Solid Medium Research Articles

Rapid colorimetric polymyxin B microelution directly from positive blood bottles: because patients with serious infections should not have to wait for results of culture-based methodologies.

To evaluate the performance of the rapid colorimetric polymyxin B microelution (RCPEm) in determining polymyxin B resistance directly from Enterobacterales-positive blood cultures. A set volume of positive blood culture bottles (diluted 1:10) was inoculated into a glucose-broth-phenol red solution (NP solution), where a polymyxin B disk was previously eluted (final concentration of 3µg/mL). Test was read each 1h for up to 4h. Color change from red/orange to yellow indicated resistant isolates. Results were compared to the reference method, broth microdilution (BMD), performed from colonies grown on solid media from the same blood culture bottle. One hundred fifty-two Enterobacterales-positive blood cultures were evaluated, 22.4% (34/152) of them resistant to polymyxin B (including 6.6% with borderline MICs). When performing directly from positive blood cultures (RCPEm-BC), specificity and sensitivity were 99.1% and 94.1%, respectively. Of note, 79.4% (27/34) of truly resistant isolates required 3h of incubation, compared to the 18 ± 2h incubation that microtiter plates of BMD demand before reading can be performed. RCPEm directly from blood cultures has great potential to be part of the routine of clinical microbiology laboratories to establish polymyxin B susceptibility, impacting outcome of patients with bloodstream infections caused by carbapenem-resistant Enterobacterales.

Discovery of new eremophilanes from the marine-derived fungus Emericellopsis maritima BC17 by culture conditions changes: evaluation of cytotoxic and antimicrobial activities

In our previous studies, the marine-derived fungus Emericellopsis maritima BC17 was found to produce new eremophilane-type sesquiterpenoids on solid media. In order to explore its potential to produce more metabolites, E. maritima BC17 was subjected to a one strain-many compounds (OSMAC) analysis leading to the discovery of three new eremophilanes (1-3) and fourteen known derivatives (4-17) in the liquid media Czapek Dox and PDB. Their structures were established by extensive analyses of the 1D and 2D NMR, and HRESIMS data, as well as ECD data for the assignment of their absolute configurations. Antitumoral and antimicrobial activities of the isolated metabolites 1, 3, 11, and 15 were investigated. PR toxin 3-deacetyl (15) exhibited cytotoxic activity against HepG2, MCF-7, A549, A2058 and Mia PaCa-2 human cancer cell lines with IC50 values ranging from 2.5 to 14.7 µM. In addition, 15 exhibited selective activity against methicillin-sensitive Staphylococcus aureus ATCC29213 at the highest concentration tested of 128 µg/mL.

In vitro microrhizome induction in Acorus calamus L., a commercially important aromatic medicinal plant

Acorus calamus L., is a leading aromatic medicinal plant that produces pungent aromatic rhizomes that are valued worldwide as an important herbal medicine and is one of the main ingredients in several polyherbal formulations used for neurological and metabolic disorders. The present investigation aims to develop an efficient protocol for in vitro microrhizome induction in A. calamus for the large-scale production of disease-free planting material for commercial purposes. In vitro derived shoots were initiated on MS medium supplemented with different concentrations of sucrose alone (3–10 %) and different concentrations of sucrose (6 % and 7 %) with varying concentrations of BAP alone (0.5–2 mg/L) and combinations with IAA (0.5 and 1 mg/L) and NAA (0.5 and 1 mg/L) were carried out for the microrhizome induction experiments. In different concentrations of sucrose used, healthy and disease-free microshoots of A. calamus were obtained in MS media supplemented with 7 % sucrose followed by 6 % sucrose. The highest shoot length (24.79 ± 0.03 cm) and microrhizome size (4.98 ± 0.03 cm length and 399.60 ± 0.37 mg fresh weight) were obtained in MS solid medium supplemented with 1 mg/L BAP and 0.5 mg/L NAA with 7 % sucrose followed by the same hormone concentration with 6 % sucrose (shoot length-23.68 ± 0.03 cm, microrhizome length- 4.68 ± 0.03 cm and fresh weight of microrhizome-376.60 ± 0.57 mg). The developed protocol can be used for large-scale production of disease-free propagules without rooting and acclimatization and enhance the production of true-to-type planting material.

Statistical theory of fluids confined in quenched disordered porous media.

We develop a theory to calculate structural correlations and thermodynamic properties of a fluid confined in a random porous solid medium (matrix). We used density functional formalism to derive an annealed averaged expression for the density profile and excess free energy of fluid arising due to random fields of a particular realization of the matrix. After performing the second average over the quenched-disordered variables, the excess free energy is organized to give one- and two-body potentials for fluid particles. The average over disorder reduces the system to an effective one-component system of fluid in which particles feel one-body (external) potential and interact via effective pair potential. The effective pair potential is a sum of the bare (the one in the pure fluid) and the matrix-induced potential. The resulting partition function involves only fluid variables. Equations are derived for fluid-fluid and fluid-matrix correlation functions and for free energy, pressure, and chemical potential of the fluid. The theory is applied to a model system of hard spheres and results for the effective pair potential, correlation functions, and thermodynamic properties are reported. The effective pair potential is found to be attractive at the contact and develops a repulsive peak before decaying to zero. Results for pair correlation function and structure factor are compared with simulation results for several fluid densities at two matrix densities. In all the cases, a very good agreement has been found.

Antimicrobial Activity of Fungal Endophytes Associated with Peperomia argyreia (Piperaceae)

The endophytic fungal biodiversity of unique plants like Peperomia argyreia (Miq.) É. Morren (Piperaceae) has antimicrobial properties and can be employed for infection treatment. Fungal isolates were obtained from appropriately treated plant tissues cultured in solid media, characterized by morphology, and identified by molecular biology using ITS and NL primers. The antimicrobial properties of fungal extracts were analyzed by combining microdilution and bioautographic assays complemented with metabolic profiling by automated thin-layer chromatography and 1H NMR techniques. Thirty-one filamentous fungi were isolated and characterized by ITS and/or D1/D2 region amplification of rDNA, identified as Thermothielavioides, Trichoderma, Cyphellophora, Cladosporium, Arcopilus, Plectosphaerella; Chaetomium, Sporothrix, Alboefibula, and Penicillium. Thermothielavioides spp. inhibited Staphylococcus aureus ATCC 25923; moreover, Penicillium westlingii P4 showed inhibitory activity on Ascochyta rabiei AR2. The bioactivity-guided fractionation of the EtOAc extract (MIC = 62.5 μg/mL) of P. westlingii P4 allowed the purification of citrinin as the main inhibitory compound (MIC = 62.5 μg/mL). Peperomia argyreia harbors a rich and diverse endophytic community able to produce bioactive molecules. Citrinin, with a minor influence of volatile compounds biosynthesized by P. westlingii P4, was responsible for the inhibition of A. rabiei AR2.

A polyphasic characterisation of Aspergillus section Flavi isolated from Malaysian and Nigerian food grains and poultry feeds by phenotypic, chemotypic, and molecular methods

The taxonomy and toxigenicity of Aspergillus section Flavi (ASF) in various regions are still under-reported, as many potentially toxic species are continuously discovered. This study aims to determine the aflatoxigenicity and identify 74 isolates of ASF from Malaysian and Nigerian food grains and feeds using phenotypic, chemotypic, and molecular methods. Isolates were screened for sclerotium morphotype and aspergillic acid production phenotypically, while an ultra-fast reverse-phase high-performance liquid chromatography (HPLC) was optimised to quantify the cyclopiazonic acid and aflatoxins produced by the isolated ASF, based on which, they were characterised into phenotypes L-strains, S-strains, and NS-strains, and chemotypes I to VI. Chemotypic data and nucleotide sequences of β-tubulin and internal transcribed spacer (ITS) genes were utilised to identify the isolates’ species and determine their phylogenetic relationships. Aflatoxigenesis on solid media and multiplex polymerase chain reactions of four essential genes (aflR, aflM, aflD, and aflP) of aflatoxin biosynthesis were used to confirm the aflatoxigenicity of each isolate. All the six chemotypes of ASF were present among the isolated strains, with the aflatoxigenic chemotypes (55.41%) and CPA-positive isolates (68.92%) being significantly higher (p < 0.05) than the non-aflatoxigenic and CPA negative chemotypes. Aflatoxins and CPA were produced in the 0.25 – 19.58 ng/g range and 29.89 – 46.55 ng/g, respectively. Chemotypic data accurately differentiate toxigenic from atoxigenic species but only identify 90% of species names. Molecular data accurately resolved species but not toxigenicity. The results highlight the need for robust regulatory measures in the concerned regions to mitigate the risk of aflatoxicosis.

Planning and design of an architectural energy supply system based on photovoltaic-thermal technology

China’s building energy consumption is growing and the energy consumption of the whole process of buildings accounts for more than 46.5% of the country’s total energy consumption, of which the proportion of energy consumption used for heating, ventilation and air conditioning is as high as 50%. Solar energy has the greatest potential to achieve energy conservation in buildings. Buildings are the best carrier for solar energy utilization because of the advantages of local collection and local application. The combination of solar energy and buildings can meet a variety of energy and health needs in buildings, among which solar photovoltaic and CSP building integration technology is an important way to reduce building energy consumption and improve indoor air quality. This article presents a PV/T-based thermal storage wall system that efficiently utilizes solar energy. It outlines the PV/T technology’s fundamental principles and provides a detailed description of the system’s construction and operation, highlighting critical components like the solid thermal storage medium and heat transfer via a heat pump. The analysis indicates improved energy utilization and favorable economic feasibility. This technology offers a practical solution for the construction industry, promoting sustainable energy practices and environmental conservation.

Assessment of antimicrobial and anti-biofilm activities of lemongrass and bay leaf extracts on microorganisms from fermented cereal-based porridges in northern Benin

Lemongrass (Cymbopogon citratus) and bay leaf (Laurus nobilis) are condiments in food processing. This study aimed to assess the antimicrobial and anti-biofilm activities of lemongrass and bay leaf extract on the microorganisms isolated from cereal-based fermented porridges produced in northern Benin. The phytochemical components of lemongrass and bay leaf extracts were analyzed qualitatively using two solvents: water and alcohol. The antimicrobial activity of the extracts was tested using the solid media diffusion method, and the minimum concentration was determined using the microdilution method. The extracts were also evaluated for their ability to inhibit biofilm formation using qualitative assays. The two plants have different phytochemical profiles. Tannins, flavonoids, mucilage, and alkaloids were detected in both plants. Cyanogenic derivatives, O-heterosides, and genin-reduced O-heterosides were absent. L. nobilis contained anthocyanins, quinone derivatives, saponosides, reducing compounds, and free anthracenes, absent in C. citratus. The inhibition diameters ranged from 4.4 to 26.3, depending on the strains and extracts, and varied significantly. The MICs for Escherichia coli ranged from 4.5 mg/ml to 7 mg/ml, staphylococci from 3.75 mg/ml to 10 mg/ml, and lactic bacteria from 2.5 mg/ml to 4.44 mg/ml. The CMB varied between 8.125 mg/ml and 14 mg/ml for E. coli, 7.5 mg/ml to 20 mg/ml for staphylococci, and 6 mg/ml to 10 mg/ml for lactic bacteria. It is worth noting that staphylococci are the most biofilm-producing strains (80%), followed by E. coli (60%), and lactic bacteria produce less biofilm (30%). The findings of this research confirm the presence of a wide range of biological activities, including antibacterial and anti-biofilm, in the aqueous and ethanolic extracts of these two aromatic plants. Further research is required to identify and isolate the active components responsible for their medicinal properties and ascertain their operation mode.

Microbiological Profile of Culture-Positive Fungal Keratitis.

To examine the microbiological profile of cases of culture-positive fungal keratitis presenting to a tertiary eye care center in eastern India. Microbiology records of all culture-positive microbial keratitis patients presenting to L V Prasad Eye Institute, Bhubaneswar, between January 2020 and December 2021, were retrospectively reviewed. Collected data included smear results of culture-positive fungal or mixed infections, the species isolated, and the time taken for organisms to grow in each media. Fungal keratitis formed 36% of all culture-positive microbial keratitis, whereas mixed infections (fungi and other organisms) formed 8.5%. The most common fungal species isolated was Fusarium spp. (25.8%). The most common bacteria involved in mixed infection with fungi was Staphylococcus spp. (54.8%). The positivity of potassium hydroxide+calcofluor white stain in detecting fungal filaments was 89.0% and that of Gram stain was 76.1%. Culture-positive cases of fungal keratitis showed most frequent growth on potato-dextrose agar (77.6%). A similar pattern was observed in culture-positive mixed infections (Sabouraud dextrose agar [SDA]: 84%). Most frequent growth of bacteria in mixed infections was seen in thioglycolate broth (54.7%). The shortest time to achieve significant fungal growth was observed in blood agar (BA) and chocolate agar (CA) (2.2/2.3 days, and 1.8/2 days for fungal keratitis and mixed infections, respectively). Filamentous hyaline fungi took the shortest time to achieve significant growth (2.8 days), whereas yeast forms took the longest (5 days). This study highlights the importance of combined use of both solid and liquid culture media, especially potato dextrose agar (PDA)/SDA and CA, to arrive at a definitive diagnosis of fungal keratitis and possible bacterial co-infection, which forms a significant proportion of cases with fungal keratitis. In resource-poor laboratories, two culture media, either SDA or PDA, along with BA, may be plated to detect mixed infections. Examination of stained smears of corneal samples provides an inexpensive method of rapid diagnosis of fungal keratitis when culture media is not available.

Morphological and Biochemical Characterization of Strains of Ralstonia solanacearum, Causal Agent of Bacterial Wilt in Tomatoes in Cameroon and Screening of Virulent Strains

Ralstonia solanacearum is a major constraint in tomato production. The aim of this work was to identify the different biovars and races of R. solanacearum which infect tomato plants in Cameroon and determine the most virulent in a gnotobiotic environment. Thirty (30) samples were collected in the field of 03 different agro-ecological zones considered as major tomato production area in Cameroon. The bacteria were isolated on modified Kelman solid medium, and identified based on morphological and biochemical characterization. The pathogenicity test was performed in a gnotobiotic environment by root inoculation. Twenty-nine (29) isolates showed colonies characteristic of virulent strains. Water, motility, catalase, KOH, Kovac oxidase, glucose reduction, and sucrose tests were positive. However, gram staining, spore production, arginine test and sulfate reduction were negative. The biovar tests carried out revealed the presence16.66% biovar 2 (bv 2) and 76.66% biovar 3(bv 3). The tobacco hypersensitivity tests carried out revealed the presence of two (02) races: race 1 and race 3 (majority). A predominance of race3 biovar 3 was observed in Cameroon. Results from pathogenicity test revealed that two (02) strains, FM6 (race 3 bv2) and BFo (race 3 bv 3) were high virulent, causing up to 100% loss of seedlings on the sixth day post-inoculation.

Bactericidal effect of bacteria isolated from the marine sponges Hymeniacidon perlevis and Halichondria panicea against carbapenem-resistant Acinetobacter baumannii.

In this study, we evaluated the antimicrobial activity of bacteria isolated from the marine sponges Hymeniacidon perlevis and Halichondria panicea against seven Acinetobacter baumannii strains, the majority of which were clinically relevant carbapenem-resistant A. baumannii strains. We observed the inhibitory activity of 18 (out of 114) sponge-isolated bacterial strains against all A. baumanii strains using medium-throughput solid agar overlay assays. These inhibitory strains belonged to the genera Lactococcus, Pseudomonas, and Vagococcus. In addition, this antimicrobial activity was validated through a liquid co-cultivation challenge using an inhibitory strain of each genus and a green fluorescent protein-tagged A. baumanii strain. Fluorescence measurements indicated that the growth of A. baumanii was inhibited by the sponge isolates. In addition, the inability of A. baumanii to grow after spreading the co-cultures on solid medium allowed us to characterize the activity of the sponge isolates as bactericidal. In conclusion, this study demonstrates that marine sponges are a reservoir of bacteria that deserves to be tapped for antibiotic discovery against A. baumanii.

Isolation, and selection of indigenous potassium solubilizing bacteria from Vietnam Mekong Delta rhizospheric soils and their effects on diverse cropping systems

Potassium (K) is an essential nutrient for plant growth, biosynthesis, and metabolism. It enhances plant tolerance to salinity and acidity by triggering various defense mechanisms. These characteristics are particularly important to agricultural production in the Vietnam Mekong River Delta, experiencing increased saltwater intrusion, precipitation extremes and unpredictable drought. This study isolated and selected indigenous potassium-solubilizing bacteria (PSB) from Vietnam Mekong Delta rhizospheric soils collected from three different ecological systems producing rice, maize, kumquat, and green onion. Experiments were conducted to evaluate the effect of indigenous PSB on rice seedlings and future potential as biostimulants for commercial field application. To isolate PSB, solid Aleksandrov medium supplemented with 0.2% muscovite was used. The soluble K was quantified using the atomic absorption method at a wavelength of 766.5 nm. Among 24 isolates, Burkholderia vietnamiensis L1.1 and Staphylococcus hominis T7.3 exhibited the highest K solubilization, releasing 567 μg L−1 and 488 μg L−1 liquid medium, respectively. The study explored the optimal conditions for K release, revealing that a temperature of 35 °C, a pH 5, and NaCl concentration up to 1 % in a medium favored K solubilization. B. vietnamiensis L1.1 significantly enhanced rice seed germination (95.3 %) compared to the control treatment without PSB (82 %). Furthermore, the combined application of B. vietnamiensis L1.1 and S. hominis T7.3 notably improved rice growth under K deficient conditions. This research suggests that these indigenous PSB can effectively increase plant available K and improve the productivity of rice and other crops grown under saline and acidic stresses.

Real-Time Detection of Yeast Growth on Solid Medium through Passive Microresonator Biosensor

This study presents a biosensor fabricated based on integrated passive device (IPD) technology to measure microbial growth on solid media in real-time. Yeast (Pichia pastoris, strain GS115) is used as a model organism to demonstrate biosensor performance. The biosensor comprises an interdigital capacitor in the center with a helical inductive structure surrounding it. Additionally, 12 air bridges are added to the capacitor to increase the strength of the electric field radiated by the biosensor at the same height. Feasibility is verified by using a capacitive biosensor, and the change in capacitance values during the capacitance detection process with the growth of yeast indicates that the growth of yeast can induce changes in electrical parameters. The proposed IPD-based biosensor is used to measure yeast drop-added on a 3 mm medium for 100 h at an operating frequency of 1.84 GHz. The resonant amplitude of the biosensor varies continuously from 24 to 72 h due to the change in colony height during vertical growth of the yeast, with a maximum change of 0.21 dB. The overall measurement results also fit well with the Gompertz curve. The change in resonant amplitude between 24 and 72 h is then analyzed and reveals a linear relationship with time with a coefficient of determination of 0.9844, indicating that the biosensor is suitable for monitoring yeast growth. Thus, the proposed biosensor is proved to have potential in the field of microbial proliferation detection.

In vitro and in vivo screening of bacterial species from contaminated soil for heavy metal biotransformation activity

Heavy metals (HMs) are widely used in various industries. High concentrations of HMs can be severely toxic to plants, animals and humans. Microorganism-based bioremediation has shown significant potential in degrading and detoxifying specific HM contaminants. In this study, we cultivated a range of bacterial strains in liquid and solid nutrient medium containing different concentrations of different HMs to select and analyze bacteria capable of transforming HMs. The bacterial strains most resistant to selected HMs and exhibiting the ability to remove HMs from contaminated soils were identified. Then, the bacterial species capable of utilizing HMs in soil model experiments were selected, and their ability to transform HMs was evaluated. This study has also generated preliminary findings on the use of plants for further removal of HMs from soil after microbial bioremediation. Alcaligenes faecalis, Delftia tsuruhatensis and Stenotrophomonas sp. were selected for their ability to grow in and utilize HM ions at the maximum permissible concentration (MPC) and two times the MPC. Lysinibacillus fusiformis (local microflora) can be used as a universal biotransformation tool for many HM ions. Brevibacillus parabrevis has potential for the removal of lead ions, and Brevibacillus reuszeri and Bacillus safensis have potential for the removal of arsenic ions from the environment. The bacterial species have been selected for bioremediation to remove heavy metal ions from the environment.

Clostridioides difficile in calves, cattle and humans from Dutch dairy farms: Predominance of PCR ribotype 695 (clade 5, sequence type 11) in cattle

BackgroundClostridioides difficile is a leading cause of infectious diarrhea in both humans and livestock. In particular, C. difficile strains belonging to sequence type (ST) 11 are common enteropathogens. The aim of this study was to determine the presence and genetic relatedness of C. difficile types in dairy cattle and calves. MethodDutch dairy farms were visited between February and December 2021. Feces was collected from adult dairy cattle and calves of two age categories (<4 weeks and 4 weeks-4 months). Fecal samples were also requested from dairy farmers, family members and employees. Fecal samples were cultured in an enrichment medium for 10–15 days and subcultured on solid media for capillary PCR ribotyping and whole genome sequencing. ResultsC. difficile was detected on 31 out of 157 (19.8%) dairy farms. The highest prevalence was found in calves <4 weeks (17.5%). None of the 99 human samples collected were positive. Thirty-seven cultured isolates belonged to 11 different PCR ribotypes (RT) of which RT695 (56.8%) and RT078/126 (16.2%) were most abundant. In the database of the Netherlands National Expertise Centre for C. difficile infections (CDI, >10.000 patient isolates), RT695 was found in only two patients with hospital-onset CDI, diagnosed in 2020 and 2021. Sequence analysis of 21C. difficile RT695 from cattle revealed that all isolates belonged to clade 5, ST11 and contained genes encoding toxin A, toxin B and binary toxin. RT695 strains carried antimicrobial resistance genes typically found in clade 5C. difficile. Groups of genetically related RT695 isolates were found between dairy farms, whereas identical strains were only present in individual farms. ConclusionsC. difficile was found in ∼20% of dairy farms with a predominance of the relatively unknown RT695. Isolates of RT695 belonged to the same clade and sequence type as RT078/126, which is recognized as an important zoonotic type.

Characterization, Comparative Assessment and Antibacterial Potential of Copper(II) Soya Complexes against Staphylococcus Aureus

Copper(II) soap complexes have been proven their activity against bacteria very effectively. Herein, the influence of biophysical and biomechanical parameters on the activity of Copper(II) soya thiourea complex was evaluated. To this aim, liquid as well as solid growth media were developed by Kirby-Bauer disc diffusion method. The antibacterial activity of Copper(II) soya thiourea complex against the Gram-positive bacterium Staphylococcus aureus was assessed in various concentration of Copper(II) Soya complexes. Copper (II) Soya complexes also resist bacterial growth at higher concentration. This review provides a board overview of Staphylococcus aureus with an emphasis on the Copper(II) soya thiourea complex

First Report of Neofusicoccum parvum causing leaf spot on Camellia japonica in China.

Camellia japonica is an important garden landscape plant in southern China. In April 2022, leaf spot symptoms were observed at the camellia garden of Jiaying University (24°32'83″N, 17 116°12'31″E) in Meizhou city, Guangdong Province, China. The initial symptoms were grayish brown spots on the leaves, as the disease progressed, the lesions were enlarged and affected the whole leaf and eventually led to the loss of its ornamental value. The disease incidence was above 15%. Leaf pieces (5 × 5 mm) from 3 diseased Camellia leaves were sterilized in 75% ethanol for 1 min, then in 1% NaOCl for 1 min; and rinsed three times with sterile water. Leaf pieces were inoculated on potato dextrose agar (PDA) medium and incubated at 25 °C. Three days later, fungal colonies initially showed a white aerial mycelium, turning gray after 5 days, and dark gray after 7 days of incubation. Conidia were single-celled, hyaline, ellipsoidal and without septa. Dimensions of conidia (n≥50) were 14.27 to 20.65 × 4.28 to 6.56 μm. The morphological characteristics matched the genus Neofusicoccum (Pavlic et al. 2009). For molecular identification, the rDNA internal transcribed spacer (ITS1, 5.8S and ITS2) region, translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2) of a representative isolate SC6-2 were amplified using the primer pairs ITS1/ITS4, EF1/EF2 and BT2a/-BT2b, respectively (Golzar and Burgess，2011). The sequences obtained were deposited in GenBank (accession nos. PP064173, PP479650 and PP082457 for ITS, tef1-α and tub2, respectively). Nucleotide BLAST analysis showed a 99.81% homology with N. parvum (519/520 bp, OQ509869; 519/520 bp, KF294003; 518/519 bp KF293989) for ITS, 100% homology with N. parvum (398/398 bp, MN318108; 398/398 bp, MK294085; 398/398 bp, MH936021) for tub2, and >99% homology with N. parvum (259/259 bp, 100%, MW390561; 263/265 bp, 99.25%，MN175952; 263/265 bp, 99.25%, MK781982) for tef1-α. The combined phylogenetic analyses (ITS, tef1-α, and tub2) showed that the sequence of the tested isolate and the corresponding sequence of N. parvum (CMW9081, SHSJ1-2) in GenBank grouped in the same branch of the phylogenetic tree. Based on morphological characters, DNA sequencing, and the phylogenetic tree, it can be determined that the pathogen was Neofusicoccum parvum. Inoculation on Camellia leaves was performed to confirm pathogenicity. Nine healthy camellia leaves were pin-pricked with a sterile needle and inoculated with mycelial plugs of isolate SC6-2. Nine other healthy leaves were pin-pricked and inoculated with noncolonized PDA plugs as control leaves. The inoculated leaves were maintained on water agar solid medium at 25°C. To keep a high-humidity environment the inoculation sites were covered by moistened cotton for 2 days. The experiment was repeated three times. Five days after inoculation, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 6 days. The fungal isolates recovered from inoculated leaves were morphologically identical to the N. parvum isolates originally recovered from symptomatic leaves collected in the field, fulfilling Koch's postulates. Neofusicoccum parvum is an aggressive pathogen that causes severe disease on important tree and woody species (Liddle et al. 2019). It has been reported that N. parvum can infect the leaves and branches of grapes (Otoya-Martinez et al. 2023), dieback on Camellia japonica (Pintos, et al. 2012), Brazilian pepperwood (Bertetti et al. 2022), mango (Giancarlo et al. 2023) and other plants. To our knowledge, this is the first report of N. parvum causing leaf spot on Camellia japonica in China.

Nitrogen-phosphorus dual-doped auricularia auricula porous carbon as host for Li-S battery.

A nitrogen-phosphorus dual-doped porous spore carbon (NP-PSC) positive electrode matrix was prepared using native auricularia auricula as solid medium based on the principle of biomass rot. Yeast was introduce and cultured by the auricularia auricula solid medium. The freeze-drying and carbonization activation processes made the materials present a three-dimensional porous spore carbon aerogel properties. Yeast fermentation transformed auricularia auricula from blocky structure to porous structure and introduced nitrogen-phosphorus dual-doping. The physical and chemical properties of the prepared materials were characterized in detail. Electrochemical performance of NP-PSC in Li-S batteries was systematically investigated. Porous structure and heteroatom-doping improved the electrochemical performance, which is much superior to conventional activated carbon materials.

Peptidoglycan modulation by Francisella tularensis

HAYDEN HESS, STUART CANTLAY, JOSEPH HORZEMPA, Department of Biomedical Sciences, West Liberty University, West Liberty, WV 26074 and MELANIE SAL, Department of Biology, West Virginia Wesleyan College, Buckhannon, WV 26201. Peptidoglycan modulation by Francisella tularensis. Peptidoglycan, a crucial component of bacterial cell walls, plays a fundamental role in maintaining cell shape, integrity, and resistance to osmotic pressure. Mycoplasmas are the only bacteria that do not produce peptidoglycan cell walls. Other peptidoglycan-producing bacteria are capable of transiently existing without a peptidoglycan as an “L-form”, but only on solid media. Preliminary observations made by our laboratory suggested that Francisella tularensis bacteria do not produce detectible levels of peptidoglycan while growing in liquid media. However, increasing salt concentration induces the production of peptidoglycan in all bacteria. In this study we confirmed and extended these observations by incubating bacteria with fluorescent D-alanine (which integrates into the peptidoglycan during synthesis). Fluorescence microscopy revealed that very few F. tularensis bacteria produce detectable peptidoglycan in low salt media, while seemingly all individual bacteria produce this layer in high salt media. Interestingly, the genome of F. tularensis encodes two distinct beta-lactamases. Here we show that both the wild-type F. tularensis LVS and beta-lactamase null mutant were both resistant to high levels of beta-lactam antibiotics in low salt liquid media. However, only the wild type and complemented mutant strains were resistant to beta-lactam antibiotics in high salt media while the beta lactamase null mutant was susceptible. These data further support the conclusion that F. tularensis does not produce, nor need, peptidoglycan in low salt liquid media. Future studies will investigate whether peptidoglycan modulation is required to avoid detection by the innate immune system.

Reliability of a Screening Method Using Antibiotic Disks to Detect Carbapenemases in Glucose-Nonfermenting Gram-Negative Microorganisms From Clinical Samples of a Regional Hospital in Southeastern Spain.

Infections by glucose-nonfermenting gram-negative bacilli (NFGNB) pose a major public health problem due to multiresistance to beta-lactam antibiotics, especially plasmid-borne carbapenemases. Their detection by microbiology laboratories is challenging, and there is a need for easy-to-use and reliable diagnostic techniques. Our objective was to evaluate an in-house screening method to presumptively detect carbapenemases in NFGNB in a simple and clinically useful manner. The study included 175 NFGNB isolates from urinary, respiratory, and rectal samples. In a triple assay, isolates were incubated at 37°C for 24 h on three solid-culture media: MacConkey II Agar, 5% Sheep Blood Columbia Agar and Mueller Hinton II Agar; meropenem (MEM) and cefepime (FEP) disks were employed for screening. Studies were then performed on the inhibition halo diameter, scanning effects, and the appearance of mutant colonies, which were compared with those observed using the colorimetric Neo-Rapid CARB Kit and immunochromatography (NG5-Test Carba and K-Set for OXA-23). Receiver operating characteristic curves were constructed for these data. Carbapenemases were expressed by 79/175 (45.1%): 19 Pseudomonas aeruginosa and 60 Acinetobacter baumannii. Optimal inhibition halo diameter cutoffs to detect this resistance on 5% sheep blood agar were as follows: 6 mm (MEM) and 6.5 mm (FEP) for P. aeruginosa (in the absence of scanning effects and mutations) and 10.5 mm (MEM) and 16 mm (FEP) for A. baumannii (even in the presence of scanning effects). The combined utilization of MEM and FEP antibiotic disks in 5% sheep blood agar, measuring their inhibition haloes, offers an effective method to predict the presence of carbapenemases as resistance mechanism in P. aeruginosa and A. baumannii.