Development of a Novel Direct Bioautography–Thin-Layer Chromatography Test: Optimization of Growth Conditions for Gram-Negative Bacteria, Escherichia coli

2011 ◽  
Vol 94 (5) ◽  
pp. 1567-1572 ◽  
Author(s):  
Edyta M Grzelak ◽  
Barbara Majer-Dziedzic ◽  
Irena M Choma
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Intermediate Precision ◽  
Mueller Hinton ◽  
Diphenyltetrazolium Bromide ◽  
Tlc Plates ◽  
Direct Bioautography ◽  
Test Optimization ◽  
Layer Chromatography ◽  
Mueller Hinton Broth

Abstract With the aim of developing a TLC-direct bioautography assay using Escherichia coli as test bacteria, various parameters influencing the viability of microorganisms on TLC plates were examined and checked for flumequine standards. The optimal times for preincubation and incubation of bacterial broth were 20 h at 37°C and 2 h at 37°C, respectively. The optimal viscosity of the broth was obtained for 0.05% agarose solution in Mueller-Hinton broth. Various incubation times of the seeded TLC plates were also tested (5 h proved to be optimal). After incubation, the plates were sprayed with 0.2% aqueous [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide] (MTT) solution and incubated for 0.5 h at 37°C. The precision of the method was evaluated by the repeatability (intraday assay) and intermediate precision (interday assay). The regression coefficients were 0.9977 and 0.9968, respectively, for intraday and interday curves. The calibration curves show good linearity in the range of 0.005–0.50 μg (0.5–50.0 μg/mL). The established LOD of flumequine equaled 0.5 μg/mL, i.e., 5 ng flumequine in the spot. The developed direct bioautography test significantly enhances the sensitivity of the TLC method.

Development of a Novel Direct Bioautography-Thin-Layer Chromatography Test: Optimization of Growth Conditions for Gram-Positive Bacteria, Bacillus subtilis

2013 ◽  
Vol 96 (2) ◽  
pp. 386-391 ◽  
Author(s):  
Edyta M Grzelak ◽  
Barbara Majer-Dziedzic ◽  
Irena M Choma ◽  
Karol M Pilorz
Keyword(s):  
Bacillus Subtilis ◽  
Antimicrobial Susceptibility ◽  
Growth Conditions ◽  
Factors Affecting ◽  
Gram Positive Bacteria ◽  
Agar Disc ◽  
Mueller Hinton ◽  
Bromide Solution ◽  
Tlc Plates ◽  
Direct Bioautography

Abstract A TLC-direct bioautography (DB) assay using Bacillus subtilis as test bacteria was developed. Various factors affecting the microorganism's viability on the TLC plates were studied and verified for the flumequine standards. The Dhenasar's method called "direct sample determination" was used for TLC; the antibiotic samples were spotted on the TLC plates and subjected to bioautography without developing with a mobile phase. The best preincubation and incubation times of bacterial broth were found to be 1 h at 37°C and 6 h at 37°C. The optimal viscosity of broth was obtained by the addition of agarose to obtain a 0.05% solution in the Mueller-Hinton broth. The best incubation time of seeded TLC plates was 17 h at 37°C. The plates were visualized by spraying with 0.2% aqueous 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide solution and incubated again for 0.5 h at 37°C. The method was validated by determination of linearity, interday and intraday precision, LOD, and LOQ. The calibration curves showed good linearity in the range 0.005–0.5 μg (0.5–50.0 μg/mL). The regression coefficients were 0.9970 and 0.9955 for intraday and interday plots, respectively. The LOD of flumequine equalled 0.5 μg/mL, i. e., 5 ng of the antibiotic in the spot. The sensitivity of the developed TLC-DB test was compared with that of the two most commonly used standard antimicrobial susceptibility assays: agar disc diffusion and agar cylinder diffusion. The obtained minimum inhibitory concentration values clearly indicate much higher sensitivity of the TLC-DB method compared to the standard antimicrobial susceptibility assays.

scholarly journals PSV-41 Antibacterial activity of ferulic acid and sodium chlorate against Escherichia coli F18 and K88 during incubations with porcine fecal bacteria

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 296-297 ◽  
Author(s):  
Claudio Arzola ◽  
Elizabeth Latham ◽  
Robin Anderson ◽  
Jaime Salinas-Chavira ◽  
Yamicela Castillo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Sodium Chlorate ◽  
Fecal Bacteria ◽  
Aerobic Incubation ◽  
E Coli ◽  
Mueller Hinton ◽  
Fecal Suspension ◽  
Porcine Feces ◽  
Mueller Hinton Broth

Abstract The influence of ferulic acid (FA) and sodium chlorate (SC) was evaluated in two trials on the growth of Escherichia coli F18 and K88 (F18 and K88) incubated with porcine fecal bacteria. Treatments were 2 levels of FA (0 and 5 mg/mL) and 2 levels of SC (0 and 10 mM/mL). In trial one, ½-strength Mueller Hinton broth mixed with porcine feces (0.5% w/v) was inoculated with a novobiocin and naladixic acid resistant F18-strain. This fecal suspension was transferred to tubes (3/treatment) and anaerobically incubated at 39 oC for enumeration at 0, 6 and 24 h using MacConkey agar supplemented with novobiocin and naladixic acid with aerobic incubation at 37 oC. An interaction (FA x SC) at 6 and 24 h was observed (P < 0.01). At 6 h of incubation, SC alone or combined with FA had the lowest counts (P < 0.05); FA alone was lower than control but higher than SC or SC+FA (P < 0.05). At 24 h, FA alone or combined with SC had the lowest counts (P < 0.05); SC was lower than control but higher than FA or SC+FA (P < 0.05). In trial 2 were used the same procedures of trial 1, except that K88 was used. There was an interaction at 6 h (P < 0.01) where the lowest counts were in FA+SC (P < 0.05). SC alone or FA alone were lower than control but higher than SC+FA (P < 0.05). There was no interaction at 24 h (P = 0.16), where FA reduced the K88 counts (P < 0.01), however it was not affected by SC (P = 0.12). In conclusion, SC reduced E. coli counts; however, at 24 h of incubation greater reductions were observed when FA alone or combined with SC was added into the incubation fluid with porcine feces.

scholarly journals Insulin Regulation of Escherichia coli Abiotic Biofilm Formation: Effect of Nutrients and Growth Conditions

Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1349
Author(s):  
Nina Patel ◽  
Jeremy C. Curtis ◽  
Balbina J. Plotkin
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Fold Increase ◽  
Growth Conditions ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
E Coli ◽  
Biofilm Production ◽  
Mueller Hinton ◽  
Static Conditions

Escherichia coli plays an important role in biofilm formation across a wide array of disease and ecological settings. Insulin can function as an adjuvant in the regulation of biofilm levels. The modulation of insulin-regulated biofilm formation by environmental conditions has not been previously described. In the present study, the effects that various environmental growth conditions and nutrients have on insulin-modulated levels of biofilm production were measured. Micropipette tips were incubated with E. coli ATCC® 25922™ in a Mueller Hinton broth (MH), or a yeast nitrogen base with 1% peptone (YNBP), which was supplemented with glucose, lactose, galactose and/or insulin (Humulin®-R). The incubation conditions included a shaking or static culture, at 23 °C or 37 °C. After incubation, the biofilm production was calculated per CFU. At 23 °C, the presence of insulin increased biofilm formation. The amount of biofilm formation was highest in glucose > galactose >> lactose, while the biofilm levels decreased in shaking cultures, except for galactose (3-fold increase; 0.1% galactose and 20 μU insulin). At 37 °C, regardless of condition, there was more biofilm formation/CFU under static conditions in YNBP than in MH, except for the MH containing galactose. E. coli biofilm formation is influenced by aeration, temperature, and insulin concentration in combination with the available sugars.

scholarly journals The result of modified hydrothermal nanotitania extract to the Escherichia coli growth.

2020 ◽  
Vol 19 (4) ◽  
pp. 705-709
Author(s):  
Ahmad Mukifza Harun ◽  
Nor Farid Mohd Noor ◽  
Mohamad Ezany Yusoff ◽  
Razif Abas ◽  
Mohammad Khursheed Alam
Keyword(s):  
Escherichia Coli ◽  
Medical Science ◽  
Positive Control ◽  
Medical Field ◽  
Mueller Hinton ◽  
Mueller Hinton Broth ◽  
Growth Of Bacteria ◽  
Coli Growth

Background: This research was planned to search for a potential of modified hydrothermal nanotitania extract in inhibiting the growth of bacteria commonly known in medical field. It is also aims to test this substance against common medical bacteria,Escherichia coli. Materials and methods: In this test, suspension of modified hydrothermal nanotitania extract (together with 0.01%, 0.03% and 0.05% silver) and undoping (positive control contains TiO2 and no silver) were prepared by mixing of TiO2 in Mueller Hinton Broth (MH) agar. The platecontaining the bacteria and TiO2 were observed after 24 hour, 48 hours and 72 hours incubation at 37oC for any growth of bacteria. Results: There was no growth of Escherichia coliin the plates containing the bacteria and modified hydrothermal nanotitania extract except in the control media. Conclusions: The finding suggested the modified hydrothermal nanotitania extractioninterfered the growth of Escherichia coli. Bangladesh Journal of Medical Science Vol.19(4) 2020 p.705-709

scholarly journals Anti-Biofilm Effect of Octenidine and Polyhexanide on Uropathogenic Biofilm-Producing Bacteria

2021 ◽  
pp. 1-7
Author(s):  
Maria Loose ◽  
Kurt G. Naber ◽  
Larry Purcell ◽  
Manfred P. Wirth ◽  
Florian M.E. Wagenlehner
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Mueller Hinton ◽  
High Dilutions ◽  
Artificial Urine ◽  
Tract Infections ◽  
Mueller Hinton Broth

<b><i>Background:</i></b> A catheter allowing a release of antibacterial substances such as antiseptics into the bladder could be a new way of preventing biofilm formation and subsequent catheter-associated urinary tract infections. <b><i>Methods:</i></b> Minimal inhibitory and bactericidal concentration (MIC/MBC) determinations in cation-adjusted Mueller-Hinton broth and artificial urine were performed for 4 antiseptics against 3 uropathogenic biofilm producers, <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, and <i>Proteus mirabilis</i>. Furthermore, effects of octenidine and polyhexanide against catheter biofilm formation were determined by quantification of biofilm-producing bacteria. <b><i>Results:</i></b> Sodium hypochlorite showed MIC/MBC values between 200 and 800 mg/L for all strains tested. Triclosan was efficient against <i>E. coli</i> and <i>P. mirabilis</i> (MIC ≤2.98 mg/L) but ineffective against <i>P. aeruginosa</i>. Octenidine and polyhexanide showed antibacterial activity against all 3 species tested (MIC 1.95–7.8 and 3.9–31.25 mg/L). Both octenidine and polyhexanide were able to prevent biofilm formation on catheter segments in a concentration dependent manner. Furthermore, adding 250 mg/L of each biocide disrupted biofilms formed by <i>E. coli</i> and <i>P. mirabilis</i>, whereas even 500 mg/L was not sufficient to completely destroy <i>P. aeruginosa</i> biofilms. <b><i>Conclusion:</i></b> Octenidine- and polyhexanide-containing antiseptics showed a broad effect against typical uropathogenic biofilm producers even in high dilutions. This study provides a basis for further investigation of the potential of octenidine and polyhexanide as prophylaxis or treatment of catheter biofilms.

In vitro Time Kill of Trimethoprim/Sulfamethoxazole against Stenotrophomonas maltophilia versus Escherichia coli using Cation Adjusted Muller Hinton Broth and ISO-Sensitest Broth

2022 ◽  
Author(s):  
Maxwell J. Lasko ◽  
Matthew L. Gethers ◽  
Jennifer L. Tabor-Rennie ◽  
David P. Nicolau ◽  
Joseph L. Kuti
Keyword(s):  
Escherichia Coli ◽  
Stenotrophomonas Maltophilia ◽  
E Coli ◽  
Optimal Dosing ◽  
Mueller Hinton ◽  
Colony Forming Units ◽  
Time Kill ◽  
Pharmacodynamic Data ◽  
Mueller Hinton Broth

Trimethoprim/sulfamethoxazole (TMP/SMZ) is considered the treatment of choice for infections caused by Stenotrophomonas maltophilia , but limited pharmacodynamic data are available to support current susceptibility breakpoints or guide optimal dosing. Time-kill studies using a TMP/SMZ concentration of 4/40 μg/mL were conducted to compare 4 S. maltophilia with 4 Escherichia coli having the same MICs (0.25/4.75-4/76 μg/mL) in cation adjusted Mueller Hinton Broth (CAMHB) and ISO-Sensitest™ broth (ISO). With the exception of the resistant isolates (4/76 μg/mL), which resulted in regrowth approaching control, TMP/SMZ displayed significantly greater killing for E. coli compared with S. maltophilia at each MIC. Against E. coli , mean changes at 24 hour were -4.49, -1.73, -1.59, and +1.83 log 10 colony forming units (CFU) for isolates with MICs of 0.25/4.75, 1/19, 2/39, and 4/74 μg/mL, respectively. The f AUC/MIC required for stasis, 1-log 10 , and 2-log 10 CFU reductions were 40.7, 59.5, and 86.3, respectively. In contrast, TMP/SMZ displayed no stasis or CFU reductions against any S. maltophilia regardless of MIC, and no pharmacodynamic thresholds were quantifiable. Observations were consistent in both CAMHB and ISO broth. These data add increasing evidence that current TMP/SMZ susceptibility breakpoints against S. maltophilia should be reassessed.

scholarly journals Recording the Presence of Peanibacillus larvae larvae Colonies on MYPGP Substrates Using a Multi-Sensor Array Based on Solid-State Gas Sensors

Sensors ◽  
2021 ◽  
Vol 21 (14) ◽  
pp. 4917
Author(s):  
Beata Bąk ◽  
Jakub Wilk ◽  
Piotr Artiemjew ◽  
Jerzy Wilde
Keyword(s):  
Gas Sensors ◽  
Culture Media ◽  
Laboratory Diagnosis ◽  
Baseline Correction ◽  
American Foulbrood ◽  
Sensor Signal ◽  
Sodium Pyruvate ◽  
Mueller Hinton ◽  
Regeneration Phase ◽  
Mueller Hinton Broth

American foulbrood is a dangerous disease of bee broods found worldwide, caused by the Paenibacillus larvae larvae L. bacterium. In an experiment, the possibility of detecting colonies of this bacterium on MYPGP substrates (which contains yeast extract, Mueller-Hinton broth, glucose, K2HPO4, sodium pyruvate, and agar) was tested using a prototype of a multi-sensor recorder of the MCA-8 sensor signal with a matrix of six semiconductors: TGS 823, TGS 826, TGS 832, TGS 2600, TGS 2602, and TGS 2603 from Figaro. Two twin prototypes of the MCA-8 measurement device, M1 and M2, were used in the study. Each prototype was attached to two laboratory test chambers: a wooden one and a polystyrene one. For the experiment, the strain used was P. l. larvae ATCC 9545, ERIC I. On MYPGP medium, often used for laboratory diagnosis of American foulbrood, this bacterium produces small, transparent, smooth, and shiny colonies. Gas samples from over culture media of one- and two-day-old foulbrood P. l. larvae (with no colonies visible to the naked eye) and from over culture media older than 2 days (with visible bacterial colonies) were examined. In addition, the air from empty chambers was tested. The measurement time was 20 min, including a 10-min testing exposure phase and a 10-min sensor regeneration phase. The results were analyzed in two variants: without baseline correction and with baseline correction. We tested 14 classifiers and found that a prototype of a multi-sensor recorder of the MCA-8 sensor signal was capable of detecting colonies of P. l. larvae on MYPGP substrate with a 97% efficiency and could distinguish between MYPGP substrates with 1–2 days of culture, and substrates with older cultures. The efficacy of copies of the prototypes M1 and M2 was shown to differ slightly. The weighted method with Canberra metrics (Canberra.811) and kNN with Canberra and Manhattan metrics (Canberra. 1nn and manhattan.1nn) proved to be the most effective classifiers.

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