Plate Count Method Research Articles

Antibiofilm of Arumanis Mango Leaves (Mangifera indica L.) Ethanol Extract Against Staphylococcus aureus in vitro


 Introduction: Staphylococcus aureus is a pathogen bacteria that forms biofilm and induces acute inflammation associated with dentoalveolar abscess, which needs antibiotics such as amoxicillin for the treatment. Due to antibiotic resistance and various side effects, natural ingredients are needed as an alternative treatment. Arumanis mango leaves (Mangifera indica L.) contains mangiferin, flavonoid, and tannin which are expected to inhibit the growth of Staphylococcus aureus and its biofilm formation. Objective: The aim of this study was to determine the antibacterial and antibiofilm effects of Mangifera indica L. leaves against Staphylococcus aureus in vitro. Methods: This study was in vitro laboratory experiment with post test only control group design. Antibacterial test was carried out by plate count method and antibiofilm test was carried out by microtiter plate biofilm assay method. Staphylococcus aureus ATCC 25923 was cultured in BHI broth in 37oC for 24h with anaerobic condition. The present study used ethanol extract of Mangifera indica L. leaves in 6 different concentrations: 3,125%, 6,25%, 12,5%, 25%, 50%, and 100%. Amoxicillin was used as positive control and DMSO 10% used as negative control. Data was analyzed with Shapiro-wilk test, One-way ANOVA test, and post hoc Tukey HSD test with p<0,05 as the level of significance. Results: The most effective concentration to inhibit the growth of Staphylococcus aureus was 100% extract compared to negative control (p<0,05). The most effective concentration against Staphylococcus aureus biofilm formation was 100% extract in 3hr incubation compared to negative control (p<0,05). Conclusion: Ethanol extract of Mangifera indica L. leaves has been shown in vitro to have antibacterial and antibiofilm properties against Staphylococcus aureus.

Terpenoids as Natural Agents against Food-Borne Bacteria—Evaluation of Biofilm Biomass versus Viability Reduction

This study aimed to analyse the antibacterial potential of limonene, terpineol, and eugenol for the biofilm reduction of food-borne E. coli, S. aureus and S. typhimurium. A microdilution test with resazurin application was used for the minimum inhibitory concentration and a colony plate count was used for the minimum bactericidal concentration. Biofilm biomass was quantified using the crystal violet assay, while biofilm viability was determined using the plate count method. The results show the highest antibacterial potential among terpenoids for eugenol, followed by terpineol and limonene. Both biomass reduction and viability are strongly dependent on the concentration of all terpenoids tested (p < 0.05). Moreover, eugenol reduced biofilm biomass most effectively (67% for E. coli), while viability was reduced most by terpineol (3.8 log CFU cm−2 for E. coli and S. aureus). The correlation coefficient for the reduction in biomass and viability was highest for eugenol (0.9) and chlorhexidine for all bacteria tested, while the lowest correlation was found for limonene (0.6). Results also demonstrate that tested terpenoids are effective as standard antimicrobial agent chlorhexidine. This suggests that eugenol has potential against food-borne biofilms as it simultaneously reduces both biomass and viability of biofilms.

Homogeneous electrochemiluminescence aptasensor based on hybridization chain reaction and magnetic separation assistance for Staphylococcus aureus

Staphylococcus aureus(S. aureus) can cause a variety of diseases,so it is essential to provide the simple and responsive identification of bacterial pathogens in clinical treatment. In this work a homogeneous aptamer electrochemiluminescence biosensor for Staphylococcus aureus was developed assisted based on hybridization chain reaction and magnetic separation assistance. The ds (Probe DNA/Aptamer) is modified on the magnetic beads. In the presence of the target the target binds to the Aptamer in ds (Probe DNA/Aptamer) exposing the Probe DNA in the ds DNA. The exposed Probe DNA triggers HCR resulting in long DNA duplexes which can be simply and efficiently enriched for the luminescent molecule Ru(phen)32+. To reduce the possibility of false positive signals Exo III was added to cut unreacted dsDNA from the magnetic beads. The ECL intensity was linearly correlated with the logarithm of S. aureus concentration in the range of 10 CFU/mL − 10 × 107 CFU/mL with a detection limit of 3 CFU/mL. The method developed in this work has good sensitivity and specificity and can well evaluate the concentration of Staphylococcus aureus in normal human serum and the results are consistent with the results obtained by the plate counting method.

Upconversion nanoparticle-based aptasensor for rapid and ultrasensitive detection of Staphylococcus aureus by low-speed centrifugation.

Opportunistic foodborne pathogens such as Staphylococcus aureus (S. aureus) can cause a wide variety of threats to public health. There is an urgent clinical need for a fast, simple, low-cost, and sensitive method. Here, we designed a fluorescence-based aptamer biosensor (aptasensor) for S. aureus detection using core-shell structured upconversion nanoparticles (CS-UCNPs) as a beacon. A S. aureus-specific aptamer was modified on the surface of CS-UCNPs for binding pathogens. The S. aureus bound to CS-UCNPs can then be isolated from the detection system by simple low-speed centrifugation. Thus, an aptasensor was successfully established for the detection of S. aureus. The fluorescence intensity of CS-UCNPs correlated with the concentration of S. aureus within the range of 6.36 × 102 to 6.36 × 108 CFU mL-1, resulting in the detected limit of S. aureus being 60 CFU mL-1. The aptasensor performed well in real food samples (milk) with a detection limit of 146 CFU mL-1 for S. aureus. Furthermore, we applied our aptasensor in chicken muscles for S. aureus detection, and compared it with the plate count gold standard method. There was no significant difference between our aptasensor and the plate count method within the detected limit, while the time for the aptasensor (0.58 h) was shorter than that of the plate count method (3-4 d). Therefore, we succeeded in the design of a simple, sensitive and fast CS-UCNPs aptasensor for S. aureus detection. This aptasensor system would have the potential for the detection of a wide range of bacterial species by switching the corresponding aptamer.

In vitro degradation and multi-antibacterial mechanisms of β-cyclodextrin@curcumin embodied Mg(OH)2/MAO coating on AZ31 magnesium alloy

It is a challenging task to prepare a coating on Mg alloys for desirable corrosion resistance, good antibacterial ability and biocompatibility. In this research work, an in-situ Mg(OH)2 coating incorporated with sodium alginate (SA) and β-cyclodextrin (β-CD)@curcumin (Cur) was formed on the surface of micro arc oxidation (MAO) coated AZ31 alloy via a low temperature hydrothermal method. Characterization techniques such as X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectrometer (FT-IR) and scanning electron microscope (SEM) were employed to characterize the chemical composition and surface morphology of the coatings. The corrosion protection ability of the coatings was monitored via electrochemical polarization, hydrogen evolution and immersion tests. Photothermal antibacterial ability and cytocompatibility of the coatings were evaluated by plate counting method under the irradiation of 808 nm-near infrared light, in vitro cytotoxicity tests (MTT) and live/dead cell staining. The results indicate that a chelation of the organic molecules led to the formation of a MAO/(β[email protected])-SA-Mg(OH)2 coating with excellent corrosion protection, multi- antibacterial ability and almost no toxicity to the cells. Especially, the coating provided photothermal performance through the light absorption of Cur, which was encapsulated by β-CD to improve its bioavailability. SA enhanced the binding force between the drug and the substrate. This novel coating designated the potential application on bioabsorbable magnesium alloys.

Performance equivalence and validation of a rapid microbiological method for detection and quantification of yeast and mold in an antacid oral suspension.

Alternative and rapid microbiological methods are effective and profitable technologies that can be safely use in the pharmaceutical industry. This report studies the efficacy of the Soleris® direct yeast and mold automated method for the quantitative detection of C. albicans and A. brasiliensis at three different microbial bioburden. Validation testing was carried out using an antacid oral suspension (aluminum hydroxide 4% + magnesium hydroxide 4% + simethicone 0.4%). Equivalence of data between detection time vs. colony-forming units was established for both, alternative and conventional methodologies. Using probability of detection, linear Poisson regression, Fisher's test, and multifactorial analysis of variance ANOVA, all results from the rapid method were prove to be in statistical agreement with the reference plating procedures. The limits of detection and quantification were statistically similar for both methods (Fisher's exact test, P >0.05), showing that the alternative method was not inferior in performance to the reference method. Essential validation parameters such as precision (standard deviation < 5, coefficient of variance < 35%), accuracy (>70%), linearity (R2 >0.9025), and ruggedness (ANOVA, P <0.05) were determined. It was proved that all test results obtained from the alternative method were in statistical agreement with the standard plate count method. Thus, this new technology was found to meet all the validation criteria needed to be considered as an alternative method for yeast and mold quantification in the antacid oral suspension tested.

A paper stick test kit for rapid detection of total microbial contaminants in fresh milk

The way to determine the total microbial contaminants in fresh milk is by the Total Plate Count (TPC) method. The TPC method requires 48 hours, high analytical skills, and high analytical costs. The aim of this research was to design a rapid detection tool for microbial contaminants in fresh milk called Paper Stick Test Kit (PSTK), which is fast, cheap, and easy. The stages of research designing PSTK are: (a) making PSTK, (b) testing the optimal time for using PSTK, and (c) making standard color maps. The results of this research are: (a) the best PSTK is made from cardboard with color indicators made from a mixture of bromothymol blue 0.3 g/250 ml solvent and NaOH 0.01 N; drying PSTK is best done in an oven with a temperature of 100oC, for 20 minutes; (b) the optimal time of application of PSTK in fresh milk is 20 seconds; and c) standard color maps are based on the correlation between microbial contaminant levels in fresh milk and color levels in PSTK dipped in microbial contaminated fresh milk.

Effect of silver sulfadiazine on mature mixed bacterial biofilms on voice prostheses

BackgroundBiofilm formation on voice prostheses disrupts the function and limits the lifespan of voice prostheses. There is still no effective clinical strategy for inhibiting or removing these biofilms. Silver sulfadiazine (SSD), as an exogenous antibacterial agent, has been widely used in the prevention and treatment of infection, however, its effect on voice prosthesis biofilms is unknown. The purpose of this study was to explore the effect of SSD on the mature mixed bacterial biofilms present on voice prostheses.MethodsQuantitative and qualitative methods, including the plate counting method, real-time fluorescence quantitative PCR, crystal violet staining, the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT) reduction assay, scanning electron microscopy, and laser confocal microscopy, were used to determine the effect of SSD on the number of bacterial colonies, biofilm formation ability, metabolic activity, and ultrastructure of biofilms in a mature mixed bacterial (Staphylococcus aureus, Streptococcus faecalis and Candida albicans) voice prosthesis biofilm model. The results were verified in vitro on mature mixed bacterial voice prosthesis biofilms from patients, and the possible mechanism of action was explored.ResultsSilver sulfadiazine decreased the number of bacterial colonies on mature mixed bacterial voice prosthesis biofilm, significantly inhibited the biofilm formation ability and metabolic activity of mature voice prosthesis biofilms, inhibited the formation of the complex spatial structure of voice prosthesis biofilms, and inhibited the synthesis of polysaccharides and proteins in the biofilm extracellular matrix. The degree of inhibition and removal effect increased with SSD concentration.ConclusionsSilver sulfadiazine can effectively inhibit and remove mature mixed bacterial voice prosthesis biofilms and decrease biofilm formation ability and metabolic activity; SSD may exert these effects by inhibiting the synthesis of polysaccharides and proteins among the extracellular polymeric substances of voice prosthesis biofilms.Graphical abstract

Ability Analysis Of Waste Milkfish (Chanos chanos) As Alternative Medium Of Nutrient Agar On Escherichia coli And Staphylococcus aureus Growth

Waste milkfish got a lot from the residual processed milkfish production of Sidoarjo. The intestines, liver, and kidneys of waste milkfish contain protein, carbohydrates, and fat. The abundant availabilities were usable as a bacterial growth medium, including Nutrient Agar. Nutrient Agar has a high price and is produced by many foreign companies that can trigger a hard availability in laboratories of domestic healthcare, especially in frontier, outermost, and underdeveloped areas. This research aims to analyze the ability of waste milkfish as an alternative medium for Nutrient Agar on the growth of Escherichia coli and Staphylococcus aureus. The ability has been observed by the characteristics and number of bacterial colonies. This research was held in the bacteriology laboratory of the Medical Laboratory Technology Department, Health Polytechnic of Surabaya health ministry. The research type was True-experimental with a post-test-only control group design. Based on the preliminary test results, the waste milkfish mass variations were found in 7 grams, 7.5 grams, 8 grams, 8.5 grams, and 9 grams. Bacteria have been inoculated by the spread plate method, bacterial colony growth has been calculated by the Total Plate Count method. The data were analyzed by the Kruskal-Wallis test with a Sig. value &lt;0.05 that there were differences in the number of Escherichia coli and Staphylococcus aureus colonies in milkfish waste media with various mass. The results showed that the best ability of waste milkfish as an alternative medium for Nutrient Agar on bacterial growth was found in 9 grams mass variation. the average number of Escherichia coli colonies were 186 × 1013 CFU/mL and Staphylococcus aureus colonies were 188 × 1013 CFU/mL.

Efek Penambahan Cuka Aren (Arenga pinnata) pada Ikan Tongkol (Euthynnus affinis) terhadap Jumlah Mikroba dan Waktu Awal Pembusukan

Tuna meat is a food ingredient with a high nutrient content, especially protein. This causes tuna meat cannot be stored for a long time, because it is easily damaged. To overcome this, you can add natural preservatives using palm vinegar (Arenga pinnata). This study aims to analyze the number of microbes and detect the initial time of decay in tuna (Euthynnus affinis) added with palm vinegar (Arenga pinnata). The research design used a completely randomized design (CRD), which consisted of 3 treatments and 3 repetitions. These 3 treatments were carried out by soaking 225 grams of tuna with palm vinegar at concentrations of 0%, 2.5% and 5% with 0 time. , 3, 6, and 9 hours. Determination of the number of microbes in this study was carried out using the Total Plate Count (TPC) method. The results showed that there was microbial contamination in tuna, and the higher the concentration of palm vinegar, the smaller the chance of microbial contamination. In the spoilage test with 6 hours of immersion of tuna meat with a concentration of 0% and 2.5%, the initial decay occurred, and at a concentration of 5%, the initial spoilage did not occur. It can be concluded that the use of palm vinegar with a concentration of 2.5% can reduce the amount of microbial contamination. Palm vinegar with a concentration of 5% can inhibit spoilage, and slow down the initial time of decay in tuna.

Ultrasensitive hairpin mediated upconversion fluorescence biosensor for Staphylococcus aureus detection in foods and waters exploiting g–C3N4–assisted catalysis

A novel g-C3N4 nanosheets (g-C3N4 NSs)-assisted upconversion fluorescent aptasensor was proposed for Staphylococcus aureus (S. aureus) detection by adopting hybridization chain reaction (HCR) as a sensitizer. Two hairpin (H1 and H2) structured DNA probes were engineered predicated on the partial complementary sequence (cDNA) of S. aureus aptamer and modified on the exterior of the upconversion nanoparticles (UCNPs), respectively. The presence of S. aureus initiated the HCR system and activated H1 and H2 probes to form a double-helix away from the g-C3N4 NSs vicinity. This led to the decrease in peroxidase-like activity (PA) of the g-C3N4 NSs and corresponding fluorescence recovery proportional to the concentration of S. aureus (10-106 cfu mL−1). The method was applied to real food samples with acceptable recoveries (91.1–101.6%) and further validated by traditional plate counting method (p > 0.05).

A hybrid and scalable nanofabrication approach for bio-inspired bactericidal silicon nanospike surfaces

Insects and plants exhibit bactericidal properties through surface nanostructures, such as nanospikes, which physically kill bacteria without antibiotics or chemicals. This is a promising new avenue for achieving antibacterial surfaces. However, the existing methods for fabricating nanospikes are incapable of producing uniform nanostructures on a large scale and in a cost-effective manner. In this paper, a scalable nanofabrication method involving the application of nanosphere lithography and reactive ion etching for constructing nanospike surfaces is demonstrated. Low-cost silicon nanospikes with uniform spacing that were sized similarly to biological nanospikes on cicada wings with a 4-inch wafer scale were fabricated. The spacing, tip radius, and base diameter of the silicon nanospikes were controlled precisely by adjusting the nanosphere diameters, etching conditions, and diameter reduction. The bactericidal properties of the silicon nanospikes with 300 nm spacing were measured quantitatively using the standard viability plate count method; they killed E. coli cells with 59 % efficiency within 30 h. The antibacterial ability of the nanospike surface was further indicated by the morphological differences between bacteria observed in the scanning electron microscopic images as well as the live/dead stains of fluorescence signals. The fabrication process combined the advantages of both top-down and bottom-up methods and was a significant step toward affordable bio-inspired antibacterial surfaces.

Effect of Inoculum Concentration on the Degradation of Diesel 2 by a Microbial Consortium

The objective was to determine the effect of inoculum concentration on the degradation of Diesel 2 by a microbial consortium called BIOT.PD001. For this, five systems were designed (in triplicate), which Contained Davis Minimum Medium, 5% Diesel 2 as a carbon source, and a suspension of the microbial consortium BIOT.PD001 (9 × 108 cells/mL) in concentrations of 2, 4, 6, 8, and 10% of the final volume. The monitoring of the degradation of Diesel 2 was carried out indirectly through the bacterial counts by the plate count method, the Biochemical Oxygen Demand (BOD5) by the Winkler Method modified according to Alsterberg, and the concentration of total fats by Gerber’s method. The retention time was 15 days. It was observed that the percentage of efficiency of the process increases as the concentration of inoculum increases, obtaining the highest percentage of efficiency (94.77%) when using 10% of inoculum (v/v), while when using inoculum concentrations of 2 and 4% (v/v), the efficiency percentages are the lowest, (68.4 and 66.6%, respectively). On the other hand, the variance analysis indicated that there is a significant difference between the averages of these values. The regression analysis indicated that the inoculum concentration significantly affects the efficiency of Diesel 2 degradation and that this is 86% explained by a linear regression model. There is a linear relationship between the inoculum concentration of the BIOT.PD001 microbial consortium and the BOD5 tend to decrease as a function of time. It is concluded that the inoculum concentration significantly affects the efficiency of the degradation of Diesel 2 by the BIOT.PD001 consortium.

Accurate quantification of total bacteria in raw milk by flow cytometry using membrane potential as a key viability parameter

Total bacterial count (TBC) is one of the most widely accepted indicators to evaluate raw milk quality. Rapidly quantifying TBC, by flow cytometry (FCM) coupled with viability indicators, is increasingly being preferred over time-consuming cultivation-based methods. However, whether viability indicators accurately identify viable bacteria, challenged by natural stresses, remains largely unexplored. Here, three typical milk-borne species were challenged with representative dairy farm disinfection conditions to identify the optimal viability parameter through multiparameter FCM, which has not been attempted so far. We demonstrated that only the membrane potential accurately distinguished between viable and dead cells under stress and exhibited considerable correlation with the plate counting method, despite that all viability parameters recognized highly active and dead cells. The membrane potential indicator, DiBAC4(3), was first combined with SYTO64 to establish a novel FCM-based method, while formulating a conversion equation for the FCM-plate counting results. Moreover, the performance of the developed method was verified using real samples, where TBC in raw milk was accurately quantified within 2 h. Together, our results demonstrated that membrane potential optimally reflected the bacterial viability in raw milk under stress and emphasized that the viability of stressed cells should be carefully identified to quantify viable bacteria through FCM.

Viability and Antibacterial Activity of Bifidobacterium bifidum in Fermented Robusta Coffee for Diarrhea Treatment

Background: Diarrhea can be treated with probiotic bacteria such as Bifidobacterium bifidum, which decreases the intestinal environment's pH to become acidic so that pathogenic bacteria cannot thrive. Objective: To make fermented coffee that can increase the number of probiotic bacteria Bifidobacterium bifidum and has antidiarrheal activity against pathogenic bacteria Escherichia coli. Methods: Robusta coffee (20.25% and 19.75%) was fermented using Saccharomyces cerevisiae, and then the probiotic bacteria Bifidobacterium bifidum was added. Unfermented coffee was compared with the same concentration (20.25% and 19.75%) to obtain four formulas. Organoleptic panelists tested all formulas to determine the best formula for fermented and non-fermented coffee. The number of Bifidobacterium bifidum and antibacterial activity was calculated on the optimum formulation using the Total Plate Count and Disc Diffusion Method. Result: The optimum formula obtained at fermented and unfermented coffee concentration was 20,25%. The number of probiotic bacteria Bifidobacterium bifidum growing in fermented and non-fermented coffee was 7.3 x 108 ± 32.4 and 3.1 x 108 ± 30.7 (p &lt; 0.05). The diameter of the inhibition zone of the best fermented and non-fermented coffee was 11.5 ± 0.5 mm and 8.5 ± 0.5 mm, respectively (p &lt; 0.05). Conclusion: Fermented coffee can increase the growth of the probiotic bacteria Bifidobacterium bifidum and has strong antibacterial activity against Escherichia coli bacteria.

Silver nanoparticles-immobilized-radiation grafted polypropylene fabric as breathable, antibacterial wound dressing

In this work, a simplistic approach for developing an antibacterial and hydrophilic non-woven polypropylene (NWPP) fabric with excellent breathability has been presented via a two-step process. In the first step, acrylic acid was introduced onto NWPP fabric using simultaneous irradiation graft polymerization and pre-irradiation graft polymerization methods. Different grafting parameters, e.g., absorbed dose, monomer concentration, homopolymer inhibitor, reaction time, etc., were studied to optimize the grafting process. Subsequently, the acrylic acid grafted NWPP (NWPP-g-AA) fabric was immobilized with silver nanoparticles (Ag NPs), prepared from silver nitrate using trisodium citrate as reducing agent. After optimizing the grafting parameters, the NWPP-g-AA and Ag NPs-(NWPP-g-AA) samples were thoroughly characterized by grafting yield, FTIR, SEM, TEM, water contact angle, water vapor transmission rate (WVTR) and mechanical properties. The Ag NP-(NWPP-g-AA) fabric samples exhibited desired hydrophilicity, breathability and mechanical strength. Ag NP-(NWPP-g-AA) fabric samples were tested for antibacterial activity using plate count method after serial dilution, and exhibited excellent antibacterial efficacy against S.aureus (gram positive) and E.coli (gram negative) bacteria. This strategy demonstrated that a hydrophilic NWPP fabric having long-lasting antimicrobial activity was developed by gamma radiation-induced graft polymerization, which offers an ideal material for antibacterial wound dressing applications.

Isothermal inactivation of Mycobacterium avium subsp. paratuberculosis in curd simulating the stretching phase in pasta-filata cheese process.

Raw milk and dairy products are usually considered the major sources of Mycobacterium avium subsp. paratuberculosis (MAP) exposure for humans. During the production process of mozzarella cheese, as well as of other pasta-filata cheeses made with pasteurized or raw milk, curd is heated and stretched by addition of hot or boiling water. This step is the critical point for the inactivation of MAP during the production process, but, to our knowledge, no studies have been published about the thermal death time values of MAP in curd. The aim of this study was to determine the inactivation kinetics of MAP in curd used to produce pasta-filata cheese in six independent experiments. The milk was inoculated with a mix of MAP strains (field and registered strains) and, with the aim to simulate the thermal treatment of the curd during the stretching step, samples of 10 g of contaminated curd were vacuum packed and treated separately at six different temperatures from 60°C to 75°C in a water bath. MAP survival was then evaluated by plate count method and inactivation parameters were estimated for determining the thermal resistance of the pathogen directly in the curd. D-values increased from 0.15 min (D75-value) to 4.22 min (D60-value) and the calculated z-value was 10.2°C. These data aid: (i) to design food thermal process treatments defining acceptance limits of critical control points to ensure safety against MAP; (ii) to predict the time/temperature combinations needed to obtain a certain MAP log reduction during the curd stretching step; (iii) to optimize or validate pasta-filata cheese process.

Microbiological quality assessment of fish origin food along the production chain in upper Blue Nile watershed, Ethiopia.

Pathogenic microorganisms can grow accidentally on fish origin human food and can be a cause of human food-borne illness. The purpose of this study was to estimate the occurrence and microbial load pattern of Escherichia coli, Salmonella, Staphylococcus aureus, and Shigella spp. along the fish origin food value chain. A total of 396 fish samples were collected by a systematic random sampling technique of cooked and raw in the three species of fish. Fish muscles were tested using selective media, followed by conventional biochemical tests. The bacterial load was assessed using a standard plate count method. Whereas the fungal load were measured by cultured in a Sabouraud's dextrose agar (SDA) medium. The overall prevalence was Escherichia coli 84 (21.21%), Salmonella 27 (6.82%), Staphylococcus aureus 19 (4.80%), and Shigella spp. 17 (4.29%). The average mean total coliform count was observed 1.2 × 102 cfu/g and 5.10 × 104 cfu/g in cooked and raw fish samples, respectively. Whereas total viable count mean of 8.05 × 104 cfu/g and 11.5 × 104 cfu/g in cooked and raw fish, respectively. The Fungal load counts under the range 5.6 × 101 cfu/g to 1.09 × 103cfu/g were observed. The study has revealed that fish food in the study area has the possibility of microbial public health risk. Hence, it could be wise to improve the knowledge of key actors from harvesting to consumption to enhance the meals protection practices and high-quality standards of fish foods.

Establishing the lower bacterial concentration threshold for different optical counting techniques

This work compares several physical and optical techniques used in fundamental research and industrial applications to detect bacteria in water. Optical techniques such as, UV-absorbance spectroscopy, laser particle counting, turbidimetry and Z-Sizer light scattering, and a direct observational physical technique, the plate count method, were compared when measuring the concentration of E.coli in tenfold dilution from a stock solution. Estimates of the detection threshold limit of E.coli for the different optical counting techniques and the relationship between colony-forming units (CFU) and tenfold dilutions was established.Optical methods have generated interest due to the rapid response of just minutes, non-destructive approach and minimal sample preparation but their use is still limited to concentrations of up to 4 Log E.coli/mL. In contrast, the plate count method is still a reliable technique for water quality analysis despite its long response time of 24 h.

Dual phage amplification-mediated multiplex detection strategies for the simultaneous detection of Salmonella enterica and Staphylococcus aureus

Multiplex bacteria detection is essential to monitor various pathogens situations where multiple pathogen species are a concern. Herein, we explored novel dual phage amplification-based strategies (PAA) combined with a multiplex detection technique for simultaneous bacteria detection in one single tube. This assay was normalized on two common foodborne pathogens: Salmonella enterica and Staphylococcus aureus. In the presence of the target bacteria, phages could specifically recognize the viable cells and replicate themselves within the cells to produce more progeny phages. After thermal lysis within 10 min, the products with DNA of the progeny phages were analyzed with multiplex detection strategies. The specificity of the multiplex detection strategies integrated with the high sensitivity of the PAA assay allowed this assay to be rapid, specific, and sensitive for simultaneous quantification of Salmonella and Staphylococcus aureus. Optimized parameters of PAA, single and multiplex qPCR were defined, and the analytical sensitivities, specificities, and stabilities of single and multiplex reactions were validated under defined experimental conditions and in real samples. The results demonstrated wide linear ranges (10–108 CFU/mL), with detection limits of 10 CFU/mL for all bacteria tested. Moreover, the total detection time could be reduced to no more than 4 h. This assay was successfully applied to real food samples consistent with the results comparable to traditional plate counting methods. Due to the rapid and simple operation, low sensitivity and cost, high specificity, and the ability to distinguish live bacteria, the proposed PAA-based multiplex qPCR assay provides an effective and promising strategy for the simultaneous detection of viable bacteria.