Conventional Plate Count Method Research Articles

Comparison of SYBR® Green qPCR assay and plate count method to describe growth of Weissella viridescens and Leuconostoc mesenteroides in pure and mixed cultivation

The current study was conducted to statistically compare the SYBR® Green quantitative polymerase chain reaction (qPCR) assay and the conventional plate counting (PC) method to construct growth curves of a cocktail of Weissella viridescens in pure culture under different isothermal storage conditions (4, 8, 14, and 30 °C) and in mixed culture with Leuconostoc mesenteroides at 8 °C. The efficiency and specificity of the qPCR standard curves were confirmed, and both methods were adequate to quantify the growth kinetics of W. viridescens at all isothermal temperatures, demonstrating a good correlation and agreement. The efficiencies of the standard curves varied between 98% and 102%. The SYBR® Green qPCR assay was also able to differentiate the growth curves of W. viridescens and L. mesenteroides in the mixed culture at 8 °C. Additionally, the SYBR® Green qPCR method was considered a faster and more sensitive alternative to construct growth curves under different isothermal conditions and differentiate morphologically similar lactic acid bacteria. Overall, the results suggest that the SYBR® Green qPCR method is a reliable and efficient tool to study microbial growth kinetics in pure and mixed cultures.

Combining the oxygen sensor based respirometry and 16S rRNA amplicon sequencing for the analysis of microbiota in commercial mince products

In this study, rapid respirometric microbial testing was combined with 16S rRNA amplicon sequencing, to assess the composition of microbiota in a total of 64 samples of commercial beef, turkey, lamb and pork mince. The O2 sensor-based respirometry system, while producing the anticipated total aerobic viable counts (TVC) data and patterns for most samples, also revealed unusual (linear) respiration profiles for some samples, mostly lamb and pork mince. The TVC values for beef mince, produced by respirometry and calculated using the available calibration equation, correlated well with the conventional plate counting method, ISO 4833-1:2013, 2013, while for the other species the correlation was less good. These effects, not observed in previous studies employing various food matrices, require further investigation. Using the same samples (crude homogenates) as in respirometry, the whole microbiome was also analysed by 16S rRNA amplicon sequencing for each mince-type. The sequencing showed an overall decrease in alpha diversity over shelf-life, with lamb and pork mince maintaining a proportion of rare taxa. Some taxa exhibited significant changes in abundance over shelf-life and after the respirometric analysis, with beef mince exhibiting a decrease in aerobic bacteria and an increase in facultative anaerobes. Beta diversity was also seen to depend on mince-type. Thus, the combined use of respirometry and sequencing techniques shows promise as a useful and unique analytical approach for food quality and safety evaluation, However, more data points and in-depth analysis are required to back up the findings of this initial study.

Comparison of the Structure of Soil Microbial Communities of Different Ecosystems Using the Microbiome Sequencing Approach

In this study, we aimed to compare the functional and taxonomic composition of soil microbial communities in different ecosystems, agricultural, natural grasslands, and old-growth forests, in the context of different environmental conditions. In this research, cultivable microbial quantification was performed by conventional plate-count techniques using different selective media. The taxonomic structure of microbe communities was evaluated using NGS metagenomic sequencing on the Illumina platform NovaSeq. The taxonomic analysis showed that individual land uses are characterized by the specific structure of communities; some taxonomic groups are specific only to agricultural, grassland, or forest ecosystems. After determining the abundance of functional groups of culturable microorganisms by the conventional plate-count method, statistically significant quantitative differences in physiological groups between the individual ecosystems were revealed. The metagenomic analysis revealed that different ecosystems are characterized by specific taxonomic groups of microorganisms and that general alpha diversity varies among individual land-use samples. Since the most unstable soil systems are agricultural, they are likely to suffer the most and will suffer more in the future from climate change than natural ones.

Enumeration of Brettanomyces in Wine Using Impedance

Brettanomyces bruxellensis is a wine spoilage concern in wineries around the world. In order to maintain wine quality during storage and ageing, it is imperative to control and monitor this yeast. Being a fastidious slow growing yeast, which requires 5 to 14 days of incubation for visible growth in agar plates, it is difficult to detect growth (colonies) by conventional agar plate count method. Yeast enumeration by impedance was investigated because previous research using other microorganisms has shown that it is potentially faster than plate counting. The relationship between plate counting and impedance detection times was investigated for Brettanomyces inoculated in red wine samples. A linear relationship between log plate count concentrations and impedance detection times was found. Incubation time was reduced from 120 h down to 0.9 and 57.7 h for samples with 6.7 × 107 and 1.8 × 102 cfu/mL, respectively, using the ‘indirect’ impedance method. The ‘direct’ method also reduced the incubation times to 9.5 and 81.9 h, for the same concentrations. The ‘indirect’ impedance method has the potential to be used by the wine industry to control and monitor the Brettanomyces numbers in wines.

Molecular Structural Evolution of Near-Infrared Cationic Aggregation-Induced Emission Luminogens: Preclinical Antimicrobial Pathogens Activities and Tissues Regeneration

Molecular Structural Evolution of Near-Infrared Cationic Aggregation-Induced Emission Luminogens: Preclinical Antimicrobial Pathogens Activities and Tissues Regeneration

Environmental antimicrobial resistance is associated with faecal pollution in Central Thailand’s coastal aquaculture region

We assessed antimicrobial resistance (AMR) potential to seven major classes of antibiotics in Central Thailand’s coastal aquaculture region using high-throughput qPCR targeting 295 antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In parallel, we used MinION next generation sequencing (NGS) of 16S rRNA gene amplicons, qPCR of faecal pollution makers, and conventional plate count methods for the comprehensive characterization of environmental microbiomes. We tested the hypothesis that aquaculture increases environmental AMR by comparing ARGs and MGEs in water and sediment samples from five aquaculture sites and their associated canals. There was no evidence from the ARG and MGE data that aquaculture is a major driver of environmental AMR in Central Thailand. Instead, the highest relative prevalence of resistance traits was found in Hua Krabue canal water influenced by urban pollution from Bangkok at the inland edge of the coastal aquaculture region. The sum of ARGs and MGEs, relative to 16S rRNA genes used as markers for overall bacterial abundance, was between 0.495 ± 0.011 and 0.498 ± 0.013 in Hua Krabue canal water, compared with at most 0.132 ± 0.005 in all the other environmental samples. Corresponding patterns were observed for most faecal pollution markers, which were also elevated in Hua Krabue canal water.

Comparative study of rapid ATP bioluminescence assay and conventional plate count method for development of rapid disinfecting activity test.

Antimicrobial activity is tested when developing disinfectants, pharmaceutical products, cosmetics, and many other consumer products. However, the plate count method, the conventional way to count the number of microorganisms, needs several days of culture. Consequently, a means of rapid microbial detection is strongly desired to replace this method. We have already developed a rapid and sensitive microbial adenosine triphosphate (ATP) detection system utilizing ATP bioluminescence, which can quantify microbial ATP within 1 h. To apply this technique to antibacterial activity tests, the ATP method should be proved equal or superior to the conventional method. In this study, we conducted disinfectant activity tests comparing the ATP method and the plate count method, using polyhexamethylene biguanide (PHMB) in different concentrations (0-10 ppm) as a model disinfectant against Staphylococcus aureus and Aspergillus brasiliensis. We found that the log reduction of intracellular ATP had a positive correlation with the log reduction of the plate count. Moreover, the ATP method was able to distinguish different conditions of injured microbial cells that were observed using scanning electron microscopy, whereas colony counting detects only culturable cells. The ATP method is thus a rapid and useful alternative to the conventional method in the field of antimicrobial activity testing.

Screening of Primer Sets for Amplification Cyclic Lipopeptide Gene of <i>Bacillus</i> Against Banana Fusarium Wilt

Cyclic lipopeptides (CLPs) are a class of natural agricultural antibiotics produced by  Bacillus , it plays an important role in the biological control using of  Bacillus . In this study, 20 strains of  Bacillus , including  Bacillus amyloliquefaciens ,  Bacillus subtilis ,  Bacillus siamese , and  Bacillus cereus  were determined the antagonistic ability against to pathogenic fungi such as Foc 4 . PCR amplification was performed on 20 strains of Bacillus and 7 soil samples, by using the designed 14 pairs of primers for CLPs-related synthetase genes, including Bacillomycin, Fengycins, Iturins, Surfactin etc, screen CLPs primers that can be used for Bacillus and soil monitoring.  The results showed that: a total of 16  Bacillus  strains have the antagonistic activity against pathogens, and microscopic observation of the crude lipopeptide extract of fermentation broth of these  Bacillus  strains can destroy the biofilm system and cell wall of Foc4 , causing the mycelium and meristems to expand and deform; 12 pairs primers of this study ( BamD-F/R, BamA-F/R, FenA-F/R, FenB-F/R, 61FenA-F/R, 61FenB-F/R, ItuA-F/R, ItuB-F/R, ItuC-F/R, ItuD-F/R, SrfAA-F/R, SrfAB-F/R ) could effective amplify CLPs gene, and find that all of test  Bacillus  strains has the ItuC gene, there is a positive relationship between the antagonistic ability of  Bacillus  and the diversity and richness of CLPs genes, through PCR amplification of total soil DNA,  it was found that the designed CLPs primers were feasible to monitor  Bacillus  in soil environment. It would be used as an important supplement to conventional plate counting and high-throughput sequencing methods, monitor and guide the application of Bacillus control soil-borne diseases of plants to provide theoretical support for further field.

A Species-Specific qPCR Method for Enumeration of Lactobacillus sanfranciscensis, Lactobacillus brevis, and Lactobacillus curvatus During Cocultivation in Sourdough

Lactobacillus sanfranciscensis, Lactobacillus brevis, and Lactobacillus curvatus are frequently isolated from cereal-based fermented foods and are the dominant lactic acid bacteria in Korean sourdough. Detection of the individual species and their enumeration during fermentation are of great importance for maintaining the quality of bakery products. Here, we developed a species-specific quantitative polymerase chain reaction (qPCR) method to monitor the population of these three bacterial species during cocultivation in sourdough. Target genes, including those encoding nucleoside hydrolase, hypothetical protein, and glyoxalase, were selected by MegaBLAST for the detection of L. sanfranciscensis, L. brevis, and L. curvatus, respectively. The specificities of PCR primer sets were verified with qPCR; constant cycle threshold (Ct) values were obtained from mixed genomic DNAs (gDNAs) of target and non-target strains. The qPCR results were unaffected by the sourdough matrix. The cell numbers derived from qPCR were 10–65% higher than those obtained using a conventional plate-counting method. The qPCR standard curves for the three Lactobacillus species were established, and their populations were successfully enumerated during sourdough propagation. This method would enable quantification of three Lactobacillus species during cocultivation in sourdough and provide useful information on microbial commensalism that is essential to obtain high-quality bread.

Unsatisfactory microbiological aspects of UHT goat milk, soymilk and dairy beverage of goat milk and soy protein: A public health issue

Abstract Spore forming bacteria can play an important role in food quality and safety as spoilage and pathogenic microorganisms due to resistance to heat-treatment. However, there are limited number of studies focused on evaluate the microbiological quality and the occurrence of these bacteria in UHT goat milk, soymilk and dairy beverage of goat milk and soy protein. In this context, 75 samples of these beverages were evaluated regarding heterotrophic mesophilic microorganisms by conventional plate count and selective methods to detect microorganisms from Bacillus cereus group and Clostridium perfringens. Population counts greater than 104 CFU.ml-1 of heterotrophic mesophilic microorganisms were observed in 80% of the lots of goat milk and 100% of the lots of soymilk and dairy beverage of goat milk and soy protein. The presence of bacteria belonging to B. cereus group was observed in 16%, 52% and 44% of goat milk, soymilk and dairy beverage of goat milk and soy protein, respectively. C. perfringens was isolated from 8% samples of UHT soymilk. The frequency of genes hblA, hblB, hblC, nheA, nheB, nheC in 29 isolates obtained from these products was 62%, 48.2%, 96.5%, 79.3%, 68.9% and 79.3%, respectively. The microbiological quality of the evaluated products was unsatisfactory.

Development of enteric polymer-based microspheres by spray-drying for colonic delivery of Lactobacillus rhamnosus GG.

Antibiotics are well-known disruptive elements of the intestinal microbiota and antibiotic-associated diarrhea appeared as the most common complication related with post-antibiotic dysbiosis. Lactobacillus rhamnosus GG (LGG) strain is very effective in preventing antibiotic-associated diarrhea in children and adults. However, as any probiotics, it is concerned by the loss of viability during storage and gastrointestinal transit. The aim of this study was to develop an encapsulation system suitable for the specific colonic delivery of LGG strain after oral administration. For this purpose, spray-dried Eudragit® S100 microparticles encapsulating LGG bacteria were developed by using an aqueous based spray-drying approach, avoiding the use of organic solvents. Carbohydrates were added to the formulation since they are widely used as protective agents of bacteria against the harmful effect of dehydration stress. Here, both Surface Enhanced Raman Scattering (SERS) and conventional plate count methods showed that carbohydrates increased the survival ratio of bacteria after spray-drying from 3 to more than 50%. Moreover, these protective agents ensured low residual moisture content thus providing great stability of the cells in the spray-dried powder during storage. Significant improvement of the cell viability in simulated gastro intestinal fluid (SGIF) was observed for encapsulated cells as compared with free LGG bacteria for which no viable cell was detectable after 1h incubation in gastric fluid only. As a consequence, 4.5×107 CFU/g of encapsulated LGG were found viable after incubation of microparticles 1h in Simulated Gastric Fluid followed by 6h in Simulated Intestinal Fluid, corresponding to less than 3 log reduction of viable cells during the 7h incubation in Simulated Gastro Intestinal Fluid. These results attested that the developed encapsulation system is suitable for its use as a bacteria carrier for specific colonic delivery.

A Modified Double Subculture Method for the Two-Mode Injuries Evaluation in a Stressed Fungal Spore Population.

In order to evaluate injury of a stressed fungal spore population, a modification of formerly presented double subculture method, which consists of both the conventional plate count method and the growth delay analysis method, was proposed. In this method, an apparent logarithmic growth kinetics was assumed and the previous kinetic model was improved to be able to estimate injured subpopulations in two different modes containing early occurring growth-independent and late occurring growth-dependent injuries, called the λ and μ injuries, respectively. Based on the kinetic theory developed here, this novel method was applied to heat-treated conidia of Cladosporium cladosporioides and these two mode injuries were evaluated.

Teicoplanin-functionalized magnetic beads for detection of Staphylococcus aureus via inhibition of the luminol chemiluminescence by intracellular catalase.

An affinity-based protocol is described for the detection of Staphylococcus aureus (S. aureus). It is utilizing teicoplanin-functionalized magnetic beads as carriers. Teicoplanin, which binds to the walls of cells of S. aureus via five hydrogen bonds, acts as the recognition agent. Captured S. aureus is magnetically separated from the sample matrix and then specifically lysedby lysostaphin which cleaves the cross-linking pentaglycine bridges of peptidoglycan in the cell wall. Lastly, S. aureus is quantified via the inhibitory effect of released intracellular catalase on a chemiluminescent (CL) system composed of peroxidase, luminol, H2O2 and p-iodophenol because catalase decomposes H2O2. S. aureus can be detected with CL response in the 140 to 1.4 × 107CFU·mL-1 concentration range and a detection limit as low as 47CFU·mL-1 at a signal-to-noise ratio of 3. The method was evaluated by analyzing spiked samples including milk, human urine and saline injection solutions. The reliability was demonstrated by a recovery test and by comparison with a conventional plate counting method. Graphical abstract An antibiotic-affinity protocol is developed to detect Staphylococcus aureus (S. aureus) by utilizing teicoplanin-functionalized magnetic beads (Teic-MBs) as carriers. S. aureus can be quantified by measuring the inhibition of luminol chemiluminescence (CL) signal by intracellular catalase.

Methicillin resistant Staphylococcus aureus (MRSA) in pastry cream products sold in Amol (Iran)

Abstract Background: Methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as a matter of antibiotic resistance that is largely developed amongst common foodborne pathogens. MRSA is being considered as an important worldwide health threat and causes considerable concern to clinicians, food products manufacturers, governments and also consumers. OBJECTIVES: The objective of this study was to detect MRSA isolated from 360 samples of pastry cream products sold in the local markets in Amol, June 2016- May 2017, by plate count method and molecular technique. METHODS: The conventional plate counting method was conducted through inoculating appropriate dilutions of samples onto the Baired Parker Agar plates. MRSA isolates were detected by PCR method using mecA primers set. The resistance of isolated MRSA strains against some antibiotics was determined. RESULTS: Out of 360 pastry cream samples tested, 41.6% (150 samples) were contaminated by S. aureus with an average count of 4.94 log CFU/g in summer; 4.72 log CFU/g in autumn, 2.74 log CFU/g in winter and 3.62 log CFU/g in spring. Eleven samples out of 360 tested (3.05%) showed positive results for the mecA gene. No MRSA isolate was identified amongst winter samples. 56% of isolated strains showed sensitivity to oxacillin, 7% of isolates were sensitive to penicillin, 23 to ampicillin, 82% to gentamicin and 33% to tetracycline. CONCLUSIONS: According to the results, monitoring and improving the hygienic conditions of food production chain and educating food handlers and staff involved in food preparation is recommended in order to prevent MRSA prevalence.

A High-Sensitivity Potentiometric 65-nm CMOS ISFET Sensor for Rapid E. coli Screening.

Foodborne bacteria, inducing outbreaks of infection or poisoning, have posed great threats to food safety. Potentiometric sensors can identify bacteria levels in food by measuring medium's pH changes. However, most of these sensors face the limitation of low sensitivity and high cost. In this paper, we developed a high-sensitivity ion-sensitive field-effect transistor sensor. It is small sized, cost-efficient, and can be massively fabricated in a standard 65-nm complementary metal-oxide-semiconductor process. A subthreshold pH-to-time-to-voltage conversion scheme was proposed to improve the sensitivity. Furthermore, design parameters, such as chemical sensing area, transistor size, and discharging time, were optimized to enhance the performance. The intrinsic sensitivity of passivation membrane was calculated as 33.2mV/pH. It was amplified to 123.8mV/pH with a 0.01-pH resolution, which greatly exceeded 6.3mV/pH observed in a traditional source-follower based readout structure. The sensing system was applied to Escherichia coli (E. coli) detection with densities ranging from 14 to 140cfu/mL. Compared to the conventional direct plate counting method (24h), more efficient sixfold smaller screening time (4 h) was achieved to differentiate samples' E. coli levels. The demonstrated portable, time-saving, and low-cost prescreen system has great potential for food safety detection.

Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matrices

Bacterial foodborne illness continues to be a pressing issue in our food supply. Rapid detection methods are needed for perishable foods due to their short shelf lives and significant contribution to foodborne illness. Previously, a sensitive and reliable surface-enhanced Raman spectroscopy (SERS) sandwich assay based on 3-mercaptophenylboronic acid (3-MBPA) as a capturer and indicator molecule was developed for rapid bacteria detection. In this study, we explored the advantages and constraints of this assay over the conventional aerobic plate count (APC) method and further developed methods for detection in real environmental and food matrices. The SERS sandwich assay was able to detect environmental bacteria in pond water and on spinach leaves at higher levels than the APC method. In addition, the SERS assay appeared to have higher sensitivity to quantify bacteria in the stationary phase. On the other hand, the APC method was more sensitive to cell viability. Finally, a method to detect bacteria in a challenging high-sugar juice matrix was developed to enhance bacteria capture. This study advanced the SERS technique for real applications in environment and food matrices.

Rapid fluorescence detection of pathogenic bacteria using magnetic enrichment technique combined with magnetophoretic chromatography.

A rapid and sensitive analytical method was developed to detect pathogenic bacteria which combined magnetic enrichment, fluorescence labeling with polyethylene glycol (PEG) magnetophoretic chromatography. As pathogenic bacteria usually exist in complex matrixes at low concentration, an efficient enrichment is essential for diagnosis. In order to capture series types of pathogenic bacteria in samples, amino-modified magnetic nanoparticles (Fe3O4@SiO2-NH2) were prepared for efficient enrichment by the electrostatic interaction with pathogenic bacteria. It was shown that the capture efficiency reached up to 95.4% for Escherichia coli (E. coli). Furthermore, quantitative analysis of the bacteria was achieved by using acridine orange (AO) as a fluorescence probe for the captured E. coli due to its ability of staining series types of bacteria and rapid labeling. In order to remove the free magnetic nanoparticles and redundant fluorescent reagent, the labeled suspension was poured into a PEG separation column and was separated by applying an external magnetic field. The presence of 100cfumL-1 E. coli could be detected for semi-quantitative analysis by observing the separation column with the naked eye, and the concentration could be further evaluated by fluorescence detection. All the above processes were finished within 80min. It was demonstrated that a good linear relationship existed between the fluorescence intensity and the concentration of E. coli ranging from 102 to 106cfumL-1, with a detection limit of 100cfumL-1 when E. coli acted as target bacteria. The recovery rate of E. coli was 93.6∼102.0% in tap water and cooked meat samples, and the RSD was lower than 7% (n=6); the result coincided with the conventional plate count method. Graphical abstract ᅟ.

The development of methods for the detection of Salmonella in chickens by a combination of immunomagnetic separation and PCRs.

Micro- and nanoimmunomagnetic beads (MIMBs and NIMBs) used for immunomagnetic separation (IMS) with PCR were studied for the rapid detection of Salmonella. The capture efficiency of the two different IMBs was evaluated by a conventional plate counting method, and the binding pattern was studied using scanning electron microscopy. The specificity of the IMBs was tested with Salmonella, Shigella flexneri, enterohemorrhagic Escherichia coli O157:H7, and Listeria monocytogenes. By comparing the pre-enrichment IMS and the IMS enrichment steps with a 5.5-H enrichment time, this study developed a rapid and sensitive method for the detection of Salmonella in chicken. The method was implemented by IMS enrichment and PCR with MIMBs and NIMBs, with a total analysis time of 8 H. We showed that the method was sensitive based on NIMBs with a detection limit of 10° CFU for Salmonella in 25 g of chicken.

A competitive quantitative polymerase chain reaction method for characterizing the population dynamics during kimchi fermentation.

The aim of this study was to develop a competitive quantitative-PCR (CQ-PCR) method for rapid analysis of the population dynamics of lactic acid bacteria (LAB) in kimchi. For this, whole chromosome sequences of Leuconostoc mesenteroides, Lactobacillus plantarum, and Lb. brevis were compared and species-specific PCR primers targeting dextransucrase, 16S rRNA, and surface layer protein D (SlpD) genes, respectively, were constructed. The tested strains were quantified both in medium and kimchi by CQ-PCR and the results were compared with the data obtained using a conventional plate-counting method. As a result, the three species were successfully detected and quantified by the indicated primer sets. Our results show that the CQ-PCR method targeting species-specific genes is suitable for rapid estimation of LAB population to be used in the food fermentation industry.

An innovative challenge test for solid cosmetics using freeze-dried microorganisms and electrical methods

Freeze-dried bacteria and fungi were used as inoculum in 28 days' PET. An electrical method was used in replacement of the conventional plate count method. The use of freeze-dried microorganisms in association with the electrical method can minimize the workload and the variability involved in PET for cosmetic powders.