Population genomics study of Vibrio alginolyticus.

Vibrio alginolyticus is a Gram-negative bacillus that causes vibriosis to human and aquatic products, including fish, shrimp and shellfish. It poses a threat to public health and causes enormous economic losses to the aquaculture industry. However, research on genetic diversity and pathogenicity-related genetic elements based on whole genome is still lacking. In this study, sixty-eight strains of V. alginolyticus were collected from four provinces of China and the whole genome sequences were obtained. Combined with 113 publicly available genome sequences downloaded from NCBI, we inferred the population structure of V. alginolyticus by using fineSTRUCTURE software, and identified the virulence and antibiotic resistance factors using the VFDB, CARD and ResFinder database. The results indicated that V. alginolyticus included two main lineages, named Lineage 1 and Lineage 2. Both lineages distributed in America and Asia, but all the European genomes were classified into Lineage 1. A single cross-ocean transmission event was inferred from one of the 12 identified clonal groups in our dataset. V. alginolyticus genome contains a variety of virulence factors, such as tlh, OmpU, and IlpA, etc. The distribution of virulence factors revealed no lineage-specificity, but some of which revealed differences in their geographical distribution. A lower frequency of VP1611, vcrD, vopD, fleR/flrC and a higher frequency of IlpA were observed in genomes of Europe than other continents. In China, a lower frequency of fleR/flrC, and no IlpA were observed in genomes from Guangxi province. Among the identified antibiotic resistance genes, TxR and fos are significantly enriched in Lineage 2. In addition, TxR is more common in genomes from Asia, compared with the American and European genomes. But in China, the frequency of TxR in Sichuan genomes is much lower than in other provinces. We also found that large fragments of plasmids or ICEs that carried multiple drug resistance genes were present in five V. alginolyticus genomes (VA24, VA28, 2014V-1011, ZJ-T and Vb1833). Based on population genomics analysis, our study delineated the population structure, distribution of virulence and antibiotic resistance related factors of V. alginolyticus, which lays a foundation for future study of genetic characters and pathogenesis mechanism of this pathogen and will improve the works on monitoring, prevention and control of this pathogen.

The Survey of Cronobacter spp. (formerly Enterbacter sakazakii) in Infant and Follow-up Powdered Formula in China in 2012.

OBJECTIVE: To determine Cronobacter spp. contamination in infant and follow-up powdered formula in China. METHODS: All of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, and analyzed for Cronobacter spp. by the Chinese National Food Safety Standard. Characterization of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE) with XbaI and SpeI restriction enzymes. RESULTS: Cronobacter spp. strains were isolated from 25 samples, and the positive rates in infant powdered formulas and follow-up powdered formulas were 0.90% (10/1011) and 1.18% (15/1271), respectively. Analysis of variable data regarding different purchasing store formats, seasonality, and production locations as well as comparison of infant versus follow-up formulas did not reveal statistically significant factors. During the sampling period, one of six surveillance zones did exhibit a statistically significant trend towards higher positive rate. PFGE characterization of Cronobacter spp. to elucidate genetic diversity revealed only three pairs of Cronobacter spp. out of 25 having the same PFGE patterns. CONCLUSION: The current investigation indicated a lower positive rate of Cronobacter spp. in the powdered formula in China. This evidence suggested contamination originating from multiple different sources during the manufacturing process.

[Prevalence and antibiogram distribution of Salmonella isolated from broiler production and processing course in four provinces, China].

OBJECTIVE: To determine the contamination condition of Salmonella in broiler breeding and slaughter processing in China and to investigate the distribution of antimicrobial resistance profiles. METHODS: Five large-scale broiler holdings and fourteen slaughterhouses were chosen to detect Salmonella in Henan, Jiangsu, Sichuan and Shandong provinces in 2010. A total of 835 anal swabs and 744 chicken carcasses were sampled to compare the difference of Salmonella contamination rate.Salmonella isolates were identified by serotyping according to Kauffmann-White scheme.The antimicrobial susceptibilities of Salmonella isolates were determined by broth microdilution method and sixteen antimicrobial agents were chosen and examined. RESULTS: In total, Salmonella isolates were recovered in 56 (6.7%) specimens among 835 collected anal swabs and 122 (16.4%) specimens among 744 broiler carcasses. Positive rate of Salmonella in broiler carcasses was higher than anal swabs (χ(2) = 36.94, P < 0.05). The dominant Salmonella serovars isolated from broiler anal swabs were S.enterica serovar Indiana and S.enterica serovar Enteritidis, accounting for 58.9% (33/56) and 32.1% (18/56) respectively. The prevalent serovars in broiler carcasses were also the two serovars and occupied 29.8% (37/124), 32.2% (40/124) respectively. Nearly 95.0% (171/180) Salmonella isolates were resistant to at least one antimicrobial, 78.3% (141/180) Salmonella strains were multi-drug resistant isolates and 20 (11.1%) Salmonella isolates were resistant to 14 antimicrobials. CONCLUSION: Our findings indicated that Salmonella contamination was common and serious in commercial broiler production and processing course in China. Salmonella contamination rate in broiler slaughter processing performance was higher than broiler flocks. Additionally, antibiotic resistance of Salmonella was in serious situation.

[Quantitative analysis of foodborne salmonella-the study of mini-modified semi solid rappaport vassiliadis most probable number method].

OBJECTIVE: To improve the mini-modified semi solid rappaport vassiliadis most probable number (mini-MSRV MPN) method for Salmonella detection. METHODS: Based on the mini-MSRV MPN method,Buffered Peptone Water (BPW) was modified as one step enrichment medium and Modified Semi Solid Rappaport Vassiliadis (MSRV) medium was ameliorated as modified MSRV for Salmonella detection under standard Salmonella addition recovery. A total of 154 raw chicken samples, 48 swabs of pheasantry and 48 poultry dung samples were collected to compare the detection results of Salmonella by using improved mini-MSRV MPN, mini-MSRV MPN and regular most probable number (MPN) method. RESULTS: Salmonella recovery was < 2.7 MPN/g when the standard Salmonella addition was at the concentration of 0.9 CFU/g when the mini-MSRV MPN method was employed. If the standard Salmonella addition were at 9.0 and 90.0 CFU/g, the recoveries of bacteria were 10.1 and 94.0 MPN/g, and the average recovery rate was 112% and 104%, respectively. Salmonella detection rate of modified mini-MSRV MPN, mini-MSRV MPN and regular MPN method was 18.4% (46/250), 5.2% (13/250) and 6.0% (15/250), respectively. The detection rate was higher for modified mini-MSRV MPN method than of the other two methods (χ(2) values were 19.68 and 17.82, respectively, all P values < 0.05). The detection quantity of Salmonella (medians were 21.0, < 2.7 and < 3.0 MPN/g, respectively). The quantity detected by modified mini-MSRV MPN method was higher than that of the other two methods (both Z values were 5.71, both P values < 0.05). CONCLUSION: Modified mini-MSRV MPN method is an accurate method for foodborne Salmonella detection.

Prevalence and risk assessment of Campylobacter jejuni in chicken in China.

OBJECTIVE: To understand the occurrence and distribution of Campylobacter jejuni in chicken in China, assess its health risk to the Chinese population, and provide recommendations for effective risk control. METHODS: Data from the National Food Safety Risk Surveillance Network on Campylobacter jejuni between 2007 and 2010 and from published articles were analyzed. Eleven parameters were used based on the whole chicken preparation process and prevalence of Campylobacter jejuni for risk assessment by using the Ross-Sumner Method. RESULTS: The detection rates of Campylobacter jejuni in raw chicken were between 0.29% and 2.28% during 2007-2010 in China (more than 20 provinces). The probability of illness caused by Campylobacter jejuni due to chicken consumption was around six out of one million consumers per day in urban areas and around one out of one million consumers per day in rural areas. Total predicted illnesses per year was about 736 000, accounting for 1.6‰ of the general population in urban areas and about 301 000, accounting for 0.37‰ of the total population in rural areas. The risk rankings of Campylobacter jejuni in chicken were 52 and 49 in urban and rural areas, respectively. CONCLUSION: A high risk score for Campylobacter jejuni in chicken was obtained in China. This result may contribute to development of food safety management strategies. Key efforts should be made to control the risk of Campylobacter jejuni in chicken in China, especially in chick breeding and chicken preparation processes.

[Virulent gene prevalence of foodborne Listeria monocytogenes in China in 2005].

OBJECTIVE: To study the virulent gene prevalence of foodborne Listeria monocytogenes (LM) isolated from China. METHODS: 78 LM isolates derived from raw meat, cooked food, aquatic products and vegetables of 13 provinces and cities.LM isolates were investigated for prevalence of virulence genes (LIPI-1 (prfA, plcA, hly, mpl, actA, plcB); LIPI-2 (inlA, inlB), and iap) by PCR method. RESULTS: 87.2% (68/78) of the isolates were prfA positive, 98.7% (77/78) of the isolates were plcA, actA and plcB positive, 97.4% (76/78) of the isolates were hly positive, 87.2% (68/78) of the isolates were mpl positive, 92.3% (72/78) of the isolates were inlA positive, 100% (78/78) of the isolates were inlB positive, 98.7% (77/78) of the isolates were iap positive. Among 21 virulent gene negative isolates, there was 7 isolates lack of two or more virulence genes. The rate of virulence genes deletion isolates from cooked meat was 31.3% (10/32), the rate of virulence genes deletion isolates from raw meat was 16.1% (5/31), the rate of virulence genes deletion isolates from vegetables was 36.4% (4/11) and rate of virulence genes deletion isolates from seafood was 50% (2/4). No significant difference was found (χ(2) = 3.721, P > 0.05). The virulence gene array-1 strains were dominant among these isolates. CONCLUSION: Among 78 LM isolates, prevalent of virulent genes were different except inlB, virulence genes of LIP-1 were deleted prevalently among isolates, virulence gene deletion patterns were diverse.

[Study on the automatic ribotyping for Enterobacter sakazakii].

OBJECTIVE: To analyze the ribotyping fingerprint of Enterobacter sakazakii (E. sakazakii) isolated from food and its typing power. METHODS: Two standard strains and twenty-eight isolates of E.sakazakii were analyzed by the DuPont Riboprinter(TM) microbial characterization system. The relevant database was established and the fingerprint patterns were analyzed with BioNumerics software. RESULTS: This system grouped two standard strains and twenty-eight E.sakazakii isolates into 26 ribotypes, and four ribotypes included two strains respectively, the other twenty-two strains showed different ribotypes. The lowest similarity was 31.58%. The number of bands by ribotyping was approximately ten and the molecular weight of these bands ranged from 1 to 50 kb. By the clustering program in BioNumerics, these isolates could be grouped into four clusters. CONCLUSION: The automatic ribotyping method is convenient and fast in E.sakazakii typing.

[The effects of acrylonitrile on cell apoptosis, proliferation and related genes expression of rat normal and tumor glial cells].

OBJECTIVE: To study the effect of acrylonitrile (ACN) to cell growth, apoptosis, proliferation and related gene expression of rat normal glial cells (DI TNC1) and tumor glial cells (C6). METHODS: The concentration of ACN on DI TNC1 and C6 were 25, 50 and 75 microg/ml. For cell growth, proliferation and apoptosis assay, the treated time was 24 hours, for microarray assay, the treated time was 4 and 24 hours. RESULTS: After treatment of DI TNC1 cell with 25,50 and 75 microg/ml ACN, the DNA synthesis index were decreased 93.1%, 81.3% and 74.9% as compared to control respectively, the apoptosis index was increased 118%, 122% and 143% as compared to controls respectively. The DNA synthesis and apoptosis indexes of C6 cell showed no change after treatment with ACN. The cell cycle and apoptosis pathway related genes, such as cyclin and p53, also showed changes after treatment with ACN. CONCLUSION: ACN inhibited the cell proliferation of DI TNC1, induced the apoptosis of DI TNC1 and had no effect on cell proliferation and apoptosis of C6 cells, and the related regulation gene expression changes further confirmed the results.

[Effects of cell growth and apoptosis of preneoplastic Syrian hamster embryo cells by green tea constituent epigallocatechin-3-gallate].

OBJECTIVE: The co-culture model of Syrian hamster embryo (SHE) normal (primary cell) and preneoplastic cells mimicking in vivo status was established and used to study the chemopreventive effects of epigallocatechin-3-Gallate (EGCG) on cell growth, proliferation, apoptosis and regulated genes expression of SHE preneoplastic cells and discussed on the mechanism of EGCG's chemopreventive effect of carcinogenesis. METHODS: The SHE cell preneoplastic and normal cells were cultured on the plates with 1:10,000, 1:1000, 1:100, 1:10 rates for 7 days, and the co-culture model was established. The different concentration of EGCG (0, 0.5, 1, 5, 10, 50 micromol/L) were used to treat the cells and the SHE cells growth assay, in situ cell apoptosis assay, in situ cell proliferation assay and microarray assay were used to determined the growth, apoptosis and proliferation of SHE preneoplastic cells. RESULTS: The co-culture model of SHE cells with the 1:100 rate between SHE preneoplastic cells and normal cells was established. 0.5, 1, 5, 10 micromol/L EGCG increased the colony growth and proliferation of SHE normal cells. In the coculture model of SHE cells with 1:200 rate, compared the the control group, 5, 10 micromol/L EGCG suppressed the growth of different size of SHE preneoplastic cells clone. The DNA proliferation index and apoptosis index in the control group were 39.3% and 6.5%, respectively. After treatment of 5, 10 micromol/L EGCG, the proliferation index were decreased to 25.6% and 24.8%, and the apoptosis index were increased to 12.65% and 14.5%. EGCG suppressed the growth and proliferation of SHE preneoplastic cells in co-culture model and increased its apoptosis. The pathway of cell apoptosis was regulated through the P53, NF-kappaB, bcl-2 signal pathway, and the pathway of cell proliferation was regulated through the growth arrest at G1/S phase of cell cycle. CONCLUSION: The selective regulation of EGCG to normal and preneoplastic cells, the interaction of EGCG, SHE normal cells and SHE preneoplastic cells in co-culture model indicate that the suppression of proliferation and induction of apoptosis of preneoplastic cells by EGCG might be the mechanism of green tea' s chemopreventive effects to tumorigenicity.

[Mechanism of AcrAB-TolC system mediated multiple antimicrobial resistance in Salmonella typhimurium].

OBJECTIVE: To confirm AcrAB-TolC system mediated the multiple antimicrobial resistance in Salmonella typhimurium. METHODS: One-step knock-out of chromosomal method was used, to disrupt chromosomal genes, in Salmonella typhimurium wild strain X2. The acrAB gene and tolC gene were knocked out and two mutants of the delta acrABX2 and delta tolCX2 were produced. RESULTS: Results of E-test to two mutation strains and their parent strain showed that delta acrABX2 and delta tolCX2 were resistant to vancomycin and susceptible to amikacin and cephalothin, which was similar to their parents isolate X2, but (acrABX2 and (tolCX2 were susceptible to other eleven antimicrobial agents, while the parents isolate X2 was resistant to those. CONCLUSION: multiple antimicrobial resistance in Salmonella typhimurium. It suggested that the AcrAB-TolC system mediated the multiple antimicrobial resistance in Salmonella typhimurium.

[Determination and analysis of cellular fatty acids composition of Burkholderia gladioli].

OBJECTIVE: To determine and analyse the composition of cellular fatty acids of Burkholderia gladioli. METHODS: The cellular fatty acids composition of different pathovar strains of Burkholderia gladioli were determined by GC method and analyzed by MIDI-FAME. RESULTS: The results showed that the composition of cellular fatty acids of these strains, including original food poison strains of, were similar and were identified as, and this confirmed the conclusion that Pseudomonas cocovenenans subsp farinofermentans and Burkholderia cocovenenan were the junior synonym of Burkholdria gladioli. It was interesting to find that fatty acids of C16: 0.20H, C18: 1.20H and C16: 1.20H were related to the Bongkrekic acid (BA) producing of Pseudomonas cocovenenans subsp farinofermentans. CONCLUSION: It provide more data for the study of the mechanism of BA production and pathogenesis.

Nanodissection of single- and double-stranded DNA by atomic force microscopy.

Nanodissection of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) has been investigated by atomic force microscopy (AFM). It is found that both ss- and dsDNA can be repeatedly dissected by an AFM tip. However, a comparison study indicates that ssDNA is a little bit more easily broken by the AFM tip than dsDNA. This is supported by the fact that the time requested to break ssDNA is shorter than that of dsDNA in the same dissection procedure under the same load. Our experiment also shows that dissection of the DNA strand is very sensitive to the load applied, and a small change of the load lead to different results.

Radial compression elasticity of single DNA molecules studied by vibrating scanning polarization force microscopy.

The radial compression properties of single DNA molecules have been studied using vibrating scanning polarization force microscopy. By imaging DNA molecules at different vibration amplitude set-point values, we obtain the correlations between radially applied force and DNA compression, from which the radial compressive elasticity can be deduced. The estimated elastic modulus is approximately 20-70 MPa under small external forces (<0.4 nN) and increases to approximately 100-200 MPa for large loads.

[Assessment of the BALB/c mice as a suitable animal model for the investigation of food allergy].

OBJECTIVE: This study was to develop a suitable model for the investigation for food allergy. METHODS: BALB/c mice were dosed by intraperitoneally with Ovalbumin, Beef serum albumin, Trypsin inhibitor and Potato acid phosphatase respectively (0.25ml 20mg/mnl) on day 0 and again on day 7. Control group was dosed with PBS. Sera form individual animals were analysed for specific IgE and Passive cutaneous anaphylaxis tests. Additionally, the level of histamine in plasma were detected. RESULTS: The high titres of specific IgE (1: 32) could be provoked in test groups compared with control group. In addition, the level of histamine in plasma of test groups was higher than that in the control group. But there was no statistical significance between group food allergen and group Potato acid phosphatase. CONCLUSION: Although allergic action of BALB/c mice could be provoked, the situation of the allergic action of BALB/c mice to the proteins was very different with the human being. The BALB/c mice could not be a suitable model for the investigation for food allergy.