Jianshun Chen

Effect of Ultrasonic Treatment on the Total Phenolic and Antioxidant Activity of Extracts from Citrus Peel

Application of ultrasound to extract a variety of biologically active compounds from plant materials has been widely investigated. However, there are few reports on the local effect of ultrasonic irradiation on the yields of these compounds. In the present article, the local effect of ultrasonic treatment on total phenolic content (TPC) and antioxidant activities (ATT) of extracts from citrus peels was investigated. To optimize the extraction process, a response surface methodology (RSM) was used to evaluate the effects of ultrasonic variables including ultrasonic power, ultrasonic time, and extraction temperature on extracts from penggan (Citrus reticulata) peel. The results showed that TPC and ATT increased on increasing ultrasonic time and temperature. The maximum of TPC and ATT by ultrasonic treatment was observed in near ultrasonic irradiation surface, in which ultrasonic power appeared to be positive effect. Furthermore, when the effect of the 3 independent variables was evaluated simultaneously using RSM, the optimal ultrasonic conditions for responses were determined as: 42 to 45 W, 23 to 25 min, 31 to 34 degrees C. The results presented here emphasized that application of ultrasound should be considered both the optimization of ultrasonic variables and available ultrasonic device.

Molecular characteristics and virulence potential of Listeria monocytogenes isolates from Chinese food systems.

In this study, we examined Listeria monocytogenes isolates from Chinese food sources in an attempt to gain further insights on the molecular characteristics and virulence potential of this important foodborne pathogen. Of the 88 L. monocytogenes food isolates recovered, 42 (47.7%) were of serovars 1/2a or 3a; 23 (26.1%) of serovars 1/2b or 3b; 15 (17.0%) of 1/2c or 3c; 6 (6.8%) of serovars 4b, 4d or 4e; and 2 (2.2%) of serovars 4a or 4c. In contrast to inlAB locus conserved in all serovars, internalin cluster between ascB and dapE varies with different serovars, with inlC2DE, inlGC2DE and inlGHE predominantly in serovars 1/2b or 4b, serovar 1/2a and serovar 1/2c. While inlF existed in all the inlGHE- and inlGC2DE-containing isolates but 17.4% of those having inlC2DE, lmo2026 existed in all the inlGHE-containing isolates but 20.0% of those bearing inlGC2DE, suggesting that inlF might have co-evolved with inlGC2DE and inlGHE while lmo2026 with inlGHE only. With the exception of serovar 4a isolate, most serovar isolates demonstrated remarkable ability to form plaques on L929 cells and produced significant mouse mortality irrespective of the internalin gene organization and whether an intact actA gene is present or not. These results indicate that majority of these food isolates may have the potential to cause human diseases if ingested via contaminated foods. Given that serovar 4b accounts for nearly half of human clinical listeriosis cases documented, the relative low proportion of serovar 4b food isolates suggests that this serovar is probably more tolerant of the adverse conditions in the hosts stomach and/or more efficient in entering host cells than serovars 1/2a, 1/2b and 1/2c.

Prevalence of Listeria in Chinese Food Products from 13 Provinces Between 2000 and 2007 and Virulence Characterization of Listeria monocytogenes Isolates

Listeriosis is a severe disease with high mortality rate, especially in immunosuppressed individuals. The causative organism Listeria monocytogenes is primarily transmitted to humans through contaminated foods. To gain an understanding of the prevalence of Listeria in Chinese food products, we reviewed relevant papers from journals published in China from 2000 to 2007. The average recovery rate of Listeria spp. was 3.7% (0.1-7.7%) in all food categories in 13 provinces, with raw meat being the leading source. L. innocua (28.9%, 271/937) and L. monocytogenes (25.3%, 237/937) were more commonly isolated, both at higher proportion in all food types. Subtyping schemes in three laboratories in different provinces revealed that the majority of the L. monocytogenes isolates belonged to lineage II (67.1%), followed by lineage I at 31.6%, including the pathogenic serovars 1/2a, 1/2b, and 4b isolates. Lineage III isolates comprising the low-pathogenic serovar 4a were rare. Knowledge of the prevalence of Listeria in various food products in different regions of China may be useful for developing intervention strategies for control of contaminations along the production chains.

Lmo0036, an ornithine and putrescine carbamoyltransferase in Listeria monocytogenes, participates in arginine deiminase and agmatine deiminase pathways and mediates acid tolerance.

Listeria monocytogenes is a foodborne pathogen causing listeriosis. Acid is one of the stresses that foodborne pathogens encounter most frequently. The ability to survive and proliferate in acidic environments is a prerequisite for infection. However, there is limited knowledge about the molecular basis of adaptation of L. monocytogenes to acid. Arginine deiminase (ADI) and agmatine deiminase (AgDI) systems are implicated in bacterial tolerance to acidic environments. Homologues of ADI and AgDI systems have been found in L. monocytogenes lineages I and II strains. Sequence analysis indicated that lmo0036 encodes a putative carbamoyltransferase containing conserved motifs and residues important for substrate binding. Lmo0036 acted as an ornithine carbamoyltransferase and putrescine carbamoyltransferase, representing the first example, to our knowledge, that catalyses reversible ornithine and putrescine carbamoyltransfer reactions. Catabolic ornithine and putrescine carbamoyltransfer reactions constitute the second step of ADI and AgDI pathways. However, the equilibrium of in vitro carbamoyltransfer reactions was overwhelmingly towards the anabolic direction, suggesting that catabolic carbamoyltransferase was probably the limiting step of the pathways. lmo0036 was induced at the transcriptional level when L. monocytogenes was subjected to low-pH stress. Its expression product in Escherichia coli exhibited higher catabolic carbamoyltransfer activities under acidic conditions. Consistently, absence of this enzyme impaired the growth of Listeria under mild acidic conditions (pH 4.8) and reduced its survival in synthetic human gastric fluid (pH 2.5), and corresponded to a loss in ammonia production, indicating that Lmo0036 was responsible for acid tolerance at both sublethal and lethal pH levels. Furthermore, Lmo0036 played a possible role in Listeria virulence.

lmo0038 is involved in acid and heat stress responses and specific for Listeria monocytogenes lineages I and II, and Listeria ivanovii.

The genus Listeria comprises two pathogenic species, L. monocytogenes and L. ivanovii, as well as four nonpathogenic species, L. innocua, L. weishimeri, L. seeligeri, and L. grayi. Within L. monocytogenes, lineages I and II are responsible for most listeriosis cases, while lineage III strains are rarely associated with human morbidity but providing important clues for Listeria evolution. The gene lmo0038, belonging to the peptidylarginine deiminase family, was involved in the optimal growth under stress conditions, including low pH and heat shock (52 degrees C), and virulence potential. Further, this gene was specific to L. monocytogenes lineages I and II and L. ivanovii with significant similarities at nucleotide and amino acid levels. A novel multiplex PCR, based on lmo0038 in combination with optimized iap migration profiles, was developed for simultaneous identification of Listeria species and discrimination of L. monocytogenes lineage III, with a detection limit down to 1.0-9.0 x 10(2) CFU/mL. This assay was evaluated by 119 suspected Listeria food-related isolates and corrected 4 and 5 misidentifications by Listeria selective agar plate screening and API system, respectively. Therefore, this one-step molecular assay provides a rapid, reliable, and inexpensive screening test to detect Listeria species-particularly, the pathogenic species in surveillance programs concerning food safety and foodborne disease cases.

Vibrio parahaemolyticus Isolates from Southeastern Chinese Coast Are Genetically Diverse with Circulation of Clonal Complex 3 Strains Since 2002

Multilocus sequence typing (MLST) was used to examine the clonal relationship and genetic diversity of 71 Vibrio parahaemolyticus isolates from clinical and seafood-related sources in southeastern Chinese coast between 2002 and 2009. The tested isolates fell into 61 sequence types (STs). Of 17 clinical isolates, 7 belonged to ST3 of the pandemic clonal complex 3, with 3 strains isolated in 2002. Although there was no apparent clonal relationship found between clinical strains and those from seafood-related sources positive with pathogenic markers, there were clonal relationships between clinical strains from this study and those from environmental sources in other parts of China. Phylogenetic analysis showed that strains of 112 STs (61 STs from this study and 51 retrieved from PUBMLST database covering different continents) could be divided into four branches. The vast majority of our isolates and those from other countries were genetically diverse and clustered into two major branches of mixed distribution (of geographic origins and sample sources), whereas five STs representing six isolates split as two minor branches because of divergence of their recA genes, which had 80%-82% nucleotide identity to typical V. parahaemolyticus strains and 73.3%-76.9% identity to the CDS24 of a Vibrio sp. plasmid p23023, indicating that the recA gene might have recombined by lateral gene transfer. This was further supported by a high ratio of recombination to mutation (3.038) for recA. In conclusion, MLST with fully extractable database is a powerful system for analysis of clonal relationship for strains of a particular region in a national or global scale as well as between clinical and environmental or food-related strains.

Genome sequence of the nonpathogenic Listeria monocytogenes serovar 4a strain M7.

This report presents the complete and annotated genome sequence of the naturally nonpathogenic Listeria monocytogenes serovar 4a strain M7, isolated from cows milk in Zhejiang province, China.

Molecular characterization and functional analysis of MyD88 in Chinese soft-shelled turtle Trionyx sinensis

Myeloid differentiation factor 88 (MyD88) is one of the key adaptor proteins to signal transduction that triggers downstream cascades involved in innate immunity. In this study, the MyD88 gene from Chinese soft-shelled turtle (Trionyx sinensis) (tMyD88) was identified, representing the fist example from reptile species. The tMyD88 has a 894-bp ORF and encodes a polypeptide of 297 amino acids including a typical death domain (DD) at the N-terminus and a conservative Toll/IL-1R (TIR) domain at the C-terminus. It was expressed at high levels in spleen, blood, lungs and liver, but marginal in kidneys and intestines of turtles challenged with live cells of Aeromonas hydrophila, as determined by real-time PCR. RAW 264.7 cells transfected with pcDNA-tMyD88 showed higher NF-κB activity than the vector control (673.78 vs 410.72, P < 0.05). Expression of proinflammatory cytokines IL-1β and TNF-α was also significantly higher in RAW 264.7 cells transfected with pcDNA-tMyD88 than those having pcDNA3.1 control vector (P < 0.01). These results indicate that tMyD88 might possess an important role in defense against microbial infection in Chinese soft-shelled turtles similar to that in mammals.

Serovar 4b Complex Predominates Among Listeria monocytogenes Isolates from Imported Aquatic Products in China

Listeria monocytogenes, the causative organism of listeriosis, is primarily transmitted to humans through contaminated food. In this study, we examined 1275 batches of aquatic products imported from 29 countries and found that 36 batches from 8 countries were contaminated by Listeria (2.8%), with L. monocytogenes accounting for 2.6% (33/1275) and L. innocua for 0.2% (3/1275). Of the 23 selected L. monocytogenes isolates (from the 33 identified), 15 (65.2%) were of serovar 4b complex (4b, 4d, or 4e), three (13.0%) of 1/2a or 3a, four (17.4%) of 1/2b or 3b, and one (4.4%) of 1/2c or 3c. Notably, four of the 23 isolates belonged to epidemic clone I (ECI) and another four were associated with epidemic clone II (ECII), two highly clonal 4b clusters responsible for most of the documented listeriosis outbreaks. In the multilocus sequence typing scheme based on the concatenated genes gyrB-dapE-hisJ-sigB-ribC-purM-betL-gap-tuf, serovar 4b complex isolates from imported aquatic products exhibited significant genetic diversity. While the four ECI isolates were genetically related to those from Chinese diseased animals, both lacking one proline-rich repeat of ActA, the four ECII isolates were located between 1/2b or 3b strains. As the L. monocytogenes isolates from imported aquatic products possessed a nearly complete set of major infection-related genes, they demonstrated virulence potential in mouse model.

Catabolite control protein a of Streptococcus suis type 2 contributes to sugar metabolism and virulence

Catabolite control protein A (CcpA) is the major transcriptional regulator in carbon catabolite repression in several Gram-positive bacteria. We attempted to characterize the role of a CcpA homologue of Streptococcus suis type 2 in sugar metabolism and virulence. Addition of glucose or sucrose to the defined medium significantly reduced the activity of raffinose-inducible α-galactosidase, cellobiose-inducible β-glucosidase, and maltose-inducible α-glucosidase of the wild-type strain by about 9, 4, and 2-3 fold, respectively. Deletion of ccpA substantially derepressed the effects of repressing sugars on α-galactosidase or β-glucosidase activity. The ccpA deletion mutant showed reduced expression of virulence genes sly and eno (P<0.05), decreased adhesion to and invasion into endothelial cells (P<0.05), and attenuated virulence to mice with significant reduction of death rate and bacterial burden in organs, as compared to the wild-type strain. Both the in vitro and in vivo defect phenotypes were reversible by ccpA complementation. Thus, this study shows that CcpA of S. suis type 2 plays an important role in carbon catabolite repression and virulence.