Brain Heart Infusion Research Articles

The anti-adherence activity and bactericidal effect of GO against Streptococcus mutans from Iraqi dental patients.

The high rate of microbes and their biological activity in the patient's mouth is a concern in the domains of dental caries and periodontal disease. The study aimed to shed light on the relationship between graphene oxide's nanoparticles (nGOs) antimicrobial properties and the growth of dental pathogenic bacteria. The forty swab samples were frequently collected from the patient's cavity mouth between November 2019 and January 2020, from patients who visited dentist clinics in Baghdad by taking swabs from mouth cavities with various dental caries with two age groups (5-17) and (18-60) from male and female to streaking them on Brain-Heart Infusion (BHI) agar, then identified by re-streaking on Mitis Salivarius Bacitracin (MSB) agar. All isolates were confirmed as Streptococcus mutans after API 20 Strep method. As well as the Colony Forming Units (CFU) were then determined after diluting the bacterial cell suspensions to obtain cell samples containing 1.5 × 108CFU/ ml. The collagen-binding adhesin (cnm) and glucosyltransferases (gtf) of S. mutans genes were identified using polymerase chain reaction (PCR) method before and after exposure to the nGOs, which were prepared in different pulse laser energy (500, 600, and 700mJ) with presence and absence of the magnetic field, and the data have been analyzing. After counting the CFU, the nGOs shows high effectiveness inhibiting the growth of S. mutans. This research provides definitive answers about the relationship between nGOs, antibacterial caries, and periodontal disease.

First study on capsular serotypes and virulence factors of Pasteurella multocida isolates from Phan Rang sheep in Vietnam.

Pasteurella multocida is considered as a main factor mediating pneumonic pasteurellosis in ruminants, including sheep. It is also a current threat to Phan Rang sheep in Vietnam. This study aimed to characterize P. multocida isolated from Phan Rang sheep, their antibiotic resistance profile, and the prevalence of some virulence-associated genes of these strains. Bacteria were isolated on brain heart infusion, 10% sheep blood agar plates, and screened by biochemical tests. The polymerase chain reaction technique was used with specific primers to identify P. multocida, the presence of virulence-associated genes, and serotypes of isolates. Antimicrobial susceptibility and biofilm formation of isolates were examined using the disk diffusion method and crystal violet-based method, respectively. A total of 41 P. multocida strains were isolated from 485 samples from clinically sick and healthy sheep. Of the isolates, 58.53% were serotype A, 9.75% were serotype B, and 31.71% were serotype D. Healthy animals were infected with serotype D only. All 15 virulence genes were identified in all strains isolated from clinically sick sheep, while strains isolated from healthy sheep carried 11/15 virulence genes tested. Among virulence-associated genes exbB, exbD, tonB, ompA, oma87, fimA, hgbA, and nanB were detected in over 90% of isolates, whereas hgbB, nanH, tbpA and pfhA were less frequent. Interestingly, pmHAS and tadD were highly prevalent in capsular type A strains, whereas the toxA gene was detected in capsular type D strains only. All of the isolated strains were fully susceptible to enrofloxacin, ciprofloxacin, neomycin, and ofloxacin. About 92.68% were susceptible to chloramphenicol and 90.24% to amikacin, but there was high resistance to erythromycin, tetracycline, and amoxicillin. Our results reveal that 53.65% of 41 isolates could produce biofilm, whereas 46.34% could not. Pasteurella multocida from Phan Rang sheep possess many virulence genes and resistance to several common antibiotics such as erythromycin, tetracycline, and amoxicillin. The results are an important warning regarding antibiotic resistance of P. multocida.

Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures.

Background: The rapid diagnostics of pathogens is essential to prescribe appropriate and early antibiotic therapy. The current methods for pathogen detection require the bacteria to grow in a culture medium, which is time-consuming. This increases the mortality rate and the global burden of antimicrobial resistance. Culture-free detection methods are still under development and are not used in the clinical routine. Therefore decreasing the culture time for accurate detection of infection and resistance is vital for diagnosis. Methods: In this study, we wanted to investigate easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation ( e.g., vitamin B12 and trace metals), that can significantly reduce the lag time (t lag). These factors were arranged in simple two-level factorial designs using Gram-positive ( Escherichia coli and Pseudomonas aeruginosa) and Gram-negative ( Staphylococcus aureus and Bacillus subtilis) bacteria, including clinical isolates with known antimicrobial resistance profiles. Blood samples spiked with a clinical isolate of E. coli CCUG17620 were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures. Results: We observed that increased incubation temperature (42°C) along with vitamin B12 supplementation significantly reduced the t lag (10 - 115 minutes or 4% - 49%) in pure clinical isolates and blood samples spiked with E. coli CCUG17620. In the case of the blood sample, PCR results also detected bacterial DNA after only 3h of incubation and at three times the CFU/mL. Conclusions: Enrichment of bacterial culture media with growth supplements such as vitamin B12 and increased incubation temperature can be a cheap and rapid method for the early detection of pathogens. This is a proof-of-concept study restricted to a few bacterial strains and growth conditions. In the future, the effect of other growth conditions and difficult-to-culture bacteria should be explored to shorten the lag phase.

In vitro comparative study of antimicrobial efficacy of endodontic sealers against common pathogens in the dental pulp.

Different types of bacterial species, Enterococcus faecalis (E. faecalis), Fusobacterium nucleatum (F. nucleatum), and Porphyromonas gingivalis (P. gingivalis), cause pulpal and periradicular diseases. Hence, the antibacterial efficacy of endodontic sealers is of utmost importance from a clinical point of view. The main objective of the study is to test the antibacterial efficacy of endodontic sealers against endo microflora belonging to E. faecalis, F. nucleatum, and P. gingivalis species. The antibacterial effectiveness of five endodontic sealers (AH plus, Apexit, EndoRez, Endomethasone, and Tubliseal) was tested by means of the agar-diffusion test (ADT) and direct contact test (DCT). The bacterial suspension of individual microorganisms was applied separately onto the agar plates for ADT. Subsequently, freshly mixed and set sealer was applied on to the sterile discs. After 48 h of incubation, the inhibition zones were measured. For DCT, the sealers were put in 96-well cell culture plates and overlaid with bacterial suspension and brain heart infusion broth. The bacterial growth density of the liquid was tested by spectrophotometry at 0, 2, 4, 6, and 24 h. The data were analyzed using ANOVA and post hoc Turkey's test. The present study showed that Endomethasone and AH Plus had a good antibacterial effect. In the ADT and DCT, Endomethasone showed the greatest antimicrobial effect against E. faecalis when compared to other endodontic sealers. In the ADT, Apexit had no antimicrobial effect on E. faecalis, whereas AH Plus had the greatest antibacterial effect on F. nucleatum and P gingivalis. Whereas in DCT, EndoRez and Endomethasone showed the greatest effect against F. nucleatum and P. gingivalis.

Expression of algD Gene in Single- and Dual-Species Biofilms of <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus Under Starvation Stress

Dual-species biofilms of Pseudomonas aeruginosa and Staphylococcus aureus generate difficultto-treat illnesses. Nutrition stress in biofilms affects physiology, microbial metabolism, and species interactions, impacting bacteria growth and survival. Furthermore, the function of alginate, which is encoded by the algD gene, in the production of biofilms has been established. The present study aimed at investigating the impact of starvation on algD gene expression in single-species biofilm of P. aeruginosa and dual-species biofilms of P. aeruginosa and S. aureus from hospital sewage. A total of six P. aeruginosa and six S. aureus isolates were obtained from the microbiology laboratory at the Department of Biology, College of Science, University of Baghdad, Iraq. These isolates are multidrug-resistant and were obtained from various hospital sewage stations in Baghdad city. P. aeruginosa and S. aureus isolates were co-cultured as singleand dual-species biofilms in full-strength brain heart infusion broth (BHIB) and 1000-fold diluted BHIB. In order to evaluate the level of expression of the algD gene in P. aeruginosa that had been treated to starvation, the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was utilized. The results demonstrated that starvation stress significantly (P< 0.05) up regulated the expression of algD in single-species biofilm (3.117 to 4.532-fold). However, starvation stress down regulated the algD expression in dual-species biofilm (0.001 and 0.901-fold). In conclusion, malnutrition up regulated algD expression in single-species P. aeruginosa biofilms but down regulated it in dual-biofilms. This work helps create biofilm-related disease treatments.

Fluoride and gallein inhibit polyphosphate accumulation by oral pathogen Rothia dentocariosa.

The uptake and storage of extracellular orthophosphate (Pi) by polyphosphate (polyP) accumulating bacteria may contribute to mineral dissolution in the oral cavity. To test the effect of potential inhibitors of polyP kinases on Rothia dentocariosa, gallein (0, 25, 50, and 100 μM) and fluoride (0, 50, and 100 ppm) were added to R. dentocariosa cultures grown in brain-heart infusion broth. At a late log growth phase (8 h), extracellular Pi was measured using an ascorbic acid assay, and polyP was isolated from bacterial cells treated with RNA/DNAases using a neutral phenol/chloroform extraction. Extracts were hydrolyzed and quantified as above. Gallein and fluoride had minor effects on bacterial growth with NaF having a direct effect on media pH. Gallein (≥25 μM) and fluoride (≥50 ppm) attenuated the bacterial drawdown of extracellular Pi by 56.7% (P<0.05) and 37.3% (P<0.01). There was a corresponding polyP synthesis decrease of 73.2% (P<0.0001) from gallein and 83.1% (P<0.0001) from fluoride. Attenuated total reflectance-Fourier-transform infrared spectroscopy validated the presence of polyP and its reduced concentration in R. dentocariosa bacterial cells following gallein and fluoride treatment. Rothia dentocariosa can directly change extracellular Pi and accumulate intracellular polyP, but the mechanism is attenuated by gallein and NaF.

Paracoccidioidomycosis: an Australian case

A 33-year-old HIV negative Brazilian engineer who had been living in Australia for three years presented to his dentist with an eight week history of a progressively enlarging maxillary gingival ulcer, suspicious for malignancy. Biopsy of the lesion showed pseudoepitheliomatous hyperplasia with round structures present in periodic acid-schiff and grocott stains suggestive of a fungal organism. A second biopsy revealed fungal organisms with a pathognomonic appearance on microscopy. The organism grew on Brain Heart Infusion agar and Sabouraud agar at 30 and 35 degrees celsius. An identification of Paracoccidioides brasiliensis was made. To our knowledge, we present the first Australian case of P. brasiliensis infection, a highly pathogenic dimorphic fungus, in order to highlight some of the key diagnostic features of this unusual infection.

Combination of growth conditions and InlB-specific dot-immunoassay for rapid detection of Listeria monocytogenes in raw milk

The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.

Formation of Listeria monocytogenes persister cells in the produce-processing environment

Persisters are a subpopulation of growth-arrested cells that possess transient tolerance to lethal doses of antibiotics and can revert to an active state under the right conditions. Persister cells are considered as a public health concern. This study examined the formation of persisters by Listeria monocytogenes (LM) in an environment simulating a processing plant for leafy green production. Three LM strains isolated from California produce-processing plants and packinghouses with the strongest adherence abilities were used for this study. The impact of the cells' physiological status, density, and nutrient availability on the formation of persisters was also determined. Gentamicin at a dose of 100 mg/L was used for the isolation and screening of LM persisters. Results showed that the physiological status differences brought by culture preparation methods (plate-grown vs. broth-grown) did not impact persister formation (P > 0.05). Instead, higher persister ratios were found when cell density increased (P < 0.05). The formation of LM persister cells under simulated packinghouse conditions was tested by artificially inoculating stainless steel coupons with LM suspending in media with decreasing nutrient levels: brain heart infusion broth (1366 mg/L O2), produce-washing water with various organic loads (1332 mg/L O2 and 652 mg/L O2, respectively), as well as sterile Milli-Q water. LM survived in all suspensions at 4 °C with 85 % relative humidity for 7 days, with strain 483 producing the most persister cells (4.36 ± 0.294 Log CFU/coupon) on average. Although persister cell levels of LM 480 and 485 were reasonably steady in nutrient-rich media (i.e., BHI and HCOD), they declined in nutrient-poor media (i.e., LCOD and sterile Milli-Q water) over time. Persister populations decreased along with total viable cells, demonstrating the impact of available nutrients on the formation of persisters. The chlorine sensitivity of LM persister cells was evaluated and compared with regular LM cells. Results showed that, despite their increased tolerance to the antibiotic gentamicin, LM persisters were more susceptible to chlorine treatments (100 mg/L for 2 min) than regular cells.

P21 A novel, fast, impedance-based antimicrobial susceptibility test for Neisseria gonorrhoeae

Abstract Objectives To demonstrate that the impedance-based fast antimicrobial susceptibility test (iFAST) can define N. gonorrhoeae susceptibility for a range of frontline antibiotics, and determine time taken to see responses. Background Gonorrhoea is the second most common bacterial sexually transmitted infection in the UK. It can cause complicated infection and infertility if not treated effectively. Resistance to frontline treatments and MDR N. gonorrhoeae infections (‘super gonorrhoea’) are increasing, fuelled by inappropriate selection of antibiotics. The current gold-standard antimicrobial susceptibility test (AST) for N. gonorrhoeae is MIC agar dilution, which is time-consuming (∼7 days) and labour-intensive. Unemo et al. 2020 (https://doi.org/10.1093/jac/dkw288) published detailed resistance profiles of WHO-reference N. gonorrhoeae strains. This project aimed to test whether these strain sensitivities against front line antibiotics ceftriaxone, ciprofloxacin and azithromycin could be defined faster using iFAST, which calculates the opacity and electrical size of individual cells by measuring change in electrical currents as single cells flow between micro-electrodes. Antibiotic-treated susceptible bacteria change electrical radius (surrogate measure of cell size) and opacity (based on properties of bacterial membranes as resistors), compared with untreated or resistant cells (Spencer et al. 2020, 10.1038/s41467-020-18902-x). Methods N. gonorrhoeae WHO-reference strains F and X were incubated overnight on standard chocolate agar plates at 37°C, 3%–5% CO2. 10 viable plate colonies were selected and inoculated in 37°C brain heart infusion media, supplemented with 0.42% sodium bicarbonate and 1% Isovitalex. After 30 min incubation at 37°C with shaking, the cell concentration was measured on iFAST and added to five concentrations of ceftriaxone, ciprofloxacin, and azithromycin (at the published agar MIC and ±2 × 2-fold concentration) or at EUCAST susceptibility breakpoint concentrations (strains F, X, O, V, K, Y), at a final cell concentration of 5 × 105 cells/mL. Cells were exposed at 37°C 250 rpm for 2, 3 and 4 h. 60 µL of 1:10 dilutions of sample were read on iFAST. MATLAB analysis software created contours around 50% of untreated cells and overlayed this on exposed population scattergrams. Results Electrical MICs (eMICs) showed differences in cell count, electrical radius and opacity between cells exposed to concentrations ≥MIC, subinhibitory concentrations, or untreated cells for all three frontline drugs. These differences were detectable after 2 h of antibiotic exposure but were clearest after 4 h. When tested at breakpoint concentrations only, iFAST accurately detected susceptibility of tested N. gonorrhoeae reference strains after 4 h exposure. Generally, susceptible cells had reduced electrical cell size and increased opacity distribution (out of contour shift) for all compounds, though there was variation due to differing modes of action. Ciprofloxacin and azithromycin had more decreased electrical cell radius with increased opacity spread. Comparatively, ceftriaxone had smaller changes in electrical radius and a larger spread of opacity readings. Conclusions iFAST is potentially a rapid and accurate method for determining N. gonorrhoeae strain susceptibility for ceftriaxone, ciprofloxacin, and azithromycin. With additional validation, it may be useful for future N. gonorrhoeae AST development, as well as investigating the effect of novel antimicrobial compounds.

Biological Evaluation of Zinc Phosphate Cement for Potential Bone Contact Applications

Zinc phosphate cement is used in dentistry to lute crowns and bridges. So far, its biocompatibility for other applications has not been studied. This paper reports the biocompatibility of zinc phosphate towards MG63 cells, testing both the material (discs; 3 mm diameter × 1 mm thick) and leachate from the cement. Cell viability was determined using an MTT assay, and cytotoxicity from the effects of leachate, studied in triplicate. Microscopy (optical and scanning electron) determined the morphology and proliferation of cells attached to zinc phosphate. ICP-OES measured element release into leachate, and anti-microbial behaviour was determined against Streptococcus pyrogenes cultured on a Brain Heart Infusion agar using cement discs (3 mm diameter × 1 mm thick). Zones of inhibition were measured after 72 h. MG63 cells proliferated on zinc phosphate surfaces and retained their morphology. The cells were healthy and viable as shown by an MTT assay, both on cement and in leachate. High levels of phosphorus but low levels of zinc were released into leachate. The cement showed minimal antimicrobial activity against S. pyogenes, probably due to the long maturation times used. Zinc phosphate cement was found to be biocompatible towards MG63 cells, which indicates that it may be capable of use in bone contact applications.

Comparison of Gold Biosensor Combined with Light Microscope Imaging System with ELISA for Detecting Salmonella in Chicken after Exposure to Simulated Chilling Condition.

In this study, the performance of a gold biosensor combined with light microscope imaging system (GB-LMIS) was comparatively evaluated against enzyme-linked immunosorbent assay (ELISA) for detecting Salmonella under simulated chilling condition. The optimum concentration of anti-Salmonella polyclonal antibodies (pAbs) was determined to be 12.5 and 100 μg/ml for ELISA and GB-LMIS, respectively. GB-LMIS exhibited a sufficient and competitive specificity toward three tested Salmonella among only. To mimic a real-world situation, chicken was inoculated with Salmonella cocktail and stored under chilling condition for 48 h. The overall growth of Salmonella under chilling condition was significantly lower than that under non-exposure to the chilling condition (p < 0.05). No significant differences in bacterial growth were observed between brain heart infusion and brilliant green broth during the enrichment period (p > 0.05). Finally, both GB-LMIS and ELISA were employed to detect Salmonella at every 2-h interval. GB-LMIS detected Salmonella with a competitive specificity by the direct observation of bacteria on the sensor using a charge-coupled device camera within a detection time of ~2.5 h. GB-LMIS is a feasible, novel, and rapid method for detecting Salmonella in poultry facilities.

Evaluation of extracellular polymeric substances matrix volume, surface roughness and bacterial adhesion property of oral biofilm

Background/purposeOral biofilms are highly structured bacterial colonies embedded in a highly hydrated extracellular polymeric substances (EPS) matrix. This study aimed to investigate the characteristics of oral biofilm at different stages of maturation. Materials and methodsOral multispecies biofilms were grown anaerobically from plaque bacteria on collagen coated hydroxyapatite discs in brain heart infusion broth for one and three weeks. The volume of live bacteria and EPS matrix of the biofilms were determined by using corresponding fluorescent probes and confocal laser scanning microscopy. Atomic force microscopy (AFM) was used to quantitatively probe and correlate cell surface adhesion force of biofilms. The surface roughness was quantified in terms of the root mean square average of the height deviations. Adhesion was measured from force–distance data for the retraction of the cell from the surface. ResultsThe volume of live bacteria and EPS of 3-week-old biofilms was higher than 1-week-old biofilms. The surface roughness value in 1-week-old biofilms was significantly higher than that in 3-week-old biofilms. AFM force–distance curve results showed that the adhesion force at the cell–cell interface was significantly more at-tractive than those at bacterial cells surface of both stages biofilms. Adhesion forces between the AFM tip and the surface of bacterial cell were fairly constant, whereas the cell–cell interface experienced greater adhesion forces in the biofilm's development. ConclusionAs oral biofilms become mature, EPS volume and cell–cell adhesion forces increase while the surface roughness decreases.

A new spray-based method for the in-vitro development of dry-surface biofilms.

The inanimate environment immediately surrounding the patient in healthcare facilities is a reservoir of microorganisms embedded in dry-surface biofilms (DSB). These biofilms, first highlighted in 2012, are increasingly studied, but currently available in-vitro models only allow for the growth of semi-hydrated biofilms. We developed a new in-vitro method under actual dehydration conditions based on the hypothesis that surface contamination is mainly due to splashes of respiratory secretions. The main objective of this study was to show that the operating conditions we have defined allowed the growth of DSB with a methicillin resistant Staphylococcus aureus strain. The second objective was to show that extended-spectrum beta-lactamase-producing Enterobacteriaceae, that is, Klebsiella pneumoniae and Enterobacter cloacae were also able to grow such biofilms under these conditions. Monobacterial suspensions in sterile artificial saliva (SAS) were sprayed onto polyethylene surfaces. Nutrients and hydration were provided daily by spraying SAS enriched with 20% of Brain Heart Infusion broth. The primary outcome was mean surface coverage measured by image analysis after crystal violet staining. The method applied to S. aureus for 12 days resulted in reproducible and repeatable DSB consisting of isolated and confluent microcolonies embedded in extracellular polymeric substances as shown in scanning electron microscopy images. Similar DSB were obtained with both Enterobacteriaceae applying the same method. No interspecies variation was shown between the three strains in terms of surface coverage. These first trials are the starting point for a 3-year study currently in process.

Gas Chromatography-Mass Spectrometry (GC-MS) Metabolite Profiling of Citrus limon (L.) Osbeck Juice Extract Evaluated for its Antimicrobial Activity Against Streptococcus mutans.

Purpose The present study aimed to determine the antimicrobial nature of Citrus limon juice extract against Streptococcus mutans and to identify its metabolic profile by gas chromatography-mass spectrometry (GC-MS) technique. Materials and methods The cariogenic bacteria S. mutans were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India, and revived on brain heart infusion (BHI) agar. The C. limon (L.) Osbeck fruits were authenticated from the University of Trans-Disciplinary Health Sciences and Technology (TDU), Bengaluru, India. The antibacterial property of lyophilized lemon juice extract (LJE) dissolved in methanol was evaluated against S. mutans (MTCC 497) by the agar well diffusion assay. GC-MS technique was employed to identify the volatile metabolite profile of the methanolic LJE sample. The metabolite masses of the respective compounds were identified using the National Institute of Standards and Technology (NIST) library. Results The methanolic LJE sample concentration from 5 to 25 mg/ml did not demonstrate antimicrobial activity, while 30 to 100 mg/ml displayed antibacterial activity against S. mutans. Chlorohexidine (100 µg/ml) was used as the positive control, while methanol was used as the negative control. Significant antimicrobial metabolites were detected in the methanolic LJE sample by GC-MS analysis. Maleic anhydride, succinic anhydride, 6-Oxa-bicyclo[3.1.0] hexan-3-one, and 3-methyl-2,5-Furandione were the key metabolites identified in the methanolic LJE sample. Conclusion The present study reports that C. limon is a potential candidate for the pharmaceutical industry as it possesses bioactive compounds demonstrating therapeutic properties. Further investigations are warranted to determine the individual and synergetic effects of identified metabolites in the methanolic LJE sample for its antimicrobial property. Special attention needs to be focussed on translational research for the development of anti-caries products from C. limon.

IMPACT OF GLYCEROL AND DIMETHYL SULFOXIDE ON THE PERSISTENCE OF PASTEURELLA MULTOCIDA DURING ITS PRESEVATION AT DIFFERENT TEMPERATURES

Bacterium Pasteurella multocida type B:2 is the causative agent of Hemorrhagic Septicemia in bovines in Pakistan. This organism is also being used for oil adjuvanted (Montanide ISA 50 V2) vaccine for mass vaccination in cattle &amp; buffaloes in Punjab, Pakistan. A study was conducted to study the impact of glycerol and dimethyl sulfoxide (DMSO) on persistence of P. multocida duringits course of storage at 0, 4 and – 20 °C. Pure seed of P .multocida was grown in Brain Heart Infusion(BHI) broth at pH 7.4 and incubated at 37 °C for 18 hrs. in shaking incubator. The growth was observed for its purity microscopically as well as culturing on MacConkey’s &amp; Nutrient agar slants. The pure culture was divided into three parts. One part was added with 5 % sterilized DMSO (final concentration) and second part was added with 10 % sterilized DMSO (final concentration) and 15 % glycerine (final concentration) while third part was kept as control without addition of DMSO and glycerine. All three parts were aliquoted (1 mL) in sterilized 1.5 mL eppendroff tubes and stored at three different temperatures i.e 0, 4 and – 20 °C. Eppendroff tubes from different temperatures were thawed in water bath (22 °C) and subjected to direct culturing on BHI broth, microscopic examination, purity testing on slants, culture turbidity / Optical Density (OD) values, dry biomass calculation and mice inoculation test for three months with 15 days intervals. The results indicated that pure P. multocida culture mixed with 10 % sterilized DMSO and 15 % sterilized glycerol stored at – 20 °C survived for three months and can be used for HS vaccine production without losing its survival.

DETECTION OF ANTIBIOTIC RESISTANT IN Escherichia coli FROM THE REPRODUCTIVE TRACT OF BALI CATTLE ON SMALLHOLDER FARM

Escherichia coli originating from animals, humans, and the environment can spread antibiotic-resistant genes and can encourage antimicrobial resistance (AMR) which is a global health problem. The purpose of this study was to detect the presence of Escherichia coli which is resistant to several antibiotics from the reproductive tract of Bali cattle on smallholder farms on the island of Lombok. This research is a cross-sectional study conducted from March to June 2021 using 8 female Bali cattle that experience reproductive disorders on community farms in Lando Village, East Lombok Regency to collect their reproductive tract fluids using an artificial insemination plastic sheet gun. Reproductive fluid samples are placed on BHIB (Brain Heart Infusion Broth) medium. Escherichia coli cultures were carried out on Eosin Methylene Blue Agar (EMBA) and identified by Gram staining and biochemical tests conducted at the Public Health and Calibration Laboratory, West Nusa Tenggara Province. Antibiotic sensitivity test on isolated Escherichia coli was carried out by disc diffusion method using 5 antibiotics including Penicillin G 10U, Oxytetracycline 30 µg, Gentamicin 10 µg, Tetracycline 30 µg, and Cefotaxime 30 µg. The results showed that 2 (two) 25% Escherichia coli bacteria were successfully cultured on EMBA media and isolated from 8 samples of the reproductive fluids of Bali cattle that were collected. The results of the Escherichia coli sensitivity test to antibiotics found that Escherichia coli samples were 100% resistant to Penicillin G, 100% resistant to Oxytetracycline, 100% resistant to Gentamicin, and 50% resistant to Tetracycline, and 100% resistant to Cefotaxime from 2 isolated Escherichia coli. This explains that Bali cattle in community farms have the potential to spread Escherichia coli which has an impact on the emergence of AMR.

Comparative Evaluation of Antimicrobial Efficacy of Simvastatin Paste and Aloe Vera Paste as a Pulpotomy MedicamentAn In-vitro Study

Introduction: Pulpotomy is the complete removal of the coronal part of the dental pulp, followed by the application of an appropriate dressing or medicament to promote healing and maintain the viability of the teeth in the dental arch. Aloe vera has anti-inflammatory, anti-bacterial properties which can be used as a pulpotomy medicament. Simvastatin, a lipid-lowering medication has recently emerged as a potential medicament in regenerative dentistry. It has an anti-inflammatory and anti-oxidant properties. Hence, it can be used as a pulpotomy medicament. Aim: To evaluate and compare the antimicrobial efficacy of Aloe Vera paste and Simvastatin paste as pulpotomy medicament against Pseudomonas aeruginosa and E. faecalis. Materials and Methods: An in-vitro study will be conducted in Department of Paediatric and Preventive Dentistry at Sharad Pawar Dental College, Wardha, Maharashtra, India, in which Aloe Vera paste and Simvastatin paste will be prepared in an aseptic environment. The antimicrobial activity of simvastatin paste and Aloe Vera paste against E.faecalis and pseudomonas will be assessed using the agar diffusion method. It will be cultured overnight in brain heart infusion broth. On blood agar media, pseudomonas aeruginosa will be inoculated. 4 mm wells will be punched out on plates with a puncher and will be instantly loaded with freshly customised test materials. The agar plates shall then be incubated in an incubator at 37°C for 24 hours and 72 hours before being analysed. Statistical analysis will be done using Chi-square test, ANOVA test and multiple comparision 2k test. Expected Results: Aloe Vera and Simvastatin in the paste form shall be able to show its antimicrobial efficacy as a pulpotomy medicament. Conclusion: Aloe Vera paste and Simvastatin paste shall provide antimicrobial efficacy against E. faecalis and pseudomonas and prove to be a better pulpotomy medicament for primary teeth.

Antimicrobial Activity of Plasma Rich in Platelets (PRP) on the Ocular Microbiota of Healthy Horses from Minas Gerais: In Vitro Study.

In equine ophthalmology, ulcerative keratitis is among the most common conditions and, in general, arises as a consequence of some trauma suffered. Secondarily, subsequent contamination by pathogenic or resident bacteria of the horse's ocular microbiota may have undesirable consequences. Under physiological conditions, the normal microbiota coexists with the immune status of the host, serving as a barrier, ensuring the health of the ocular surface, and inhibiting the proliferation of pathogens. However, in the imbalance of immune barriers, the normal microbiota can become pathogenic and lead to infection, acting as an opportunistic agent. The present study aims to demonstrate the antimicrobial effect of platelet-rich plasma (PRP), its time of action, and its correlation with the concentration of its same components in vitro on Staphylococcus sciuri, a bacterium with high prevalence in the normal ocular microbiota of horses in the municipality of Minas Gerais. For the preparation of the PRP, eight adult Quarter Horse (QH) horses were used. The individual PRP was prepared by the double centrifugation protocol, and then, the PRPs were added to a pool, followed by testing their interaction in culture with Brain Heart Infusion (BHI) broth at different dilutions against five strains collected from different animals. After 3, 6, 12, and 18 hours, the colony formation units (CFU) count on a 5% horse blood agar plate was evaluated for each time point. Our study showed that Staphylococcus sciuri, the resident microorganism of the ocular conjunctival microbiota of horses, is more susceptible when compared to the standard strain "American Type Culture Collection" (ATCC-29213) Staphylococcus aureus, a pathogenic microorganism, which was used for the validation of our study. The antibacterial effect shown in this study was bacteriostatic for up to 6 hours. The most concentrated PRP dilutions, 1 : 1 and 1 : 2, were also most effective, suggesting that the antibacterial effect is volume dependent.

Characterization and virulence of Streptococcus agalactiae deficient in SaeRS of the two-component system.

There are a variety of regulatory systems in bacteria, among which the two-component system (TCS) can sense external environmental changes and make a series of physiological and biochemical reactions, which is crucial for the life activities of bacteria. As a member of TCS, SaeRS is considered to be an important virulence factor in Staphylococcus aureus, but its function in tilapia (Oreochromis niloticus)-derived Streptococcus agalactiae remains unknown. To explore the role of SaeRS in regulating virulence in the two-component system (TCS) of S. agalactiae from tilapia, ΔSaeRS mutant strain and CΔSaeRS complementary strain were constructed by homologous recombination. The results showed that the abilities of growth and biofilm formation of ΔSaeRS strain were significantly decreased when cultured in a brain heart infusion (BHI) medium (P < 0.01). Also, the survival rate of the ΔSaeRS strain in blood was decreased when compared with the wild strain S. agalactiae THN0901. Under the higher infection dose, the accumulative mortality of tilapia caused by the ΔSaeRS strain was significantly decreased (23.3%), of which THN0901 and CΔSaeRS strains were 73.3%. The results of competition experiments in tilapia showed that the invasion and colonization abilities of the ΔSaeRS strain were also dramatically lower than those of the wild strain (P < 0.01). Compared with the THN0901, the mRNA expression levels of virulence factors (fbsB, sip, cylE, bca, etc.) in the ΔSaeRS strain were significantly down-regulated (P < 0.01). SaeRS is one of the virulence factors of S. agalactiae. It plays a role in promoting host colonization and achieving immune evasion during the infection of tilapia, which provides a basis for exploring the pathogenic mechanism of S. agalactiae infected with tilapia.