Genotypic Characterization of Methicillin-resistant Staphylococcus aureus Isolated from Pigs and Retail Foods in China.

OBJECTIVE: To investigate the genotypic diversity of Methicillin-resistant Staphylococcus aureus (MRSA) isolated from pigs and retail foods from different geographical areas in China and further to study the routes and rates of transmission of this pathogen from animals to food. METHODS: Seventy-one MRSA isolates were obtained from pigs and retail foods and then characterized by multi-locus sequencing typing (MLST), spa typing, multiple-locus variable number of tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. RESULTS: All isolated MRSA exhibited multi-drug resistance (MDR). Greater diversity was found in food-associated MRSA (7 STs, 8 spa types, and 10 MLVA patterns) compared to pig-associated MRSA (3 STs, 1 spa type, and 6 MLVA patterns). PFGE patterns were more diverse for pig-associated MRSA than those of food-associated isolates (40 vs. 11 pulse types). Among the pig-associated isolates, CC9-ST9-t899-MC2236 was the most prevalent clone (96.4%), and CC9-ST9-t437-MC621 (20.0%) was the predominant clone among the food-associated isolates. The CC9-ST9 isolates showed significantly higher antimicrobial resistance than other clones. Interestingly, CC398-ST398-t034 clone was identified from both pig- and food-associated isolates. Of note, some community- and hospital-associated MRSA strains (t030, t172, t1244, and t4549) were also identified as food-associated isolates. CONCLUSION: CC9-ST9-t899-MC2236-MDR was the most predominant clone in pigs, but significant genetic diversity was observed in food-associated MRSA. Our results demonstrate the great need for improved surveillance of MRSA in livestock and food and effective prevention strategies to limit MDR-MRSA infections in China.

Molecular and Epidemiological Characterization of Infant Botulism in Beijing, China.

Laboratory-based pathogen isolation, identification, and toxicity determination were performed on samples from a suspected case of infant botulism. Mice injected with cultures generated from the enema sample and ingested Powered infant formula (PIF) presented typical signs of botulism. Antitoxins to polyvalent botulinum neurotoxins (BoNTs) and monovalent BoNT type B antitoxin had protective effects. Clostridium botulinum isolated from the enema and residual PIF samples were positive for type B toxin. Pulsed-field gel electrophoresis (PFGE) revealed that the two strains of C. botulinum isolated from the two samples produced indistinguishable pulsotypes. These findings confirmed this case of type B infant botulism associated with the ingestion of PIF contaminated by type B C. botulinum spores.

Antimicrobial Resistance, Virulence Profile, and Molecular Characterization of Listeria monocytogenes Isolated from Ready-to-eat Food in China, 2013-2014.

We aimed to investigate the potential pathogenic profile and antibiotic resistance of Listeria monocytogenes isolated from ready-to-eat food in China. Antimicrobial resistance was determined by broth microdilution following the Clinical and Laboratory Standards Institute protocol. Molecular serotyping, virulence, and resistance genes were identified using PCR. Multi-locus sequence typing was performed on resistant strains. A total of 11.53% (113/980) isolates were resistant, from which 82.3% (93/113) harbored all the virulence genes tested. The resistant strains were subtyped into 18 sequence types (STs), from which ST2, ST5, ST8, and ST9 were involved in listeriosis. This study indicated that several L. monocytogenes isolates from ready-to-eat foods in China have pathogenic potential and are resistant to antibiotics, including antibiotics used as medicines by humans for listeriosis treatment.

[Phenylpropanoid Constituents of Smilax trinervula].

Objective: To study the phenylpropanoid constituents of Smilax trinervula. Methods: The chemical constituents were isolated and purified by macroporous adsorption resin chromatography, gel chromatography and high performance liquid chromatography. Their structures were identified by spectroscopic analysis and comparison with literatures. Results: Nine phenylpropanoid compounds were isolated and their structures were identified as( +)-lyoniresin-4-yl ß-D-glucopyranoside( 1),(-)-8'-epilyoniresin-4-yl ß-glucopyranoside( 2),( +)-lyoniresin-4'-yl ß-glucopyranoside( 3),(-)-lyoniresinol-2α-O-ß-D-glucopyranoside( 4),( +)-lyoniresinol( 5),icariol A2( 6),icariol A2-4-O-ß-D-glucopyranoside( 7),7S,7'S,8R,8'R-icariol A2-9-O-ß-D-glucopyranoside( 8) and( +)-syringaresinol-4'-O-ß-D-glucopyranoside( 9). Conclusion: All the compounds are isolated from Smilax genus for the first time.

Modification and evaluation of Brucella broth based Campylobacter jejuni transport medium.

Reliable transport of Campylobacter jejuni isolates is critical to microbial epidemiology research, especially in developing countries without a good temperature control mailing system. Various factors, including oxygen, temperature, transport medium composition, could affect the survival of C. jejuni. In this study, the protective effects of different ingredients in C. jejuni transport media at 4 °C and 25 °C and under aerobic condition were quantitatively evaluated respectively. The results showed that enriched medium, supplementation with 5% blood and being kept at 4 °C could improve the viability of different C. jejuni strains during transport. In addition, supplementation with 25 mmol/L L-fucose in Wang's transport medium could significantly improve the survival of C. jejuni at both 4 °C and 25 °C. To the best of our knowledge, this is the first report to evaluate the protective effect of L-fucose in enriched C. jejuni transport medium which is feasible in developing countries without an effective cold chain mailing system. These data will be good reference for C. jejuni transport medium improvement in future.

Progress of nanopreparation technology applied to volatile oil drug delivery systems.

Traditional Chinese medicine volatile oil has a long history and possesses extensive pharmacological activity. However, volatile oils have characteristics such as strong volatility, poor water solubility, low bioavailability, and poor targeting, which limit their application. The use of volatile oil nano drug delivery systems can effectively improve the drawbacks of volatile oils, enhance their bioavailability and chemical stability, and reduce their volatility and toxicity. This article first introduces the limitations of the components of traditional Chinese medicine volatile oils, discusses the main classifications and latest developments of volatile oil nano formulations, and briefly describes the preparation methods of traditional Chinese medicine volatile oil nano formulations. Secondly, the limitations of nano formulation technology are discussed, along with future challenges and prospects. A deeper understanding of the role of nanotechnology in traditional Chinese medicine volatile oils will contribute to the modernization of volatile oils and broaden their application value.

Characterization of Staphylococcus aureus isolated from clinical specimens by matrix assisted laser desorption/ionization time-of-flight mass spectrometry.

OBJECTIVE: To develop a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approach to identify Staphylococcus aureus (S. aureus) and differentiate methicillin-resistant S. aureus (MRSA) from methicillin-sensitive S. aureus (MSSA). METHODS: A total of 100 S. aureus strains isolated from clinical specimens and farm workers were collected and analyzed by MALDI-TOF-MS. And data obtained were interpreted with biotyper software. RESULTS: Ninety-two strains were identified by MALDI-TOF-MS as S. aureus at a level of secure genus and probable species, and 4 strains were identified at probable genus after their cultivation, spectral collection and data preprocessing. One strain was identified as S. aureus with lower score. It was revealed that identification of S. aureus by MALDI-TOF-MS was highly correlated with typing by biochemical and serological methods with an accuracy as high as 97%. The biotyper cluster analysis showed that 100 isolates were divided into 2 types at the distance level of 400. Higher peak intensity in the mass of both 3784 Da and 5700 Da was observed in MRSA, whereas that was absent from MSSA. CONCLUSION: MALDI-TOF-MS is considered a simple, rapid and highly reproducible technique with high-throughput and accuracy for the identification of S. aureus and it can reliably differentiate MRSA from MSSA.

First report on identification and genomic analysis of a novel porcine circovirus (porcine circovirus 4) in cats.

Porcine circovirus type 4 (PCV4) is an emerging circovirus, which has been detected in domestic pigs across various provinces in China and Korea. In this study, we aimed to investigate whether cats are susceptible to PCV4. For this purpose, we collected 116 cat samples from animal hospitals in Sichuan Province, China, between 2021 and 2022. Using a SYBR Green-based real-time PCR assay, we detected PCV4 in 5 out of the 116 clinical samples, indicating a positive rate of 4.31% (5/116) and confirming the presence of PCV4 in cats from Sichuan Province, China. Moreover, we successfully sequenced and analyzed the complete genome of one PCV4 strain (SCGA-Cat) along with 60 reference sequences deposited in the GenBank database. SCGA-Cat exhibited high nucleotide homology (98.2-99.0%) with PCV4 strains from other species, including dogs, pigs, dairy cows, and fur animals. Notably, the SCGA-Cat strain from cats clustered closely with a PCV4 strain derived from a pig collected in Fujian Province, China. To the best of our knowledge, this study represents the first report on the molecular detection of PCV4 in cats worldwide, which prompted us to understand the genetic diversity and cross-species transmission of the ongoing PCV4 cases. However, further investigations are needed to explore the association between PCV4 infection and clinical syndromes in cats.

[Development of an in vitro digestion model for assessing the bioaccessibility of aflatoxin B group in foods].

OBJECTIVE: To develop and validate an in vitro digestion model for assessing the bioaccessibilities of some important mycotoxins of aflatoxin B group (aflatoxin B(1) and aflatoxin B(2), AFB(1) and AFB(2)). METHODS: Using simulating gastrointestinal physiological digestion process, the effects of digestion time (long, medium and short), the fasting and feeding status (fasting, between fasting and semi-feeding, semi-feeding, between semi-feeding and feeding, feeding states), the volume and pH (high, medium and low) of digestive solution, as well as other food ingredients ingested along with aflatoxin B group from mixed foods on bioaccessiblities of AFB(1) and AFB(2) in the mouth, stomach and small intestine were studied. The optimal technical parameters of the model were identified and the model was validated with mycotoxin adsorbents. RESULTS: The optimal conditions of AFB(1) releasing from the ingested foods at the highest concentration in gastrointestinal tract were as follows: digestion time of 6 min, 1.5 h and 2.5 h in mouth, stomach and duodenum, respectively; the optimal pH values of 1.1 and 7.5 for gastric juice and duodenal fluid; the volume of 7, 13, 12 and 6 ml for saliva, gastric juice, intestinal fluid and bile, respectively; the optimal conditions of AFB(2) releasing from the ingested foods at the highest concentration in gastrointestinal tract were as follows: digestion time of 6 min, 2.5 h and 2.5 h in mouth, stomach and duodenum, respectively; the optimal pH values of 1.1 and 7.8 for gastric juice and duodenal fluid; the volume of 5, 12, 13 and 6 ml for saliva, gastric juice, intestinal fluid and bile, respectively. The bioaccessibilities of both AFB(1) and AFB(2) were highest at the fasting state (83.1% and 89.3% respectively). The bioaccessibilities decreased with the increasing of stomach contents, but the changes in bioaccessibility were not significant when the stomach contents reached the semi-feeding state or more. From semi-feeding to feeding state, the biocessibilities of AFB(1) decreased from 72.8% to 71.5% and AFB(2) decreased from 78.3% to 76.9%. Chlorophyll and activated charcoal were the strongest absorbent in reducing the bioaccessibilities of AFB(1) and AFB(2), and the bioaccessibilities decreased to 0.8% and 1.3% respectively. CONCLUSION: The in vitro digestion model developed in the present study is stable and reproducible, and meets the requirements for assessing the bioaccessibilities of AFB(1) and AFB(2) in foods.

Dietary iodine intake in the Chinese population.

OBJECTIVE: To evaluate dietary iodine intake and its potential risks among the Chinese population. METHODS: Individual dietary iodine intake was calculated using food consumption data multiplying by iodine concentration in foods, table salt and drinking water, followed by summing, and then compared with the corresponding age-specific reference values, including Upper Intake Level (UL) and Recommended Nutrient Intake (RNI). RESULTS: In areas with water iodine concentration (WI) lower than 150 µg/L, 80.8% of residents had iodine intake between the RNI and UL, 5.8% higher than UL, and the remaining (13.4%) lower than RNI if iodized salt was consumed. However, in the uniodized salt consumption scenario, only 1.0% of residents between RNI and UL, 1.4% higher than UL, and a large part of residents (97.6%) lower than RNI. In areas with WI higher than 150 µg/L, all residents had iodine intake between RNI and UL if iodized salt was consumed, except 10.5% and 24.9% of residents higher than UL in areas with WI at 150-300 µg/L and higher than 300 µg/L respectively. However, in the uniodized salt consumption scenario, only 1.5% and 1.7% of residents had higher iodine intake than UL respectively. CONCLUSION: The findings suggested that in general, the dietary iodine intake by the Chinese population was appropriate and safe at the present stage. People in areas with WI lower than 150 µg/L were more likely to have iodine deficiency. While people in areas with WI higher than 150 µg/L were more likely to have excessive iodine intake if iodized salt was consumed.

[Natural occurrence of masked deoxynivalenol and multi-mycotoxins in cereals from China harvested in 2007 and 2008].

OBJECTIVE: To elucidate the natural occurrence of masked deoxynivalenol (DON-3-G) and other multi-mycotoxins in cereals from parts of China. METHODS: A total of 446 corn and wheat samples harvested in 2007 and 2008 collected from Henan, Hebei, Guangxi, Anhui, Sichuan, Chongqing and Jiangsu provinces were analyzed for DON-3-G and other multi-mycotoxins (including deoxynivalenol (DON), zearalenone (ZEN), nivalenol (NIV), et al) by UPLC-MS/MS. RESULTS: Corn and wheat samples were mainly contaminated by DON and its derivatives as well as ZEN.88% (169/192) of wheat samples were positive for DON (range: 1.5 - 590.7 µg/kg; median: 30.8 µg/kg); 22.9% (44/192) of wheat samples were contaminated with ZEN (range: 1.7 - 3425.0 µg/kg; median: 8.0 µg/kg) and six samples contained ZEN concentration higher than the ZEN tolerance limit of 60 µg/kg. DON was detected in 50.5% (103/204) corn samples (range: 1.6 - 4374.4 µg/kg; median: 94.9 µg/kg); Seven samples contained DON exceeding the tolerance limit of 1000 µg/kg for DON. Additionally, ZEN was found in 41.7% (85/204) corn samples with the concentration between 1.6 µg/kg and 4808.7 µg/kg (median: 48.5 µg/kg) and there were 37 corn samples with ZEN level in the excess of tolerance limit for ZEN (60 µg/kg). DON-3-G was detected in corn and wheat samples for the first time in China with the median level of 21.4 µg/kg and 34.6 µg/kg for wheat and corn, respectively. Wheat was more heavily contaminated with DON-3-G than both 3-acetyl-DON (3-A-DON, median: 4.1 µg/kg) and 15-acetyl-DON (15-A-DON, median: 3.1 µg/kg) (t values were 5.111 and 5.966, respectively, both P values < 0.01). While, the level of 15-A-DON (median: 48.6 µg/kg) in corn was higher than 3-A-DON (median: 6.8 µg/kg) (t = -3.579, P < 0.01). The concentration of DON, DON-3-G, 3-A-DON, 15-A-DON and ZEN in corn were higher than that in wheat (Z values were -3.492, -1.960, -2.467, -8.711 and -6.272, respectively, all P values < 0.05). Wheat (median: 29.0 µg/kg) contained higher NIV in comparison with corn (median: 18.2 µg/kg, Z = -2.086, P < 0.05). CONCLUSION: Wheat and corn samples from parts of China were contaminated with multi-mycotoxins and DON was the predominant;in comparison of wheat, corn was more heavily contaminated with DON, DON-3-G, 3-A-DON, 15-A-DON and ZEN.

[Development of pulsed field gel electrophoresis and application for characterization and identification of Lactobacillus and Streptococcus thermophilus].

OBJECTIVE: To develop a pulsed field gel electrophoresis (PFGE) method for molecular typing of Lactobacillus and Streptococcus thermophilus (S. thermophilus) and to apply it in identification and characterization of both bacteria isolated from yoghurt collected from Beijing supermarket. METHODS: The five most useful restriction enzymes including Apa I, Not I, Sfi I, Xba I and Sma I were chosen to cut DNA of 52 strains of Lactobacillus, S. thermophilus as well as associated standard bacteria strains. The endonucleases and electrophoresis conditions for PFGE analysis were optimized and applied in molecular typing of Lactobacillus and S.thermophilus isolates. Cluster analysis based on the PFGE data was conducted. The identification results of PFGE were compared with those obtained in biochemical and 16s ribosomal RNA PCR identification tests. RESULTS: Not I was suitable for L. bulgaricus, L. fermentum and L. delbrueckii digestion. While Apa I was an appropriate endonuclease for S. thermophilus, L. acidophilus and L. casei digestion. The results of molecular typing indicated that 24 strains of L.bulgaricus and 15 strains of S. thermophilus were grouped into 8 types by PFGE method, respectively. While 7 strains of L.acidophilus were grouped into 3 types and 2 strains of L. delbrueckii were grouped into 2 different PFGE types. CONCLUSION: The results of PFGE analysis are in compliance with those of 16s rRNA PCR and biochemical identification. The PFGE method developed in this study is suitable for molecular characterization of both Lactobacillus and S. thermophilus.

Quantification of Cross-Contamination of Campylobacter jejuni during Food Preparation in a Model Kitchen in China.

ABSTRACT: Numerous outbreak investigations and case-control studies of campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a high risk factor for infection and illness. In this study, the cross-contamination and transfer rates of Campylobacter jejuni from chicken to ready-to-eat food were determined in various food handling scenarios. Skinless raw chicken breasts were artificially contaminated with C. jejuni and diced on cutting boards of three different materials. Whether cold water, cold water with detergent, or hot water was used, statistically significant differences were found between the transfer rates of C. jejuni to unwashed and washed cutting boards or hands, respectively. When both kitchen knife and cutting board were reused after dicing the artificially contaminated chicken, the transfer rates of C. jejuni to cucumber cut on bamboo, wooden, and plastic cutting boards were 16.28, 12.82, and 5.32%, respectively. The transfer rates from chicken to bread, a large lift-up water faucet handle, and a small twist faucet handle via unwashed hands were 0.49, 4.64, and 3.14%, respectively. This research provides scientific evidence that various types of contaminated kitchenware and cook's hands are vital potential vehicles for the cross-contamination of Campylobacter from raw chicken to ready-to-eat food and emphasizes the importance of timely and proper cleaning to prevent cross-contamination during food handling; therefore, high-quality consumer education to reduce the risk of foodborne infection is urgent and necessary.

Distinguishing Smilax glabra and Smilax china rhizomes by flow-injection mass spectrometry combined with principal component analysis.

Flow-injection mass spectrometry (FIMS) coupled with a chemometric method is proposed in this study to profile and distinguish between rhizomes of Smilax glabra (S. glabra) and Smilax china (S. china). The proposed method employed an electrospray-time-of-flight MS. The MS fingerprints were analyzed using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) with the aid of SIMCA software. Findings showed that the two kinds of samples perfectly fell into their own classes. Further predictive study showed desirable predictability and the tested samples were successfully and reliably identified. The study demonstrated that the proposed method could serve as a powerful tool for distinguishing between S. glabra and S. china.

[Study on maximum limits for Salmonella in milk and milk products].

At present, the Chinese microbiological maximum levels for foods are different from several countries of the world. As China joined in the WTO, such levels, on the one hand, are not helpful for quality control and the protection for health, on the other, they are also not benefit to the export. To give information for revising the Chinese microbiological maximum levels for foods, this article took example for the Salmonella maximum levels in milk and milk products and all factors associated with their establishment were reviewed.

[Development of monoclonal antibody against fumonisin B1 and ELISA-kit of quantitative analysis for fumonisin B1].

OBJECTIVE: To detect the contamination level of fumonisin B1 in grain, and to develop rapid detection kit that possess patent of China. METHODS: Hybridoma cell line excreting monoclonal antibody against fumonisin B, was produced using B cell hybridoma technique and develop a rapid, sensitive, quantitative ELISA-kit for detection fumonisin B1. RESULTS: The monoclonal antibody used in the ELISA-kit were tested for subtype as IgG1 and its affinity constant was 8.3 x 10(-8) mol/L. The monoclonal antibody obtained in the present study was highly specific to fumonisin B1 , because no cross reactions between the monoclonal antibodies against fumonisin B1 with the analogues of fumonisin B, were found. The limited concentration of the ELISA-kit was 5 ng/ml, linear range was 50-500 ng/ml, the linear equation was Y = -0.582 x +1.793( = 0.99, P < 0.05). The recovery rate of maize on the level of 50n g/ml, 200 ng/ml and 500 ng/ml was among 71.89%-112.95%. The kit can be stored at normal tempertature in ten months at least. The coefficient of variant winthin-laboratory and between-laboratory was less than 20%.

Relationship between Intrauterine Bacterial Infection and Early Embryonic Developmental Arrest.

BACKGROUND: Early embryonic developmental arrest is the most commonly understudied adverse outcome of pregnancy. The relevance of intrauterine infection to spontaneous embryonic death is rarely studied and remains unclear. This study aimed to investigate the relationship between intrauterine bacterial infection and early embryonic developmental arrest. METHODS: Embryonic chorion tissue and uterine swabs for bacterial detection were obtained from 33 patients who underwent artificial abortion (control group) and from 45 patients who displayed early embryonic developmental arrest (trial group). RESULTS: Intrauterine bacterial infection was discovered in both groups. The infection rate was 24.44% (11/45) in the early embryonic developmental arrest group and 9.09% (3/33) in the artificial abortion group. Classification analysis revealed that the highest detection rate for Micrococcus luteus in the early embryonic developmental arrest group was 13.33% (6/45), and none was detected in the artificial abortion group. M. luteus infection was significantly different between the groups (P < 0.05 as shown by Fisher's exact test). In addition, no correlation was found between intrauterine bacterial infection and history of early embryonic developmental arrest. CONCLUSIONS: M. luteus infection is related to early embryonic developmental arrest and might be one of its causative factors.

Risk assessment of dietary exposure to aluminium in the Chinese population.

In order to address the issue of excessive intake of aluminium (Al) from Al-containing food additives in the Chinese diet, this study conducted a dietary exposure assessment of Al in the general population based on the national surveillance data of Al content in foods and national food consumption data. It was found that the mean dietary exposure of the whole Chinese population to Al from Al-containing food additives was 1.795 mg kg‒1 bw week‒1, not exceeding the PTWI, while high dietary exposures (e.g., 97.5th percentile) to Al were 7.660 and 2.103-2.903 mg kg‒1 bw week‒1 for children, respectively, both exceeding the PTWI. It was found that the dietary exposure to Al for 32.5% of the total Chinese population and 42.6% of children aged 4-6 years exceeded the PTWI. Wheat flour and wheat-based products are the main source of dietary A l exposure (85% of the total intake); and puffed foods are the major source of Al intake for children. These findings suggested that consumption of Al-containing food additives could be a health concern for consumers with high food consumption (97.5th percentile) and children under the age of 14 years.

[Natural occurrence of citrinin in Monascus products].

OBJECTIVE: In order to survey the natural occurrence of citrinin in Monascus products. METHODS: A total of 114 samples with either solid or liquid phases, collected from markets or delivered by the food production factories were analyzed by HPLC for citrinin. RESULTS: The results revealed that 68 (59.65%) samples were positive for citrinin with the levels between 0.18 and 1739.23 mg/kg (211.61 mg/kg for the average and 6.62 mg/kg for the median, respectively). The concentration of cirtrinin in various Monascus products is different. Twenty-five red pigment samples of Monascus were heavily contaminated with citrinin at the level ranged from 0.85 - 1739.93 mg/kg (average 508.40 mg/kg, median 169.88 mg/kg). Twelve of 19 red rice samples fermented by Monoscus species were not citrinin-free but 83.33% (10/12) with the citrinin levels below 10 mg/kg. The level of citrinin in 84% (21/25) health meals of Monascus is less than 6 mg/kg. CONCLUSION: The domestic Monascus products were contaminated by citrinin and the ratio of color value/citrinin in 15 (13.16%) samples exceeds the Japanese national standard.