Modeling the Effect of Pediocin Application on the Growth and Survival of Listeria monocytogenes

Polydopamine-modified 3D flower-like ZnMoO4 integrated MXene-based label-free electrochemical immunosensor for the food-borne pathogen Listeria monocytogenes detection in milk and seafood

The absence of standardised procedures for minimum inhibitory concentration (MIC) testing of antimicrobial agents against veterinary mycoplasma and ureaplasma species (Mollicutes) has made it difficult to compare results originating from different laboratories. This report, prepared on behalf of the International Research Programme on Comparative Mycoplasmology (IRPCM), offers guidelines and recommendations for veterinary MIC testing of these organisms in an effort to rectify this problem. The subjects discussed include suitable media for broth and agar MIC assays, storage and preparation of antimicrobial agents, standardisation of mycoplasma inocula for MIC tests, validation of equipment, incubation conditions, and determination of MIC end points. A standard medium for all veterinary mycoplasma MIC tests cannot currently be recommended, owing to the diversity of nutritional requirements of different mycoplasma species. Instead mycoplasma broths or agars giving optimal growth of specific mycoplasmas or ureaplasmas are recommended, as suboptimal growth may lead to falsely low MIC results. The importance of using standardised mycoplasma inocula, for assays using either solid or liquid media is stressed. The growth phase may be less important as lag phase and logarithmic phase cultures of Mycoplasma gallisepticum, M. synoviae, M. bovis and M. hyopneumoniae have given very similar results in liquid MIC assays. The liquid method of Tanner and Wu and the agar method described by Hannan et al. are compared and described in detail. Methods for calculating MIC50s and MIC90s are described and the interpretation of results discussed. Methods for assessing mycoplasmacidal (MMC) activity of antimicrobial agents are also described. Adoption of these guidelines should lead to more consistent MIC results being obtained between laboratories.

Listeria monocytogenes, a ubiquitous bacterium in nature, can lead to human listeriosis through food consumption. Listeriosis is a rare, preventable, and treatable foodborne disease but can cause hospitalizations and fatalities. We reviewed the literature published in China to better understand the prevalence of L. monocytogenes in food products, incidence of human listeriosis, and characteristics of L. monocytogenes strains in China. The average prevalence of L. monocytogenes in Chinese food products in 28 provinces was 4.42%, with the highest prevalence of 8.91% in meat-poultry products, followed by aquatic animals, Chinese salad and salad, rice and flour products, and so on. Two hundred fifty-three invasive listeriosis cases were reported from 2011 to 2016 in 19 provinces, and the overall case-fatality rate was 25.7% with no deaths reported of pregnant women and children. L. monocytogenes strains were generally susceptible to most antibiotics, with ampicillin and penicillin G still effective in treatment. The predominant sequence types (STs) in food were ST9 and ST8, while clinically ST87 was most common ST in China. The national human listeriosis pilot surveillance started in 2013, and a total of 133 listeriosis cases have been collected until now. On the basis of the surveillance program, further research should be conducted to uncover the reason for the prevalence and pathogenic mechanism of the highly epidemiological hypervirulent ST87 strains in China.

با توجه به تاثیرات منفی نگهدارنده های شیمیایی بر سلامت مصرف‌کنندگان، توجه مراجع قانونی و صنایع غذایی بر کاربرد اسانس‌ها و عصاره‌های گیاهی بعنوان نگهدارنده‌های طبیعی در مواد غذایی متمرکز شده است. در این پژوهش تاثیر اسانس‌های ریحان و مریم گلی کبیر بر رشد لیستریا مونوسیتوژنز و آسپرژیلوس فلاووس طی دوره نگهداری پنیر سفید ایرانی مورد مطالعه قرار گرفت. روش‌های بکار رفته در این تحقیق مشتمل بر تعیین حداقل غلظت مهارکنندگی رشد (MIC)، حداقل غلظت باکتری‌کشی (MBC) و حداقل غلظت قارچ‌کشی (MFC) اسانس‌ها، تعیین تاثیر غلظت‌های مختلف اسانس‌ها بر رشد لیستریا و آسپرژیلوس در پنیر طی دوره نگهداری محصول بود. ترکیبات اصلی اسانس مریم گلی کبیر شامل لینالیل استات، لینالول و اسانس ریحان شامل لینالول و آلفا-کادینول بود.MIC و MBC اسانس مریم گلی، به ترتیب، معادل 015/0% و 02/0% و اسانس ریحان برابر با 05/0% و 06/0% برای لیستریا بود. همچنین، MIC و MFC در برابر آسپرژیلوس برای اسانس مریم گلی معادل 5/0% و 65/0% و برای اسانس ریحان برابر با 6/0% و 8/0% بدست آمد. غلظت 35/0% اسانس مریم گلی و 5/0% اسانس ریحان از تولید اسپور توسط قارچ در محیط کشت جلوگیری نمود. اسانس مریم گلی در غلظت 1% طی دوره نگهداری پنیر از رشد آسپرژیلوس بطور کامل بازداری نمود و جمعیت لیستریا را نسبت به شاهد log 6 کاهش داد. اسانس ریحان تاثیر ضدمیکربی ضعیف‌تری نسبت به اسانس مریم گلی کبیر نشان داد.

A PCR Method Based on 16S rRNA Sequence for Simultaneous Detection of the Genus Listeria and the Species Listeria monocytogenes in Food Products

The antimicrobial effect of several products including commercial formulations currently used in sugar and alcohol factories was determined by adapted MIC (Minimal Inhibitory Concentration) test on Saccharomyces cerevisiae and on natural contaminants Lactobacillus fermentum and Leuconostoc mesenteroides. The MIC test by macrodilution broth method was adapted by formulating of the culture medium with cane juice closely simulating industrial alcoholic fermentation must. Acid penicillin V (MIC 0.10-0.20 µg/ml) and clindamycin (MIC 0.05-0.40 µg/ml) were most effective against bacterial growth in 24 h. Among the chemicals, sulphite (MIC 10-40 µg/ml), nitrite (MIC 50 µg/ml). Methyldithiocarbamate was efficient only on L. fermentum (MIC 2.5 µg/ml) and S. cerevisiae (MIC 5.0 µg/ml). Thiocianate (MIC 1.2-5.0 µg/ml), bromophenate (MIC 9-18 µg/ml) and n- alkyldimethylbenzylammonium cloride (MIC 1-8 µg/ml) affected S. cerevisiae at similar inhibitory concentration for L. mesenteroides or L. fermentum. Formaldehyde was more effective on bacteria (MIC 11.5 - 23 µg/ml) in both pH (4.5 and 6.5) than yeast (MIC 46-92 µg/ml). Several tested formulated biocides seriously affect S. cerevisiae growth in the similar dosages of the bacterial inhibition, so these products should be avoided or used only in special conditions for the bacterium control of fermentation process. For this step, the control of these contaminants by antibiotics are more suitable and effective.

Background: Listeria monocytogenes is one of the major causes of infections in developing countries. The aim of the present study was to investigate chemical composition and the combined effect of Mentha spicata essential oil with nisin against L. monocytogenes at different temperatures (4, 9 and 14°C), pHs (5, 6 and 7) and NaCl concentrations (1, 2 and 4g/100ml) in in vitro condition. Methods: Chemical composition of the essential oil was evaluated by GC-MS analysis. Minimum inhibitory concentration (MIC) values of the essential oil and nisin were determined by using broth micro-dilution test. The Differences in Population (DP) assay and Fractional Inhibitory Concentration (FIC) index were applied to investigate their synergistic effects. Results: The main components of the essential oil were carvone (78.76%) and limonene (11.50%). MIC values of the essential oil and nisin against L. monocytogenes were 320IU/ml and 160µl/ml, respectively. Concentration of 80μl/ml essential oil in combination with 160IU/ml nisin significantly (P<0.001) inhibited the bacterium at all pHs. Also, concentration of 40μl/ml and 80 essential oil in combination with 80 and 160IU/ml nisin significantly (P<0.001) inhibited the bacterium at all temperatures. 2g/100ml and 4g/100 ml NaCl concentration enhanced the sensitivity of L. monocytogenes toward four combinations. Reduction in the pH and incubation temperature and increasing of salt content led to enhance the anti-listerial effects of the essential oil and nisin. Conclusion: nisin and M. spicata essential oil could be considered as potential strong antimicrobials that can be used for the growth inhibition of L. monocytogenes in food products.

Antimicrobial activity of Thymus zygis essential oil against Listeria monocytogenes and its application as food preservative

Background The growth and susceptibility testing of anaerobic bacteria presents many unique challenges. The Clinical Laboratory Standards Institute (CLSI) recommends performing minimum inhibitory concentration (MIC) testing for C. difficile through agar dilution (AD) assay, which carries logistical and time burdens compared to broth microdilution (BMD) methods. In this project we aim to assess the intra-and inter-laboratory reproducibility of MIC testing for C. difficile. Methods A total of 30 C. difficile isolates underwent MIC testing using AD and BMD techniques. To test intra-lab reproducibility, proficiency testing to develop a reproducible MIC testing process using 18 isolates was implemented across two multi-disciplinary labs, which was then validated in a prospective cohort of 116 isolates. MICs obtained by each method and lab were compared and essential agreement (EA) and major and minor error rates were calculated. To combat the labor and time-intensive demands of AD testing, automation with Integra Assist Plus was tested. Times for plate set up using a technician versus the Integra Assist Plus were measured twice and the average times were compared. Results AD and BMD yielded discordant resistant/susceptible results in 16.7% (5/30) of isolates tested. During proficiency testing, intra-lab comparison of AD MICs yielded 88.9% (16/18) EA and no disagreements occurred with more than 1 dilution difference. EA improved in the larger cohort to 93.9% (109/116), of which minor and major disagreements occurred in 17% (21/116) and 25% (29/116), respectively. A total of 10’4” minutes were saved using automation (12’44” mean) versus a technician (24’40” mean). Conclusion Here we present the process undertaken to ensure the rigor and reproducibility of C. difficile susceptibility testing. We developed a procedure that yields accurate results from two different labs, minimizes cost, and lowers the overall time required. Our future research will include validation with a larger sample and more academic partners. Disclosures Kevin W. Garey, PharmD, MS, Acurx: Grant/Research Support|cidara: Advisor/Consultant|cidara: Grant/Research Support|Paratek: Grant/Research Support|Seres Health: Grant/Research Support|Summit: Grant/Research Support.

One of the widely used microbiological methods to determine the toxicity of chemicals, catalysts, and other types of materials is the minimum inhibitory concentration (MIC) test. The present study aims to investigate the influence of composition of composite materials based on TiO2 and their particle size as well as bacterial type and shape based on the MIC values reported in the literature. The results show that among the 36 articles selected, most of the studies used Escherichia coli (E. coli) (26) and Staphylococcus aureus (S. aureus) (19) bacteria to determine MIC values. This study revealed that the MIC in values below 70 µg ml−1 for S. aureus was lower than that for E. coli bacteria (below 200 µg ml−1). Importantly, MIC value decreased from 60.6 to 7.66 µg ml−1 with decrease in the size of nanoparticles. It follows from the increased surface area for smaller-sized particles, thus increased interaction with bacteria during MIC test.

Nanozyme-catalyzed and zwitterion-modified swabs based for the detection of Listeria monocytogenes in complex matrices

Bitter gourd (Momordica charantia L) is a plant that contains quite a lot of compounds such as alkaloids, flavonoids and saponins in its fruit. Based on this, bitter melon has considerable potential to be used as an antibacterial for Streptococcus mutans. The bacterium Streptococcus mutans belongs to a group of round-shaped Gram-positive bacteria that cause dental caries. This study aims to determine the antibacterial effectiveness of several concentrations of ethanol extract of bitter gourd against Streptococcus mutans bacteria. Antibacterial testing of the ethanol extract of bitter gourd was carried out by measuring the Diameter of Inhibitory Area (DDH) through the paper disc diffusion method and the Minimum Inhibitory Concentration (MIC) through the agar dilution method. DDH testing was carried out on bitter melon extract concentrations of 70%, 60%, 50%, 40%, 30% and amoxicillin 30 pg/mL as positive control and distilled water as negative control. While MIC testing was carried out at concentrations of 30%, 35%, 40%, 45%, 50%, 55% and 60%. The results showed that the ethanol extract of bitter melon was less effective as an antibacterial against Streptococcus mutans, because the inhibition zone formed on the DDH test was not absolute or did not form a perfect circle. However, in MIC it can be seen that at a concentration of 50% the ethanol extract of bitter gourd fruit can inhibit it slightly and at a concentration of 60% it is seen that there is no bacterial growth, so it can be concluded that MIC is at a concentration of 60%. The results showed that the ethanol extract of bitter melon was less effective as an antibacterial against Streptococcus mutans, because the inhibition zone formed on the DDH test was not absolute or did not form a perfect circle. However, in MIC it can be seen that at a concentration of 50% the ethanol extract of bitter gourd fruit can inhibit it slightly and at a concentration of 60% it is seen that there is no bacterial growth, so it can be concluded that MIC is at a concentration of 60%. The results showed that the ethanol extract of bitter melon was less effective as an antibacterial against Streptococcus mutans, because the inhibition zone formed on the DDH test was not absolute or did not form a perfect circle. However, in MIC it can be seen that at a concentration of 50% the ethanol extract of bitter gourd fruit can inhibit it slightly and at a concentration of 60% it is seen that there is no bacterial growth, so it can be concluded that MIC is at a concentration of 60%.

Enhancing detection of Listeria monocytogenes in food products using an enzyme

The Minimal Inhibitory Concentration (MIC) and the Nathan's Agar Well Diffusion (NAWD) tests are bacterial antimicrobial susceptibility predictors. Some suggest that the NAWD is not as reliable as the MIC test. We compared the MIC and NAWD tests as to how well they agree to bacterial sensitivity or resistance and predicted clinical outcome of burn wound infections. Using 65 bacterial isolates from burned patients, the MIC and NAWD tests agreed in 60.0% of the isolates (vs. a perfect agreement of 100%, p less than 0.001), implying that these tests are not interchangeable. From 18 burned patients treated with nitrofurazone or mafenide acetate, 28 infectious isolates were evaluated. The outcome of these infections was correctly predicted by NAWD in 92.8% and the MIC in 72.0% of the cases (p less than 0.05). It seems that for burns treated with topical antimicrobials, the NAWD is a more reliable predictor of bacterial susceptibility.

The roots of the minimum inhibitory concentration (MIC) determination go back to the early 1900s. Since then, the test has undergone modifications and advancements in an effort to increase its dependability and accuracy. Although biological investigations use an ever-increasing number of samples, complicated processes and human error sometimes result in poor data quality, which makes it challenging to replicate scientific conclusions. Automating manual steps using protocols decipherable by machine can ease procedural difficulties. Originally relying on manual pipetting and human vision to determine the results, modern broth dilution MIC testing procedures have incorporated microplate readers to enhance sample analysis. However, current MIC testing procedures are unable to simultaneously evaluate a large number of samples efficiently. Here, we have created a proof-of-concept workflow using the Opentrons OT-2 robot to enable high-throughput MIC testing. We have further optimized the analysis by incorporating Python programming for MIC assignment to streamline the automation. In this workflow, we performed MIC tests on four different strains, three replicates per strain, and analyzed a total of 1,152 wells. Comparing our workflow to a conventional plate MIC procedure, we find that the HT-MIC method is 800% faster while simultaneously boasting a 100% accuracy. Our high-throughput MIC workflow can be adapted in both academic and clinical settings since it is faster, more efficient, and as accurate than many conventional methods.