Selective Media Research Articles

Isolation, Characterization, and Selection of Bacillus sp. from Shallot Rhizosphere that Inhibits Fusarium oxysporum Growth

Bacillus sp. is a Plant Growth Promoting Rhizobacteria (PGPR) species that lives in the rhizosphere. This bacterium can produce antifungal compounds that suppress pathogenic fungi, such as Fusarium oxysporum. The aim of this research was isolate, characterize, and obtain Bacillus sp. that can inhibit F. oxysporum which causes twisted disease in shallots. Isolation was done by sampling 10 g of soil from shallot roots, placing it in 9 mL of sterile water, and carrying out a series of dilutions. The first dilution was incubated at 80 °C for 20 minutes to obtain Bacillus sp. on the selective medium HiChrome. Colony morphology, shape, cell color, Gram staining, catalase and endospore staining were observed from obtained isolates. An antagonist test was done to determine the inhibitory effects of isolates against the pathogenic fungus F. oxysporum. Four isolates of Bacillus sp. were obtained with irregular colony shape, dull white color without muccus, edges varying in shape between lobate and undulate, and raised elevation. The results of the antagonist test showed that the four isolates had the ability to inhibit the growth of the pathogenic fungus F. oxysporum with the greatest inhibition shown by isolate BM1 at 30.12%, with an antibiosis mechanism and hyphae swelling.

Molecular, Genetic, and Biochemical Characterization of OXA-484 Carbapenemase, a Difficult-to-Detect R214G Variant of OXA-181.

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests.

Prevalence, genetic characteristics, and antimicrobial resistance of staphylococcal isolates from oral cavity and skin surface of healthy individuals in northern Japan

BackgroundOral cavity is an ecological niche for colonization of staphylococci, which are a major bacterial species causing community-acquired infections in humans. In this study, prevalence, and characteristics of staphylococci in oral cavity and skin of healthy individuals were investigated in northern Japan. MethodsSaliva from oral cavity and swab from skin surface of hand were collected and cultured on selective media. Species of the isolates were identified genetically, and ST was determined for S. aureus and S. argenteus. Genes associated with antimicrobial resistance were detected by PCR. ResultsAmong 166 participants, a total of 75 S. aureus isolates were obtained from 61 individuals (37 %), and recovered more frequently in oral cavity (n = 48) than skin (n = 27). Among 23 STs identified in S. aureus isolates, ST8 (CC8), ST15 (CC15), and ST188 (CC1) were the most common (10 isolates each), with STs of CC1 being dominant (17 isolates). Methicillin-resistant S. aureus (MRSA) was isolated in the skin of two individuals and belonged to ST1 and ST6. Resistance to erythromycin and gentamicin associated with erm(A) and aac(6’)-Ie-aph(2”)-Ia, respectively, was more commonly found in ST5 and ST8 isolates. One S. argenteus isolate (ST2250, mecA-negative) was recovered from oral cavity of a participant (0.6 %). A total of 186 isolates of coagulase-negative staphylococci (CoNS) were recovered from 102 participants and identified into 14 species, with S. warneri being the most common (n = 52), followed by S. capitis (n = 42), S. saprophyticus (n = 20) and S. haemolyticus (n = 19). mecA was detected in S. saprophyticus, S. haemolyticus, and S. caprae, while arginine-catabolic mobile element (ACME) in only S. capitis and S. epidermidis. ConclusionS. aureus was more prevalent in oral cavity than skin surface, belonging to three major STs, with CC1 being a dominant lineage. The prevalence of antimicrobial resistance was distinct depending on CoNS species.

From Swiffers to Solutions: The Impact of Environmental Sampling in the Veterinary Medical Center at OSU

Background: In 2018, the Ohio State University College of Veterinary Medicine (OSU CVM) implemented an Antimicrobial Stewardship Program, central to which was the integration of an environmental surveillance (ES) program. The ES focuses on pathogens recognized as urgent threats to public health by the Centers for Disease Control and Prevention. The pathogens currently targeted include carbapenemase-producing Enterobacterales (CPE), Salmonella spp., methicillin-resistant Staphylococcus spp. (MRSs), vancomycin resistant Enterococcus spp., and enrofloxacin resistant Pseudomonas aeruginosa. Identification of these pathogens allows the hospital to be aware of the local environmental microflora which can act as a sentinel for disease in the hospital, potentially causing healthcare associated infections. Therefore, the objective of this program is to identify resistant bacterial pathogens, characterize their resistance profiles, analyze prevalence patterns, and initiate infection control interventions where needed in the OSU VMC. Method: From January 2018 through December 2023, a total of 5449 samples were collected from approximately 86 locations across the OSU VMC encompassing the small animal, equine, and farm animal sections. A majority (64%, n=3561) of samples were collected from the small animal hospital, with the farm animal section contributing 1055 samples and the equine section 899. Areas sampled were frequented by both humans and animals, as well as surfaces exclusively touched by humans. Samples were collected using Swiffers® and processed through selective culture media. Result: Approximately half (52%, n=2890) of the samples collected represented human-touch only surfaces. A total of 3794 bacterial isolates were recovered, with an overall low prevalence for all targeted pathogens. Prevalence of CPE was 2% (n=103), with Enterobacter species being the most common. Recovery of MRSs was 8.5% (n=464) and Salmonella species was 1% (n=47). Conclusion: Through this initiative, the equine division of the OSU VMC collaborated with the antimicrobial stewardship team to enhance their Salmonella fecal and ES practices. In 2019, ES was critical in identifying persistent CPE and extended-spectrum cephalosporin-resistant Enterobacteriaceae in the ICU and surrounding areas of the small animal hospital. Effective measures were taken to halt the spread of ESC among patients and eliminate CPE in the environment. With the discovery of a new CPE in early 2023 in the small animal ICU and nearby areas, the program initiated targeted ES and cleaning and disinfection protocols, to identify contaminated areas and control disease transmission. These efforts have increased patient safety, health, and well-being, demonstrating how ES can be an important tool for infection control and prevention in veterinary settings.

Estimation of IL-8 and TNF-α Levels in Pediatric Diarrhea Patients Infected with Enterohemorrhagic E. coli O157:H7

Abstract Background: Verotoxins are bacterial virulence factors produced by E. coli O157:H7, transmitted by the fecal-oral route. Objectives: The aim of this article was to diagnose E. coli O157:H7 which causes diarrhea and sometimes develops into HUS, which considers pig health problems and estimates the levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α in the sera of pediatric patients infected with Enterohemorrhagic E. coli compared to the control group. Materials and Methods: Stool and blood samples were collected from 421 pediatric patients with diarrhea, ranging in age from birth to 13 years old, from March to October 2022. Samples were collected from Al Noor Teaching Hospital, Babylon Hospital for Pediatric and Gynecology, Hilla, Iraq. E. coli O157:H7 was cultured on eosin methylene blue (EMB) and Sorbitol MacConkey agar (SMA), confirmed by biochemical test and cultured on HiCrome E. coli O157:H7 selective medium which was an agar base supplemented with cefixime tellurite agar. Serum from 30 pediatric diarrhea patients infected with E. coli O157:H7 compared with 30 healthy children as control group used to determine serum levels of IL-8 and TNF-α by sandwich ELISA. Results: The results revealed that out of the total 421 samples used in this study, E. coli O157H:7, represented 7% (30 of 421) stool samples. This 30serum samples of infected children as well as 30 samples from healthy children subjected to the estimate serum level of IL-8 and TNF-α which record significant differences P ≤ 0.01 and P ≤ 0.05 to this cytokines in different age group; the mean of IL-8 level was 283.62 ± 17.8 pg/mL (7–9 years), and the mean of TNF-α was 208.62 ± 28.7 pg/mL (10–13 years) comparative with the control group of 80.58 ± 15.4pg/mL and 32.50 ± 7.5 pg/mL, respectively, and also result showed an increased mean level of IL-8 than TNF-α in the male comparative with female 195.19 ± 10.4 pg/mL and 159.05 ± 12.4 pg/mL, respectively, comparative with the control group. The result showed no significant differences in IL-8 and TNF-α between watery diarrhea (192.43 ± 24.3 pg/mL and 136.05 ± 20.4 pg/mL) and bloody diarrhea (189.02 ± 22.5 pg/mL and 123.80 ± 13.5 pg/mL), and also result showed significant increase of mean sera level of IL-8 than TNF-α in formula feeding children comparative with breastfeeding children (187.87 ± 19.5 pg/mL and 119.93 ± 17.4 pg/mL, respectively). Conclusion: The finding of this study suggested that increased levels of IL-8 and TNF-α are present in all age groups, in male comparative with female, and also in pediatric diarrhea feeding by formula than breastfeeding and no differences of this cytokine according to consistency of diarrhea. These results contribute to using the immune profile as a serological marker for diagnosing diarrhea caused by E. coli O157:H7 in comparison with the control group.

Comparative Analysis of the Mass Spectra of Mycobacterium abscessus Complex Strains Isolated on Various Nutrient Media.

Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry. The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains. A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15. MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.

Inhibitory effects of essential oils on the growth of grain storage molds and the formation of aflatoxin in stored organic corn grains

AbstractBackgroundMold contamination in post‐harvest grain storage degrades grain quality and produces mycotoxins, causing economic losses and health issues. This study evaluated the inhibitory effects of six essential oils (cinnamon, clove, eugenol, orange terpenes, oregano, and thyme oil) on total and specific mold genera growth using potato dextrose agar (PDA) and selective media plates, respectively, at different concentrations of EOs (0–0.8 mg/mL). The antifungal index (AI) of each treatment were measured after 7 days incubation at 22°C. The fungicidal activity of EOs in stored organic corn grains was tested using a simulated fumigation method, and the aflatoxin contents in the corn grains were determined biweekly for 6 weeks.ResultsResults indicate that the tested EOs, except orange terpenes, significantly inhibited mold growth. Overall, eugenol demonstrated the highest effectiveness against total mold growth with cinnamon oil following closely behind. The cinnamon oil also consistently showed highest AI and lowest IC50 (0.065 mg/mL) against the growth of Aspergillus and Penicillium followed by clove oil with IC50 being 0.12 mg/mL. The Fusarium spp. was more sensitive to EOs, particularly cinnamon oil (IC50 = 0.006 mg/mL), and eugenol and oregano oil (IC50 = 0.01 mg/mL). In the simulated fumigation tests, cinnamon, oregano, and thyme oils at 0.05 mL/25 g corn significantly inhibited the mold growth and aflatoxin production at water activity 0.85, and temperature 25 and 35°C over a period of 42 days storage.ConclusionThe EOs tested in this study except orange terpenes can potentially serve as alternatives to toxic synthetic fungicides/fumigants for organic corn grain protection during storage.

Filtered handheld far-ultraviolet disinfection device in reducing environmental pathogens from high-touch clinical surfaces

Background: Healthcare-acquired infections (HAIs) continue to be a major challenge. In fact, an increased risk of HAIs has been linked to high-touch surfaces contaminated with multidrug-resistant organisms (MDROs), and enhanced environmental disinfection is linked to reduced HAI rates. Recently, more focus has been placed on emerging disinfection technologies, such as UV light-producing portable device that emits light at a wavelength of 222 nm, which has previously demonstrated germicidal capabilities at short contact times. In this study, we aim i) to evaluate the efficacy of a filtered far-UV-C handheld device (FFUHH) to reduce bacterial loads on high-touch surfaces in clinical workrooms in a cancer center, and ii) to isolate, identify and establish a genetic relationship between these environmental clinically significant pathogens and the ones recovered from patients. Methods: Samples were collected weekly on a rotating schedule over a 24-week period from five high-touch items (dictation device, mouse, armchair, desk, and keyboard) in multiple clinical work rooms on hematologic malignancy and stem cell transplant units. Contact plates for colony count and swabs were collected pre- and post-intervention with the FFUHH on standardized adjacent areas respectively for each surface. The swabs were enriched and cultured on selective media to isolate clinically significant pathogens. Whole genome sequencing (WGS) was then performed on environmental pathogens validated by MALDI-TOF as well as clinical samples collected from patients in the same unit around the time of environmental sample collection. Results: A total of 440 plates, 220 pre- and 220 post-interventions, were collected and analyzed. The highest mean colony count pre-treatment was detected from the armchairs and the lowest for the keyboards. The mean reduction of colony forming units (CFUs) ranged between 53% for the keyboard and 83% for the mouse. The reduction was statistically significant across all surfaces with P values &lt; 0.05, except for the keyboard (Figure 1). We isolated many pathogens of the human microbiota identified by MALDI-TOF such as Micrococcus luteus, S. capitis as well as methicillin-resistant S. epidermidis, S. haemolyticus and S. hominis. We also identified several Candida parapsilosis, Pseudomonas stutzeri, one Listeria grayi and one Acinetobacter baumanni. Finally, WSG allowed us to further characterize an environmental multi-drug resistant S. epidermidis ST5 strain associated with patient bacteremia, and ST16 strains detected on surfaces both pre- and post-FFUHH treatment. Conclusion: The FFUHH effectively reduced the microbial burden on high-touch surfaces in clinical workrooms on hematologic malignancy and stem cell units.

The polymicrobial nature of the oral cavity and claws of cats diagnosed by mass spectrometry and next-generation sequencing

Close contact between cats and humans increases the risk of transmission of zoonotic pathogens, through bites and scratches due to the complexity of microorganisms in the oral and nail microbiotas of felines. This study investigated the presence of bacteria and fungi in the oral cavity and claws of 100 apparently healthy cats using conventional and selective microbiological culture media, and next-generation sequencing (NGS) and mass spectrometry (MALDI-TOF MS). Furthermore, antimicrobial susceptibility testing of bacteria isolates was performed by disc diffusion method. In total, 671 bacteria and 33 yeasts were identified by MALDI-TOF MS. Neisseria animaloris (10.8 %), Staphylococcus felis (8.5 %), and Pasteurella multocida (7 %) were the most prevalent bacteria in oral cavity samples (n = 343), while the most common yeast (n = 19) was Candida albicans (68.4 %). Staphylococcus pettenkoferi (13.4 %), Staphylococcus felis (6.4 %), and Staphylococcus simulans (5.8 %) were the prevalent bacteria identified in the claw samples (n = 328), while Rhodotorula mucilaginosa (57.2 %) was the most common yeast (n = 14). NGS predominantly identified the genera Moraxella, Neisseria, Pasteurella, and Fusobacterium in oral cavity samples, whereas enterobacteria and staphylococci were prevalent in nail bed samples. In addition, the genera Capnocytophaga and Bartonella were identified, which have been described in serious human infections secondary to feline aggressions. Levofloxacin, marbofloxacin, and amoxicillin/clavulanic acid were the most effective drugs against the main groups of bacteria identified. Multidrug resistance was observed in 17 % of the bacterial isolates. Furthermore, three staphylococci harboring the methicillin resistance gene mecA were identified. We highlight the complexity of microorganisms inhabiting the oral/claw microbiotas of cats, the high resistance rate of the isolates to conventional antimicrobial agents, and the zoonotic risk of aggressions caused by bites and scratches from domestic cats.

Rapid detection of pathogenic E. coli based on CRISPR Cas system.

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.

Bakteryjna redukcja siarczanów jako odpowiednia metoda usuwania siarczanów z kwaśnych wód kopalnianych

The aim of this work was to investigate of the sulfates removal from acid mine drainage (AMD) by biological method using sulfate-reducing bacteria (SRB). A sample of AMD out-flowing from the Pech shaft of the abandoned and flooded Smolník deposit in Slovak Republic was studied. The untreated AMD (with contents metals) and treated AMD i.e. after of the metals removing by the synthetic sorbent Slovakite were used. The base of the biological sulfates elimination was the sulfates bacterial reduction under influence of SRB genera Desulfovibrio. These bacteria realize the sulfates reduction to hydrogen sulfide at the simultaneously oxidation of energetic substrate. Standard selective nutrient medium DSM-63 and modified selective nutrient medium DSM-63 (without sulfates contents) with standard amount of sodium lactate (as energetic substrate) were used in the experiments with untreated AMD. Standard selective nutrient medium DSM-63 and modified selective nutrient medium DSM-63 with standard, double and triple amount of sodium lactate were used in the experiments with treated AMD. In the case of untreated AMD has been reached low removing of sulfates - 20% efficiency (standard medium) and 26% (modified medium). The formation of heterogeneous precipitates containing metals pointed to the need for treatment of AMD by the initial removal of metals by sorption on Slovakite sorbent and subsequent removal of sulfates by bacterial reduction. The results of experiments with AMD treated in this way showed 49%, 70% and 88% efficiency of sulfate removal when using sodium lactate in standard, double and triple amounts. The results of this work suggest that ratio of substrate quantity and sulfate concentration is one of the key parameter of sulfate reducing condition. However, the price of the energy substrate is also an important factor. Therefore, subsequent experiments will be focused on the use of the more affordable substrates (e.g. whey) or mixed bacterial culture of SRB, which will also be able to use the products of decomposition of basic energetic substrates.

Evaluation of Three Different Selective Media for Enumeration of Clostridium perfringens in Untreated and Treated Wastewater.

Current and emerging legislation in North Carolina and other regions calls for the enumeration of Clostridium perfringens as a surrogate indicator for protozoan parasites in various types of waters. Past studies that have evaluated selective media for the detection of this bacterium have provided limited, conflicting, and inconclusive results. In this study, membrane filtration was used to enumerate C. perfringens as culturable spores or total culturable cells in 19 samples of untreated and 25 samples of partially treated wastewaters on 3 candidate media, Tryptose Sulfite Cycloserine Agar (TSC), CP ChromoSelect Agar (CPCS), and membrane Clostridium perfringens Agar (m-CP) in parallel, and the results were compared. Presumptive isolates from each agar were further subjected to phenotypic confirmation tests for acid phosphatase production and stormy fermentation to further determine the performance of each agar. The CPCS agar was determined to have the highest enumerative capacity of total C. perfringens cells when compared to both TSC agar and m-CP agar (p-value < 0.05), but there was no significant difference in its ability to detect spores when compared to TSC agar (p-value > 0.05). The overall specificity of CPCS agar as determined by agreement of results from both confirmation tests was 0.81, while the specificity of TSC agar was only 0.28. Based on its performance, ease of preparation and use and consistency of colony characteristics, CPCS agar is recommended as the preferred medium for C. perfringens enumeration in wastewater.

Exploring microbial lipases: Screening and identification for biocatalyst potential in bioethanol synthesis from glycerol-based biodiesel waste

Lipases are hydrolytic enzymes that break triglycerides into free fatty acids and glycerol. Typically, these enzymes are produced by microorganisms and used as industrial biocatalysts. Microorganisms isolated from oil-based environmental samples were screened for their lipase-producing ability, collecting twenty bacteria and eighteen fungal strains. Through selective media and morphological identification, various microorganisms were identified, including Pseudomonas sp. (12 strains), Escherichia coli (4 strains), Serratia (2 strains), carotenoid-producing bacteria (1 strain), an unknown isolate bacterium, Aspergillus sp. (6 strains), Aspergillus niger (1 strain), Aspergillus flavus (1 strain), Penicillium sp. (3 strains), Saccharomyces sp. (2 strains), Filamentous Black Fungus (2 strains), Omphalina sp. (1 strain), Rhizopus sp. (1 strain), and Trichoderma sp. (1 strain). Based on lipolytic index results, six bacteria exhibited high lipolytic indices, with Pseudomonas sp. having the highest index. After multi-screening, three lipolytic bacteria belonging to the genera Pseudomonas sp., Serratia sp., and Escherichia coli, as well as three lipolytic fungi belonging to the genera Saccharomyces sp., Trichoderma sp., and Aspergillus flavus, respectively, demonstrated the highest lipolytic activity when screened on a lipase assay, reaching 6.3 and 5.01 mU/g at 72 h of incubation. Consequently, these selected microorganisms were chosen for further study on bioethanol synthesis from glycerol. The study has revealed that the isolated bacteria and fungi strains have the potential to be efficient lipase producers.

“Phylogenomic insights into brucellaceae: The Pseudochrobactrum algeriensis case”

The genus Pseudochrobactrum encompasses free-living bacteria phylogenetically close to Ochrobactrum opportunistic pathogens and to Brucella, facultative intracellular parasites causing brucellosis, a worldwide-extended and grave zoonosis. Recently, Pseudochrobactrum strains were isolated from Brucella natural hosts on Brucella selective media, potentially causing diagnostic confusions. Strikingly, P. algeriensis was isolated from cattle lymph nodes, organs that are inimical to bacteria. Here, we analyse P. algeriensis potential virulence factors in comparison with Ochrobactrum and Brucella. Consistent with genomic analyses, Western-Blot analyses confirmed that P. algeriensis lacks the ability to synthesize the N-formylperosamine O-polysaccharide characteristic of the lipopolysaccharide (LPS) of smooth Brucella core species. However, unlike other Pseudochrobactrum but similar to some early diverging brucellae, P. algeriensis carries genes potentially synthetizing a rhamnose-based O-polysaccharide LPS. Lipid A analysis by MALDI-TOF demonstrated that P. algeriensis LPS bears a lipid A with a reduced pathogen-associated molecular pattern, a trait shared with Ochrobactrum and Brucella that is essential to generate a highly stable outer membrane and to delay immune activation. Also, although not able to multiply intracellularly in macrophages, the analysis of P. algeriensis cell lipid envelope revealed the presence of large amounts of cationic aminolipids, which may account for the extremely high resistance of P. algeriensis to bactericidal peptides and could favor colonization of mucosae and transient survival in Brucella hosts. However, two traits critical in Brucella pathogenicity are either significantly different (T4SS [VirB]) or absent (erythritol catabolic pathway) in P. algeriensis. This work shows that, while diverging in other characteristics, lipidic envelope features relevant in Brucella pathogenicity are conserved in Brucellaceae. The constant presence of these features strongly suggests that reinforcement of the envelope integrity as an adaptive advantage in soil was maintained in Brucella because of the similarity of some environmental challenges, such as the action of cationic peptide antibiotics and host defense peptides. This information adds knowledge about the evolution of Brucellaceae, and also underlines the taxonomical differences of the three genera compared.

The Near-Gapless Penicillium fuscoglaucum Genome Enables the Discovery of Lifestyle Features as an Emerging Post-Harvest Phytopathogen.

Penicillium spp. occupy many diverse biological niches that include plant pathogens, opportunistic human pathogens, saprophytes, indoor air contaminants, and those selected specifically for industrial applications to produce secondary metabolites and lifesaving antibiotics. Recent phylogenetic studies have established Penicillium fuscoglaucum as a synonym for Penicillium commune, which is an indoor air contaminant and toxin producer and can infect apple fruit during storage. During routine culturing on selective media in the lab, we obtained an isolate of P. fuscoglaucum Pf_T2 and sequenced its genome. The Pf_T2 genome is far superior to available genomic resources for the species. Our assembly exhibits a length of 35.1 Mb, a BUSCO score of 97.9% complete, and consists of five scaffolds/contigs representing the four expected chromosomes. It was determined that the Pf_T2 genome was colinear with a type specimen P. fuscoglaucum and contained a lineage-specific, intact cyclopiazonic acid (CPA) gene cluster. For comparison, a highly virulent postharvest apple pathogen, P. expansum strain TDL 12.1, was included and showed a similar growth pattern in culture to our Pf_T2 isolate but was far more aggressive in apple fruit than P. fuscoglaucum. The genome of Pf_T2 serves as a major improvement over existing resources, has superior annotation, and can inform forthcoming omics-based work and functional genetic studies to probe secondary metabolite production and disparities in aggressiveness during apple fruit decay.

MDB-72. BREFELDIN A (BFA) INDUCES CD133 PROTEOLYTIC PROCESSING IN MEDULLOBLASTOMA STEM CELLS (MBSCS)

Abstract BACKGROUND Medulloblastoma (MB) is a malignant brain tumor occurring in children, characterized by elevated resistance to conventional chemotherapy. Medulloblastoma stem cells (MBSCs) represent a fraction of the tumor cell population which has been associated with poor prognosis and refractoriness to conventional therapies. CD133 (Prominin 1, PROM1) is a transmembrane protein serving as a cancer stem cell marker associated with cancer progression. Brefeldin A (BFA) is an antibiotic disrupting the Golgi apparatus, thus triggering Endoplasmic reticulum (ER) stress signaling pathways. BFA cancer-inhibitory properties have been already described in cancer of different tissue origin. However, the effects of BFA on MBCSs have not been investigated yet. Thus, the aim of this study was to characterize the response of MBSCs to BFA and the effects induced by BFA on CD133. METHODS Human group 3 MB (G3MB) cell lines were grown in stem selective medium (B27™) and treated with 0.3 uM BFA for 24h before collection. Apoptosis, clonogenicity and CD133 expression were evaluated. CD133 was immunoprecipitated and the corresponding bands were excised and analyzed by nanoLC-MS/MS. Raw data were processed using PEAKS XPro. RESULTS Our results show that BFA-treated cells induced the overexpression of a CD133 isoform that was missing the N-terminus region. Moreover, we found a downregulation of CD133 activated intracellular signalling together with a reduced exposition on cell membrane. CONCLUSIONS We report here for the first time, a N-terminus proteolytic processing of CD133, potentially associated to the cell trafficking effects induced by BFA. Finally, this study suggests a proof of principle for the design of novel molecules with similar structures and mechanism of action of BFA.

Isolation and Molecular Confirmation of Staphylococcus aureus from Bovine Mastitis in Various Locations of Tamil Nadu, India

This present investigation was done for the Isolation and Molecular confirmation of Staphylococcus aureus from Bovine mastitis-infected cows in various Tamil Nadu, India locations. Two hundred milk samples, ranging from 15 to 20 ml, were collected aseptically from cows in farms in and around Tamil Nadu and surrounding areas after discarding the first few streaks of milk from 2021 to 2023. We used the CMT test to check the milk samples. Following a thorough mixing of the milk samples delivered to the laboratory, one loop containing the infected milk sample was plated on Nutrient agar and incubated at 37 ˚C for 18 to 24 hours. To identify the Staphylococcus aureus, the isolated bacterial colonies were preliminarily identified by Microscopic examination (Gram staining and Motility test), Plating in Selective medium (Mannitol Salt Agar) and Biochemical tests. The DNA of the bacteria was extracted and Polymerase Chain Reaction (PCR) was done to detect the presence of the nuc gene. Of the 200 milk samples obtained from nursing cows, 37.5 % were from Mastitis-positive animals; of these, 61.3 % were in the sub-clinical stage, while 38.7 % were in the clinical stage. Only 33 (16.5 %) of the 200 milk samples tested positive for Staphylococcus aureus. A total of 26 out of 75 animals (34.7 %) with Mastitis, and 7 out of 125 animals (5.6 %) without Mastitis. Fifteen (57.7 %) of the twenty-six Staphylococcus aureus isolates found in cows with mastitis were from clinical cases, whereas eleven (42.3%) were from sub-clinical cases. All of the Staphylococcus aureus isolates examined contained the 279 bp nuc gene, according to the conventional PCR findings. In conclusion, the research has shown that sub-clinical mastitis is more common than clinical mastitis and that Staphylococcus aureus was substantially more often isolated from mastitis milk, particularly in clinical instances.

An assessment of various disinfectants using the Kirby-Bauer Method with disc diffusion to determine their effectiveness against locally isolated pathogens

This study aimed to assess the effectiveness of various disinfectants against locally isolated pathogens using the Kirby-Bauer Method with disc diffusion. Indigenous strains of Salmonella typhimurium, E. coli, Campylobacter, Citrobacter freundii, and Staphylococcus aureus were obtained from the bacterial depository bank and sub-cultured on their respective selective media. A 0.5 McFarland Turbidity Standard was prepared, and eight commercially available disinfectants were tested for efficacy. The results of this study will inform the selection of appropriate disinfectants for use in preventing the spread of disease in various settings, including home sanitation, healthcare, and industrial manufacturing processes. The disinfectants used in this study produced different results against the targeted pathogens. Hydrogen peroxide and Formalin produced larger zones of inhibition, while Povidone Iodine and Hydrochloric acid produced intermediate zones. Ethanol, Methanol, and Dettol produced smaller .zones of inhibition. Benzalkonium Chloride was effective only against S. aureus, while all other indigenous isolates resisted it. Keywords: Disinfectants, Sal. typhimurium, E.coli, Campylobacter, Citrobacter freundii, Staphylococcus auerus, Antibiotic sensitivity test

Antibiotic susceptibility and genome analysis of Enterococcus species isolated from inpatients in one hospital with no apparent outbreak of vancomycin-resistant Enterococcus in Japan.

To prevent nosocomial infection, it is important to screen for potential vancomycin-resistant Enterococcus (VRE) among patients. In this study, we analyzed enterococcal isolates from inpatients in one hospital without any apparent outbreak of VRE. Enterococcal isolates were collected from inpatients at Hiroshima University Hospital from April 1to June 30, 2021 using selective medium for Enterococci. Multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing were performed. A total of 164 isolates, includingEnterococcus faecium (41 isolates), Enterococcus faecalis (80 isolates), Enterococcus raffinosus (11 isolates), Enterococcus casseliflavus (nine isolates), Enterococcus avium (12 isolates), Enterococcus lactis (eight isolates), Enterococcus gallinarum (two isolates), and Enterococcus malodoratus (one isolate), were analyzed. We found one vanA-positive E. faecium, which was already informed when the patient was transferred to the hospital, ninevanC-positive E. casseliflavus, and twovanC-positive E. gallinarum. E. faecium isolates showed resistance to ampicillin (95.1%), imipenem (95.1%), and levofloxacin (87.8%), and E. faecalis isolates showed resistance to minocycline (49.4%). Ampicillin- and levofloxacin-resistant E. faecium had multiple mutations in penicillin-binding protein 5 (PBP5) (39/39 isolates) and ParC/GyrA (21/36 isolates), respectively. E. raffinosus showed resistance to ampicillin (81.8%), imipenem (45.5%), and levofloxacin (45.5%), and E. lactis showed resistance to ampicillin (37.5%) and imipenem (50.0%). The linezolid resistance genes optrA and cfr(B) were found only in one isolate of E. faecalis and E. raffinosus, respectively. This study, showing the status of enterococci infection in hospitalized patients, is one of the important information when considering nosocomial infection control of VRE.

Using Bio-inline Reactor to Evaluate Sanitizer Efficacy in Removing Dual-species Biofilms Formed by Escherichia coli O157:H7 and Listeria monocytogenes

The efficacy of a sanitizer in biofilm removal may be influenced by a combination of factors such as sanitizer exposure time and concentration, bacterial species, surface topography, and shear stresses. We employed an inline biofilm reactor to investigate the interactions of these variables on biofilm removal with chlorine. The CDC bioreactor was used to grow E. coli O157:H7 and L. monocytogenes biofilms as a single species or with Ralstonia insidiosa as a dual-species biofilm on stainless steel, PTFE, and EPDM coupons at shear stresses 0.368 and 2.462 N/m2 for 48 hours. Coupons were retrieved from a CDC bioreactor and placed in an inline biofilm reactor and 100, 200, or 500 ppm of chlorine was supplied for 1- and 4 min. Bacterial populations in the biofilms were quantified pre- and posttreatment by plating on selective media. After chlorine treatment, reduction (Log CFU/cm2) in pathogen populations obtained from three replicates was analyzed for statistical significance. A 1-min chlorine treatment (500 ppm), on dual-species E. coli O157:H7 biofilms grown at high shear stress of 2.462 N/m2 resulted in significant E. coli O157:H7 reductions on SS 316L (2.79 log CFU/cm2) and PTFE (1.76 log CFU/cm2). Similar trend was also observed for biofilm removal after a 4-min chlorine treatment. Single species E. coli O157:H7 biofilms exhibited higher resistance to chlorine when biofilms were developed at high shear stress. The effect of chlorine in L. monocytogenes removal from dual-species biofilms was dependent primarily on the shear stress at which they were formed rather than the surface topography of materials. Besides surface topography, shear stresses at which biofilms were formed also influenced the effect of sanitizer. The removal of E. coli O157:H7 biofilms from EPDM material may require critical interventions due to difficulty in removing this pathogen. The inline biofilm reactor is a novel tool to evaluate the efficacy of a sanitizer in bacterial biofilm removal.