Tryptone Soya Agar Research Articles

Microwave Oven Application for the Preparation and Sterilization of Microbiological Culture Media: A Feasible Method with An Adapted Water Bath and Perforable Cap

There is currently increased interest in the use of alternatives to autoclaved culture media, in order to maintain the properties of the media, while saving energy and time. In this study, we assess a new system for culture media preparation, using a conventional microwave with a water bath and a glass bottle with a rubber cap that allows depressurization. Sterilization, using the proposed system (1000 W, 3 to 20 min), was compared with autoclaving for the preparation of tryptone soy agar (TSA), tryptone soy broth (TSB), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). Microwave exposure for 7 min yielded sterile TSA plates. The productivity of both sterilization methods was assessed using the pour plate method, and significant increases in the growth of certain micro-organisms after using a microwave were observed for every culture medium, especially those that were sterilized by boiling (VRBG). The kinetics of microbial destruction showed that Escherichia coli and Bacillus subtilis spores were destroyed after 3 and 7 min in a microwave, respectively, while three decimal reductions were obtained for Geobacillus stearothermophilus spores after 15 min in an autoclave. This new sterilization method could be a feasible, rapid, and economical method to prepare microbiological media, with a quality similar to that obtained through autoclaving.

First report of Xanthomonas campestris pv. campestris causing black rot on oilseed rape (Brassica napus L.) in France.

In October 2022, v-shaped necrotic lesions were observed on the leaf margins of field-grown winter oilseed rape (WOSR), Brassica napus L., in western France (Ille-et-Vilaine (35) and Maine-et-Loire (49) departments). Disease incidence on volunteers and cultivated WOSR was generally low (5-10 %) but occasionally up to 80% on some fields. Leaf sections sampled from the margin of necrotic leaf tissue were dilacerated in sterile deionized water and the extract was spread onto tryptone soya agar (TSA) with cycloheximide (100 mg.L-1) and Polyflor (Syngenta, France) (2ml.L-1, containing 5 mg.L-1 propiconazole) then incubated at 28°C for 2 days. Colonies were yellow-pigmented, mucoid, and convex, which are morphological characteristics of Xanthomonas spp. colonies. The partial fyuA and gyrB gene sequences were amplified for eight isolated strains (CFBP 9155, CFBP 9156, CFBP 9157, CFBP 9158, CFBP 9159, CFBP 9161, CFBP 9162, and CFBP 9163) using primers of Fargier et al. (2011), and sequenced (Genoscreen, France). The sequences were deposited under numbers OR232891 to OR232898 for fyuA and OR634932 to OR634939 for gyrB. BLASTN analysis of the sequenced fyuA amplicon showed 100% identity and query coverage with the fyuA fragment of Xanthomonas campestris pv. campestris (Xcc) CFBP 6865R (Bellenot et al., 2022). BLASTN analysis of the sequenced gyrB amplicon showed two allelic forms: one showed 100% identity and query coverage with the gyrB fragment of Xcc strain CFBP 6865R (Bellenot et al., 2022), the other one showed 100% identity and query coverage with the type strain Xcc CFBP 5241 (ATCC33913) (Vorhölter et al., 2003). Moreover, two qPCR tools were used to identify the strains successfully as Xcc (Köhl et al., 2011; Rezki et al., 2016) which target the same gene encoding a hypothetical protein and whose primers overlap. The pathogenicity of the eight isolated strains was validated using a bacterial suspension (108 CFU.ml-1) for i) leaf spraying until runoff onto the leaf surfaces of WOSR plants previously maintained at saturated humidity for 48 hours, ii) wound-leaf inoculation of the two youngest true leaves with scissors that had been dipped into the bacterial suspension. Both tests were performed on 3-week-old WOSR plants of the Aviso (INRAE) genotype. Deionized water was used as negative control. Strains CFBP 5241 and the strain CFBP 4954 (Fargier et al., 2007) were used as positive controls for disease expression. Tested plants (seven for spray inoculation and four for wound-leaf inoculation per strain and control condition) were incubated in a greenhouse at 20°C/24°C (night/day). Isolated strains and the strain CFBP 4954 caused yellow lesions with both inoculation methods that necrotized starting about 10 days post inoculation (dpi). The spots coalesced within 14 dpi to form necrotic areas. The type strain CFBP 5241 caused mild symptoms, with only yellow lesions that did not coalesce. Plants inoculated with water remained symptomless. To complete Koch's postulate, re-isolations were achieved. Re-isolated strains on TSA showed the same colony morphology as described above. All re-isolated strains were identified as Xcc based on partial gyrB sequencing and Xcc specific qPCR test (Rezki et al., 2016). This first report in France and the recent identification in Serbia (Popović et al., 2013) may illustrate the emergence of the disease on this crop in Europe. The prevalence and consequences of this disease should be evaluated over a wider geographic area.

Rapid Detection of the Aeromonas sp. group using Conventional PCR at the Aquaculture Technology Development Center, Cangkringan, Sleman, Special Region of Yogyakarta

The catfish is a freshwater cultivated fish that can be found all over Indonesia. However, the problems that occur in general are usually attacks by pathogens from the Aeromonas sp group. This research aims to optimize the rapid detection of the Aeromonas sp group using PCR at the Aquaculture Technology Development Center, Cangkringan, Sleman, special region of Yogyakarta. The research was conducted by survey with purposive sampling. Isolation was carried out using two growth media, namely Tryptone Soy Agar (TSA) and Glutamate Starch Phenile (GSP). Colonies that had characteristics of the Aeromonas sp group were subjected to PCR amplification using specific primers for the Aeromonas sp group. The results of the amplification resulted in DNA that matched the target, namely 953bp, which showed that the sample belonged to the Aeromonas sp group. Based on this technique, the level of accuracy in detecting pathogens is higher than conventional methods.

Isolation and Characterization of Antimicrobial Peptides Isolated from Brevibacillus halotolerans 7WMA2 for the Activity Against Multidrug-Resistant Pathogens.

Multidrug resistance to pathogens has posed a severe threat to public health. The threat could be addressed by antimicrobial peptides (AMPs) with broad-spectrum suppression. In this study, Brevibacillus halotolerans 7WMA2, isolated from marine sediment, produced AMPs against Gram-positive and Gram-negative bacteria. The AMPs were precipitated by ammonium sulfate 30% (w/v) from culture broth and dialyzed by a 1kDa membrane. Tryptone Soy Agar (TSA) was used for the cultivation and resulted in the largest bacteria-inhibiting zones under aerobic conditions at 25°C, 48h. An SDS-PAGE gel overlay test revealed that strain 7WMA2 could produce AMPs of 5-10kDa and showed no degradation when held at 121°C for 30min at a wide pH 2-12 range. The AMPs did not cause toxicity to HeLa cells with concentrations up to 500µg/mL while increasing the arbitrary unit up to eight times. The study showed that the AMPs produced were unique, with broad-spectrum antimicrobial ability.

Marnyiella aurantia, gen. nov., sp. nov., a novel bacterial species of the family Weeksellaceae that could produce flexirubin type pigments.

Two Gram-stain-negative, non-spore-forming, rod-shaped, and obligately aerobic bacteria, designated strains CX-624T and cx-311, were isolated from soil samples in Qinghai Province, China. The two strains grew best at 28 °C on the plate with Tryptone soya agar (TSA). Cells formed circular, convex, translucent, smooth, and orange colonies with approximately 1.0 mm diameter after 2 days of incubation on TSA at 28 °C. The strains were oxidase-negative and catalase-positive. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0, and major polar lipids included phosphatidylethanolamine, an unidentified aminophospholipid, four unidentified lipids and an aminolipid. MK-6 was the sole menaquinone in strain CX-624T. Comparative analysis of the nearly full-length 16S rRNA gene sequences showed strains CX-624T and cx-311 were member of the family Weeksellaceae, with the highest similarity to Kaistella haifensis H38T (96.66 %), Epilithonimonas pallida DSM 18015T (96.59 %), and Chryseobacterium gambrini DSM 18014T (96.53 %). Both phylogenetic analysis of the 16S rRNA gene and 177 core genes revealed that strains CX-624T and cx-311 formed an independent clade. Average nucleotide identity values (< 72.64 %), average amino-acid identity values (<72.61 %) and digital DNA-DNA hybridization (< 21.10 %) indicated that the strains CX-624T and cx-311 should constitute a novel genus. The DNA G+C contents of strains CX-624T and cx-311 were 43.0 mol% and 42.7 mol%. According to the data obtained in this study, strain CX-624T represents a novel species belonging to a novel genus of the Weeksellaceae, for which the name Marnyiella aurantia gen. nov., sp. nov. is proposed. The type strain is CX-624T (=GDMCC 1.1714T = JCM 33925T).

Jeotgalibaca caeni sp. nov., isolated from biochemical tank sludge.

A Gram-stain-positive, coccoid-shaped, non-spore-forming, facultatively anaerobic bacterium, designated YN-L-12T, was isolated from the activate sludge of a pesticide plant. Colonies on tryptone soya agar were small, white, opaque and circular. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YN-L-12T belonged to the genus of Jeotgalibaca, and showed the highest similarity to Jeotgalibaca arthritidis 1805-02T (97.0 %), followed by Jeotgalibaca ciconiae H21T32T (96.5 %), Jeotgalibaca porci 1804-02T (95.6 %) and Jeotgalibaca dankookensis EX-07T (95.4 %). The strain grew at 15-37 °C (optimum, 30 °C), with 0-6.5 % (w/v) NaCl (optimum, 0.5 %) and at pH 7-9 (optimum, pH 7.5). The major fatty acids were C18 : 1 ω9c, C16 : 1 ω9c and C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, glycolipid and an unidentified lipid. The DNA G+C content of the strain was 41.1 mol%. Average nucleotide identity values between strain YN-L-12T and J. arthritidis 1805-02T and J. ciconiae H21T32T were 72.8 and 72.3 %, respectively. The digital DNA-DNA hybridization values between YN-L-12T and J. arthritidis 1805-02T and J. ciconiae H21T32T were 24.1 and 20.3 %, respectively. According to the results of phenotypic, chemotaxonomic and phylogenetic analyses, strain YN-L-12T represents a novel species of the genus Jeotgalibaca, for which the name Jeotgalibaca caeni sp. nov. is proposed, with strain YN-L-12T (=KCTC 43533T=CCTCC AB 2022400T) as the type strain.

Establishment of a Single Temperature Incubation Approach for Environmental Monitoring Samples with Focus on Mold Recoveries.

A robust environmental monitoring program is essential to properly estimate and identify microorganisms in cleanrooms, ensuring that microbial contamination remains acceptably low, and a good state of control is maintained in the manufacturing areas. The incubation conditions are important to support optimal microbial recoveries, considering that there is no single culture medium, temperature and incubation time that can recover all microorganisms. Particularly molds are quite sensitive microorganisms, and some species may have very specific nutritional and environmental needs. In this study, a two-phase approach was used to identify a single incubation-temperature approach that could recover most of the cleanroom microbial flora with focus on molds. Phase 1 included a growth promotion study performed in the laboratory using pharmacopeial and in-house strains, comparing different media (SDA and TSA) at single or dual incubation-temperature approaches for 5 or 6 days. Phase 2 was based on an in situ study where sampling was performed in different areas of a pharmaceutical facility, comparing the recoveries at different incubation conditions. In addition, extension studies of phase 1 and phase 2 were performed to get a better understanding of growth requirements for in-house molds. The results show that an incubation on Tryptone Soy Agar (TSA) at 25-30° for 3-4 days was able to recover most tested microorganisms in phase 1 and a large variety of microorganisms in phase 2, indicating that the single incubation-temperature is an optimal approach for the recovery of microorganisms in cleanrooms. Exceptions were noted for one strain of the species Cutibacterium acnes, a microaerophilic bacterium for which anaerobiosis and higher temperatures were needed, and two mold strains (Sistotrema brinkmannii and Stereum hirsutum), indicating that those molds required a specific media (Sabouraud Dextrose Agar, SDA) for their proliferation. The results showed that TSA incubated at single or dual incubation-temperature approach cannot compensate for the absence of SDA for some environmental molds that may be atypical in cleanrooms. Therefore, in addition to TSA, certain monitoring with SDA at e.g., cleanroom entrance points may be beneficial to recover molds with very specific nutritional requirements.

Isolation of Probiotic Strains from Fishes Cultured in Sewage-fed Ponds

The research for the need of probiotics in aquaculture is increasing with the demand for environment friendly aquaculture. The present study aimed at to isolate probiotic strains from the gut of carps cultured in sewage-fed ponds. The fish gut was dissected longitudinally and the entire gut content was plated onto deMann Rogosa Sharp (MRS) Agar for allowing the Lactic Acid Bacteria (LAB) to manifest itself as white pinpoint colonies. The MRS Agar plates containing LAB were then over-layed with osft Tryptone Soya Agar (TSA) containing pathogenic Pseudomonas cells at 105 nos/ml. Those LAB which produced a clear inhibitory zone with a diameter more than 1 mm larger than that of the colony itself was judged to be a tentative probiotics strain. During the present study, four strains (SM1, SM2, ST1 and ST2) gave a clear inhibitory zone and were classified as tentative probiotic strains. The four tentative probiotic strains were further confirmed of its probiotic effect against three pathogenic strains of Pseudomonas, Aeromonas and Edwardsiella by antagonistic assay by cross streak method and among four only two strains SM2 and ST1 exhibited an inhibition of 2 mm and 1 mm, respectively against the three pathogenic test strains and confirmed their status as probiotic strains.

Antimicrobial Efficacy of a Novel Automated Irrigation Device As Compared to Conventional Needle Irrigation Against Enterococcus faecalis: An In Vitro Study.

Aim The present study aims to compare the antibacterial efficacy of a novel automated endodontic irrigation device with that of a manual irrigation technique. Materials and methods The present study considered 45 extracted single-rooted teeth. After sectioning the teeth, the specimens were inoculated with Enterococcus faecalis (E. faecalis) bacteria for three weeks. Instrumentation was done using ProTaper Gold rotary files to size 50 and 5% taper. Based on the irrigation protocol, the experimental samples were divided into Group I: control (normal saline irrigation) (n = 15); Group II: conventional needle irrigation (n = 15); and Group III: automated irrigation (n = 15). The irrigation in Group III was done using the automated irrigation device. After the experimental irrigation, the pre-weighted dentinal shaving was collected in Eppendorf tubes, diluted 10 times, and incubated in the Petri dish with tryptone soy agar (TSA) for 48 hours. Finally, the colony-forming unit (CFU) counts were assessed. IBM SPSS Statistics for Windows version 23.0 (Armonk, NY, USA, IBM Corp.) was used for data analysis. Intergroup comparisons were made using the non-parametric Kruskal-Wallis test. Results The mean CFU count (CFU/ml) for the groups was: Group 1 (normal saline irrigation): 3.67x101; Group 2 (manual irrigation): 2.69× 101; Group 3 (automated irrigation): 1.57× 101. A statistically significant reduction in E. faecalis CFU count was seen among the groups assessed (p<0.01). The automated irrigation group exhibited the most substantial reduction in E. faecalis CFU count. Group 2 showed a significant difference compared to Group 1 (p<0.05). Conclusions The novel automated endodontic irrigation device was superior to manual irrigation in reducing E. faecalis CFU counts. This indicates that the automated irrigation device has the potential to enhance bacterial elimination efficacy during endodontic treatment.

Chitinophaga nivalis sp. nov., isolated from forest soil in Pyeongchang, Republic of Korea.

Rod-shaped Gram-stain-negative, aerobic bacterial strains, designated PC14 and PC15T, were isolated from a forest soil sample collected in Pyeongchang county, Gangwon-do, Republic of Korea. Strains PC14 and PC15T grew at 15-37 °C (optimum, 28-30 °C in tryptone soya agar and Mueller-Hinton agar), hydrolysed chitin and casein, and tolerated pH 8.5 and 2 % (w/v) NaCl. The strains were most closely related to members of the genus Chitinophaga, namely Chitinophaga arvensicola DSM 3695T (98.4 %), Chitinophaga longshanensis Z29T (98.3 %), Chitinophaga ginsengisegetis Gsoil 040T (97.8 %), Chitinophaga polysaccharea MRP-15T (97.8 %) and Chitinophaga niastensis JS16-4T (97.7 %). The type strain grew well on conventional commercial media in the laboratory, including tryptone soya agar, Mueller-Hinton agar, Reasoner's 2A agar, nutrient agar and Luria-Bertani agar. The major polar lipid profile comprised phosphatidylethanolamine, an unidentified aminolipid and unidentified polar lipids. The major respiratory quinone was menaquinone-7. The main fatty acids were iso-C15:0, C16:1 ω5c, C16:0 3-OH, iso-C15:0 3-OH and iso-C17:0 3-OH. The DNA G+C content of the isolated strain based on the whole genome sequence was 46.6 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strains PC14 and PC15T and the reference type strains ranged from 71.0 to 76.5 %, and from 20.3 to 20.7 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, strain PC15T could be differentiated phylogenetically and phenotypically from the recognized species of the genus Chitinophaga. Therefore, strain PC15T is considered to represent a novel species, for which the name Chitinophaga nivalis sp. nov. is proposed. The type strain is PC15T (=KACC 22893T=JCM 35788T).

Combination of the electronic nose with microbiology as a tool for rapid detection of Salmonella

Salmonella is one of the most important foodborne pathogens and its analysis in raw and processed products is mandatory in the food industry. Although microbiological analysis is the standard practice for Salmonella determination, these assays are commonly laborious and time-consuming, thus, alternative techniques based on easy operation, few manipulation steps, low cost, and reduced time are desirable. In this paper, we demonstrate the use of an e-nose based on ionogel composites (ionic liquid + gelatine + Fe3O4 particles) as a complementary tool for the conventional microbiological detection of Salmonella. We used the proposed methodology for differentiating Salmonella from Escherichia coli, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Staphylococcus aureus in nonselective medium: pre-enrichment in brain heart infusion (BHI) (incubation at 35 °C, 24 h) and enrichment in tryptone soy agar (TSA) (incubation at 35 °C, 24 h), whereas Salmonella differentiation from E. coli and P. fluorescens was also evaluated in selective media, bismuth sulfite agar (BSA), xylose lysine deoxycholate agar (XLD), and brilliant green agar (BGA) (incubation at 35 °C, 24 h). The obtained data were compared by principal component analysis (PCA) and different machine learning algorithms: multilayer perceptron (MLP), linear discriminant analysis (LDA), instance-based (IBk), and Logistic Model Trees (LMT). For the nonselective media, under optimized conditions, taking merged data of BHI + TSA (total incubation time of 48 h), an accuracy of 85% was obtained with MLP, LDA, and LMT, while five separated clusters were presented in PCA, each cluster corresponding to a bacterium. In addition, for evaluation of the e-nose for discrimination of Salmonella using selective media, considering the combination of BSA + XLD and total incubation of 72 h, the PCA showed three separated and well-defined clusters corresponding to Salmonella, E. coli, and P. fluorescens, and an accuracy of 100% was obtained for all classifiers.

Physical parameters influence the microbial quality of indoor air in research laboratories: A report from Malaysia

The quality of air conditioning and ventilation systems in laboratories is vital to ensure optimum health of the users and guarantee the accuracy of experiments conducted. Moreover, recent studies show that the average quality of air conditioning and ventilation systems in labs can pose health risks to occupants. The study aims to examine the relationship between physical parameters and microbial pollutants in research laboratories' indoor air quality (IAQ) by quantifying and characterising the bacteria and fungi in the indoor environment of research laboratories using the settle plate method; whereby our Petri dishes containing media are exposed to the air for sample collection. This study was conducted over the course of 3 ​day ​at three different research laboratories: Virology Lab (VL), Genetic Lab (GL), and Aquatic Lab (AL). The physical parameters such as air temperature, humidity and carbon dioxide were measured using FLUKE 975 air meter, whereas the microbial parameters were assessed using the settle plate method with Tryptone Soya agar and Sabouraud Dextrose agar for the bacterial and fungal count, respectively. Our results showed that the high temperature and relative humidity, exceeded the standard threshold for temperature (23–26 ​°C) and relative humidity (40–70%) set by the Department of Occupational Safety and Health (DOSH). Additionally, because of poor air conditioning and ventilation system maintenance, there were high total bacterial counts in GL (CFU/m3 234.34 ​± ​30.93) and total fungal counts in VL (CFU/m3 589.34 ​± ​53.03). We also showed that the most commonly isolated bacteria and fungi in research laboratories were Staphylococcus spp. (72.2%) and Cladosporium spp. (76.7%). In conclusion, we reported that there was a correlation between the physical parameters and microbial pollutants in the research laboratories. Therefore, regular monitoring of indoor air quality (IAQ) parameters and maintenance of the air conditioning and ventilation system should be carried out to ensure a good and safe indoor environment in research laboratories.

Culturomics and Circular Agronomy: Two Sides of the Same Coin for the Design of a Tailored Biofertilizer for the Semi-Halophyte Mesembryanthemum crystallinum

According to the EU, the global consumption of biomass, fossil fuels, metals, and minerals is expected to double by 2050, while waste will increase by 70%. In this context, the Circular Economy Action Plan (CEAP) intends to integrate development and sustainability. In this regard, tailored biofertilizers based on plant growth-promoting bacteria (PGPB) can improve plant yield with fewer inputs. In our project, an autochthonous halophyte of the Andalusian marshes, namely Mesembryanthemum crystallinum, was selected for its interest as a source of pharmaceuticals and nutraceuticals. The aim of this work was to use a culturomics approach for the isolation of specific PGPB and endophytes able to promote plant growth and, eventually, modulate the metabolome of the plant. For this purpose, a specific culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard tryptone soy agar (TSA) and MA in order to obtain two independent collections. A higher number of bacteria were isolated on TSA than in MA (47 vs. 37). All the bacteria were identified, and although some of them were isolated in both media (Pseudomonas, Bacillus, Priestia, Rosellomorea, etc.), either medium allowed the isolation of specific members of the M. crystallinum microbiome such as Leclercia, Curtobacterium, Pantoea, Lysinibacillus, Mesobacillus, Glutamicibacter, etc. Plant growth-promoting properties and extracellular degrading activities of all the strains were determined, and distinct patterns were found in both media. The three best bacteria of each collection were selected in order to produce two different consortia, whose effects on seed germination, root colonization, plant growth and physiology, and metabolomics were analyzed. Additionally, the results of the plant metabolome revealed a differential accumulation of several primary and secondary metabolites with pharmaceutical properties. Overall, the results demonstrated the feasibility of using “low cost media” based on plant biomass to carry out a culturomics approach in order to isolate the most suitable bacteria for biofertilizers. In this way, a circular model is established in which bacteria help plants to grow, and, in turn, a medium based on plant wastes supports bacterial growth at low prices, which is the reason why this approach can be considered within the model of “circular agronomy”.

Laser Capture Microdissection, Culture Analysis, and Bacterial Sequencing to Evaluate the Microbiota of Focal Duodenal Necrosis in Egg Layers.

Focal duodenal necrosis (FDN) is a common intestinal disease of table egg layers. In this research we aimed to identify the bacteria commonly found in FDN lesions as seen with histopathological analysis. Fifty-nine ethanol-fixed duodenum samples were collected from egg layers on eight FDN-affected farms, and 42 samples had typical FDN lesions. Excision of bacteria-containing lesions using laser capture microdissection was performed, followed by 16S rRNA gene sequencing of extracted DNA for bacterial identification. Bacterial sequencing analysis revealed no consistent bacterial species identified from samples with FDN. However, analysis of the relative phylum abundance revealed differences in the duodenal microbiota between layers with FDN and healthy birds. There were differences in the abundance of Proteobacteria, Firmicutes, and Actinobacteria between FDN-positive and FDN-negative control samples compatible with intestinal dysbiosis. In addition, 10 duodenal samples with FDN lesions were collected for bacteriological analysis, yielding 47 colonies on tryptone soy agar, MacConkey agar, and blood agar plates. Using 16S rRNA gene PCR, 39/47 (53.8%) colonies were identified as Escherichia coli. PCR for E. coli virulence genes identified 21/39 (53.8%) E. coli isolates as avian pathogenic E. coli-like. PCR analysis for 19 E. coli virulence genes associated with intestinal disease strains including inflammatory bowel disease found 11/39 (28.2%) isolates containing more than 10 of these virulence genes. In conclusion, FDN appears to be a multifactorial inflammatory intestinal disease associated with intestinal dysbiosis, and Gram-negative bacteria including E. coli may contribute to the pathogenesis of this disease.

Comparison of the Efficiency of Selected Disinfectants against Planktonic and Biofilm Populations of Escherichia coli and Staphylococcus aureus.

The aim of this study is to compare the efficacy of selected food disinfectants on planktonic populations of Staphylococcus aureus and Escherichia coli and on the same microorganisms (MOs) incorporated in a biofilm. Two disinfectants were used for treatment: peracetic acid-based disinfectant (P) and benzalkonium chloride-based disinfectant (D). Testing of their efficacy on the selected MO populations was performed using a quantitative suspension test. The standard colony counting procedure was used to determine their efficacy on bacterial suspensions in tryptone soy agar (TSA). The germicidal effect (GE) of the disinfectants was determined based on the decimal reduction ratio. For both MOs, 100% GE was achieved at the lowest concentration (0.1%) and after the shortest exposure time (5 min). Biofilm production was confirmed with a crystal violet test on microtitre plates. Both E. coli and S. aureus showed strong biofilm production at 25 °C with E. coli showing significantly higher adherence capacity. Both disinfectants show a significantly weaker GE on 48 h biofilms compared to the GE observed after application of the same concentrations on planktonic cells of the same MOs. Complete destruction of the viable cells of the biofilms was observed after 5 min of exposure to the highest concentration tested (2%) for both disinfectants and MOs tested. The anti-quorum sensing activity (anti-QS) of disinfectants P and D was determined via a qualitative disc diffusion method applied to the biosensor bacterial strain Chromobacterium violaceum CV026. The results obtained indicate that the disinfectants studied have no anti-QS effect. The inhibition zones around the disc therefore only represent their antimicrobial effect.

Effect of Bitter Leaf Extract (Andrographis paniculata) against Edwardsiella tarda Bacteria In vitro

This study aimed to determine the antibacterial activity of bitter leaf extract (A. paniculata) against the growth of E. tarda bacteria. This test was carried out using Kirby Bauer method (disc test) using five different concentrations, namely 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, and 250 mg/L in TSA (Tryptone Soya Agar) and incubated for 2x24 hours using a temperature of 30 oC. The positive control used was chloramphenicol (5 mg/L), while the negative control used PBS. Bitter leaf extract contains active compounds that have bacteriostatic antibacterial properties. After incubation for 24 hours the concentration of 250 mg/L showed the highest inhibition zone with a diameter of 7.82 mm and decreased at 48 hours to 7.46 mm. After being incubated for 24 hours, the concentration of 250 mg/L obtained an effectiveness value of 57.08% which is the best effectiveness, and there was a decrease in the 48th hour to 54.45%.

Recovery of Naturally Occurring Human Borne Microbial Contamination from Settle Plates Exposed in a Unidirectional Airflow Workstation for 4 Hours

The ability of irradiated 90 mm diameter tryptone soya agar settle plates, exposed for 4 hours in a unidirectional airflow (UDAF) workstation to recover microbial contamination, was investigated. The investigation was completed by direct contamination of numerous plates with naturally occurring microbe-carrying particles (MCPs) dispersed from a person within an enclosed environment. Half of these plates were subsequently exposed in a UDAF workstation for 4 hours and then incubated. The other half, contaminated with a similar number of MCPs, were not exposed in the workstation and provided the controls. Investigation of the reduction of plate weight during exposure was also completed to help understand the influence of plate media water content to any loss of microbial recovery. Following incubation, the numbers of recovered micro-organisms were compared and it was determined that the average number of recovered test plate colonies was reduced by 8.7% compared to the control plates and the exposure of the plates to UDAF for 4 hours reduced the plate weight, associated with loss of the media water, by an average of 12.3%. It was concluded that the ability of the plates to recover micro-organisms, following a 4 hour exposure to UDAF, was not significantly reduced. Key words: Microbiological settle plate desiccation, media dehydration, unidirectional airflow (UDAF), 4 hour exposure, microbe-carrying particles (MCPs), environmental monitoring, settle plate sampling.

A Culturomics-Based Bacterial Synthetic Community for Improving Resilience towards Arsenic and Heavy Metals in the Nutraceutical Plant Mesembryanthemum crystallinum.

Plant-growth-promoting bacteria (PGPB) help plants thrive in polluted environments and increase crops yield using fewer inputs. Therefore, the design of tailored biofertilizers is of the utmost importance. The purpose of this work was to test two different bacterial synthetic communities (SynComs) from the microbiome of Mesembryanthemum crystallinum, a moderate halophyte with cosmetic, pharmaceutical, and nutraceutical applications. The SynComs were composed of specific metal-resistant plant-growth-promoting rhizobacteria and endophytes. In addition, the possibility of modulating the accumulation of nutraceutical substances by the synergetic effect of metal stress and inoculation with selected bacteria was tested. One of the SynComs was isolated on standard tryptone soy agar (TSA), whereas the other was isolated following a culturomics approach. For that, a culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard TSA and MA media, stablishing two independent collections. All bacteria were tested for PGP properties, secreted enzymatic activities, and resistance towards As, Cd, Cu, and Zn. The three best bacteria from each collection were selected in order to produce two different consortiums (denominated TSA- and MA-SynComs, respectively), whose effect on plant growth and physiology, metal accumulation, and metabolomics was evaluated. Both SynComs, particularly MA, improved plant growth and physiological parameters under stress by a mixture of As, Cd, Cu, and Zn. Regarding metal accumulation, the concentrations of all metals/metalloids in plant tissues were below the threshold for plant metal toxicity, indicating that this plant is able to thrive in polluted soils when assisted by metal/metalloid-resistant SynComs and could be safely used for pharmaceutical purposes. Initial metabolomics analyses depict changes in plant metabolome upon exposure to metal stress and inoculation, suggesting the possibility of modulating the concentration of high-value metabolites. In addition, the usefulness of both SynComs was tested in a crop plant, namely Medicago sativa (alfalfa). The results demonstrate the effectiveness of these biofertilizers in alfalfa, improving plant growth, physiology, and metal accumulation.

Biological activity of volatiles produced by the strains of two Pseudomonas and two Serratia species.

Volatile compounds emitted by bacteria can play a significant role in interacting with microorganisms, plants, and other organisms. In this work, we studied the effect of total gaseous mixtures of organic as well as inorganic volatile compounds (VCs) and individual pure volatile organic compounds (VOCs: ketones 2-nonanone, 2-heptanone, 2-undecanone, a sulfur-containing compound dimethyl disulfide) synthesized by the rhizosphere Pseudomonas chlororaphis 449 and Serratia plymuthica IC1270 strains, the soil-borne strain P. fluorescens B-4117, and the spoiled meat isolate S. proteamaculans 94 strain on Arabidopsis thaliana plants (on growth and germination of seeds). We demonstrated that total mixtures of volatile compounds emitted by these strains grown on Luria-Bertani agar, Tryptone Soya Agar, and Potato Dextrose Agar media inhibited the A. thaliana growth. When studied bacteria grew on Murashige and Skoog (MS) agar medium, volatile mixtures produced by bacteria could stimulate the growth of plants. Volatile compounds of bacteria slowed down the germination of plant seeds; in the presence of volatile mixtures of P. fluorescens B-4117, the seeds did not germinate. Of the individual VOCs, 2-heptanone had the most potent inhibitory effect on seed germination. We also showed that the tested VOCs did not cause oxidative stress in Escherichia coli cells using specific lux-biosensors. VOCs reduced the expression of the lux operon from the promoters of the katG, oxyS, and soxS genes (whose products involved in the protection of cells from oxidative stress) caused by the action of hydrogen peroxide and paraquat, respectively.

Effect of Giving Jackfruit (Artocarpus heterophyllus) Leaves Crude Extract as An Alternative Antibacterial of Edwardsiella tarda Bacteria In Vitro

Barriers to cultivation activities are disease attacks, one of which is caused by infection with the bacterium Edwardsiella tarda. Treatment efforts can be made using natural ingredients such as jackfruit (A. heterophyllus) leaves. This study aims to determine the content of antibacterial compounds in the jackfruit (A. heterophyllus) leaves crude extract and their effect on Edwardsiella tarda bacteria in vitro. The inhibition test used disc test with five different concentrations of jackfruit (A. heterophyllus) leaves crude extract with three replications: 75 mg/L, 150 mg/L, 225 mg/L, 300 mg/L and 375 mg/L with a comparison using two types of controls (positive with 5 mg/L chloramphenicol antibiotics and negative with no treatment) using Tryptone Soy Agar (TSA) media and incubated for 2x24 hours. The results of phytochemical screening proved that the jackfruit (A. heterophyllus) leaves crude extract contains flavonoids, alkaloids, tannins and saponins. Treatment E with a dose of 375 mg/L gave the highest inhibition of 7.80 mm and treatment A with a dose of 75 mg/L gave the lowest inhibition of 4.38 mm. The relationship between the treatment given to the test parameter in the form of the resulting inhibitory power obtained the equation y = 0.0111x + 3.6297 with an R2 value of 0.974 which means 97% of the treatment given in the form of crude extract of jackfruit (A. heterophyllus) leaves affects inhibition of bacteria E. tarda.