length profiles are significantly influenced by microcarrier concentrations and cell culture growth. Higher microcarrier concentrations led to higher average shear stress and Kolmogorov values. The cultured HEK-293 cells after seven days of growth also had higher average shear stress and Kolmogorov values than at the day of seeding, indicating an impact caused by the cells’ metabolism and biomass. Overall, the results indicated that assuming water-like properties underestimates shear stress and Kolmogorov length scales, especially at zones of lower shear rates due to the observed shear thinning behavior. Thus, careful monitoring of dynamic viscosity of cell cultures and proper control of mixing parameters are critical to deliver the desired mixing conditions for optimized cell growth especially during scale-up production operations.

Deciphering Hyperammonia-Producing Bacteria (HAB) in the Rumen of Water Buffaloes (Bubalus bubalis) and Their Inhibition through Plant Extracts and Essential Oils

Hyperammonia-producing bacteria (HAB) are a class of microbes present in the stomach of ruminants, responsible for the rapid rate of ammonia production from protein degradation beyond the capacity of these animals for their utilization. Thus, ruminant nutritionists are interested in decreasing ruminal protein degradation and ammonia genesis by focusing on controlling the activity of HAB. The investigations of the present study were carried out to determine predominant hyperammonia-producing bacteria in the rumen of buffaloes, their isolation and characterization, as well as the inhibition of these isolates with various sources of plant secondary compounds (tannins, saponins, and essential oils). Studies employing high-throughput sequencing of amplicons of the 16S rRNA gene from genomic DNA recovered from enrichment culture of HAB of buffalo rumina indicated that, at the phylum level, Proteobacteria (61.1 to 68.2%) was the most predominant HAB. Acidaminococcus was most predominant among the identified genera. In vitro experiments were conducted with enrichment culture of buffalo rumen contents incubated with different types of feed additives such as essential oils (eucalyptus oil, lemon grass oil, and clove oil) and extracts of plants (Sapindus mukorossi fruits and Ficus bengalensis leaves), each at graded dose levels. The reduction in ammonia production by clove and lemon grass oils was evident due to the presence of major bioactive compounds, especially eugenol and limonene, which have strong antimicrobial activity. However, clove oil and Indian soapberry/reetha (Sapindus mukorossi) fruit were found to be promising and effective in reducing the growth, protease production, and ammonia production of HAB culture.

Anti-Hair Loss Effects of the DP2 Antagonist in Human Follicle Dermal Papilla Cells

Prostaglandin D2 (PGD2) levels are high in the balding areas of human scalps, and PGD2 has been found to inhibit hair growth. It is known that the inhibition of the PGD2 receptor can promote hair growth by preventing hair follicles from entering the catagen phase. Thus, we identified an antagonist of DP2, the receptor for PGD2, as a potential treatment for hair loss using an AI-based DeepZema® drug development program. In this study, we identified that the DP2 antagonist (DP2A) could ameliorate alopecia in human follicle dermal papilla cells (HFDPCs) that were stimulated by dihydrotestosterone (DHT), a known molecule related to hair loss. We observed that the DP2A promoted wound healing efficiency and increased alkaline phosphatase levels in the HFDPCs that were damaged with DHT. In addition, we found that the DP2A diminished the reactive oxygen species (ROS) levels generated in the DHT-damaged HFDPCs. We confirmed that the DP2A effectively recovered the membrane potential of mitochondria in these cells. We also demonstrated that the DP2A enhanced the phosphorylation levels of both Akt and ERK in the HFDPCs that were damaged with DHT. Notably, we revealed that the DP2A slightly enlarged the three-dimensional spheroid size in these cells and confirmed that the DP2A improved hair growth in the organ culture of human hair follicles. Taken together, we suggest that DP2A has therapeutic effects on HFDPCs that are damaged by DHT and holds promise as a potential treatment for treating hair loss.

Evaluation for the Effect of Heat Stable Enterotoxin (a) Produced by Enterotoxigenic Escherichia coli on Different Cancer Cells In Vitro

This study was conducted for evaluating the cytotoxic effect of heat stable enterotoxin a (STa) produced by enterotoxigenic Escherichia coli on the proliferation of primary cancer cell cultures, obtained from tumor samples that were collected from (13) cancer patients and as follows: (five colon cancer patients, two bladder cancer patients, two breast cancer patients, two stomach cancer patients and two lung cancer patients), and on normal cell line (rat embryonic fibroblast / REF) (in vitro) with the use of different concentrations starting from (1) mg/ml and ending with (0.0002) mg/ml by making two fold serial dilutions by using the 96- well microtiter plate, and in comparison with negative (PBS) and positive (MMC, at concentration of 10 µg/ml) controls . Results showed that, after (24) hours of exposure to STa, the growth of all primary cancer cell cultures obtained from colon cancer patients was inhibited by STa treatment and this inhibition was concentration dependent. Also it was shown that the cytotoxic effect of the high concentration of STa was close to that seen after MMC treatment. While no differences were seen in the growth of all primary cancer cell cultures that were obtained from the other cancer patients, which mean that STa treatment neither inhibit nor enhanced their growth. At the same time STa did not show or has any cytotoxic effect on the normal cell line (REF).

SPECIES IDENTIFICATION AND IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING OF ASPERGILLUS ISOLATED FROM VARIOUS CLINICAL SAMPLES

Objectives: Aspergillus is ubiquitous mold species present in environment in the form of spores. Infection due to Aspergillus is uncommon in immunocompetent individuals unless they possess any abnormality or have undergone any treatment with corticosteroids. In immunocompromised individuals, infection by this fungus is common among people with prolonged neutropenia, transplants, and human immunodeficiency virus infection. The symptoms are diverse, ranging from allergic reactions to invasive aspergillosis and life-threatening complications. The aim of this study is to isolate and identify Aspergillus species obtained from various clinical specimens and perform antifungal susceptibility testing (AFST). Methods: A total of 200 isolates in which positive direct microscopic findings correlated well with culture growth for Aspergillus were included. Antifungal susceptibility was performed by micro broth dilution technique according to clinical laboratory standard institute M38-A2 guidelines. Results: Aspergillus flavus was found to be predominant species followed by Aspergillus fumigatus. AFST was performed, where all Aspergillus strains exhibited low minimum inhibitory concentration (MIC) and minimum effective concentration (MEC) values for most of antifungal drugs tested except for one strain of A. flavus, which exhibited high MIC for voriconazole and high MEC for caspofungin. Conclusion: Polyenes, azoles, and echinocandin resistance are emerging in many parts of the world. Therefore, continued application of AFST combined with morphological characterization for species identification is critical in detecting the emergence of resistance among various Aspergillus species to reduce mortality, morbidity, and overcome treatment failure.

Organogenesis in a Broad Spectrum of Grape Genotypes and Agrobacterium-Mediated Transformation of the Podarok Magaracha Grapevine Cultivar.

We present data on the ability for organogenesis in 22 genotypes of grapevine and developed a direct organogenesis protocol for the cultivar Podarok Magaracha and the rootstock Kober 5BB. The protocol does not require replacement of culture media and growth regulators, and the duration is 11 weeks. The cultivation of explants occurs on modified MS medium with the addition of 2.0 mg L-1 benzyladenine and indole-3-butyric acid (0.15 mg L-1 for the rootstock Kober 5BB or 0.05 mg L-1 for the cultivar Podarok Magaracha). The direct organogenesis protocol consists of three time periods: (1) culturing explants for 2 weeks in dark conditions for meristematic bulk tissue, (2) followed by 4 weeks of cultivation in light conditions for regeneration, and (3) 5 weeks of cultivation in dark conditions for shoot elongation. Based on this protocol, conditions for the Agrobacterium-mediated transformation of the Podarok Magaracha cultivar were developed with an efficiency of 2.0% transgenic plants per 100 explants. Two stably transformed lines with integration into the genome of the pBin35SGFP plasmid construction, confirmed by Southern blotting, were obtained.

Probiotic Milk Enriched with Protein Isolates: Physicochemical, Organoleptic, and Microbiological Properties.

Incorporating plant protein isolates into milk can enhance probiotic culture growth by providing essential nutrients and altering the physicochemical properties of fermented milk. This study investigated the effects of adding 1.5% or 3.0% soy, pea, and whey protein isolates on the growth of Lacticaseibacillus casei and Lactobacillus johnsonii monocultures, as well as the physicochemical (acidity, syneresis, color) and organoleptic properties of fermented milk during 21 days of refrigerated storage. The results showed that 1.5% SPI and WPI did not significantly alter milk acidity compared to controls. Still, pH increased with 1.5% and 3.0% PPI. Storage time significantly affected pH in L. casei fermented milk. The initial addition of WPI at 1.5% and 3.0% reduced syneresis in L. casei fermented milk compared to other samples. Color components were significantly influenced by isolates. Initial L. casei cell counts were lower with SPI (LCS1.5 and LCS3) and 1.5% PPI (LCP1.5) compared to controls. Increasing isolate concentration from 1.5% to 3% enhanced L. johnsonii growth in WPI-milk but reduced L. casei in LCW3 compared to LCW1.5. Only increased pea protein concentration significantly increased L. casei growth. Probiotic populations generally were reduced during extended storage. Moreover, isolates impacted milk organoleptic evaluation. This research demonstrates the potential of protein isolates in creating health-promoting and diverse fermented products and offers insights into their interaction with probiotic cultures to advance functional food technologies.

Morphological and Molecular Diversity of Colletotrichum gloeosporioides Causing Yam (Dioscorea alata L.) Anthracnose Disease in Côte d’Ivoire

Yam is a plant of great importance for the Ivorian people in terms of food, nutrition, economy, and socio-cultural value. However, its production is threatened by several biotic factors, including anthracnose, one of the most destructive diseases affecting yams. The objective of this study was to assess the morphological and molecular diversity of Colletotrichum gloeosporioides isolates responsible for anthracnose in yam (Dioscorea alata) in Côte d'Ivoire. Twenty-seven previously isolated strains were collected from Plant Health Laboratory of Nangui ABROGOUA University and analyzed. The study examined the cultural (coloration, growth mode, texture, and colony diameter), microscopic (conidial shape, size, and spore concentration), and molecular characteristics of these isolates. Significant morphological diversity was observed among the isolates. Macroscopically, three major groups were identified based on colony coloration (whitish, orange, and grayish). Colony textures varied, presenting cottony, flaky, downy, or filamentous appearances, with growth patterns ranging from concentric and radiating to regular rings. Colony diameters ranged from 37 mm to 82 mm. Microscopically, sporulation varied significantly among isolates, ranging from 1.1 × 10⁵ to 85.4 × 10⁵ spores, as did conidial size, which ranged from 10.54 to 19.29 µm in length and 3.93 to 6.43 µm in diameter. Molecular analysis through comparison with GenBank nucleotide sequences revealed homology rates of 99-100% and varying proximities between the rDNA sequences of the isolates. In conclusion, the characterization of these isolates provides valuable insights for improving disease control strategies for yam anthracnose.

A-264 Validation of a Novel Real-Time PCR Assay for Identification of Mycobacterium species Isolates Recovered from Clinical Culture

Abstract Background Historically, clinical mycobacteriology laboratories used the nucleic acid hybridization-based AccuProbe assay manufactured by Hologic, Inc., to rapidly rule in or rule out Mycobacterium tuberculosis (MTB) and members of the M. avium-intracellulare complex (MAC). The clinical value of this test was in its ability to rapidly rule in/out MTB from positive broth cultures and allow the initiation of appropriate antimicrobial therapy and infection prevention practices. Recent discontinuation of this commercial assay left a gap in testing available for the rapid identification of these organisms, which allowed us to develop an in-house real-time PCR test. Methods A replacement assay was developed by Labcorp R & D based on real-time PCR amplification of extracted nucleic acids from positive culture growth. Aliquots from positive AFB VersaTREK broth cultures were heat inactivated, and nucleic acids were extracted using the MagNA Pure 24 (MP24) instrument (Roche). Real-time PCR using published primer probe sets (Rocchetti, et al 2016) was performed on the QuantStudio™ 7 (QS7) instrument (Applied Biosystems®). The assay was designed with three independent primer/probe sequence targets: one for MTB Complex (IS6110), one for MAC (ITS), and one pan-target for the Mycobacterium genus (16s rRNA). Results Procedural verification was performed using 100 residual clinical specimens previously identified using the Hologic AccuProbe® assay. Categorical concordance was 97% (97/100). The three discrepant results were identified as MAC positive by PCR; however, these were not identified as MAC on the predicate assay. Analytical sensitivity for the PCR assay was established to be 1,000 CFU/mL for each target. Analytical specificity was assessed in vitro for nine Mycobacterium species, with no cross-reactivity observed. In vitro analysis of target detection in the presence of yeast (Candida glabrata) was also performed, and we saw no inhibition. Analysis of the primer/probe sets for all targets was performed in silico using XploreDB database of microbial genomes, with confirmed 100% matches for both MAC and MTB genomes compared to hundreds of species. By comparison, the PAN-Myco primer set showed decreased performance, having three mismatches (two on forward primer and one on the probe). As this target sequence is used as an Internal Control (IC) this was not problematic for overall assay performance. The assay demonstrated excellent precision (%CV) for each target: MTB = 0.74%, MAC = 1.10%, PAN-Myco = 3.19%. Conclusions Utilizing the depth of molecular methodologies available in our laboratories, we developed a rapid method for identification of MAC and MTB from positive broth cultures, which had excellent performance compared to the predicate assay. The PCR ID LDT test is now available and allows testing to continue with no interruption in patient care.

Clinical evaluation of a multiplex PCR-based test for joint infection: a prospective diagnostic accuracy study of forty-nine patients

PurposeThe purpose of this study was to evaluate the diagnostic accuracy (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)) of the PCR-based BioFire® Joint Infection Panel (BJI Panel) against microbiological culture growth for patients suspected of having a native or prosthetic joint infection.MethodsSynovial fluid and tissue biopsies were prospectively collected from patients from June 2022 to June 2023. The results of the BJI Panel were compared with those of culture growth.Results51 samples were included. Including all pathogens, the sensitivity was 69%, the specificity 89%, the PPV 73% and the NPV 86%. Including only pathogens in the BJI Panel, the sensitivity was 100%, the specificity 90%, the PPV 73% and the NPV 100%.ConclusionThe BJI Panel has a high accuracy for detecting the pathogens in its panel, but the absence of important common pathogens from the panel reduces its sensitivity and NPV. With a short turnaround time and precise pathogen detection, the BJI Panel has the potential to add value as a complementary diagnostic method.

The Impact Of Social Media On Consumer Behavior And Its Implications For Business Growth

Social media has had a significant influence on consumer behavior and corporate growth since its inception in the late 1990s. Mostly used websites like Facebook and Twitter, which transformed online communication and information sharing, were made possible by early platformes like Friendster and Six Degrees. Social media became even more accessible as smartphone usage increased in the 2010s, which fueled the growth of influencer culture and brand communities. Peer reviews and genuine voices are now trusted by consumers, which has shifted power away from brands and highlighted the value of real-time feedback and tailored marketing. Improved customer relationship management, better marketing techniques, and insightful data have all resulted from this evolution for firms. Through direct audience engagement, improved brand reputation management, and market exploration, businesses can propel growth in a highly competitive environment. The advanced function of social media in shaping consumer behavior and its ensuing effects on corporate success are examined in this article. Social media's ongoing development is changing how customers find, assess, and buy goods. Through an analysis of recent data and trends, this study demonstrates the ways in which social media influences brand loyalty, purchase decisions, and general consumer participation. According to the research, companies that successfully use social media can improve their market visibility, cultivate clientele, and increase revenues

The Chitinous Skeleton of Ianthella basta Marine Demosponge as a Renewable Scaffold-Based Carrier of Antiseptics

The chitinous skeleton of the marine demosponge Ianthella basta exhibits a unique network-like 3D architecture, excellent capillary properties, and chemical inertness, making it highly suitable for interdisciplinary research, especially in biomedical applications. This study investigates the potential of renewable I. basta chitinous scaffolds for drug delivery and wound dressing. The scaffolds, characterized by a microtubular structure, were impregnated with selected commercially available antiseptics, including solutions with hydrophilic and hydrophobic properties. Evaluations against selected clinical strains of bacteria, as well as fungi, demonstrated significant zones of growth inhibition with antiseptics such as brilliant green, gentian violet, decamethoxine, and polyhexanide. Notably, the antibacterial properties of these antiseptic-treated chitin matrices persisted for over 72 h, effectively inhibiting microbial growth in fresh cultures. These findings highlight the considerable potential of I. basta chitin scaffolds as sustainable, innovative biomaterials for controlled drug release and wound dressing applications.

Bio-based polylactic acid labware as a sustainable alternative for microbial cultivation in life science laboratories

Single-use plastics (SUPs) in life science laboratories account for approximately 5.5 million tonnes of waste per year globally. Of SUPs used in life science laboratories, Petri dishes, centrifuge tubes, and inoculation loops are some of the most common. In order to reduce the reliance on petrochemical-based SUPs in the life science research laboratory and minimize the negative environmental impacts associated with SUPs, this research investigates the applicability of polylactic acid (PLA) in single-use labware as a replacement for petrochemical-based plastics. PLA is one of the most well-studied biodegradable plastics that can be produced from sustainable resources. Commercially available PLA was used to 3D print a select range of labware to test the suitability of PLA-based material for routine microbiology work. An injection moulded PLA-based Petri dish was also designed and produced, for increased optical clarity. The biocompatibility was tested against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus epidermidis) strains of bacteria. The PLA-based labware did not negatively impact the cell growth, viability, and metabolic activity of the bacterial cultures. The injection moulded PLA Petri dish showed a reduced colony forming unit count for the Gram-negative E. coli strain compared to the polystyrene Petri dish, ∼1.5 × 109 CFU/mL and ∼3.0 × 109 CFU/mL respectively, during late-exponential growth. The colony counts were, however, in the same order of magnitude. This observed difference may be due to the internal environment inside the Petri dish, hence the internal O2 concentration, humidity, and temperature during bacterial growth were investigated. This work demonstrates, for the first time, a full successful workflow of bacterial growth using a sustainable bioplastic, providing a pathway to reducing the environmental impacts of SUPs in life science laboratories.

Identification of novel antistaphylococcal hit compounds.

Staphylococcus aureus is one of the most common nosocomial biofilm-forming pathogens worldwide that has developed resistance mechanisms against majority of the antibiotics. Therefore, the search of novel antistaphylococcal agents with unexploited mechanisms of action, especially with antibiofilm activity, is of great interest. Seryl-tRNA synthetase is recognized as a promising drug target for the development of antibacterials. We have carried out molecular docking of compounds with antistaphycoccal activity, which were earlier found by us using phenotypic screening, into synthetic site of S. aureus SerRS and found seven hit compounds with low inhibitory activity. Further, we have performed search of S. aureus SerRS inhibitors among compounds which were previously tested by us for inhibitory activity toward S. aureus ThrRS, that belong to the same class of aminoacyl-tRNA synthetases. Among them six hits were identified. We have selected four compounds for antibacterial study and found that the most active compound 1-methyl-3-(1H-imidazol-1-methyl-2-yl)-5-nitro-1H-indazole has MIC values toward S. aureus multidrug-resistant clinical isolates ranging from 78.12 to 156.2 µg/ml. However, this compound precipitated during anti-biofilm study. Therefore, we used 3-[N'-(2-hydroxy-3-methoxybenzylidene)hydrazino]-6-methyl-4H-[1,2,4]triazin-5-one with better solubility (ClogS value = 2.9) among investigated compounds toward SerRS for anti-biofilm study. It was found that this compound has a significant inhibitory effect on the growth of planktonic and biofilm culture of S. aureus 25923 with MIC value of 32 µg ml-1. At the same time, this compound does not reveal antibacterial activity toward Esherichia coli ATCC 47076. Therefore, this compound can be proposed as effective antiseptic toward multidrug-resistant biofilm-forming S. aureus isolates.

Production and utilization of pseudocobalamin in marine Synechococcus cultures and communities.

Cobalamin influences marine microbial communities because an exogenous source is required by most eukaryotic phytoplankton, and demand can exceed supply. Pseudocobalamin is a cobalamin analogue produced and used by most cyanobacteria but is not directly available to eukaryotic phytoplankton. Some microbes can remodel pseudocobalamin into cobalamin, but a scarcity of pseudocobalamin measurements impedes our ability to evaluate its importance for marine cobalamin production. Here, we perform simultaneous measurements of pseudocobalamin and methionine synthase (MetH), the key protein that uses it as a co-factor, in Synechococcus cultures and communities. In Synechococcus sp. WH8102, pseudocobalamin quota decreases in low temperature (17°C) and low nitrogen to phosphorus ratio, while MetH did not. Pseudocobalamin and MetH quotas were influenced by culture methods and growth phase. Despite the variability present in cultures, we found a comparably consistent quota of 300 ± 100 pseudocobalamin molecules per cyanobacterial cell in the Northwest Atlantic Ocean, suggesting that cyanobacterial cell counts may be sufficient to estimate pseudocobalamin inventories in this region. This work offers insights into cellular pseudocobalamin metabolism, environmental and physiological conditions that may influence it, and provides environmental measurements to further our understanding of when and how pseudocobalamin can influence marine microbial communities.

Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells, which was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomic analysis of the whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile.