Tryptic Soy Agar Research Articles

Transfer of Salmonella enterica and Enterococcus faecium from food-contact surfaces to stone fruits

Contaminated food-contact surfaces are a potential route for spreading microorganisms to stone fruit during postharvest handling. The objective of this study was to investigate the factors that affect the transfer of bacteria from food contact surfaces to stone fruits. Coupons (1 × 1 cm) of polyurethane (PU) or polyvinyl chloride (PVC) were inoculated with rifampin-resistant variants of Salmonella (five-strain cocktail) or Enterococcus faecium NRRL B-2354 at ~5 or ~ 7 log CFU/cm2. Inoculated coupons (n = 8–11) were attached to a texture analyzer, and uniform contact conditions (5 N, 5 s) were used to explore the impact of bacterial species, inoculation level, donor surface, the presence of dried peach juice or wax, recipient produce commodity, and the dryness of inoculum. Whole fruits were transferred to 20 mL of 0.1 % peptone, rubbed for 2 min, and then the diluent was plated onto trypticase soy agar supplemented with rifampin at 50 μg/mL. Whole fruits were enriched when populations were anticipated to fall below the limit of detection (1.6 log CFU/fruit). At an inoculum of ~5 log CFU/coupon, Salmonella and E. faecium were recovered from the fruit by enrichment but not by plating. At ~7 log CFU/coupon, transfer rates, i.e., ratio of populations on recipient fruit to donor surface, were not significantly (P ≥ 0.05) influenced by either bacterial species (Salmonella [0.26 % ± 0.77 %] versus E. faecium [0.068 % ± 0.071 %]) or donor surface (PU [0.085 % ± 0.098 %] versus PVC [0.16 % ± 0.16 %]). The rates of transfer of E. faecium from contaminated PU to peaches (0.050 % ± 0.031 %), nectarines (0.066 % ± 0.076 %), and onion skins (0.048 % ± 0.059 %) were not significantly different. The mean transfer rates of E. faecium increased significantly (P < 0.05) in the presence of dried wax (18 % ± 16 %) or peach juice (1.3 % ± 2.6 %) on the PU surface compared with the control (0.080 % ± 0.086 %). The transfer rates of E. faecium from contaminated surfaces were also significantly influenced by the drying time post-inoculation; the drier the inoculum, the lower the transfer rates. The presence of residues or moisture on food contact surfaces facilitated the transfer of microorganisms during dry handling of fresh stone fruits. The results underscore the importance of implementing adequate cleaning, sanitation and, where appropriate, drying of equipment surfaces to effectively remove organic residues and mitigate the risks of cross-contamination.

Effects of Sodium Chloride, Sodium Lactate, and Sodium Diacetate on the Growth Probabilities of Salmonella Spp. and Staphylococcus Aureus

ABSTRACTSalmonella spp. and Staphylococcus aureus have been linked to foodborne illnesses caused by the consumption of processed meat products. This study examined the growth probabilities of these two pathogens as affected by sodium chloride (salt), sodium lactate, and sodium diacetate in a solid medium for using these three additives to improve the microbial safety of processed meat. Sterilized tryptic soy agar (TSA, 200 μL) formulated with a combination of salt (3%–8%, aw 0.98–0.93), lactate (0%–2.4%), and diacetate (0%–0.25%) and inoculated with Salmonella spp. or S. aureus was dispersed into 96‐well microplates and incubated at 37°C for 7 days. After incubation, a well showing any Salmonella spp. or S. aureus colonies was denoted as a growth event, otherwise a no‐growth event. The number of growth events for each formulation was recorded. The effects of the additives on the growth event were analyzed by logistic regression to identify the growth and no‐growth boundaries and the formulations that may prevent the growth of Salmonella spp. or S. aureus. For Salmonella spp., the observed minimum no‐growth concentrations in TSA were 3% salt with 0.8% lactate+0.2% diacetate or 1.6% lactate+0.1% diacetate, 4% salt with 2.4% lactate, 5% salt with 0.25% diacetate, 6% salt with 0.8% lactate+0.15% diacetate, 7% salt with 0.8% lactate or 0.15% diacetate, and 8% salt alone. For S. aureus, the concentrations were 3% salt with 2.4% lactate+0.2 diacetate, 5% salt with 1.6% lactate+0.2% diacetate, 7% salt with 0.8% lactate+0.25% diacetate, and 8% salt with 0.8% lactate+0.20% diacetate or 1.6% lactate+0.15% diacetate. These no‐growth formulations also inhibited the growth of both pathogens in cooked meat samples. Mathematical models were developed to describe the effects of the additives on the growth probabilities of Salmonella spp. and S. aureus. Findings from this study may be used for formulating refrigerated and shelf‐stable meat products to reduce Salmonella spp. and S. aureus risk.

First report of Pectobacterium peruviense as the causal pathogen of blackleg and soft rot in Solanum tuberosum cv. Superchola in Cañar, Ecuador.

Potato (Solanum tuberosum) is grown in Ecuador's Andean region at altitudes from 2,200 to 3,600 m.a.s.l., and the most commercially significant cultivar is Superchola, which constitutes 60% of total production, covering around 11,000 hectares (MAG, s.f. 2022). From November 2022 to January 2023, dark, water-soaked lesions progressing upwards from the base of the stems were observed on Superchola potato plants in Cañar, Ecuador (2°23'56.4''S 78°59'13.2''W). Approximately 20 plants in a hectare showed symptoms. Symptomatic stems from five plants were collected, surface-disinfected with 2% sodium hypochlorite, followed by 70% ethanol, and thoroughly rinsed with sterile distilled water. Plant tissue sections (1 cm²) were homogenized in 10 mM MgCl₂, serially diluted, and plated on Tryptic Soy Agar (TSA). After 48 hours of incubation at 28°C, creamy-white, yellowish, and white round colonies appeared. Streak plating was performed, yielding 14 purified bacterial isolates named M1 to M14. For the pathogenicity tests, each purified isolate was inoculated into healthy potato tubers and stem pieces (5 cm) using the prick inoculation method described by Ma et al. (2018) with modifications. Surface-sterilized tubers and stems were wounded using autoclaved toothpicks, into which 5µl of a bacterial suspension, carrying ~1 x 106 CFU/ml of bacteria per strain, was deposited into a 5 mm deep wound. Three independent replicates were conducted. Only one isolate, designated as M3, presented maceration on potato tubers, and dark, watered-soaked lesions appeared on the stem 48 h post-inoculation with strain. Other bacterial isolates showed no symptoms in either stems or tubers. Pathogenicity tests were confirmed on healthy, 4-week-old potato plants grown in a 50% perlite and 50% peat moss substrate. Inoculations were done using two methods: (1) toothpick piercing, where 5 µl of saline solution containing ~1 x 106 CFU/ml of bacteria was applied to a wound approximately 5mm deep (Ma et al. 2018), and (2) immersion of the plant foliage in a bacterial suspension of 1x 108 CFU/ml in saline solution for 10 min Controls used bacteria-free toothpicks (wounded) and sterile 10 mM MgCl₂ solution (unwounded). Plants were enclosed in plastic containers to maintain high humidity and grown at 15/25°C (day/night) with a 12 h photoperiod. Five days post-inoculation, blackleg symptoms were observed in the stems of the infected area, which turned black and rotten (wounded and unwounded). In all cases, negative controls remained symptomless. To complete Koch's postulates, bacteria were reisolated from symptomatic potato stems and showed the same morphology. To identify the isolate M3, whole genome sequencing using Oxford Nanopore technology was performed. Three marker genes were extracted, and a 5,734-bp sequence was concatenated from dnaX (2073 bp), recA (1,074bp), and leuS (2,583bp) (GenBank accession nos. PQ177849, PQ177847 and PQ177848, respectively). The concatenated sequences of M3 and type strains were used to construct a multilocus Bayesian inference phylogenetic tree using BEAST version 1.8.4. Isolate M3 grouped with Pectobacterium peruviense, showing a 100% sequence identity with the type strain P. peruviense IFB5232(Drummond et al. 2012; Portier et al. 2019; Toth et al. 2021; Waleron et al. 2018). To our knowledge, this is the first report of P. peruviense causing blackleg and soft rot in potato plants in Ecuador. Further research is essential, but this information could assist in monitoring the pathogen's spread and developing disease management strategies.

Evaluation of Commercially Available Sanitizers Efficacy to Control Salmonella (Sessile and Biofilm Forms) on Harvesting Bins and Picking Bags

This study evaluated the efficacy of five commercially available sanitizers to reduce Salmonella (sessile and biofilm forms) count on experimentally inoculated materials representative of harvesting bins and picking bags in the fresh produce industry. Sessile Salmonella cells were grown onto tryptic soy agar to create a bacterial lawn, while multistrain Salmonella biofilms were grown in a Centers for Disease Control and Prevention (CDC) reactor at 22 ± 2 °C for 96 h. Samples were exposed to 500 ppm free chlorine, 500 ppm peroxyacetic acid (PAA), 75 psi steam, and 5% silver dihydrogen citrate (SDC) for 30 sec, 1, or 2 min or 100 ppm chlorine dioxide gas for 24 h. Sanitizer, surface type, and application time significantly affected the viability of Salmonella in both sessile and biofilm forms (P < 0.05). All treatments resulted in a significant reduction of Salmonella when compared to the control (P < 0.05). Chlorine dioxide gas was the most effective treatment in both sessile and biofilm forms regardless of the type of surface, and it achieved a 5-log reduction. PAA at 500 ppm applied for 2 min was the only liquid sanitizer that resulted in a greater than 3-log reduction in all surfaces. Scanning electronic microscopy demonstrated the porous surface nature of nylon and wood, compared to HDPE, impacted sanitizer antimicrobial activity. Understanding the efficacy of sanitizers to control Salmonella on harvesting bins and picking bags may improve the safety of fresh produce by increasing available sanitizing treatment.

Study of some physicochemical and bacteriological characteristics of drinking water in three selected rural communities in Wamba, Nasarawa State, Nigeria.

Access to safe drinking water remains a challenge in rural Nigeria. This study was carried out to determine physicochemical and bacteriological characteristics of drinking water in three rural communities in Wamba, Nasarawa State. A cross-sectional survey was conducted, 5 water samples were in respective cases collected from stream, well and borehole sources. Standard techniques were used for physicochemical analysis. Total viable counts, Total Staphylococcal counts, Total coliforms &amp; Feacal coliforms in the samples were obtained using pour plate method, growth on Mannitol salt agar, growth on MacConkey agar and Eosin Methylene Blue agar respectively. Stream Water had higher values for physicochemical parameters. Stream water exhibited the highest bacterial counts, followed by well and borehole water. The Bacteria isolated were Escherichia coli, Staphylococcus aureus, Pseudomonas spp, Klebsiella spp, Proteus spp and Salmonella spp. The acid lability attributes of the isolates were determined using Nutrient Agar buffered with (4-(2- Hydroxyethyl)-1- piperazineethanesulfonic acid) and Lipase activity was assessed using Trypticase soy agar supplemented with 1% Tween 80. Statistical analysis was performed using SPSS-PC statistical package, with a p-value of &lt; 0.05 it was considered statistically significant. These findings indicate contamination of drinking water sources, posing health risk to consumers and it shows the need for water treatment, monitoring &amp; public health education in rural communities.

The Determination of the Rapid and Effective Activity of an Air Sanitizer against Aerosolized Bacteria Using a Room-Sized Aerobiology Chamber.

Air sanitization is an important non-pharmaceutical intervention for mitigating the risk of indoor pathogen spreading. A dipropylene glycol-containing air sanitizer was tested against aerosolized Staphylococcus aureus and Klebsiella pneumoniae. The bacteria, suspended in a soil load, were aerosolized using a six-jet Collison nebulizer with pressurized air. The 25-m3 (~900 ft3) aerobiology chamber was maintained at 22 ± 2 °C and 50 ± 5% relative humidity per the U.S. Environmental Protection Agency's 2012 Guidelines on air sanitizers. An initial 2-min air sample was collected from the chamber using a slit-to-agar sampler containing 150-mm Petri plates, with Trypticase soy agar (TSA) containing neutralizers to quench the microbicidal activity of the air sanitizer, to determine the initial bacterial challenge in the air. The air sanitizer was sprayed into the chamber from pressurized cans. Additional air samples were collected from the chamber over 10 min to detect surviving bacteria. The TSA plates were then incubated aerobically at 36 ± 1 °C for 90 ± 4 h and scored for bacterial colony-forming units. A 30-s spray of the air sanitizer reduced infectious S. aureus and K. pneumoniae titers by 3.0 log10 (99.9%) in 3.2 ± 0.3 min and 1.2 ± 0.0 min, respectively. Based on these findings, the EPA granted registration of the air sanitizer as the first product of its kind for indoor air sanitization.

Optimization of Screening Media to Improve Antimicrobial Biodiscovery from Soils in Undergraduate/Citizen Science Research-Engaged Initiatives.

Background/Objectives: Research-engaged academic institutions offer the opportunity to couple undergraduate education/citizen science projects with antimicrobial biodiscovery research. Several initiatives reflecting this ethos have been reported internationally (e.g., Small World, Tiny Earth, MicroMundo, Antibiotics Unearthed). These programs target soil habitats due to their high microbial diversity and promote initial screening with non-selective, nutrient media such as tryptic soy agar (TSA). However, evaluation of published outputs to date indicates that isolate recovery on TSA is consistently dominated by the genera Bacillus, Pseudomonas, and Paenibacillus. In this study, we evaluated the potential of soil extract agar to enhance soil isolate diversity and antibiosis induction outcomes in our undergraduate Antibiotics Unearthed research program. Methods: We comparatively screened 229 isolates from woodland and garden soil samples on both tryptic soy agar (TSA) and soil extract agar (SEA) for antimicrobial activity against a panel of clinically relevant microbial pathogens. Results: On one or both media, 15 isolates were found to produce zones of clearing against respective pathogens. 16S rRNA gene sequencing linked the isolates with three genera: Streptomyces (7), Paenibacillus (6), and Pseudomonas (2). Six of the Streptomyces isolates and one Pseudomonas demonstrated antimicrobial activity when screened on SEA, with no activity on TSA. Furthermore, incorporation of the known secondary metabolite inducer N acetyl-glucosamine (20 mM) into SEA media altered the pathogen inhibition profiles of 14 isolates and resulted in broad-spectrum activity of one Streptomyces isolate, not observed on SEA alone. In conclusion, SEA was found to expand the diversity of culturable isolates from soil and specifically enhanced the recovery of members of the genus Streptomyces. SEA was also found to be a superior media for antibiosis induction among Streptomyces isolates when compared to TSA. It was noted that Paenibacillus isolates' antibiosis induction demonstrated a strain-specific response with respect to the growth media used. Conclusions: The authors propose SEA inclusion of in soil screening protocols as a cost-effective, complementary strategy to greatly enhance outcomes in undergraduate/citizen science-engaged antimicrobial biodiscovery initiatives.

Transfer of beef bacterial communities onto food-contact surfaces.

Food spoilage and pathogenic bacteria on food-contact surfaces, especially biofilm-forming strains, can transfer to meats during processing. The objectives of this study were to survey the bacterial communities of beef cuts that transfer onto two commonly used food-contact surfaces, stainless steel (SS) and high-density polyethylene (HDPE) and identify potentially biofilm-forming strains. Top round, flank, chuck, and ground beef were purchased from 3 retail stores. SS and HDPE coupons (approximately 2cm × 5cm) were placed on beef portions (3h, 10°C), after which, the coupons were submerged halfway in PBS (24h, 10°C). Bacteria from the beef cuts and coupon surfaces (n = 3) were collected, plated on tryptic soy agar plates and incubated (5 days, 25°C). Bacterial isolates were identified by 16S rRNA gene amplicon sequencing and assayed for biofilm formation using a crystal violet binding (CV) assay (72h, 10°C). Additionally, beef and coupon samples were collected for bacterial community analysis by 16S rRNA gene amplicon sequencing. Sixty-one of 972 beef isolates, 29 of 204 HDPE isolates, and 30 of 211 SS isolates were strong biofilm-formers (Absorbance>1.000 at 590 nm in the CV assay). Strong-binding isolates identified were of the genera Pseudomonas, Acinetobacter, Psychrobacter, Carnobacterium, and Brochothrix. Coupon bacterial communities among stores and cuts were distinct (p < 0.001, PERMANOVA), but there was no distinction between the communities found on HDPE or SS coupons (p > 0.050, PERMANOVA). The bacterial communities identified on the coupons may help determine the communities capable of transferring and colonizing onto surfaces, which can subsequently cross-contaminate foods.

Antimicrobial effects of a multimodal wound matrix against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa in an in vitro and an in vivo porcine wound model.

Chronic non-healing wounds pose significant challenges due to an elevated inflammatory response caused in part by bacterial contamination (Physiol Rev. 2019;99:665). These wounds lead to billions being spent in the health care system worldwide (N Engl J Med. 2017;376:2367, Int J Pharm. 2014;463:119). We studied the in-vitro and in-vivo antimicrobial effects of a multimodal wound matrix (MWM) against two common wound pathogens, Methicillin-Resistant Staphylococcus aureus (MRSA USA300) and Pseudomonas aeruginosa ATCC 27312 (PA27312) (Int Wound J. 2019;16:634). The in-vitro study conducted was a zone of inhibition test with the two microbes at 104 Log CFU/mL inoculated on Tryptic soy agar with 5% sheep blood (TSAII) plates. Treatments used were MWM, Mupirocin (Positive control for MRSA), Silver Sulfadiazine (Positive Control for PA), Petrolatum and Sterile Saline (both serving as Negative Controls). Treatments were allowed to diffuse into the agar for 3 h and then were incubated for 24 h at 37°C. The in-vivo study utilized a deep dermal porcine wound model (22 × 22 × 3 mm) created on six animals. Three animals were inoculated with MRSA USA300 and the other three with PA27312 with each allowing a 72-h biofilm formation. After 72 h, baseline wounds were assessed for bacterial concentration and all remaining wounds were treated with either MWM alone, Silver Treatment or Untreated Control. Wounds were assessed on days 4, 8 and 12 after treatment application for microbiological analysis. In-vitro, MWM exhibited significant inhibition of MRSA USA300 and PA27312 growth when compared to negative controls (p ≤ 0.05). Likewise, in-vivo, the MWM-treated wounds exhibited a significant (p ≤ 0.05) bacterial reduction compared to all other treatment groups, especially on days 8 and 12 for both pathogens. MWM demonstrated promise in addressing colonized wounds with biofilms. Additional studies on MWM's benefits and comparisons with existing treatments are warranted to optimize wound care strategies (Adv Wound Care. 2021;10:281).

Antimicrobial, Optical, and Mechanical Properties of Saliva-Contaminated Silver–Zeolite Nanoparticle-Incorporated Dental Acrylic Resins

Background and Purpose: This study aimed to evaluate the flexural strength, color change and antimicrobial effect of silver–zeolite nanoparticles (NPs) in acrylic resin materials. Methods: Fifty-six disc-shaped acrylic resin samples were divided into four groups (n = 7) according to concentrations of silver–zeolite NPs (0%, 2%, 4%, 5%). Discs were contaminated with C. albicans and S. mutans. The antimicrobial effect was tested by inoculating contaminated discs on Tryptic soy agar (TSA), Sabouraud Dextrose Agar (SDA), Tryptic soy broth (TSB), and Sabouraud dextrose broth (SDB). Forty rectangular 65 × 10 × 2.5 mm acrylic resin specimens were also classified into four groups (n = 10) according to concentrations of silver–zeolite NPs. For the color change, L, a, and b values of rectangular specimens were examined with a spectrophotometer. A three-point bending test was also performed using a Devotrans device to determine the flexural bond strength of rectangular specimens. Scanning electron microscope analysis (SEM/EDX analysis) was also performed. Results: In this study, the antimicrobial effect increased with the concentration of silver–zeolite NPs added to acrylic resin discs. In our study, adding 2% silver–zeolite NPs was more effective against C. albicans. The antimicrobial effect against S. mutans increased with concentration of silver–zeolite NPs (&lt;0.001). The colonization of C. albicans was significantly reduced by silver–zeolite NPs. A significant increase was observed in the color change as the nanoparticle percentage ratio increased (p &lt; 0.001). The flexural strength values of the groups containing 2% and 4% nanoparticles were found to be clinically acceptable. Conclusions: The study showed that bacterial and fungal colonization is significantly reduced by adding silver–zeolite nanoparticles to acrylic resin discs. Based on its antimicrobial, physical, and mechanical properties, we recommend adding 2% silver–zeolite nanoparticles to the acrylic resin material for optimal results.

Dolosigranulum savutiense sp. nov., isolated from human upper respiratory samples collected in Botswana.

Four strains (MSK211, MSK294T, MSK312, MSK433) of a novel Dolosigranulum species were cultured from nasopharyngeal swabs collected from mother-infant dyads in southern Botswana. These strains grew optimally on tryptic soy agar with 5% sheep blood solid medium and in fastidious bacteria broth. Colonies on tryptic soy agar with 5% sheep blood agar appeared grey or white with a flat, smooth surface and variable alpha haemolysis. Cells were Gram-positive, non-spore-forming, non-motile cocci that lacked catalase or oxidase activity. Major fatty acids were C16 : 0 (palmitic acid), C18 : 1 ω9c (oleic acid), and C18 : 0 (stearic acid). Analyses of 16S rRNA gene sequences identified these strains as belonging to the genus Dolosigranulum (family Carnobacteriaceae), which currently contains only a single validly published species (Dolosigranulum pigrum). Whole-genome sequencing revealed that the genomes of these strains are 1.98-2.07 Mbp in size and have a G+C content of 39.6-39.9 mol%. Comparisons of these genomes to publicly available genomes of D. pigrum yielded average nucleotide identities and in silico DNA-DNA hybridization values of 92.3-92.9% and 49.1-51.4%, respectively. These results indicate that these strains represent a novel species of Dolosigranulum, for which we propose the name Dolosigranulum savutiense sp. nov., with the type strain MSK294T (=DSM 117171T=JCM 36673T).

Patulibacter defluvii sp. nov., Isolated from a Wastewater Treatment Plant in Guangzhou City, China.

A novel Gram-stain-positive, aerobic, catalase-positive, oxidase-negative, non-motile, and rod-shaped bacterium with ibuprofen-degrading capacity, designated DM4T, was isolated from the sewage of a wastewater treatment plant (WWTP) in Guangzhou city, China. Strain DM4T grew optimally at 0% (w/v) NaCl, pH 5.0-7.0, and 30°C, forming white colonies on trypticase soy agar. C18:1ω9c, C18:2ω9.12c and C15:1ω10c were the predominant fatty acids. Results of 16S rRNA gene alignment and phylogenetic analysis indicated that strain DM4T belonged to the genus Patulibacter, was closely related to Patulibacter medicamentivorans DSM 25692T (98.5%) and P. brassicae KCTC 39817T (98.1%). Strain DM4T had a genome size of 5.33Mbp, and the DNA G + C content was 75.0%. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridisation (dDDH) values between strain DM4T and P. medicamentivorans were 85.2%, 83.9%, and 29.0% respectively, while those between strain DM4T and P. brassicae were 78.5%, 71.3%, and 22.2%, respectively. Strain DM4T could significantly degrade ibuprofen by almost 80% after 84h of incubation, and the degradation kinetics was well fitted with the first-order kinetics. Evidence from phenotypic, phylogenetic and chemotaxonomic analyses support that strain DM4T (= GDMCC 1.4574T = KCTC 59145T) represents a new species of the genus Patulibacter, for which the name Patulibacter defluvii sp. nov. is proposed.

Influence of phenotypic variation of Paenibacillus polymyxa E681 on growth promotion in cucumbers.

The goal of the current study is to better understand how bacteria may adapt to survive under adverse environmental conditions by altering and improving their phenotypes. In this study, we report the consequences of phenotypic variation in Paenibacillus polymyxa E681 (E681), a plant growth-promoting rhizobacterium (PGPR), isolated from winter barley root that has a variety of advantageous effects on crop plants. In our previous study, two different types of bacterial cells in E681 were distinguished. We used the term F-type for the variant that doesn't produce endospores and B-type for the endospore-producing wild type. Under the circumstances of our experiment, the cucumber rhizosphere soil and the surface of the seeds produced phenotypic variance. On tryptic soy agar (TSA) plates, the B-type spontaneously converted into the F-type, but the reverse was not reversible. Intriguingly, the plant growth promotion test displayed that cucumber seedlings treated with F-type cells had characteristics resembling those of the untreated control. Whereas, growth promotion of cucumber seedlings treated with B-type depends on temperature conditions. In particular, an increased growth promotion was observed at a low temperature of 20°C. The phenotypic change from B-type to F-type did not occur at 20°C for 6 days in the growth curve analysis of E681, but it did occur on the fourth and second days at 30 and 37°C, respectively. Therefore, before using PGPR strains as a bacterial inoculant for sustainable agriculture, it is imperative to resolve phenotypic variance in these strains.

Soil Bacteria Community Composition Indicating Soil Quality in Different Tillage Practices

Organic tillage practice of current study used fully cattle manure, while semi-organic tillage used cattle manure and 10% of synthetic fertilizers as transition to reduce chemical inputs in order to improve sustainable production and ecosystem health. Despite the important role of soil microbes in soil of agriculture, we still have limited understanding of the complex response of microbial composition to organic and semi-organic tillage practices. The aim of this work was to evaluate the impact of use of organic and semi-organic tillage practices on bacteria abundance, richness and functional diversity of microbial communities. Analysis of soil bacteria using nutrient agar by serial dilutions of soil suspension was performed to quantify bacteria abundance. They were further identified and categorized into IAA-producing bacteria and non IAA-producing bacteria groups in Tryptic Soy Agar (TSA). Bacteria abundance was significantly higher in semi-organic tillage than organic tillage. Even though bacteria richness did not show statistically significant, beneficial effect of organic tillage was reflected in higher bacteria richness in term of isolates. The assessment of their functional diversity on producing IAA hormone demonstrated higher IAA-producing bacteria than non IAA-producing bacteria in both tillage practices, indicating the availability of rich substrate through introduction of cattle manure improve soil quality and beneficial for soil bacteria. We observed that among all physicochemical properties, C-organic indicated highly significant, higher in organic tillage than semi-organic tillage. It has been proposed that the availability of organic carbon may favour bacteria as resource of energy. Keywords: Bacteria community composition; IAA-producing bacteria; Organic tillage; Semi-organic tillage

Bacterial community of kimchi added with seafood based on culture-dependent investigations

Previously, microbial communities of five commercial kimchi added with seafood and one kimchi without seafood were analyzed using a culture-independent (CI) method. In the current study, microbial communities of the same samples were analyzed using a culture-dependent (CD) method with two media: tryptic soy agar (TSA) and Lactobacilli de Man, Rogosa and Sharpe (MRS) agar. MRS agar showed a higher proportion of lactic acid bacteria, while TSA showed a higher proportion of Bacillus species. Leuconostoc mesenteroides became dominant over time except in kimchi added with hongeu (HBK, okamejei kenojei). In the case of HBK, Bacillus was dominant. The low pH of HBK was confirmed by cell size and heat treatment under pH 4–7 conditions that Bacillus could be present in the form of spores. With the CD method, only Lactococcus lactis, Leu. citreum, and Weissella cibaria were detected. With the CI method, only Pediococcus inopinatus was detected. A notable finding was that Leu. mesenteroides was more abundant than Latilactobacillus sakei with the CD method, whereas it was similar or lower with the CI method. This discrepancy was confirmed to be due to different rates of DNA recovered from the two strains. This shows that the assay method may influence the detection of these two strains.

Neisseria leonii sp. nov., isolated from the nose, lung, and liver of rabbits.

A taxogenomic study of three strains (3986T, 51.81, and JF 2415) isolated from rabbits between 1972 and 2000 led to the description of a new Neisseria species. The highest sequence similarity of the 16S rRNA gene was found to Neisseria animalis NCTC 10212T (96.7 %). The 16S rRNA gene similarity above 99 % and average nucleotide identity (ANI) values above 96 % among the strains, indicated that they belong to the same species. At the same time, the strains shared ANI values below 81 % and dDDH values below 24 % with all described Neisseria species. In the bac120 gene phylogenetic tree, the three strains clustered near Neisseria elongata and Neisseria bacilliformis in the Neisseria clade. However, the Neisseria clade is not monophyletic, and includes the type strains of Morococcus cerebrosus, Bergeriella denitrificans, Kingella potus, Uruburuella suis, and Uruburuella testudinis. Neisseria shayeganii clustered outside the clade with members of the genus Eikenella. Amino acid identity (AAI) values were calculated, and a threshold of 71 % was used to circumscribe the genus Neisseria. According to this proposed AAI threshold, strains 3986T, 51.81, and JF 2415 were placed within the genus Neisseria. The cells of the three strains were Gram-stain-negative diplococcobacilli and non-motile. Optimal growth on trypticase soy agar occurred at 37 °C and pH 8.5 in aerobic conditions. Notably, all strains exhibited indole production in the API-NH test, which is atypical for Neisseria and the family Neisseriaceae. The strains exhibited a common set of 68 peaks in their MALDI-TOF MS profiles, facilitating the swift and accurate identification of this species. Based on genotypic and phenotypic data, it is proposed that strains 3986T, 51.81, and JF 2415 represent a novel species within the genus Neisseria, for which the name Neisseria leonii sp. nov. is proposed (type strain 3986T=R726T=CIP 109994T=LMG 32907T).

Identification of Escherichia coli strains using MALDI-TOF MS combined with long short-term memory neural networks.

The current study aims to develop a new technique for the precise identification of Escherichia coli strains, utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with a long short-term memory (LSTM) neural network. A total of 48 Escherichia coli strains were isolated and cultured on tryptic soy agar medium for 24 hours for the generation of MALDI-TOF MS spectra. Eight hundred MALDI-TOF MS spectra were obtained per strain, resulting in a database of 38,400 spectra. Fifty percent of the data was utilized for LSTM neural network training, with fine-tuned parameters for strain-level identification. The other half served as the test set to assess model performance. Traditional PCA dimension reduction of MALDI-TOF MS spectra indicated 47 out of 48 strains to be unclassifiable. In contrast, the LSTM neural network demonstrated remarkable efficacy. After 20 training epochs, the model achieved a loss value of 0.0524, an accuracy of 0.999, a precision of 0.985, and a recall of 0.982. When tested on the unseen data, the model attained an overall accuracy of 92.24%. The integration of MALDI-TOF MS and LSTM neural network markedly enhances the identification of Escherichia coli strains. This innovative approach offers an effective and accurate tool for MALDI-TOF MS-based strain-level identification, thus expanding the analytical capabilities of microbial diagnostics.

Bacterial Contamination of Environmental Surfaces of Veterinary Rehabilitation Clinics.

The presence of potentially pathogenic bacteria on veterinary clinic surfaces may be problematic. In this study, we collected swab samples (Fisherbrand, double transport swabs with Stuart's liquid medium) and water samples from five veterinary rehabilitation clinics. Swabs and water samples were transported to a microbiology lab for processing. At the lab, swabs were used to inoculate Hardy's Cdiff Banana Broth (for Clostridium difficile [Cdiff]) and five different types of bacterial growth media, including Hardy CHROM MRSA agar (methicillin-resistant Staphylococcus aureus [MRSA] and S. pseudintermedius [SIM]), mannitol salt agar (S. aureus [SA]), eosin methylene blue agar (enterics [ENT]), Pseudomonas isolation agar (Pseudomonas spp. [PS]), and tryptic soy agar [TSA] (non-specific). The most prominent presumptive species cultured was Cdiff (on nearly 55% of swabs). Bacillus spp. and enteric bacteria were encountered on nearly 35% of swabs, with MRSA and SIM on just over 10% of swabs. The most contaminated sample site was harnesses/life jackets used with the underwater treadmill (33% of swabs). The underwater treadmill water had total bacterial counts from 1,600 to 2,800 cfu/mL. Of all presumptive bacterial species detected, SIM tends to be more pathogenic for dogs. Targeted cleaning/disinfecting in these clinics could help reduce risks for both animals and caregivers utilizing these clinics.

IMMUNOSTIMULANT EVALUATION OF NEEM LEAVES AGAINTS NON-SPECIFIC IMMUNE OF TILAPIA INFECTED BY A. hydrophila

The aim of this study was to find out how neem leaf extract affected the immune systems of Tilapia (O. niloticus) that had A. hydrophila in their bodies. This included looking at its phytochemistry, antibacterial properties, bioactive compounds, and ability to make the immune system stronger. The extraction uses methanol. The phytochemical test of neem leaf extract includes flavonoids, alkaloids, tannins, saponins, and triterpenoids. We obtained the isolate A. hydrophila from the Jepara Brackish Water Aquaculture. Before use, we store these bacteria in Trypticase Soy Agar (TSA) media at 4 oC and Trypticase Soy Broth (TSB) subcultures. re use. Test antibacterial using published We confirmed flavonoids, tannins, saponins, and triterpenoids through phytochemical tests. firmed. The test against A. hydrophila bacteria found that 100% of the extract was the most effective concentration at stopping the growth of A. hydrophila bacteria. After feeding A. hydrophilla bacteria to Tilapia (O. niloticus), the extract of neem leaf (A. indica) significantly boosted the immune system. bserved.

Anti-Listeria efficacy of peroxyacetic acid in bacterial buffered solution and on bell peppers

This study aims to 1) evaluate the efficacy of peroxyacetic acid (PAA) to inactivate Listeria monocytogenes in 0.1 % buffered-peptone-water; and 2) evaluate the reduction and mitigation of cross-contamination on peppers. In study-1, aliquots of 1.0-ml PAA solutions (133, 265, and 448 ppm) were added to the first 6 wells of 8-strip-deep well microplates. Then, 0.1 ml of serially diluted nalidixic-acid-resistant (NaL) L. monocytogenes was added and mixed immediately with a multichannel pipette. After exposure for 0, 5, 15, 30, and up to 120-s, 1 ml of 2 × D/E neutralized solution was added to terminate the reaction followed by spread-plating onto tryptic soy agar +200 ppm NaL and incubated at 35 °C for 48 h. In study-2, 5 inoculated red peppers were triple-washed with 15 uninoculated green peppers with 0, 23, 53, and 96 ppm of PAA and analyzed using MPN-method. L. monocytogenes counts were <0.3 log10 CFU/ml after exposure to 133, 265, and 448 ppm of PAA for 120, 90 and 60 s, respectively and fit the Linear and Weibull Models. Applying 23–96 ppm of PAA reduced L. monocytogenes by 2.03–3.32 log10MPN/g and transferred cross-contaminated cell counts by 2.36–3.17 log10MPN/g, with no differences (P > 0.05) compared to the water only treatment. Results suggested that applying allowable concentrations (23–96 ppm) of PAA during triple-wash does not show promising anti-Listeria activity on peppers. Future studies are needed to determine the minimum concentrations of the PAA solution required to effectively reduce and mitigate microbial cross-contamination of Listeria monocytogenes on peppers.