Bacterial Culture Research Articles

A single-center study on the distribution and shifting trend of infectious keratitis pathogens from 2018 to 2022

Objective: To investigate the pathogen species, composition, and distribution characteristics of infectious keratitis pathogens in Shandong Province and its surrounding areas. Methods: In this cross-sectional study, patients with keratitis who underwent corneal sampling and microbiological culture at the Shandong Eye Hospital from January 1, 2018 to December 31, 2022 were included. Under topical anesthesia, the edge of the lesion was scraped by an experienced physician. The samples were inoculated on blood agar and Sabouraud dextrose agar plates, separately for bacterial and fungal culture and identification. If necessary, the samples were inoculated on a non-nutrient agar medium with Escherichia coli for Acanthamoeba culture. Bacterial isolates were identified using Vitek 2 compact or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fungal isolates were identified based on morphological characteristics or sent to a company for sequencing in cases of difficult identification. The epidemiological data of the patients, pathogen species and number (counting as 1 strain if the same strain was isolated from multiple corneal specimens of the same patient), culture positivity rate, and seasonal distribution were recorded. Differences in pathogen positivity rates among different seasons were analyzed using the chi-square test. Results: Among the 4, 024 patients with infectious keratitis during the study period, there were 2 510 males (62.3%) and 1 514 females (37.6%), aged from 46 days to 94 years. Positive microbial culture results were achieved in 2, 363 patients (58.7%), including 906 cases (38.3%) with bacterial positivity, 1 231 cases (52.1%) with fungal positivity, 28 cases (1.2%) with Acanthamoeba positivity, and 198 cases (8.4%) with mixed fungal and bacterial infections. A total of 2 561 strains were isolated, including 1 104 bacterial strains. The most common bacteria were coagulase-negative Staphylococcus spp. (623/1 104, 56.4%), followed by Streptococcus spp. (131/1 104, 11.9%) and Pseudomonas aeruginosa (68/1 104, 6.2%). The most common fungi were Fusarium spp. (634/1 429, 44.4%), followed by Aspergillus spp. (279/1 429, 19.5%) and Alternaria spp. (229/1 429, 16.0%). Bacterial keratitis was more common in summer and autumn, while fungal keratitis was more common in autumn and winter. Conclusions: Among infectious keratitis cases in Shandong Eye Hospital, Fusarium species were predominant fungal pathogens, while coagulase-negative Staphylococcus predominated in bacterial pathogens. Both fungal and bacterial corneal infections showed seasonal variations.

A liquid static culture using a gas-permeable film bag contributes to microbiology

We propose a simple tool for liquid static culture using a copolymer film with high gas permeability. The film bags were successfully used to culture microorganisms Escherichia coli, Komagataella phaffii (methylotrophic) and Bacillus sp. (biofilm-forming), with cells cultured under physical stress-free conditions with sufficient oxygen supply. Similar growth curves and plasmid productivity were observed for liquid shake and film bag E. coli cultures. The early growth response of the film bag culture following colony inoculation of liquid media differed from conventional shake cultures. Our results indicate that a gas-permeable film bag is a promising liquid culture tool and provides novel microbiology materials.

First Report of Rhizopus arrhizusi causing rot of Dictyophora rubrovolvata in China.

Dictyophora rubrovolvata, as an edible fungus with high medicinal value, is widely cultivated in several provinces in China (Hang et al. 2012). However, between December 2023 and March 2024, a rot disease occurred in the main production area in Fengxian District, Shanghai, China (N30°93', E121°49'). The disease incidence was 25% in the affected 1.33-ha growing area. High temperatures (>25℃) and poor ventilation provide favorable conditions for the spread of this disease. The disease mainly occurs at the stage of fruiting bodies formation of D. rubrovolvata. When the epidermis is damaged and broken, it becomes infested with mold, which then produces a layer of moldy rot with pus. The infected D. rubrovolvata tissues at the edge of the lesions were isolated, surface sterilized and cultured on potato dextrose agar (PDA) at 30 ℃ under dark conditions. Pure cultures were obtained by single-spore isolation. After 3 days, isolates were transferred to Czapek Yeast agar (CYA) (Samson et al, 2014). On CYA, the fungal colony consisted of white flocculent hyphae. Scanning electron microscopy analysis showed that the mycelium was white, and the internodes of the stolons formed characteristic pseudoroots, from which upwardly clustered erect, unbranched sporocarp peduncles expanded apically to form rounded sporocarp sacs, within which sporocarp spores were produced. (Hariprasath P, 2019). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS) and intergenic spacer (IGS) gene of the isolate were amplified by PCR (White et al. 1990; Liu XY. 2008). The resulting sequence was deposited in GenBank with accession PP951880 and PQ001670, respectively. PCR results and morphological observations indicated the isolated strain was a pure culture and the strain was designated as DIC01. Comparative results showed that the sequences with accession numbers MT603964.1 and DQ990323.1 showed high homology of 99.15% and 98.96% to the ITS and IGS sequences of Rhizopus arrhizusi DIC01, respectively. Phylogenetic analysis with ITS and IGS genes of the isolated strain and 7 Rhizopus spp. strains were performed using MEGAX with Neighbor-Joining (NJ) method. Based on the results of growth habits, morphological observations, and phylogenetic analysis, the pathogen was identified as R. arrhizusi. A spore suspension of the R. arrhizusi DIC01 (1 x107 conidia/mL) was inoculated back to healthy D. rubrovolvata. Five healthy fruit bodies of D. rubrovolvata were injected, and another five healthy morels were treated with potato dextrose broth (PDB) as controls. D. rubrovolvata was incubated at 25°C and 90% relative humidity without ventilation for 5 days. The pathogen successfully infected the D. rubrovolvata, which developed white moldy lesions similar to those of natural diseases. The controls remained healthy without any symptoms. The pathogen was reisolated from the affected lesions and identified as R. arrhizusi DIC01 based on its morphological characteristics and phylogenetic marker genes. R. arrhizusi has been reported to cause endothelial cell damage and mycelial invasion into blood vessels, leading to thrombosis and tissue necrosis. (Hariprasath P, 2019). To our knowledge, this is the first report of R. arrhizusi causing rot disease of D. rubrovolvata. This study confirmed that R. arrhizusi is the pathogenic fungus responsible for rotting disease in D. rubrovolvata farms in Fengxian, Shanghai.

How Restrictive Legislation Influences Antimicrobial Susceptibility in Selected Bacterial Isolates from the Canine Vagina

Antimicrobial stewardship is one of the cornerstones in the battle against antimicrobial resistance. Restrictive legislation aims to foster antimicrobial stewardship. Prophylactic prescription of antimicrobials is still a widespread practice in canine breeding management to prevent suspected infectious infertility. The aim of this study was to evaluate the efficacy of restrictive legislation in Germany (Veterinary Home Pharmacy Ordinance, TÄHAV) based on resistance profiles of common bacterial isolates from the vaginal tract by comparing the resistance situation before (time frame (TF1)) and after (TF2) its amendment. In total, results of 13,373 antimicrobial susceptibility tests of bacterial isolates of Escherichia coli (n = 5209), beta-hemolytic streptococci (n = 4010), and Staphylococcus (Staph.) intermedius group (n = 4154) derived from canine vaginal swabs were assessed. Antimicrobial susceptibility testing was performed on pure cultures using the broth microdilution method. Susceptibility to selected antimicrobials was evaluated. Susceptibility of Escherichia coli generally increased within TF2 with, however, a significant increase in the number of non-susceptible isolates to cefalexin (p < 0.0001). Beta-hemolytic streptococci exhibited good susceptibility to most antimicrobials. Susceptibility developed ambivalently within the Staphylococcus intermedius group. Despite an overall positive effect of increased susceptibility, an increase in non-susceptibility to single antimicrobials was detected, possibly indicating a need for refinement of the legislation.

Rocky Mountain Spotted Fever Encephalitis and "Starry Sky" Pattern on MRI: A Case Report.

Rocky Mountain Spotted Fever (RMSF) is a tick-borne disease caused by Rickettsia rickettsii (R. rickettsii). RMSF presents after a tick bite with fever, rash, and headache but can also cause more serious neurological manifestations. We report a case of RMSF encephalitis presenting with altered sensorium and rapid progression to coma, fever, and petechial rash, and an magnetic resonance imaging (MRI) brain notable for a "starry sky" pattern. A 61-year-old woman presented with confusion and fever and was diagnosed with a urinary tract infection. Two days later, she became comatose. MRI brain revealed lacunar infarcts in the right centrum semiovale and splenium of the corpus callosum. Lumbar puncture was notable for neutrophilic pleocytosis and elevated protein with negative bacterial and viral cultures. Empiric meningitis therapy was initiated, and she was transferred to our institution. On transfer, she was febrile, comatose, and had a diffuse petechial rash. Repeat MRI brain demonstrated diffuse, innumerable punctate foci of diffusion restriction with susceptibility-weighted signal attenuation throughout cerebral hemispheres in a "starry sky" pattern. Skin biopsy revealed perivascular lymphocytic infiltrates. Serologic RSMF antibody titers were obtained, and doxycycline was initiated for presumed RMSF encephalitis. The family opted to pursue palliative measures, given no clinical improvement. RSMF titers and postmortem PCR from brain tissue were positive for R. rickettsii. This case report highlights the clinical presentation of RMSF encephalitis. RMSF encephalitis should be suspected in a patient presenting with encephalopathy, fever, petechial rash, and MRI brain findings of diffuse punctate foci of diffusion restriction and susceptibility-weighted signal attenuation in a "starry-sky" pattern.

The Efficacy of Orange Terpene and Bacillus mycoides Strain BM103 on the Control of Periwinkle Leaf Yellowing Phytoplasma.

Phytoplasmas are obligate phytopathogenic bacteria belonging to the class Mollicutes. The pathogens, transmitted by insect vectors, associated with hundreds of plant diseases worldwide. Due to the regulation on banning use of antibiotics and limited efficacy of the traditional disease management manners, an eco-friendly alternative is needed. Given that terpene and probiotics have antibiotic activity and the ability to induce systemic resistance, in this study, the effectiveness of orange terpene and a Bacillus mycoides strain, BM103, was evaluated in periwinkle plants infected with periwinkle leaf yellowing (PLY) phytoplasma derived from a shoot-tip tissue culture system. Weekly drenching of 1,000 ppm diluted orange terpene emulsion or pre-activated strain BM103 liquid culture dilution exhibited the ability to inhibit PLY phytoplasma accumulation. The expression of the genes associated with plant defense response and flower development was upregulated after treatment. Moreover, pre-treatment of orange terpene or strain BM103 delayed PLY infection via cleft-grafting inoculation. While orange terpene did not suppress the symptoms, strain BM103 did result in a milder symptom expression that might partially attribute to its plant growth-promoting characteristics. Additionally, the pre-activation of strain BM103 may contribute to its efficacy. Taken together, this research indicates that orange terpene and B. mycoides BM103, with the ability to rapidly induce plant defense responses, could potentially be developed into biological control materials as preventive agents or biofertilizers.

Impact of bacterial co-isolation on treatment initiation and long-term prognosis of patients with nontuberculous mycobacterial pulmonary disease: analysis of a prospective cohort study.

Nontuberculous mycobacterial pulmonary disease (NTM-PD), a chronic respiratory condition, presents a growing challenge globally. Uncertainties exist regarding the impact of concurrent bacterial co-isolation on treatment initiation and long-term prognosis. This study analysed data from participants enrolled in an ongoing prospective observational cohort study on NTM-PD (NCT01616745) between 1 July 2011, and 31 December 2022, who provided sputum samples for bacterial culture at enrolment. Identification of potential pathogenic microorganisms (PPMs) was defined as a positive bacterial culture. Clinical characteristics were compared between NTM-PD patients with Pseudomonas, non-pseudomonal PPMs, and those without PPM co-isolation. Cox proportional hazard regression models were employed to assess the association of bacterial co-isolation with rates of NTM-PD treatment initiation and all-cause mortality. Overall, 453 patients (median age, 62 years; 30% male) were included in the analysis. PPMs were co-isolated in 77 patients (17%), including 13 with Pseudomonas species. Co-isolation of Pseudomonas was associated with a significantly higher erythrocyte sedimentation rate (P = 0.02) and St. George's Respiratory Questionnaire score (P = 0.01). Non-pseudomonal PPM co-isolation was significantly associated with a higher likelihood of NTM-PD treatment initiation (adjusted hazards ratio [aHR], 1.56, 95% confidence interval [CI], 1.03-2.36, P = 0.036), whereas co-isolation of Pseudomonas was independently correlated with increased all-cause mortality (aHR, 3.25, 95% CI, 1.08-9.84, P = 0.037). Our findings emphasize the importance of microbial surveillance, as bacterial co-isolation affects treatment initiation and prognosis in patients with NTM-PD.

Introduction of 7-day amotosalen/ultraviolet A light pathogen-reduced platelets in Honduras: Impact on platelet availability in a lower middle-income country.

Honduras became the first lower middle-income country (LMIC) to adopt amotosalen/UVA pathogen-reduced (PR) platelet concentrates (PCs) as a national platelet safety measure in 2018. The Honduran Red Cross (HRC) produces ~70% of the national platelet supply using the platelet-rich plasma (PRP) method. Between 2015 and 2018, PCs were screened with bacterial culture and issued as individual, non-pooled PRP units with weight-based dosing and 5-day shelf-life. PR PCs were produced in six-PRP pools with a standardized dose (≥3.0 × 1011), no bacterial screening and 7-day shelf-life. Gamma irradiation and leukoreduction were not used. PC production and distribution data were retrospectively analysed in two periods. Period 1 (P1) included 3 years of PRP PCs and a transition year (2015-18). Period 2 (P2) included 5 years of PR PCs (2019-23). PC doses were standardized to an equivalent adult dose for both periods. Descriptive statistics were calculated. HRC produced 10% more PC doses per year on average in P2 compared to P1. Mean annual waste at HRC declined from 23.9% in P1 to 1.1% in P2. Two urban regions consumed 96% of PC doses in P1 and 88.3% in P2. PC distributions increased in 14/18 regions. Standardized dosage, PR and 7-day shelf-life increased PC availability, reduced waste, eliminated bacterial screening and avoided additional costs for arboviral testing, leukoreduction and irradiation. Access to PC transfusion remains limited in Honduras; however, the conversion to pooled PR PCs illustrates the potential to sustainably expand PC distribution in an LMIC.

Screening for contamination with genetically modified organisms in food, feed and their additives obtained in retail and online shops in Latvia

The aim of the study was to screen for contamination of food and feed products with genetically modified organisms (GMO) in several sample groups obtained from retail and online shops. The total number of samples was 171, including 67 of food, 26 feed, 27 additive, and 51 samples of dried pollen for human consumption. Detection and quantification of GMO in food and feed was performed according to the approved quantitative real-time PCR methods. Fermentation product samples were analyzed using screening for antimicrobial resistance genes, 16S RNA and ITS1 sequencing, isolation of bacterial cultures and whole genome sequencing. In total, GMO contamination was found in 12 samples (in 10.45% of food and in 19.23% of feed samples). A lower proportion of GMO containing samples were from the European Union (10.26 %) vs. samples from outside (17.02%), but none with ingredients of both origins. Non-compliant were three food and five feed samples. Of the 27 samples of food and feed additives and food supplements, 10 samples were possibly non-compliant, as they contained live bacteria with antimicrobial resistance genes, and seven of them contained possibly recombinant DNA. The study indicates presence of GMO contamination that emphasizes the need for further monitoring.

Palygorskite improves growth performance and prevents liver damage in avian pathogenic Escherichia coli-challenged broiler chickens at an early age.

Avian pathogenic Escherichia coli (APEC) is a major bacterial infection that causes economic losses in the global poultry industry. Palygorskite (PAL) has been shown to enhance growth performance while improving antioxidative and anti-inflammatory properties of broilers. This study evaluated the protective effects of PAL on growth performance and liver function in broilers subjected to APEC challenge. A total of 320 one-day-old male Arbor Acres chicks were divided into four groups with eight replicates of ten birds each, based on a 2×2 factorial arrangement (basal diet or 5g/kg PAL-supplemented diet) and inoculation (bacterial culture medium or APEC). PAL increased body weight gain (BWG) prior to APEC challenge (P < 0.05). However, APEC caused losses in BWG, feed intake (FI), and feed efficiency, along with increased relative hepatic weight, hepatic pathology scores, and hepatic-cell apoptosis rate (P < 0.05). Compared to normal birds, APEC increased interleukin (IL)-1β, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), and nitric oxide (NO) levels, as well as lysozyme (LZM) and myeloperoxidase (MPO) activities, while decreasing total antioxidant capacity (T-AOC) and IL-10 levels, and total superoxide dismutase (T-SOD) and catalase (CAT) activities in both serum and liver, APEC also raised alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, but reduced total protein (TP), albumin (ALB), immunoglobulin (Ig) A, IgG, and IgM levels in serum (P < 0.05). Moreover, APEC increased hepatic mRNA level of IL-1β, IFN-γ, TNF-α, nuclear factor kappa B, and inducible nitric oxide synthase (iNOS), while inhibited mRNA level of IL-10 (P < 0.05). In contrast, PAL increased BWG and FI, alleviated hepatic-cell apoptosis rate during the challenge period (P < 0.05). An incorporation of PAL reduced triglyceride and NO contents, ALT and AST activities, while increasing TP, ALB, IL-10, IgG, and IgM levels in serum, enhancing serum T-SOD and CAT activities, elevating hepatic T-AOC and CAT activities, inhibiting hepatic MDA accumulation, and reducing IL-1β levels and LZM activity in both liver and serum (P < 0.05). An interactive effect was found for hepatic TNF-α and iNOS mRNA expression, in which PAL inhibited their mRNA expression in APEC-challenged birds (P < 0.05). Overall, PAL addition partially mitigated the negative impact of APEC challenge on growth performance and liver function of broiler chicks at an early age.

Prevalence of antibiotic-resistant enterobacteriaceae in domestic wastewater and associated health risks in reuse practices

The use of wastewater for non-potable purposes is an important alternative for addressing water scarcity, especially in developing regions. However, minimizing the risks, particularly those associated with emerging contaminants that may induce resistance among pathogens in wastewater, is crucial. This study assessed the occurrence of antibiotic-resistant bacteria in untreated wastewater used for agricultural purposes and evaluated the quantifiable health risks associated with this practice in Tamale, Ghana. The resistance of some Enterobacteriaceae, such as E. coli, Klebsiella, and Salmonella-Shigella, to four commonly used antibiotics in Ghana was assessed using a conventional microbiological culture approach and the Kirby Bauer disk diffusion method. A Quantitative Microbial Risk Assessment (QMRA) was performed to estimate the health risks associated with two distinct scenarios of wastewater reuse: (1) accidental ingestion of contaminated wastewater and soil, and (2) consumption of vegetables irrigated with wastewater. This approach applied a Monte Carlo simulation based on 10,000 interactions and identified E. coli O157:H7 as the reference pathogen. Among Enterobacteriaceae, Klebsiella pneumoniae, Salmonella-Shigella and E. coli were isolated, in concentrations exceeding the limit recommended by the World Health Organization (103 CFU/100 ml). All the isolated bacteria were resistant to metronidazole (5 μg). Thirty-three per cent of Klebsiella pneumoniae isolates were intermediate/moderately susceptible, and all other bacteria were resistant to amoxicillin (30 μg). All Klebsiella pneumoniae and the majority of Salmonella-Shigella (69.8 %) isolates were resistant to trimethoprim-sulfamethoxazole (25 μg) and tetracycline (30 μg). When assessing health risks, the mean annual probability of infection associated with consuming vegetables irrigated with wastewater varied between 5.14 × 10−2 and 9.79 × 10−1 per person per year. Conversely, for the accidental ingestion scenario, the probability was 1.00 per person per year. In these scenarios, the probability of illness ranged from 1.29 × 10−2 to 2.4 × 10−1 and 2.5 × 10−1 per person per year. The health risks posed by these findings surpass the maximum threshold prescribed by the World Health Organization, thereby emphasizing the need for prompt mitigation strategies.

Biofilm infections of endobronchial valves in COPD patients after endoscopic lung volume reduction: a pilot study with FISHseq

Endoscopic lung volume reduction (ELVR) using endobronchial valves (EBV) is a treatment option for a subset of patients with severe chronic obstructive pulmonary disease (COPD), suffering from emphysema and hyperinflation. In this pilot study, we aimed to determine the presence of bacterial biofilm infections on EBV and investigate their involvement in lack of clinical benefits, worsening symptomatology, and increased exacerbations that lead to the decision to remove EBVs. We analyzed ten COPD patients with ELVR who underwent EBV removal. Clinical data were compared to the microbiological findings from conventional EBV culture. In addition, EBV were analyzed by FISHseq, a combination of Fluorescence in situ hybridization (FISH) with PCR and sequencing, for visualization and identification of microorganisms and biofilms. All ten patients presented with clinical symptoms, including pneumonia and recurrent exacerbations. Microbiological cultures from EBV detected several microorganisms in all ten patients. FISHseq showed either mixed or monospecies colonization on the EBV, including oropharyngeal bacterial flora, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus spp., and Fusobacterium sp. On 5/10 EBV, FISHseq visualized biofilms, on 1/10 microbial microcolonies, on 3/10 single microorganisms, and on 1/10 no microorganisms. The results of the study demonstrate the presence of biofilms on EBV for the first time and its potential involvement in increased exacerbations and clinical worsening in patients with ELVR. However, further prospective studies are needed to evaluate the clinical relevance of biofilm formation on EBV and appropriate treatment options to avoid infections in patients with ELVR.

Acetate-producing bacterium Paenibacillus odorifer hampers lung cancer growth in lower respiratory tract: an in vitro study.

Lung cancer accounts for the large majority of cancer incidence and mortality worldwide for decades. The dysbiotic microbiome and its metabolite secretions in the gut have been regarded as the dominant biological factors in oncogenesis, development, and progression, adding probiotic components of which have come to be potential therapeutic regimes. However, there still exists little knowledge about whether probiotic microorganisms in lower airways inhibit lung cancer by lung microenvironment remodulation. In this study, we performed bioinformatics analysis from previous sequencing data and specific microbiome databases to identify the potent protective microbes in lower airways, followed by bacterial cultivation and morphological verifications in vitro. We found that Paenibacillus odorifer was correlated closely with the anti-tumorous by-product acetic acid in lower respiratory tract. Additionally, the enrichment of this microorganism in the health, rather than in lung neoplasms from public data sets, further confirmed its protective activity in preserving pulmonary homeostasis. Colony cultivation of this strain and targeted metabolite analysis indicated that Paenibacillus odorifer proliferation was weakened at 37°C but lasted longer than it did at the optimal temperature. And performing as a candidate origin of acetic acid, this strain was liable to inhibit the growth of lung cancer cells in time- and dose-dependent approaches which was validated by colony formation assays. These results suggested that Paenibacillus odorifer functions as a candidate probiotic in lower airways to restrict lung cancer cell growth by releasing protective molecules, indicating a potential preventive microbial strategy.IMPORTANCEVarious types of microorganisms in lower respiratory tracts protect local homeostasis against oncogenesis. Although extensive efforts engaged in gut microbiome-mediated pulmonary carcinogenesis, emerging evidence suggested the crucial role of microbial metabolites from respiratory tracts in modulating carcinogenesis-related host inflammation and DNA damage in lung cancer, which was still not fully understood in lower respiratory tract microbes and its metabolite-mediated microecological environment homeostasis in preventing or alleviating lung cancer. In this study, we analyzed the lower respiratory tract microbiome and SCFAs expression among different lung segments from the same participants, further identifying that Paenibacillus odorifer was correlated closely with anti-tumorous by-product, acetate acid in lower respiratory tract by multi-omics analysis. And previous experiments showed this strain could inhibit the growth of lung cancer cells in vitro. These findings indicated that Paenibacillus odorifer in lower respiratory tracts might perform as a candidate probiotic against lung carcinogenesis by releasing protective factor acetate, which further presented a promising diagnostic and interventional approach in clinical settings of lung cancer.

Growth of sulfate-reducing Desulfobacterota and Bacillota at periodic oxygen stress of 50% air-O2 saturation

BackgroundSulfate-reducing bacteria (SRB) are frequently encountered in anoxic-to-oxic transition zones, where they are transiently exposed to microoxic or even oxic conditions on a regular basis. This can be marine tidal sediments, microbial mats, and freshwater wetlands like peatlands. In the latter, a cryptic but highly active sulfur cycle supports their anaerobic activity. Here, we aimed for a better understanding of how SRB responds to periodically fluctuating redox regimes.ResultsTo mimic these fluctuating redox conditions, a bioreactor was inoculated with peat soil supporting cryptic sulfur cycling and consecutively exposed to oxic (one week) and anoxic (four weeks) phases over a period of > 200 days. SRB affiliated to the genus Desulfosporosinus (Bacillota) and the families Syntrophobacteraceae, Desulfomonilaceae, Desulfocapsaceae, and Desulfovibrionaceae (Desulfobacterota) successively established growing populations (up to 2.9% relative abundance) despite weekly periods of oxygen exposures at 133 µM (50% air saturation). Adaptation mechanisms were analyzed by genome-centric metatranscriptomics. Despite a global drop in gene expression during oxic phases, the perpetuation of gene expression for energy metabolism was observed for all SRBs. The transcriptional response pattern for oxygen resistance was differentiated across individual SRBs, indicating different adaptation strategies. Most SRB transcribed differing sets of genes for oxygen consumption, reactive oxygen species detoxification, and repair of oxidized proteins as a response to the periodical redox switch from anoxic to oxic conditions. Noteworthy, a Desulfosporosinus, a Desulfovibrionaceaea, and a Desulfocapsaceaea representative maintained high transcript levels of genes encoding oxygen defense proteins even under anoxic conditions, while representing dominant SRB populations after half a year of bioreactor operation.ConclusionsIn situ-relevant peatland SRB established large populations despite periodic one-week oxygen levels that are one order of magnitude higher than known to be tolerated by pure cultures of SRB. The observed decrease in gene expression regulation may be key to withstand periodically occurring changes in redox regimes in these otherwise strictly anaerobic microorganisms. Our study provides important insights into the stress response of SRB that drives sulfur cycling at oxic-anoxic interphases.1UtHR5dBbf2f8WaGLBYUtHVideo

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of InVitro Cultivation and Membrane Integrity Methods.

Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%. Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

Foliar blight, leaf spot, and stem blackening diseases caused by Stemphylium beticola, Alternaria alternata, and Alternaria atra on dragon's head (Lallemantia iberica) in Iran

Dragon's head, Lallemantia iberica, is an oilseed crop adapted to be grown in cold-dry areas. In the growing season of 2022–23 a severe foliar blight, leaf spot, and stem blacking symptoms were observed in commercial fields in northern and northwestern Iran, leading to more than 50 % yield loss. Infected samples were collected and transferred to the laboratory. Following the isolation of fungal agents, five representative isolates were selected for further studies. Morphological studies, based on conidial shape, conidiation, and other mycological characteristics, were carried out on a pure culture established from a single spore. Furthermore, multigene phylogenetic analyses were performed using sequences of translation elongation factor 1-α (TEF1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Internal Transcribed Spacer (ITS) rDNA genes for Alternaria isolates, along with calmodulin (cmdA) for the Stemphylium isolate. The morphological and molecular studies identified the fungal isolates as Alternaria atra, Alternaria alternata, and Stemphyliium beticola. Pathogenicity tests on the commercial cultivar ‘Sara’ indicated that all isolates were pathogenic to dragon's head, but they differed significantly in aggressiveness. To the best of our knowledge, this is the first report of the association of these fungal species with an important foliar disease on this crop in Iran and worldwide.

Advanced Electrochemical Biosensing toward Staphylococcus aureus Based on the RPA-CRISPR/Cas12a System and Conductive Nanocomposite.

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that poses significant challenges to food safety. Herein, a sensitive and specific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed for evaluating S. aureus. In the presence of S. aureus, the extracted target DNA fragments are efficiently amplified by recombinase polymerase amplification (RPA). The designed crRNA, binding to Cas12a, effectively recognizes the target fragment cleaving hpDNA. The signal molecule of hpDNA is cleaved from the sensing interface, resulting in a reduction of current response. Under optimal experimental conditions, the developed electrochemical biosensor exhibits remarkable sensitivity in detecting S. aureus. The linear range for quantifying S. aureus in pure culture is 1.04 × 101-1.04 × 108 CFU/mL, with a detection limit as low as 3 CFU/mL. In addition, the biosensor enables the accurate and sensitive detection of S. aureus in milk within a linear range of 1.07 × 101-1.07 × 107 CFU/mL. The electrochemical biosensor enhances anti-interference capability owing to the specific amplification of RPA primers and the single-base recognition ability of crRNA. The RPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference capability, precision, and sensitivity, thereby establishing a robust foundation for real-time monitoring of microbial contamination.

Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Two Clarireedia spp., the Causal Agent of Dollar Spot of Turfgrass.

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental United States. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe, and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount of purified DNA needed for detection was 0.05 ng μl-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot-infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple, and reliable detection in 1.25 h. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific method for the detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.

First Report of Fusarium concentricum Causing Leaf Blight, Shoot Blight, Flower and Fruit Rot on Kadsura coccinea in China.

Kadsura coccinea (Lem.) A. C. Smith is a traditional medicinal plant grown in south China. Lignans and terpenoids extracted from the root, fruit and stem were found to have anti-proliferative, anti-HIV, anti-hepatitis, anti-oxidant, neuroprotective, and other pharmacological attributes for treating rheumatoid arthritis and gastroenteric et al. disorders (Yang et al., 2020). However, little is known about the biotic disorders of this evergreen climbing shrub. In surveys carried out in a 15- hectare -orchard located 23°59'55''N, 113°55'13''E, all K. coccinea plants were observed exhibiting an array of symptoms including leaf blight, shoot blight, flower rot and fruit rot from May to July of 2023, with disease incidences of 17% , 18%, 16% and 28%, respectively. A greyish -brown blight appeared predominantly on the leaf margins or tips. Light to dark brown lesions on the shoots, flower calyxes and fruits were slightly sunken, irregularly shaped, and watery, usually with white aerial mycelium. For pathogen isolation, infected tissues were cut into fragments of about 5 mm in diameter, surface sterilized with 75% ethanol for 15 s and 1% NaClO for 2 min, and then rinsed three times with sterilized distilled water. Filter paper-dried tissues were placed onto potato dextrose agar (PDA) and incubated at 28°C for 5 days. Seventy-one morphologically similar colonies were produced from 100 tissue fragments, with other tissues lacking colonies or with different colonies. Genomic DNA of 10 randomly selected isolates were extracted from 5-day-old pure cultures. Molecular characterization of the 10 subcultured strains was analyzed by sequencing three regions i.e. translation elongation factor 1-alpha (TEF-1), beta-tublin (TUB), and RNA polymerase Ⅱ second largest subunit (RPB2), amplified using primers EF1/EF2 (O'Donnell et al., 1998), T1/T22 (O'Donnell and Cigelnik 1997), and RPB2-5f2/RPB2-7cr (Liu et al. 1999). Maximum likelihood and Bayesian Inference analysis based on the dataset of the combined three sequences by MEGA 11.0 showed that nine strains were consistently identified as F. concentricum. The three sequences (OR632200, OR632199, and OR754282) of a representative strain (SGXF1) shared 99.42%, 99.62%, and 99.80% identity with those of the type strain CBS450. 97. The colonies were white to pale buff with serrated edges and sparse aerial mycelium that initially formed a loose, white cottony texture of 5-10 mm in height. After 7 d's culture at 26°C in an incubator with a photic door, dense and abundant fluffy reddish-white aerial mycelium covered the entire PDA medium of 9 cm-diameter, with alternating pale orange and reddish-grey concentric rings at center with diffusible pigments. Catenate microconidia were obovoid to fusoid-shaped, mostly 0-septate, with a flattened base, (10.98 ± 0.83) x (3.41 ± 0.15) μm (n=20), and were produced on both mono-and polyphialides, whereas macroconidia were curved and long, with a slightly beaked apical cell and a basal cell, mainly 3- to 5-septate, and (33.80 ± 0.81) x (4.31 ± 0.12) μm (n=20) in size. These morphological characteristics further indicates it to be F. concentritum (Nirenberg and O'Donnell, 1998; Xiao et al., 2023). To determine pathogenicity, three 1-y-old seedlings were wound inoculated by conidial suspension (1 × 106 conidia/mL) on different tissues and incubated at 25°C in a greenhouse with 10 replications. At 2 d after inoculation (DAI), the treated flowers gradually turned from fresh red and green to black at calyxes, with the black area then gradually expanding. At 6 DAI, part of the flowers turned black, accompanied by white mycelium and yellowish powdery spots. Similarly, leaves, stems, and fruits turned from green to dark brown at infection sites within 7 DAI. Black to brown, uneven necrotic areas, and reddish spots were observed under a dissecting microscope, with white mycelium attached at 6 DAI. The symptoms observed in lab were similar to those observed in the field, whereas three control plants remained asymptomatic. Fungal colonies identical to initial isolations were recovered from artificially infected tissues and confirmed to be F. concentricum again, thereby completing Koch's postulates. This report is the first to document F. concentricum causing disease on K. coccinea. Appropriate measures will be developed based on this study to protect this economically important plant in the field.

Prevalence and Antimicrobial Susceptibility Patterns of Wound and Pus Bacterial Pathogens at a Tertiary Care Hospital in Central Riyadh, Saudi Arabia

The long history and extensive use of antibiotics have caused resistant bacterial pathogens to emerge, increasing mortality and morbidity. The current study was designed to see the prevalence of aerobic bacterial isolates with their antimicrobial resistance pattern from out- and inpatients requested for wound or pus culture. Retrospective study conducted at a tertiary care hospital in central Riyadh from January 2023 to December 2023. Samples were collected and inoculated onto the appropriate media following standard guidelines. Bacterial pathogens were identified by the Vitek2 compact system. Antimicrobial susceptibility was tested using the Kirby–Bauer disk diffusion method as well as by MIC determination through the Vitek2 compact. A total of 1186 subjects were included in the study with a bacterial isolation rate of 691 (58.3%). Out of these, 155 positive cultures had incomplete information or anaerobic or fungal growth and were excluded from the study. With a slight female predominance (54.9%), the majority of subjects (72.2%) were outpatients, and over half of the isolates (55.2%) were Gram-positive. The most common isolate was Staphylococcus spp. (44.4%), followed by E. coli (13.6%) and P. aeruginosa (12.9%). The highest resistance was reported against penicillin followed by fusidic acid against Gram-positive bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in 40.5% of Staphylococcus aureus (S. aureus) isolates. Amikacin was the most susceptible antibiotic against all Gram-negative isolates. MDR Gram-negative bacteria accounted for 51.9% of wound infection isolates (95% CI: 45.95 to 58.33) while 6.3% (95% CI: 4.39 to 8.86) were XDR (nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories). A high prevalence of bacterial isolates, with S. aureus as the predominant pathogen, showed high rates of multidrug resistance. This highlights the importance of monitoring antibiotic choices for prophylaxis and treatment in the study area.