Cheng Tang

Phenotypic characterization and prevalence of enterotoxin genes in Staphylococcus aureus isolates from outbreaks of illness in Chengdu City.

Staphylococcus aureus produces a spectrum of enterotoxin that is recognized as the main reason for causing staphylococcal food poisoning. The aim of the current study was to investigate the phenotypic characteristics and enterotoxin genotypes of S. aureus isolated from food poisoning sufferers. On the basis of the amplification of 16S rRNA and nuc gene specific to S. aureus assay and the phenotype (hemolytic activity, thermal stable nuclease [Tnase] test, and biofilm formation), all isolates were identified as S. aureus. To genotypically characterize S. aureus isolates, genes encoding staphylococcal enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser, and seu) were investigated by using polymerase chain reaction technique. The results showed that the eight isolates of S. aureus had different enterotoxin genotypic characteristics, which was the main cause of food poisoning. One isolate contained 10 enterotoxin genes, and the other 7 isolates carried 3 or more enterotoxin genes. The frequency of the newly identified enterotoxin genes (seg-seu) was higher than classical genes (sea-see). Overall, multi-gene detection rates were 75% (for sek, ser, and seu); 50% (for sea and sem); 37.5% (for sen, seg, and sei); and 12.5% (for seb, sec, sed, and sej), respectively. The see and seh gene were not detected in any isolates. The current study provided the exact distribution of enterotoxin genes in eight S. aureus strains from food poisoning sufferers, which indicated that the pathogenicity of the newly identified enterotoxin should be highlighted. The need for prevention of food poisoning occurrences caused by enterotoxin of S. aureus should be reinforced.

Characteristics of volatile organic compounds produced from five pathogenic bacteria by headspace-solid phase micro-extraction/gas chromatography-mass spectrometry.

The characteristics of volatile compounds from five different bacterial species, Escherichia coli O157:H7, Salmonella Enteritidis, Shigella flexneri, Staphylococcus aureus, and Listeria monocytogenes, growing, respectively, in trypticase soy broth were monitored by headspace solid‐phase micro‐extraction/gas chromatography‐mass spectrometry. The results showed that most volatile organic compounds (VOCs) of five pathogens started to increase after the sixth to tenth hour. Methyl ketones and long chain alcohols were representative volatiles for three Gram‐negative bacteria. The especially high production of indole was characterized to E. coli O157:H7. The production of 3‐hydroxy‐2‐butanone was indicative of the presence of two Gram‐positive bacteria. Both 3‐methyl‐butanoic acid and 3‐methyl‐butanal were unique biomarkers for S. aureus. The population dynamics of individual pathogen could be monitored using the accumulation of VOCs correlated with its growth. And these five pathogens could be distinguishable though principle component analysis of 18 volatile metabolites. Moreover, the mixed culture of S. aureus and E. coli O157:H7 was also investigated. The levels of 3‐methyl‐butanal and 3‐methyl‐butanoic acid were largely reduced; while the level of indole almost unchanged and correlated with E. coli O157:H7 growth very well. The characteristics of volatiles from the five foodborne pathogens could lay a fundamental basis for further research into pathogen contamination control by detecting volatile signatures of pathogens.

Surveillance study of enterotoxin genes in Staphylococcus aureus isolates from goats of different slaughterhouses in Sichuan, China

Staphylococcus aureus causes a number of diseases in humans and animals, and is the most common etiological agent of foodborne illnesses. The agent produces staphylococcal enterotoxins (SEs), which are the main cause of food poisoning. The aim of the present study was to characterize the distribution of genes encoding staphylococcal enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sem, sen, ser and seu) in S. aureus strains isolated from goats slaughtered in four different slaughterhouses in Sichuan, China. The presence of the target 16S rDNA (Staphylococcus genus specific) and nuc gene (S. aureus species specific) was used to determine the isolates to be S. aureus species. Of the 19 S. aureus isolates tested, 18 (95%) were found to be positive for three or more SEs gene (3–7 SEs genes) by polymerase chain reaction (PCR) amplification. The most frequent gene was seu (17/19, 89.5%), followed by seg (14/19, 73.6%), sen (10/19, 52.6%), sei (9/19, 47.3%), and sed (9/19, 47.3%). None of the isolates harbored the genes encoding seb, see, and seh. Among the classical enterotoxigenic strains, the occurrence of sed gene was highest (47.4%) followed by ea (36.8%) and sec (31.6%). The occurrence of the newly identified enterotoxin genes (seg-seu) was higher than that of traditional genes (sea-see). According to the present results, the S. aureus strains isolated from goats seem to be, at least at this stage, of importance as vectors causing staphylococcal food poisoning.

Identification and measurement of staphylococcal enterotoxin M from Staphylococcus aureus isolate associated with staphylococcal food poisoning

Staphylococcus aureus produces a wide variety of staphylococcal enterotoxins (SEs, SEA to SEX), which are responsible for staphylococcal food poisoning. This study is aimed to establish a system to detect staphylococcal enterotoxin M (SEM) protein in food matrixes. In the present study, sem gene was characterized in a S. aureus isolate H4 associated with food poisoning. The amino acid sequence of the deduced SEM protein was same as that of previously identified SEM from S. aureus 04‐02981. Subsequently, mature SEM protein was expressed in Escherichia coli BL21 (DE3) cells and purified with a Ni‐NTA spin column. The polyclonal and monoclonal antibody against it were prepared. Using these antibodies, a highly sensitive, specific sandwich enzyme‐linked immunosorbent assay (ELISA) system was developed capable of detecting SEM in milk, meat and rice. Cross‐reactivity with SEB, SEI and SEK in this method was insignificant. Quantification of SEM secretion in vitro using this novel capture ELISA revealed that SEM was mainly secreted during the transition from the exponential to the stationary phase. Furthermore, sem gene and SEM protein production were screened by PCR and the developed ELISA system. The results indicated that there were two SEM+ strains of 19 S. aureus isolates originating in cold dishes and humans suffering from food poisoning. The investigations make it possible to assess SEM in food hygiene supervision in near future.

Identification and measurement of staphylococcal enterotoxin-like protein I (SEll) secretion from Staphylococcus aureus clinical isolate.

Staphylococcus aureus (Staph. aureus) produces a wide variety of staphylococcal enterotoxins (SEs) and staphylococcal enterotoxin‐like (SEl) proteins, which are the most causative agents of staphylococcal food poisoning. In contrast to classical SEs (SEA to SEE), the relationship between the novel SEs/SEls (SEG to SElX) and staphylococcal food poisoning is not elucidated. This study is aimed to establish a system to detect staphylococcal enterotoxin‐like protein I (SElI) for analysis of staphylococcal food poisoning.

Prevalence and genomic characteristics of canine kobuvirus in southwest China

To investigate canine kobuvirus (CaKoV) infection in southwest China, 107 fecal samples were collected from dogs with obvious diarrhea in Sichuan and Chongqing regions, China. CaKoV infection was detected in 54 diarrheic samples (50.46%) by RT-PCR targeting a partial fragment (504 bp) of the 3D gene. Comparison of these partial 3D gene sequences from 14 of these CaKoV-positive samples show 95.4%–99.0% nucleotide (nt) identity within this group, and nt identities ranging from 93.1% to 98.2% with previously reported CaKoV 3D gene sequences. Additionally, we amplified five VP1 gene sequences and analyzed the inferred phylogeny. Amino acid (aa) identities of the five VP1 gene sequences were 81.5%–89.4% with those previously reported. Furthermore, one complete CaKoV genome was successfully obtained from a positive sample and designated SMCD-59/CHN/2015. This genome consisted of 8,184 nt, and shared 92.9%–96.6% nt identity (97.6%–98.1% aa identity) with other reported CaKoV genomes. This study provides proof that CaKoV circulates in diarrheic dogs in southwest China, and that these viruses exhibit unique genetic characteristics.

First detection of Nebovirus and Norovirus from cattle in China

Neboviruses and genogroup III noroviruses (NoVsGIII) are causative agents of calf diarrhea. The purpose of this study was to investigate the presence of neboviruses and noroviruses in cattle in China. Twenty-eight diarrhea fecal samples collected from 5 different farms were analyzed by RT-PCR. The results showed that 3 nebovirus positive samples were detected on 2 farms, with two strains being related to Bo/DijonA216/06/FR strain and the other one clustering with NB-like strains. Meanwhile, 3 norovirus positive samples were detected on 3 farms, all of which belonged to genotype 1. Our results confirmed the presence of neboviruses and NoVsGIII in China for the first time, and supported the presence of a novel “DijonA216-like” nebovirus genotype.

Identification and genomic characterization of a novel CRESS DNA virus from a calf with severe hemorrhagic enteritis in China

Abstract In this study, a novel circular replication-associated protein (Rep)-encoding single stranded (CRESS) DNA virus was discovered in diarrheic sample of a calf with severe hemorrhagic enteritis. The virus, named Bo-Circo-like virus CH, has a circular genome with 3909 nucleotides (nt). Six putative open reading frames (ORFs) were identified, including Rep, capsid (Cap) and four proteins of unknown function. Both the genome size and the number as well as the organization of encoded ORFs, Bo-Circo-like virus CH is most closely related to Po-Circo-like virus 21 detected in pig faeces. A preliminary survey using specific primers for the Rep region showed that 5.3% (4/75) of diarrheic samples were positive for Bo-Circo-like virus, and all 42 healthy samples were negative. In conclusion, our results indicate that Bo-Circo-like virus CH may represent a new virus in bovine. Further investigation is needed to determine the relationship between the virus infection and diarrhea.

The effect of rfaD and rfaF of Haemophilus parasuis on lipooligosaccharide induced inflammation by NF-κB/MAPKs signaling in porcine alveolar macrophages

In Haemophilus parasuis, the rfa cluster has been identified as a virulence-associated factor that is involved in lipooligosaccharide (LOS) biosynthesis. In this study, we assessed the roles of rfaD and rfaF genes in H. parasuis SC096 on LOS-induced pro-inflammatory factors and the related signaling pathways in porcine alveolar macrophages (PAMs) by real-time PCR and western blotting. The results showed that the LOSs of both rfaD and rfaF mutants (ΔrfaD-LOS and ΔrfaF-LOS) significantly decreased the mRNA expression of pro-inflammatory factors (IL-1α, IL-1β, IL-6, IL-8 and TNF-α) in PAMs compared with H. parasuis SC096 LOS (WT-LOS). Furthermore, in ΔrfaD-LOS- and ΔrfaF-LOS-treated cells, IκBα degradation was significantly inhibited and levels of phospho-p65 and phospho-p38 were significantly reduced in PAMs. These findings suggested that the rfaD and rfaF genes mediated LOS induction of pro-inflammatory cytokines in PAMs by regulating the NF-κB and MAPKs signaling pathways during H. parasuis infection.

Prevalence and genome characteristics of canine astrovirus in southwest China

The aim of this study was to investigate canine astrovirus (CaAstV) infection in southwest China. We collected 107 faecal samples from domestic dogs with obvious diarrhoea. Forty-two diarrhoeic samples (39.3 %) were positive for CaAstV by RT-PCR, and 41/42 samples showed co-infection with canine coronavirus (CCoV), canine parvovirus-2 (CPV-2) and canine distemper virus (CDV). Phylogenetic analysis based on 26 CaAstV partial ORF1a and ORF1b sequences revealed that most CaAstV strains showed unique evolutionary features. Interestingly, putative recombination events were observed among four of the five complete ORF2 sequences cloned in this study, and three of the five complete ORF2 sequences formed a single unique group, suggesting that these strains could be a novel genotype. We successfully sequenced the complete genome of one CaAstV strain (designated 2017/44/CHN), which was 6628 nt in length. The features of this genome include putative recombination events in the ORF1a, ORF1b and ORF2 genes, while the ORF2 gene had a continuous insertion of 7 aa in region II compared with the other complete ORF2 sequences available in GenBank. Phylogenetic analysis showed that 2017/44/CHN formed a single group based on genome sequences, suggesting that this strain might be a novel genotype. The results of this study revealed that CaAstV circulates widely in diarrhoeic dogs in southwest China and exhibits unique evolutionary events. To the best of our knowledge, this is the first report of recombination events in CaAstV, and it contributes to further understanding of the genetic evolution of CaAstV.