Identification of Enterococcus spp. from food sources by matrix-assisted laser desorption ionization-time of flight mass spectrometry and characterization of virulence factors, antibiotic resistance, and biofilm formation.

The continuous detection of multi-drug-resistant enterococci in food source environments has aroused widespread concern. In this study, 198 samples from chicken products, animal feces, raw milk, and vegetables were collected in Japan and Egypt to investigate the prevalence of enterococci and virulence characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for species identification and taxonomic analysis of the isolates. The results showed that the rates of most virulence genes (efaA, gelE, asa1, ace, and hyl) in the Japanese isolates were slightly higher than those in the Egyptian isolates. The rate of efaA was the highest (94.9 %) among seven virulence genes detected, but the cylA gene was not detected in all isolates, which was in accordance with γ-type hemolysis phenotype. In Enterococcus faecalis, the rate of kanamycin-resistant strains was the highest (84.75 %) among the antibiotics tested. Moreover, 78 % of E. faecalis strains exhibited multi-drug resistance. Four moderately vancomycin-resistant strains were found in Egyptian isolates, but none were found in Japanese isolates. MALDI-TOF MS analysis correctly identified 98.5 % (68/69) of the Enterococcus isolates. In the principal component analysis dendrogram, strains isolated from the same region with the same virulence characteristics and similar biofilm-forming abilities were characterized by clustered distribution in different clusters. This finding highlights the potential of MALDI-TOF MS for classifying E. faecalis strains from food sources.

Comparison of prevalence, characterization, antimicrobial resistance and pathogenicity of foodborne Listeria monocytogenes in recent 5 years in Japan.

This study investigated the prevalence, serotype, antimicrobial resistance (AMR), virulence potential, and biofilm formation of Listeria monocytogenes isolated in 2022 in Japan and compared their profiles with those of isolates in 2012 and 2017. A total of 85 chicken samples were randomly collected from different supermarkets in Fukuoka in 2022. L. monocytogenes were isolated by conventional method and characterized by MALDI-TOF MS. Among 85 samples tested in 2022, 9 (10.6%) were positive for L. monocytogenes and 17 strains were isolated from the positive samples. The isolates were serotyped as 1/2b (41.2%), 3a (29.4%), 3b (23.5%) and 1/2a (5.9%). Antimicrobial susceptibility tests of the 2022 isolates showed susceptibility to majority of the antibiotics, except cefoxitin, oxacillin, and fosfomycin. Compared to the previous surveillance results, the prevalence of L. monocytogenes in 2022 (10.6%) was significantly lower (p < 0.05) than those of the isolates in 2017 (24%) and 2012 (52.9%). The distribution of serotypes 1/2a and 1/2b decreased over time, and serotype 4b was not detected in the 2022 isolates. The proportion of multidrug resistant strains in 2022 (16.7%) was significantly lower than those in 2012 (46.7%) and 2017 (82.6%). Moreover, a total of 36 isolates (12 isolates/ year) were used to detect the virulence genes (hlyA, plcA, clpC, and inlA) and biofilm-forming capacity. Most of the isolates from different years harboured four virulence genes. The biofilm formation of the 2022 isolates was significantly weaker (p < 0.05) than those of the 2012 and 2017 isolates. Thus, despite the low rates of contamination in chicken meat and AMR of the isolates, virulent L. monocytogenes contamination in food should still be acknowledged.

Characterization and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from chicken and pork.

The prevalence and antimicrobial resistance (AMR) profile were investigated in Campylobacter jejuni and Campylobacter coli in chicken and pork in Fukuoka, Japan in 2019. Their AMR profiles were compared with those of C. jejuni and C. coli strains isolated in 2013. A total of 53 chicken and 14 pork samples were collected from different supermarkets in Fukuoka in 2019. Campylobacter spp. were isolated by conventional method and characterized by PCR and MALDI-TOF MS. Among 53 chicken samples tested in 2019, 24.5% and 5.7% were positive for C. jejuni and C. coli, respectively, and three (21.4%) of 14 pork samples were positive for C. coli, but not C. jejuni. From the positive samples, 13 and six strains of C. jejuni and C. coli were isolated, respectively. Antimicrobial susceptibility test against 12 different antimicrobials were performed on 48 isolates (43 C. jejuni and five C. coli) from chicken in 2013 and 19 isolates (13 C. jejuni from chicken, three C. coli from chicken and three C. coli from pork) in 2019 using the disk diffusion method. All the C. jejuni and C. coli isolated in 2013 and 2019 were highly resistant to cefazolin and sulfamethoxazole/trimethoprim. Among the C. jejuni isolates from chickens, 25.6% of 2013 isolates were resistant to nalidixic acid, ciprofloxacin, and levofloxacin, and 7% to ampicillin and minocycline, while 30.8% of the isolates were resistant to minocycline, 23.1% to nalidixic acid, ciprofloxacin, and levofloxacin, and 15.4% to ampicillin in 2019. Among the C. coli isolates, 80% of isolates from chickens in 2013, and 33.3% from chicken and 100% from pork in 2019 were resistant to nalidixic acid, ciprofloxacin, and levofloxacin. The frequency of multi-drug resistant (MDR) C. jejuni and C. coli strains from chickens in 2019 were 30.8% and 33.3%, respectively, which were lower than those isolated in 2013 (37.2% and 100%, respectively). One C. jejuni and two C. coli isolates from 2013 were resistant to six antibiotics. However, two C. jejuni and one C. coli isolate from chickens in 2019 were resistant to seven and five antibiotics, respectively. All the C. coli isolates from pork in 2019 were resistant to five antibiotics. The high frequency of AMR strains in C. coli isolates from pork suggests that appropriate use of antimicrobials is required in swine husbandry.

Morphological Changes in Spores during Germination in Bacillus cereus and Bacillus subtilis.

Processes from spore germination to outgrowth were observed in detail using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for Bacillus cereus and Bacillus subtilis. At 15 and 30 min after germination induction, SEM observation and SEM-EDX analysis of Bacillus spores prepared by freeze substitution showed that spherical structures including compounds having the same elemental ratio as that of the spore were observed on the surface of the spores. The results suggested the leakages of the cellular materials from the spores. At 360 min, B. cereus spores in outgrowth phase elongated with hemispherical structures at the end of the long side of the cells. The discoid structures with a hole (20-30 nm diameter) in the center was observed at 360 min. Confocal laser scanning microscopy after staining with fluorescence-labeled anti-spore antibodies showed that the hemispherical and discoid structures originated from the spore coat. These structures broke down after detached from the cells in outgrowth phase.

[Evaluation of Microbial Spoilage in PET Bottled Soft Drinks by Direct Drinking].

In order to evaluate microbial growth in opened PET bottled soft drinks, inoculation tests were carried out using type and reference strains of various microorganisms. Microorganisms were inoculated into a 500 mL PET bottle containing 250 mL of various soft drinks followed by incubation until 1 week at 4, 25, 35℃ without shaking, and 35℃ with shaking. The microbial counts were measured over time and compared with the results of the previous study "Studies on Contaminants in Soft Drink"2)-4). As a result, similar growth patterns were observed in the combination of tomato juice with Lactobacillus fermentum, sports drink with Candida albicans, and mineral water with Klebsiella pneumoniae. However, in green tea, mixed herb tea, orange juice and coffee with milk, the growth of microorganisms generally tended to be weaker than those of the previous studies. It was considered that components in the soft drinks inhibited the growth of the microorganisms. From the above results, the proliferative properties of type and reference strains in soft drinks were clearly different from the spoiled soft drinks isolates. The results in this study indicated that attention must be paid in the safety evaluation.

Baicalein Inhibits Stx1 and 2 of EHE: Effects of Baicalein on the Cytotoxicity, Production, and Secretion of Shiga Toxins of Enterohaemorrhagic Escherichia coli.

Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers.

Evaluation of repetitive-PCR and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid strain typing of Bacillus coagulans.

In order to establish rapid and accurate typing method for Bacillus coagulans strains which is important for controlling in some canned foods and tea-based beverages manufacturing because of the high-heat resistance of the spores and high tolerance of the vegetative cells to catechins and chemicals, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and repetitive-PCR (rep-PCR) were evaluated. For this purpose, 28 strains of B. coagulans obtained from various culture collections were tested. DNA sequence analyses of the genes encoding 16S rRNA and DNA gyrase classified the test strains into two and three groups, respectively, regardless of their phenotypes. Both MALDI-TOF MS and rep-PCR methods classified the test strains in great detail. Strains classified in each group showed similar phenotypes, such as carbohydrate utilization determined using API 50CH. In particular, the respective two pairs of strains which showed the same metabolic characteristic were classified into the same group by both MALDI-TOF MS and rep-PCR methods separating from the other strains. On the other hand, the other strains which have the different profiles of carbohydrate utilization were separated into different groups by these methods. These results suggested that the combination of MALDI-TOF MS and rep-PCR analyses was advantageous for the rapid and detailed typing of bacterial strains in respect to both phenotype and genotype.

Development of novel monoclonal antibodies directed against catechins for investigation of antibacterial mechanism of catechins.

Catechins are major polyphenolic compounds of green tea. To investigate mechanism for antibacterial action of catechins, 11 monoclonal antibodies (MAbs) were raised against a 3-succinyl-epicatechin (EC)-keyhole limpet hemocyanin (KLH) conjugate. Amino acid sequences of variable regions determined for MAbs b-1058, b-1565, and b-2106 confirmed their innovative character. MAb b-1058 strongly interacted with its target substances in the following order of magnitude: theaflavin-3,3'-di-O-gallate (TFDG)>theaflavin-3-O-gallate (TF3G)≥theaflavin-3'-O-gallate (TF3'G)>gallocatechin gallate (GCg)>penta-O-galloyl-ß-d-glucose (PGG)>epigallocatechin gallate (EGCg), as determined using surface plasmon resonance (SPR) on MAb-immobilized sensor chips. The affinity profiles of MAbs b-1058 and b-2106 to the various polyphenols tested suggested that flavan skeletons with both carbonyl oxygen and hydroxyl groups are important for this interaction to take place. S. aureus cells treated with EGCg showed green fluorescence around the cells after incubation with FITC-labeled MAb b-1058. The fluorescence intensity increased with increasing concentrations of EGCg. These MAbs are effective to investigate antibacterial mechanism of catechins and theaflavins.

Development of Rapid Identification and Risk Analysis of Moniliella Spp. in Acidic Processed Foods.

The number of spoilage incidents in the food industry attributable to a species of the genus Moniliella has recently increased, but the risk of food spoilage has not yet been evaluated. The purpose of this study was to develop a method to rapidly identify high-risk species and to conduct a risk analysis study of Moniliella spp. Acetic acid resistance of M. acetoabutens and ethanol resistance of M. suaveolens were higher than for other Moniliella species. All examined strains of M. acetoabutens developed a high tolerance to acetic acid by being cultured twice in liquid media containing low concentrations of acetic acid. These findings indicate that M. acetoabutens and M. suaveolens are high-risk species for food spoilage and must be discriminated from other fungi. We developed species-specific primers to identify M. acetoabutens and M. suaveolens using the polymerase chain reaction (PCR) to amplify the D1/D2 domain of 28S rDNA. The PCR using the primer sets designed for M. acetoabutens (Mac_F1/R1) and M. suaveolens (Msu_F1/R1) was specific to the target species and did not detect other fungi involved in food spoilage or environmental contamination. This method is expected to be effective for the monitoring of raw materials and components of the food production process.

Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis.

Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.

Method for rapid detection and identification of chaetomium and evaluation of resistance to peracetic acid.

In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the ß-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

Method for identifying heat-resistant fungi of the genus Neosartorya.

Species of the genus Neosartorya are heat-resistant fungi that cause the spoilage of heat-processed acidic foods due to the formation of heat-resistant ascospores, and they produce mycotoxins, such as fumitremorgins and gliotoxin. Their anamorphs are phylogenetically and morphologically very close to Aspergillus fumigatus, which has never been reported as a spoilage agent in heat-processed food products. Therefore it is important to discriminate between the species of Neosartorya and A. fumigatus in the food industry. In the present study, we examined ß-tubulin and calmodulin genes to identify Neosartorya and A. fumigatus at the species level and found a region for specifically detecting these species. We succeeded in developing the PCR method of differentiating and identifying Neosartorya and A. fumigatus using specific primer sets. Moreover, we developed specific primer sets to identify Neosartorya species, N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri, and N. spinosa-complex, which are important in food spoilage; these fungi vary in heat resistance and productivity of mycotoxins, depending on the species. PCR using these primer sets did not detect other fungi involved in food spoilage and environmental contamination. These identification methods are rapid and simple with extremely high specificity.

[Evaluation of the immunochromatography kit for detection of emetic-toxin producing Bacillus cereus].

We evaluated the sensitivity and specificity of an immunochromatography kit, Single-path Emetic Tox Mrk (Merck), which targets a marker protein for the detection of Bacillus cereus that produces emetic toxin. Strains were isolated after outbreaks of food poisoning, and from retail prepared foods and food products. The strains were examined for the presence of the emetic toxin-synthetase gene by PCR. All 58 emetic strains isolated from the food poisoning cases showed a positive reaction in the immunochromatography kit. No emetic strains gave false negative result. Among 47 non-emetic strains, only two strains isolated from the food poisonings and one strain isolated from food products showed a false positive reaction in the test. We concluded that this method has high sensitivity and specificity. The test can be used for detection of emetic toxin-producing B. cereus not only from food poisoning cases, but also in food products.

Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride.

We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy.

A rapid method for identifying Byssochlamys and Hamigera.

Heat-resistant fungi, genera Byssochlamys, Talaromyces, Neosartorya, and Hamigera, contribute significantly to the spoilage of heat-processed acidic foods, due to the formation of heat-resistant ascospores. Here, we first evaluated the differences in the beta-tubulin gene between Byssochlamys and Hamigera and developed specific primers to identify the Byssochlamys species fulva, nivea, and spectabilis, and Hamigera. Using primers designed for B. fulva and B. nivea (B1F/1R), specific PCR products were detected for B. fulva and B. nivea, as well as B. langunculariae and B. zollerniae, two closely related species. Similarly, the Pae4F/4R-1 and H2F/2R primers produced specific PCR products for B. spectabilis and Hamigera, respectively. Using these three primer sets, strains involved in acidic food spoilage and environmental contamination were not detected. The detection limits of all primer sets were 1 ng of DNA by PCR and 10 pg of DNA by nested PCR. Each PCR assay was specific, even if the sample was contaminated 1,000-fold by other fungal DNA. Thus, this method has proved to possess an extremely high degree of specificity.