Junni Tang

Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens

Aims:  To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively).

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.

Characterization of Adhesin Genes, Staphylococcal Nuclease, Hemolysis, and Biofilm Formation Among Staphylococcus aureus Strains Isolated from Different Sources

Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms, leading to nosocomial infection and cross-contamination of foods. The current study was focused on the detection of adhesin genes, staphylococcal nuclease and hemolytic activities, and biofilm formation among the isolates of S. aureus from different sources. Fifteen adhesin genes (bap, bbp, clfA, clfB, cna, ebpS, fib, fnbA, fnbB, eno, icaAD, icaBC, sasG, sasC, pls) involved in S. aureus cell aggregation and biofilm accumulation were detected by polymerase chain reaction using specific primer. The activities of staphylococcal nuclease and hemolysis were analyzed by using toluidine blue-DNA agar and sheep blood agar for each strain. The ability of biofilm formation among different S. aureus strains was tested by using the glass tube method and microtiter-plate method. Our results showed the diversity of biofilm formation from different sources. Some isolates were strong biofilm producers; some were weak biofilm producers; and some were nonbiofilm producers. Staphylococcal nuclease and hemolysis seem to play a certain inhibitory role in biofilm formation. The adhesin genes varied among different S. aureus strains. The bap gene was not present in any strains. The bbp gene was only detected in one strain. The detection rates of other adhesin genes were as follows: clfB and sasG (100%); cna, eno, fib, and ebpS (93.75%); fnbA, icaAD, and icaBC (87.50%); fnbB (68.75%); sasC (31.25%); clfA (25%); and pls (12.50%), respectively. The variation between phenotypic and genotypic characterization may be due to the heterogeneity in the genetic origins. There was no direct correlation in distribution of adhesin genes and biofilm formation, which indicates that a single gene or subset of genes cannot be utilized as a biofilm indicator for morphology. Our results also indicated that biofilm formation might be affected by many factors, which brings new challenges to the prevention of this serious pathogen due to biofilm-related infection and contamination.

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.

Antimicrobial susceptibility and presence of resistance & enterotoxins/enterotoxin-likes genes in Staphylococcus aureus from food

ABSTRACT The aim of this study was to investigate the antibiotic resistance profiles and staphylococcal enterotoxins/enterotoxin-likes genes (se/sel) of Staphylococcus aureus from food. About 93 S. aureus strains were screened and 43 strains harbored se/sel genes, which were from raw meat, pickles, cooked meat, egg products, bean products, aquatic products, grains and fresh vegetables, respectively. Among the 21 se/sel genes, selx gene was found in 30/93 (32.26%) strains, followed by seb, ser, sea, sec, selp, sell and set. About 50/93 strains carried 10 antibiotic resistance genes. The prevalence rate of norA, chlA and grlA genes was relatively high, followed by ermC, tetA, aac6ʹ/aph2”, ermB, tetM, msrA and blaZ. Sixty-two strains of the 93 S. aureus isolates (62/93, 66.67%) were multidrug-resistant (≥3 antimicrobial agents).

Allograft Inflammatory Factor-1 Mediates Macrophage-Induced Impairment of Insulin Signaling in Adipocytes

Background/Aims: Allograft inflammatory factor-1 (AIF-1) is an inflammatory cytokine produced mainly by macrophages within human white adipose tissue. Its expression is increased in obese subjects and positively correlated with insulin resistance. The purpose of this study is to characterize the regulatory role of AIF-1 in insulin signaling of adipocyte. Methods: AIF-1 was over-expressed via transfection of AIF-1 cDNA into murine RAW 264.7 macrophages, and the constitutive expression of AIF-1 was decreased via transfection of targeting siRNA. Murine 3T3L1 adipocytes were treated with macrophage-conditioned medium or AIF-1 protein. Intracellular lipid accumulation was assayed by oil red O stain. Reactive oxygen species production was determinated by a flow cytometer and adipokine secretion was measured with ELISA. Glucose uptake was detected using the glucose oxidase method and insulin-signal-transduction related molecules were analyzed by Western blot. Results: Short term (48 h) AIF-1 treatment slightly promoted intracellular lipid storage in differentiating 3T3L1 cells. The protein stimulated reactive oxygen species production, provoked TNFα, IL6, resistin, but suppressed adiponectin release and insulin-stimulated glucose uptake both under normal basal and insulin resistance conditions. Furthermore, AIF-1 induced NF-κB activation, inhibited PPARγ expression, GLUT4 translocation to plasma membrane and Akt phosphorylation. Conclusion: Macrophage-derived AIF-1 up-regulated reactive oxygen species production, adipokine TNFα, IL6, resistin release, and inhibited adiponectin secretion. Moreover, it suppressed insulin-stimulated glucose uptake by down-regulating insulin signaling. Thus, AIF-1 could be related to obesity-related diseases.

Comparative Effects of Food Preservatives on the Production of Staphylococcal Enterotoxin I from Staphylococcus aureus Isolate

Staphylococcal enterotoxin I (SEI) is associated with staphylococcal food poisoning, but little is known about different food preservatives on the production of SEI. In this study, the effect of different food preservatives (sodium nitrite, polylysine, chitosan, and tea catechin) on the bacteria growth, sei gene expression, and extracellular SEI production of Staphylococcus aureus isolate H4 was detected in tryptone soya broth (TSB) culture. Our results showed that all of these preservatives depressed S. aureus H4 growth and the order of inhibitory effect was 0.8 g/L tea catechin > 6 g/L chitosan > 0.25 g/L polylysine > 0.4 g/L tea catechin > 0.15 g/L sodium nitrite. Furthermore, 0.25 g/L polylysine or 0.15 g/L sodium nitrite did not significantly alter sei gene transcription, while 6 g/L chitosan obviously increased the relative mRNA level of sei gene expression. 0.4 g/L tea catechin remarkably inhibited sei gene transcription. In addition, 0.15 g/L sodium nitrite and 6 g/L chitosan significantly enhanced SEI secretion. 0.25 g/L polylysine, especially 0.4 g/L tea catechin, sharply inhibited the level of SEI secretion. The results indicated that tea catechin not only suppressed Staphylococcus aureus growth, but also inhibited SEI production and secretion, suggesting that tea catechin may be better than sodium nitrite, polylysine, or chitosan for keeping the food from the contamination of SEI. These investigations would be useful for food industry to provide safer food products due to S. aureus enterotoxins-related control strategy.