[The Validity of the Poisson Distribution to Analyze Microbial Colony Counts on Agar Plates for Food Samples].

Microbial colony counts of food samples in microbiological examinations are one of the most important items. The probability distributions for the colony counts per agar plate at the dilution of counting had not been intensively studied so far. Recently we analyzed the colony counts of food samples with several probability distributions using the Pearson's chi-square value by the "traditional" statistics as the index of fit [Fujikawa and Tsubaki, Food Hyg.Saf.Sc., 60, 88-95 (2019)]. As a result, the selected probability distributions depended on the samples. In this study we newly selected a probability distribution, namely a statistical model, suitable for the above data with the method of maximum likelihood from the probabilistic point of view. The Akaike's Information Criterion (AIC) was used as the index of fit. Consequently, the Poisson model were better than the negative binomial model for all of four food samples. The Poisson model was also better than the binomial for three of four microbial culture samples. With Baysian Information Criterion (BIC), the Poisson model was also better than these two models for all the samples. These results suggested that the Poisson distribution would be the best model to estimate the colony counts of food samples. The present study would be the first report on the statistical model selection for the colony counts of food samples with AIC and BIC.

Transitional phase Gd-EOB-DTPA-enhanced MRI: Visual assessment of hepatic function and fibrosis based on uptake rate of Gd-EOB-DTPA.

PURPOSE: The aim of the study is to investigate the feasibility for hepatic function and fibrosis visual assessment using transitional phase imaging based on the uptake process of Gd-EOB-DTPA. MATERIALS AND METHODS: We retrospectively selected 105 consecutive patients who underwent Gd-EOB-DTPA enhanced MRI examination at 1.5 Tesla for intrahepatic lesion evaluation from June 2020 to June 2022. Data were classified into two groups defined by the signal intensity (SI) difference in the hepatic vein against that of the hepatic parenchyma at transitional phase as follows: High and Iso-SI group: hepatic vein SI equal to or greater than the hepatic parenchymal SI; and Low-SI group: hepatic vein SI lower than hepatic parenchymal SI. We evaluated whether significant differences in ALBI score, FIB-4, APRI and LSR of hepatobiliary phase between two groups. We measured cut-off values between two groups in all items according to receiver operating characteristic analysis. Furthermore, the inter-reader reproducibility of the visual assessment on transitional phase images between two readers was evaluated using the ICC. RESULTS: The visual assessment results were as follows: High and Iso- and Low-SI groups included 48, 57, patients, respectively. Significant differences were observed in ALBI score, FIB-4, APRI and LSR between two groups. The cut-off values of ALBI score, FIB-4, APRI and LSR were -2.69, 2.28, 0.49 and 2.15. ICC of transitional phase image visual assessment between two readers was 0.86. CONCLUSIONS: Hepatic function and fibrosis might be assessed by visual assessment of transitional phase images in Gd-EOB-DTPA enhanced MRI.

Counseling (ro)bot as a use case for 5G/6G.

This paper presents a counseling (ro)bot called Visual Counseling Agent (VICA) which focuses on remote mental healthcare. It is an agent system leveraging artificial intelligence (AI) to aid mentally distressed persons through speech conversation. The system terminals are connected to servers by the Internet exploiting Cloud-nativeness, so that anyone who has any type of terminal can use it from anywhere. Despite a promising voice communication interface, VICA shows limitations in conversation continuity on conventional 4G networks. Concretely, the use of the current 4G networks produces word dropping, delayed response, and the occasional connection failure. The objective of this paper is to mitigate these issues by leveraging a 5G/6G slice inclusive of mobile/multiple edge computing (MEC). First, we propose and partly implement the enhanced and advanced version of VICA. Servers of enhanced versions collaborate to increase speech recognition reliability. Although it significantly increases generated data volume, the advanced version enables a recognition of the facial expressions to greatly enhance counseling quality. Then, we propose a quality assurance mechanism using multiple levels of catalog, as well as 5G/6G slice inclusive of MEC, and conduct experiments to uncover issues related to the 4G. Results indicate that the number of speech recognition errors in Internet Cloud is more than twofold compared to edge computing, implying that quality assurance using 5G/6G in conjunction with VICA Counseling (ro)bot has higher efficiency.

[Application of the log-Logistic Model to Dose Response Relation in Microbial Risk Assessment].

Microbial risk assessment in food safety is a valuable tool to reduce the risks of infection by pathogens. The dose-response relation is aimed to establish the relationship between the dose of a pathogen that populations are exposed to and the probability of the adverse health effect by the pathogen. Among many dose-response models ever proposed, the exponential and beta-Poisson models have been internationally applied, but the decision on which model is selected between them solely depends on the goodness of fit to specific data sets. On the other hands, the log-logistic model, one of the alternative models, has been little studied on the dose-response relation. In the present study, thus, the application of the log-logistic model to dose-response relation was studied with hypothetical and experimental data sets of infection (or death), comparing to the above two models. Here the experimental data sets were for pathogenic organisms such as pathogenic Escherichia coli, Listeria monocytogenes, and Cryptosporidium pavrum. Consequently, this model successfully fit to those data sets in comparison to the two models. These results suggested that log-logistic model would have the potential to apply to the dose-response relation, similar to the exponential and beta-Poisson models.

[Staphylococcal Enterotoxin A Production and Inactivation in Bread during the Production Processes].

Staphylococcus aureus food poisoning is caused by the intoxication of staphylococcal enterotoxin (SE) produced in foods. Staphylococcal food poisoning is mostly due to staphylococcal enterotoxin type A (SEA) among SEs. There have been many studies on the growth and SEA production of S. aureus in various foods, but few studies in bread. Thus, the SEA production by S. aureus in dough during fermentation and the SEA inactivation in dough during baking were studied in the normal production processes of bread in this study. No growth of S. aureus or SEA production in dough, whose total weight was about 470 g, was observed during the fermentation at 25 and 35℃ for four hr, suggesting that the risk of SEA production in dough during fermentation under these conditions would be negligible. Any SEA injected at 6.0 and 0.56 ng/g in dough could not be detected after 20 and 10 min of baking at 200℃, respectively. These results showed that the baking process, which was completed in 25 min, was enough to inactivate SEA at those doses of SEA in the dough. The results on the production and inactivation of SEA in dough during the production processes in this study would be useful information on microbiological food safety of bread making.

Effects of climatic elements on Salmonella contamination in broiler chicken meat in Japan.

The effects of climatic elements on Salmonella contamination of chicken meat were investigated. Logistic regression analysis was performed to evaluate the association between Salmonella isolation, for 240 chicken samples purchased from March 2015 to February 2017, and climatic elements, over 65 days of chicken rearing. Salmonella was isolated from 143 samples (59.6%), and the most dominant serovars identified were Infantis (77/240, 32.1%) and Schwarzengrund (56/240, 23.3%). Previous studies have reported S. Schwarzengrund contamination of broiler chickens only in western Japan; however, in the present study, S. Schwarzengrund was also isolated from meat produced in eastern Japan-20% (12/60) in the C prefecture to 36.4% (8/22) in the Y prefecture-suggesting that S. Schwarzengrund-contaminated areas have expanded towards eastern Japan. Air temperature showed a significant negative association with S. Schwarzengrund isolation for chicken meat produced during periods with rising temperature (spring and summer) [odds ratio (OR), 0.894 to 0.935; P<0.01]. Moreover, the risk of S. Schwarzengrund contamination of chicken meat was higher during spring (OR, 3.951; P=0.008) and winter (OR, 4.071; P=0.006) than during summer. Effects of climatic elements and differences in contamination risk across seasons were not observed for any Salmonella serovars and only S. Infantis, which could be attributed to differences in transmission patterns and vehicles among Salmonella serovars. These findings are valuable for understanding the dynamics of S. Schwarzengrund dissemination in broiler farms.

Antibacterial Activity of Lactic Acid Bacteria to Improve Shelf Life of Raw Meat.

Lactic Acid Bacteria （LAB） are generally recognized as safe. It has been used to increase the shelf-life of fermented products, and its antimicrobial action is based on the metabolites secretions, such as lactic acid, hydrogen peroxide, reuterin, bacteriocins and the like-bacteriocins substances. It has been proven that LAB are able to inhibit deteriorating bacteria of raw meat, but improper handling of live cultures could lead to spoilage. So, the use of their bacteriocins, small antimicrobial peptides, could be an alternative. Besides reducing the number of spoilage bacteria, it seeks to inhibit pathogenic bacteria such as Salmonella, enterohemorrhagic Escherichia coli and Listeria. The food industry uses few bacteriocins and now bacterial resistance has been reported. For that reason, the search of novel bacteriocins produced by LAB is a priority. Moreover, the natural microbiota of meat could be a reservoir of LAB.

Effects of Lactic Acid and Salt on Enterotoxin A Production and Growth of Staphylococcus aureus.

Food poisoning caused by Staphylococcus aureus is responsible for staphylococcal enterotoxin (SE) produced in foods. Staphylococcal food poisoning is mostly caused by staphylococcal enterotoxin type A (SEA) among SEs. Growth/no growth for S. aureus under various environmental conditions was well studied with a logistic regression model so far. Recently we successfully described the boundaries of SEA production and growth of S. aureus in broth at various temperatures and salt concentrations with the model. In this study, the effects of lactic acid and salt on SEA production and growth of S. aureus was quantitatively studied. Consequently the boundaries of SEA production and growth of S. aureus cocktail in broth at various combinations of salt concentrations and pH values that were adjusted with lactic acid were successfully described with a logistic regression model. Here the cocktail was incubated in stationary culture at 30 °C and 10 °C. The maximum toxin production and cell growth of the cocktail were observed both at 5% salt in the pH range from 4.5 to 7.0. Also, the characteristics of individual strains of the cocktail in SEA production and growth at 30 °C and 10 °C were found to be specific to the strains. The present study revealed the effect of lactic acid and salt on SEA production and growth of S. aureus as well as the variety of SEA production and growth of S. aureus strains. These results would become useful information in food industry to prevent staphylococcal food poisoning. PRACTICAL APPLICATION: Boundaries of enterotoxin A production/no production and growth/no growth of staphylococcal cocktail at various combinations of pHs adjusted with lactic acid and salt concentrations were well described with a logistic regression model. The maximum toxin production and cell growth were observed both at 5% salt in the pH range from 4.5 to 7.0. A variety of the toxin production and cell growth were observed in terms of pH and salt concentration among individual strains of the cocktail.

[Characteristics of Microbial Colony Counts on Agar Plates for Food and Microbial Culture Samples].

Microbial colony counts of concern of food products are one of the most important items in microbiological examinations. The distributions of colony counts per agar plate of food samples are considered to be reflected with microbial cell distributions in food homogenates. However, (i) the probabilistic distributions of the colony counts per agar plate at the dilution of counting and (ii) the relationship between the colony counts per plate and the number of agar plates for food samples have not been intensively studied so far. In this study, therefore, these two points were studied with raw food samples of raw minced beef and chicken and raw milk and microbial culture samples of Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae. Among four major probabilistic distributions, it was found that aerobic plate counts per plate of the foods were well described with negative binomial, Poisson, and normal distributions and that the colony counts per plate of microbial cultures were described well with binomial, Poisson, and normal distributions. The effect of the number of agar plates on the estimation of the mean of colony counts per plate of a sample was then studied with the data randomly resampled from the experimental data. The resampled data showed that with more number of plates the mean of counts fluctuated less and the coefficients of variation of colony counts per plate decreased further, which were coincident to the estimated by the central limit theory. Our study would provide useful information on the characteristics of colony counts per plate of food samples which are routinely examined.

[Application of an Estimation Method of Microbial Concentration of a Food Sample from Qualitative Data].

Microbial concentrations of thermally processed foods are mostly low. Recently we proposed a stochastic method based on the MPN technique for estimating microbial concentration in a food product whose concentration is low. Namely, with the number of target pathogen-positive samples in the total samples of a food product by a qualitative examination, the concentration of the target microbe in the product was estimated with the stochastic method. However the credible interval of the estimate with the method was not yet studied. Also the method was not validated with foods yet. The present study showed the 95% credible interval of the estimation by the method with the maximum likelihood analysis. Also the present study showed the reliability of the method experimentally by using a powdered formulae for infants product and a liquid food model injected with Salmonella cells. This method could estimate as low as seven and two cells/1,000 g or 1,000 mL, respectively. Next, the estimated concentrations and 95% credible intervals of Enterobacteriaceae in fresh meat for eating raw were demonstrated at various Enterobacteriaceae-positive numbers of the sample with the present method. Further, the characteristics of the present method on the sample size and the aliquot of sample were clarified. These results suggested that the present method could be a good tool to verify microbiological safety of food products.

Comprehensive Study of the Boundaries of Enterotoxin A Production and Growth of Staphylococcus aureus at Various Temperatures and Salt Concentrations.

Staphylococcus aureus food poisoning, which is still a serious health problem worldwide, is caused by the intoxication of staphylococcal enterotoxin (SE). Among many types of SE, staphylococcal enterotoxin type A (SEA) is known to be the most responsible for S. aureus food poisoning worldwide. Several researchers have reported the modeling of growth/no growth boundaries for S. aureus with a logistic regression model. In this study, the boundaries of SEA production and S. aureus growth in broth were first performed with the logistic regression model, to evaluate the effects of environmental factors of temperature and salt. Consequently, the boundaries of SEA production and growth of S. aureus in temperature and salt concentration could be produced with the model in shaking and stationary cultures. The area where S. aureus cells would grow, but not produce SEA could be shown between the boundaries of SEA production and growth. Internal and external validations showed that the model could well describe and predict experimental results. Further, the maximum concentrations of SEA and cell population under various conditions of temperature and salt concentration were also compared between the shaking and stationary cultures. These results obtained in this study would become useful information in food industry to prevent S. aureus food poisoning outbreaks. PRACTICAL APPLICATION: Production/no production of staphylococcal enterotoxin A at various temperatures and salt concentrations in shaking and stationary cultures could be estimated well with a logistic regression model. Growth/no growth of staphylococcal cells under the same conditions was also well estimated with the model. The area where S. aureus cells would grow, but not produce SEA could be shown with the model. This present analysis would provide useful information in food industry to prevent staphylococcal food poisoning outbreaks.

Characteristics of Staphylococcal Enterotoxin A Production and Growth of Staphylococcus aureus in Shaking and Stationary Cultures.

Staphylococcal food poisoning, which is still a serious health problem worldwide, is caused by staphylococcal enterotoxin (SE) . Among many types of SE, staphylococcal enterotoxin type A (SEA) is known to be the most responsible for staphylococcal food poisoning. Production by SEA-producing strains in shaking culture has been studied by many investigators, whereas the kinetical differences between shaking and stationary cultures have not been studied intensively. Therefore, this difference was studied at various temperatures from 14℃ to 46℃ in the present study. Consequently the maximum SEA concentration and populations of SEA producer in shaking culture was higher than those in stationary culture. Of interest, however, the productivity, which is the maximum SEA amount produced by one cell, in shaking cultures was lower than that in stationary culture. Kinetic analysis clarified that SEA gene expression in staphylococcal cells preceded toxin production at optimal temperature. Next, several SEA producers were studied for the maximum toxin production at various temperatures from 14℃ to 42℃ in shaking culture. Consequently all strains showed different patterns, suggesting that the characteristics of SEA production of these strains would be strain-specific. These results in this study would provide useful, basic information to prevent staphylococcal food poisoning outbreaks.

Estimation of Microbial Concentration in Food Products from Qualitative, Microbiological Test Data with the MPN Technique.

Microbial concentration in samples of a food product lot has been generally assumed to follow the log-normal distribution in food sampling, but this distribution cannot accommodate the concentration of zero. In the present study, first, a probabilistic study with the most probable number (MPN) technique was done for a target microbe present at a low (or zero) concentration in food products. Namely, based on the number of target pathogen-positive samples in the total samples of a product found by a qualitative, microbiological examination, the concentration of the pathogen in the product was estimated by means of the MPN technique. The effects of the sample size and the total sample number of a product were then examined. Second, operating characteristic (OC) curves for the concentration of a target microbe in a product lot were generated on the assumption that the concentration of a target microbe could be expressed with the Poisson distribution. OC curves for Salmonella and Cronobacter sakazakii in powdered formulae for infants and young children were successfully generated. The present study suggested that the MPN technique and the Poisson distribution would be useful for qualitative microbiological test data analysis for a target microbe whose concentration in a lot is expected to be low.

Degradation of staphylococcal enterotoxin A by a Pseudomonas aeruginosa isolate from raw milk.

Recently, we found that staphylococcal enterotoxin A (SEA)-producing Staphylococcus aureus strains produced SEA in raw milk with microbial contaminants at high temperatures like 40 °C only. Moreover, the concentration of SEA produced in raw milk gradually decreased after the peak. The reason(s) for SEA degradation in raw milk was studied in this study. Degradation of SEA spiked in raw milk was observed at 40 °C, but not at 25 °C. A Pseudomonas aeruginosa isolate from raw milk degraded SEA spiked in broth at 40 °C. A sample partially purified with a chromatographic method from culture supernatant of the isolate degraded SEA. Two main proteolytic bands were observed in the sample by zymographic analysis with casein. These results suggested that the SEA in raw milk might be degraded by a protease(s) produced by the P. aeruginosa isolate. This finding might be the first report on SEA degradation by a proteolytic enzyme(s) derived from Pseudomonas bacteria to our knowledge.

Prediction of Competitive Microbial Growth.

　Prediction of competitive microbial growth is becoming important for microbial food safety. There would be two approaches to predict competitive microbial growth with mathematical models. The first approach is the development of a growth model for competitive microbes. Among several candidates for the competition model considered, the combination of the primary growth model of the new logistic (NL) model and the competition model of the Lotka-Vorttera (LV) model showed the best performance in predicting microbial competitive growth in the mixed culture of two species. This system further successfully predicted the growth of three competitive species in mixed culture. The second approach is the application of the secondary model especially for the parameter of the maximum cell population in the primary growth model. The combination of the NL model and a polynomial model for the maximum population successfully predicted Salmonella growth in raw ground beef. This system further successfully predicted Salmonella growth in beef at various initial concentrations and temperatures. The first approach requires microbial growth data in monoculture for analysis. The second approach to the prediction of competitive growth from the viewpoint of microbial food safety would be more suitable for practical application.

The Growth Kinetics of Salmonella Enteritidis in Raw Ground Beef.

The growth kinetics of Salmonella Enteritidis in raw beef has been little studied so far. Thus, this study aimed to clarify the growth kinetics of the pathogen in ground beef using a growth model. When Salmonella cells inoculated at various initial doses into ground beef were incubated at a given temperature (24℃), the maximum population (Nmax) of the microbe at the stationary phase varied with the doses. This relationship was expressed with a polynomialequation for Nmax using the initial dose. The combination of the growth model and the polynomial equation successfully predicted Salmonella growth at a given initial dose. When Salmonella cells inoculated in ground beef were incubated at various constant temperatures, the growth curves of the pathogen and natural microflora (NM) were well described with the growth model. The rate constant of growth and the Nmax values for Salmonella and NM were then analyzed kinetically. From these results, growth curves of Salmonella and NM in ground beef stored at dynamic temperatures were successfully predicted. Competition between Salmonella and NM in ground beef was also found during the storage. This study could give usable information on the growth of Salmonella and NM in ground beef at various temperatures.

Prediction of the Growth of Salmonella Enteritidis in Raw Ground Beef at Various Combinations of the Initial Concentration of the Pathogen and Temperature.

Recently we clarified the growth kinetics of Salmonella Enteritidis in raw ground beef at various temperatures with our growth model. Based on those results, this study aimed to build a new methodology to predict the growth of Salmonella in ground beef at given initial concentrations of the pathogen and temperatures. Namely, the maximum cell population of Salmonella at various combinations of its initial concentration and temperature was developed with a polynomial equation. The rate constants of Salmonella growth at various temperatures were estimated with the square root model studied in our recent study. A new system consisting of our growth model, the polynomial equation, and the square root model successfully predicted the growth of Salmonella inoculated at given concentrations in beef at constant and dynamic temperatures. The growth of natural microflora in beef at those temperature patterns were also successfully predicted with the growth model.

Urethra-sparing high-intensity focused ultrasound for localized prostate cancer: Functional and oncological outcomes.

OBJECTIVES: To evaluate longitudinal changes in urinary function and quality of life, and the oncological outcomes of patients treated with urethra-sparing high-intensity focused ultrasound for localized prostate cancer. METHODS: Patients with negative findings in the urethra and the anterior urethral zone using transrectal ultrasound-guided targeted biopsies, and magnetic resonance imaging, received urethra-sparing or whole-gland high-intensity focused ultrasound as the primary therapy for localized prostate cancer without transurethral resection of the prostate. Longitudinal changes in urinary function and quality of life, and the oncological outcomes of the patients were analyzed retrospectively. RESULTS: The median follow-up times for urethra-sparing and whole-gland high-intensity focused ultrasound were 36 and 30 months, respectively. Comparing the patients treated with urethra-sparing high-intensity focused ultrasound (n = 45) with those treated with whole-gland high-intensity focused ultrasound (n = 65), there were significant differences in the International Prostate Symptom Score (P = 0.014) at 3 months, International Prostate Symptom Score quality of life (P = 0.033) at 3 months, maximum urinary flow rate (mL/s; at 3 months, P = 0.010; at 6 months, P = 0.038) and residual urine volume (mL; at 3 months, P < 0.0001; at 6 months, P = 0.016; at 12 months, P = 0.028). For quality of life, there were significant differences in Functional Assessment of Cancer Therapy - General (at 3 months, P = 0.022) and Functional Assessment of Cancer Therapy - Prostate (at 3 months, P = 0.028; at 6 months, P = 0.034). There were no significant differences in oncological outcomes regarding negative biopsy rates on follow up (91% vs 92%; P = 0.8) or biochemical disease-free survival rates (86.7% vs 89.2%; P = 0.7). CONCLUSIONS: Urethra-sparing high-intensity focused ultrasound might prevent prolonged bladder outlet obstruction, and could be a treatment option for localized prostate cancer.

Morphological analysis of the effects of intraoperative transrectal compression of the prostate during high-intensity focused ultrasound for localized prostate cancer.

OBJECTIVES: To evaluate the effects of transrectal compression of the prostate for intra-operative prostatic swelling and intraprostatic point shift during high-intensity focused ultrasound treatment of localized prostate cancer. METHODS: Patients treated with whole-gland high-intensity focused ultrasound as primary monotherapy for localized prostate cancer were enrolled in the study. Using the standard and compression method, the volumes of degassed water in the balloon covering the high-intensity focused ultrasound probe were 50 mL and 80-160 mL, respectively. To identify prostatic swelling and shift during high-intensity focused ultrasound and the volume occupied by the non-enhanced area, three-dimensional prostate models were reconstructed using ultrasound and contrast-enhanced magnetic resonance imaging. RESULTS: In comparison with the standard (n = 40) and compression (n = 48) methods, intraoperative increase in the prostate volume (21% vs 5.3%; P = 0.044), intraprostatic point shift (4 mm vs 2 mm, P = 0.040 in the transition zone; 3 mm vs 0 mm; P = 0.001 in the peripheral zone) and the volume occupied by the non-enhanced area (89% vs 96%; P = 0.001) were significantly suppressed. The biochemical disease-free survival rate in patients treated using the compression method was significantly improved relative to the standard method (92.6% vs 76.5%; P = 0.038). Regarding complications, there was no significant difference in the rate of urethral stricture (P = 0.9), urinary tract infection (P = 0.9), incontinence (P = 0.3), erectile dysfunction (P = 0.9) or recto-urethral fistula between the patients treated using the standard and compression methods. CONCLUSIONS: Intraoperative transrectal compression suppresses intraoperative increase in the prostate volume and intraprostatic point shift during high-intensity focused ultrasound, having the potential to achieve precise whole-gland and lesion-targeted focal therapy.

Prediction of competitive microbial growth in mixed culture at dynamic temperature patterns.

A novel competition model developed with the new logistic model and the Lotka-Volterra model successfully predicted the growth of bacteria in mixed culture using the mesophiles Staphylococcus aureus, Escherichia coli, and Salmonella at a constant temperature in our previous studies. In this study, we further studied the prediction of the growth of those bacteria in mixed culture at dynamic temperatures with various initial populations with the competition model. First, we studied the growth kinetics of the species in a monoculture at various constant temperatures ranging from 16℃ to 32℃. With the analyzed data in the monoculture, we then examined the prediction of bacterial growth in mixed culture with two and three species. The growth of the bacteria in the mixed culture at dynamic temperatures was successfully predicted with the model. The residuals between the observed and predicted populations at the data points were <0.5 log at most points, being 83.3% and 84.2% for the two-species mixture and the three-species mixture, respectively. The present study showed that the model could be applied to the competitive growth in mixed culture at dynamic temperature patterns.