Occurence of potentially enterotoxigenic Bacillus cereus in infant milk powder

Considering that powdered infant milk formula effectively supports the growth of numerous pathogens, this study investigates the prevalence of potentially pathogenic Bacillus cereus in dried milk products by evaluating the occurrence of B. cereus and the presence of virulence-associated genes. The approach consisted of enriching, isolating and biochemical identifying isolates, followed by PCR assays aimed at the hbl (C, D, A, B), nhe (A, B, C) and cytK enterotoxin genes coding HBL complex, NHE complex and cytotoxin K, respectively. Among cytK-positive strains, the discrimination of two different forms for cytotoxin K, cytK-1 and cytK-2 was performed. Bacillus cereus was detected in powdered infant milk formula samples. All the strains harbored at least one gene of the cytK, HBL and NHE enterotoxins. Because of an increasing trend in invasive infections by B. cereus in infants and immunocompromised children, our PCR findings highlight the need to implement an adequate control plan in order to guarantee the health of potentially fragile consumers. From a hygiene point of view, intensive and continuous monitoring of potentially pathogenic B. cereus may be crucial for powdered infant milk formula safety and even recommended in order to assess the infant health risk, as proposed by Commission Regulation (EC) no. 1441/2007 on microbiological criteria for foodstuffs. Furthermore, the detection in this study of B. licheniformis, B. subtilis and B. mycoides strains raises significant health issues regarding Bacillus spp. in powdered infant milk formula.     

Effect of Ethylenediaminetetraacetic Acid (EDTA) on Growth from Spores of Bacillus cereus

Germination and growth from spores of toxigenic B. cereus strain USDA 201 in BHI broth containing Na²EDTA were studied. Reduced growth was effected by low EDTA concentrations (< 300 ppm). Little or no growth was observed after incubation for 48 hr in broth containing from 300–1,000 ppm of EDTA EDTA did not affect percent spore germination, or release of Ca following heat activation and subsequent incubation. However, delayed colony development was observed as EDTA concentrations increased in the broth, and atypically small colonies formed on Plate Count Agar. Growth inhibition by 500 ppm EDTA was evident over the pH range 5–9, with the highest spore resistance observed at pH 7. Addition of Fe, Zn, and Ca to the media reversed the growth inhibitory action, whereas Mg was less effective.     

蜡样芽胞杆菌生长特性及大蒜精油对其抑菌活性的研究

主要研究了37℃条件下3种蜡样芽胞杆菌在营养肉汤中的生长状况,建立了37℃下蜡样芽胞杆菌在营养肉汤中的Boltzmann牛长模犁,3条牛长曲线相关系数鼯均大于0．97;检测了不同培养时间蜡样芽胞杆菌的产芽胞情况,结果表明1号菌株和14号菌株较早产芽胞,培养相同时间,产芽胞数：1号菌株,4号菌株〉标准菌株;采用牛沣杯法和平板计数法研究了大蒜精油对蜡样芽胞杆菌的抑制效果,结果表明浓度为lO。的人蒜精油对3种蜡样芽胞杆菌都有很好的抑制效果,3种菌株中l号菌株最难抑制。     

Lipid reserve dynamics and magnification of persistent organic pollutants in spawning sockeye salmon (Oncorhynchus nerka) from the Fraser River, British Columbia

Pacific sockeye salmon (Oncorhynchus nerka) can travel several hundred kilometers to reach native spawning grounds and fulfill semelparous reproduction. The dramatic changes in lipid reserves during upstream migration can greatly affect internal toxicokinetics of persistent organic pollutants (POPs) such as PCBs, PCDDs, and PCDFs. We measured lipid content changes and contaminant concentrations in tissues (liver, muscle, roe/gonads) and biomarker responses (ethoxyresorufin O-deethylase or EROD activity and CYP1A levels) in two Pacific sockeye salmon stocks sampled at several locations along their spawning migration in the Fraser River, British Columbia. Muscle lipid contents declined significantly with increasing upstream migration distance and corresponded to elevated lipid normalized concentrations of PCBs and PCDD/Fs in spawning sockeye. Post-migration magnification factors (MFs) in spawning sockeye ranged between 3 and 12 and were comparable to model-predicted MFs. sigmaPCBs(150-500 ng x g(-1) lipid), sigmaPCDD/Fs (1-1000 pg x g(-1) lipid) and 2,3,7,8-TCDD toxic equivalent or TEQ levels (0.1-15 pg x g(-1) lipid) in spawning sockeye were relatively low and did not affect hepatic EROD activity/CYP1A induction. Despite a 3-fold magnification, TEQ levels in eggs of spawning Fraser River sockeye did not exceed 0.3 pg x g(-1) wet wt, a threshold level associated with 30% egg mortality in salmonids. PCBs in Fraser River sockeye are comparable to previous levels in Pacific sockeye. In contrast to Pacific sockeye from more remote coastal locations, PCDDs and PCDFs in Fraser River sockeye were generally minor components (<25%) of TEQ levels, compared to dioxin like PCB contributions (>75%). The data suggest that (i) the Fraser River is not a major contamination source of PCBs or PCDD/Fs and (ii) marine contaminant distribution, food-chain dynamics, and ocean-migration pathway are likely important factors controlling levels and patterns of POPs in returning Pacific sockeye. We estimate an annual chemical flux entering the Fraser River of up to 150 g for sigmaPCBs and 40 mg for sigmaPCDD/ Fs via returning sockeye. The results indicate that historical concentrations of PCBs and PCDD/Fs remain a potential threat to organism and ecosystem health on the west coast of Canada.     

PCB congeners in Lake Michigan coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tshawytscha) salmon

We determined PCB congener concentrations in coho and chinook salmon collected in two Lake Michigan tributaries during the fall of 1996. Chinook salmon were larger than coho salmon and contained higher concentrations of the 78 PCB congeners we detected. There were no differences between male and female chinook or coho salmon in size or their PCB concentrations. Among individual fish, we found little evidence for a relationship between congener concentrations and percent lipid; however, congener concentrations did show a generally positive relationship with salmon size. Fish and macroinvertebrate congener concentrations are clearly related, and PCB congeners biomagnify approximately 20-30-fold as they flow from macroinvertebrates, two trophic levels below salmon, to the salmon. Slopes of regressions of salmonid congener concentrations on macroinvertebrate congener concentrations within homologs indicated that the degree of biomagnification generally increased with the degree of congener chlorination, although this pattern was much stronger for Mysis than for Diporeia. Log Kow and categorical variables for coplanar and "toxic" PCBs were not significant additional model terms, indicating that bioaccumulation of PCB congeners was not statistically related to these physicochemical attributes of the PCBs. The distribution of homologue PCBs shifts from a distinct predominance of hexachlorobiphenyls in macroinvertebrates to pentachlorobiphenyls and hexachlorobiphenyls in the salmon.     

培养条件对蜡样芽孢杆菌生物被膜生长的影响

以常见的食源性致病菌蜡样芽孢杆菌为研究对象,采用超声平板计数法对其生物被膜形成过程中的不同影响因素进行了研究。结果表明:在37℃、富营养环境、中性及弱碱性条件下,蜡样芽孢杆菌易形成大量生物被膜;添加低浓度的NaCl和葡萄糖均可促进生物被膜的形成,但促进作用不明显;添加高浓度的NaCl和葡萄糖对蜡样芽孢杆菌生物被膜生长有显著抑制作用。     

Production of Bacillus cereus emetic toxin (cereulide) in various foods

To determine the role of Bacillus cereus as a potential pathogen in food poisoning, the production of an emetic toxin (cereulide) by B. cereus was quantified in various food sources. The amount of emetic toxin in 13 of 14 food samples implicated in vomiting-type food poisoning cases ranged from 0.01 to 1.28 microg/g. A vomiting-type strain, B. cereus NC7401, was inoculated into various foods and incubated for 24 h at 20, 30, and 35 degrees C. In boiled rice, B. cereus rapidly increased to 10(7)-10(8) cfu/g and produced emetic toxin at both 30 and 35 degrees C. In farinaceous foods, the production of emetic toxin was as high as that in the food samples implicated in food poisoning. Low levels of emetic toxin were detectable in egg and meat and their products and a small quantity of toxin was detectable in liquid foods such as milk and soymilk when not aerated. Bacterial growth and toxin production was inhibited in foods cooked with vinegar, mayonnaise, and catsup, supposedly by the decreased pH of acetic acid. This is the first report that has quantified emetic toxin of B. cereus in various foods.     

Biocontrol of psychrotrophic enterotoxigenic Bacillus cereus in a nonfat hard cheese by an enterococcal strain-producing enterocin AS-48

Bacillus cereus is a food poisoning bacterium of great concern, especially in milk products. In this study, we describe the efficient control of the psychrotrophic and toxigenic strain B. cereus LWL1 in milk and in a nonfat hard cow's cheese by the enterocin AS-48 producer strain Enterococcus faecalis A-48-32 (Bac+). No viable B. cereus cells were detected after 72 h incubation in milk coinoculated with the AS-48-producing strain and B. cereus. Diarrheic toxin production was also markedly inhibited by the Bac+ strain to eightfold lower levels compared with control cultures of B. cereus. In cheeses manufactured by inoculation with a commercial starter (about 6.8 log CFU/ml) and B. cereus (about 4 log CFU/ml), the latter reached 6.27 log CFU/g after 5 days of maturation, and approximately 8 log CFU/g after 15 days. However, in cheeses made from milk inoculated with the starter along with a mixture of E. faecalis-B. cereus (2/1 ratio), counts of B. cereus decreased by approximately 1.0, 2.0, 4.32, and 5.6 log units with respect to control cheeses after 5, 10, 15, and 30 days of ripening, respectively. Growth of E. faecalis A-48-32 was associated with enterocin AS-48 production and persistence in cheese. Interestingly, growth of starter cultures was not affected by the Bac+ strain, and neither was lactic acid production. These results clearly indicate that E. faecalis A-48-32 produced satisfactory amounts of bacteriocin in cheese and support the potential use of AS-48-producing strains as culture adjuncts to inhibit B. cereus during cheese manufacture and ripening.     

Real-time-PCR-System zum Nachweis von Bacillus cereus (emetischer Typ) in Lebensmitteln

Bacillus cereus is a Gram-positive, facultative anaerobic, spore-performing bacterium. Some B. cereus strains have the ability to produce two different types of toxins: (a) diarrhoeic toxin: the disease is similar to a C. perfringens toxin-infection; it is caused by a heat-labile protein, (b) emetic toxin: the disease is similar to Staphylococcus-aureus intoxikation; it is caused by a heat-stable protein. Statements about the frequency of B. cereusfood-poisonings are difficult because a reporting system for this disease is missing in Germany and there exists no valid methodology to diagnose this disease with conventional microbiological or biochemical methods. Nevertheless B. cereus, beside S. aureus, seems to be the most important bacterium to cause food-associated intoxications. The detection of emetic toxin directly in food is difficult, time-consuming and expensive (HPLC-MS) and so a real-time-PCR assay including an Intern Amplification Control (IAC) for the detection of emetic B. cereus was developed and tested with different food matrices and routine samples. The assay is a rapid and reliable detection system for the routine laboratory to confirm in combination with microbiological methods (plating, colony-counting or enrichment) the suspicion of a food-associated B. cereus intoxication. This is a great progress from the epidemiological and legal point of view, particularly as a first diagnosis can be given after 30 hours. In addition it will be possible to make a risk assessment for different food products in order to protect the consumers’ health, because investigations can be performed more easily and frequently.

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Zusammenfassung: Bacillus cereus z?hlt zu den gram-positiven, fakultativ anaeroben, sporenbildenden Bakterien. Einige Bacillus cereus-St?mme sind in der Lage, zwei unterschiedliche Arten von Toxinen zu bilden: (a) diarrhoeisches Toxin: Die Erkrankung ?hnelt einer Toxin-Infektion durch C. perfringens, ausgel?st durch ein hitzelabiles Protein, (b) emetisches Toxin: Die Erkrankung ?hnelt einer Staphylokokken-Intoxikation, ausgel?st durch ein niedermolekulares hitzestabiles Protein. Aussagen über die H?ufigkeit von Bacillus cereus-bedingten Krankheitsausbrüchen sind schwierig, da keine generelle Meldepflicht besteht und die Diagnosestellung mittels konventioneller mikrobiologischer Methoden nicht m?glich ist. Man geht aber davon aus, dass Bacillus cereus neben Staphylococcus aureus zu den h?ufigsten Lebensmittel-Intoxikationserregern z?hlt. Da sich der direkte Toxinnachweis aus dem Lebensmittel für das emetische Toxin nach wie vor als schwierig und teuer erweist (HPLC-MS), wurde ein real-time-PCR-System einschlie?lich einer Internen Amplifikationskontrolle (IAC) zum Nachweis von Bacillus cereus, emetischer Typ etabliert, an unterschiedlichen Lebensmittelmatrices und anschlie?end an Probenmaterial aus dem t?glichen Probenaufkommen getestet. Das real-time-PCR-System hat sich als eine schnelle und zuverl?ssige Nachweismethode für die Routinediagnostik erwiesen, mit der sich in Kombination mit mikrobiologischen Methoden (Kontaktplattenverfahren, Keimzahlbestimmung, Anreicherungsverfahren) der Verdacht auf eine lebensmittelbedingte Bacillus cereus-Intoxikation (emetisches Toxin) best?tigen l?sst. Dies stellt aus epidemiologischer und lebensmittelrechtlicher Sicht einen gro?en Fortschritt dar, zumal eine erste Verdachtsdiagnose bereits nach 30 Stunden gestellt werden kann. Ebenso ist es m?glich, durch weitergehende Untersuchungen eine Risikoabsch?tzung für unterschiedliche Produktgruppen im Sinne des gesundheitlichen Verbraucherschutzes zu treffen.

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Eingegangen: 19. Januar 2007     

Growth and toxin profiles of Bacillus cereus isolated from different food sources

Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail. Polymerase chain reaction (PCR) using primers detecting cold shock protein A gene signatures (cspA), showed that none of the strains were the newly suggested species in the B. cereus group, B. weihenstephanensis, comprising psychrotolerant cereus strains, although one of the strains grew at 4 degrees C, two at 6 degrees C and seven grew at 7 degrees C. One of the two strains that grew at 6 degrees C had a maximum growth temperature of 42 degrees C, while the remaining 10 strains all grew at temperature of 43 degrees C or higher. Only three strains grew at 48 degrees C. At 42 degrees C, the generation time varied between 11 and 34 min. Spore germination was much faster for the two strains that grew at 6 degrees C than for the other nine strains in milk at 7 degrees C and 10 degrees C. All strains were cytotoxic and contained the non-haemolytic enterotoxin gene (nhe), 10 strains contained the enterotoxin T gene (bceT), and only six had the gene (hbl) encoding haemolytic enterotoxin. Two strains showed some microheterogeneity in the nhe operon. but contained all three genes. We can conclude that true B. cereus strains can have growth profiles as expected for B. weihenstephanensis, and that nhe and bceT were not correlated with growth profiles. However, the two psychrotolerant strains with minimal growth temperature of 4 degrees C and 6 degrees C did not contain hbl, as judged from our PCR results.     

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n=17), strains connected to diarrheal foodborne outbreaks (n=40) and food-environment strains (n=43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 degrees Celsius. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 degrees Celsius and 49% at 7 degrees Celsius (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 degrees Celsius, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 degrees Celsius and a lower germination, particularly at 7 degrees Celsius, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 degrees Celsius, 37 degrees Celsius, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods.     

Hypersalinity acclimation increases the toxicity of the insecticide phorate in coho salmon (Oncorhynchus kisutch)

Previous studies in euryhaline fish have shown that acclimation to hypersaline environments enhances the toxicity of thioether organophosphate and carbamate pesticides. To better understand the potential mechanism of enhanced toxicity, the effects of the organophosphate insecticide phorate were evaluated in coho salmon (Oncorhynchus kisutch) maintained in freshwater (<0.5 g/L salinity) and 32 g/L salinity. The observed 96-h LC50 in freshwater fish (67.34 ± 3.41 μg/L) was significantly reduced to 2.07 ± 0.16 μg/L in hypersaline-acclimated fish. Because organophosphates often require bioactivation to elicit toxicity through acetylcholinesterase (AChE) inhibition, the in vitro biotransformation of phorate was evaluated in coho salmon maintained in different salinities in liver, gills, and olfactory tissues. Phorate sulfoxide was the predominant metabolite in each tissue but rates of formation diminished in a salinity-dependent manner. In contrast, formation of phorate-oxon (gill; olfactory tissues), phorate sulfone (liver), and phorate-oxon sulfoxide (liver; olfactory tissues) was significantly enhanced in fish acclimated to higher salinities. From previous studies, it was expected that phorate and phorate sulfoxide would be less potent AChE inhibitors than phorate-oxon, with phorate-oxon sulfoxide being the most potent of the compounds tested. This trend was confirmed in this study. In summary, these results suggest that differential expression and/or catalytic activities of Phase I enzymes may be involved to enhance phorate oxidative metabolism and subsequent toxicity of phorate to coho salmon under hypersaline conditions. The outcome may be enhanced fish susceptibility to anticholineterase oxon sulfoxides.     

Differentiation of the rainbow trout (Oncorhynchus mykiss) from Atlantic salmon (Salmon salar) by the AFLP-derived SCAR

A species-specific SCAR marker for rainbow trout, which was used to detect adulteration and fraudulent labeling in Atlantic salmon products, has been developed based on the AFLP analysis and evaluated in this study. The SCAR marker could be amplified and visualized in 1% agarose gel in all tested rainbow trout samples and absent in all salmon samples. Using DNA admixtures, the detection of 1% (0.5 ng), 10% (5 ng) rainbow trout DNA in Atlantic salmon DNA for fresh and processed samples, respectively was readily achieved. The molecular approach was sensitive and demonstrated to be a rapid and reliable method for identifying frauds in salmon products and could be extended for applications of species identification in food industry.     

Growth inhibitory effects of kimchi (Korean traditional fermented vegetable product) against Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus

Kimchi is a unique Korean traditional vegetable product that is fermented by lactic acid bacteria (LAB) and is mainly consumed as a side dish with boiled rice. Its main ingredients are brined Chinese cabbage, red pepper powder, and fermented fish sauce, and these are combined with many spices such as garlic, green onion, ginger, and some seaweed. The relationship between the concentration of LAB or the pH and the growth of three gram-positive foodborne pathogens (Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus) was evaluated. Heat treatment (HT; 85 degrees C for 15 min) or neutralization treatment (NT; pH 7.0) was conducted on day 0 (0-D group) and day 3 (3-D group) of incubation. The pH in the control group and the NT group dropped sharply to 4.12 to 4.30 after 2 days of incubation and slightly decreased thereafter, whereas the pH in the control group and HT group stayed at 7.0 during incubation. LAB were not detected in the HT kimchi during incubation. B. cereus in the NT-0-D, NT-3-D, and HT-3-D groups was reduced by 1.5 to 3.1 log CFU/ml but increased slightly in the HT-0-D group. L. monocytogenes in HT-3-D and NT-3-D groups disappeared after 5 days of incubation, and S. aureus in the NT-0-D group disappeared after 4 days. These findings indicate that growth of all the foodborne pathogens was inhibited by NT-0-D, HT-3-D, and NT-3-D, but B. cereus was not inhibited by HT-0-D. Thus, growth of LAB in kimchi is an important factor in the control of foodborne pathogens.     

蜡状芽孢杆菌(Bacillus celreus)污染及其对食品安全的影响

蜡状芽孢杆菌是常见的食品污染菌，在工业化社会中正成为日益重要的食物病原菌。它能产生一种呕吐毒素和多种肠毒素，主要引发呕吐和腹泻型食物中毒。与蜡状芽孢杆菌同属的苏云金杆菌也能产生类似的肠毒素，近来发现它可能与食品中毒有关。我国应当加强对蜡状芽孢杆菌的监测和研究。     

Antimicrobial activity of carvacrol toward Bacillus cereus on rice

The antimicrobial activity of carvacrol, a compound present in the essential oil fraction of oreganum and thyme, toward the foodborne pathogen Bacillus cereus on rice was studied. Carvacrol showed a dose-related inhibition of growth of the pathogen. Concentrations of 0.15 mg/g and higher inhibited the growth and the extent of inhibition depended on the initial inoculum size. To decrease the input of carvacrol on the taste and flavor of the product, a combined treatment with the structure analog cymene was tested. Due to the smell and taste of carvacrol at high concentrations, carvacrol was combined with cymene, a natural antimicrobial compound with a similar structure. A synergistic effect was observed when 0.30 mg/g carvacrol was combined with 0.27 mg/g cymene. Finally it was demonstrated that a common taste enhancer like soya sauce also increased the antimicrobial action of carvacrol toward B. cereus. The antimicrobial activity of carvacrol with cymene or soya sauce was influenced by the addition of NaCl.     

Detection of Bacillus cereus on selected retail chicken products

Samples from five chicken meat products, obtained at retail stores, were evaluated for the presence of Bacillus cereus. The products tested were as follows: breaded, fully cooked, frozen nuggets (NUGGETS); breaded, fully cooked, frozen tenders (TENDERS); fully cooked, frozen, white-meat fajita-style strips (STRIPS); raw, refrigerated, boneless, skinless, marinated breast fillets (FILLETS); and raw, refrigerated, cut-up, tray-pack bone-in parts (PARTS), either split breasts or thighs. Four packages of each item were obtained on three different days (n = 60). Frozen and refrigerated products were held overnight in their respective environments as appropriate; then packages were opened aseptically, and a total of 25 g of tissue was excised from multiple pieces within a package. The 25-g samples were enriched in 225 ml of Trypticase soy-polymixin broth for 18 to 24 h at 30 degrees C and then plated on mannitol-egg yolk-polymixin agar and incubated for 18 to 24 h at 30 degrees C. Colonies characteristic of B. cereus were chosen and replated for isolation on mannitol-egg yolk-polymixin agar. Suspect colonies were confirmed as Bacillus spp. by Gram stain, hemolysis on blood agar, and a biochemical test strip. Isolates were further confirmed as B. cereus using Bacteriological Analytical Manual procedures, including tests for motility, rhizoid growth, hemolysis, and protein toxin crystal production. B. cereus was detected in 27 of 60 total samples. By product, the prevalence levels were as follows: NUGGETS, 11 of 12 positive; TENDERS, 8 of 12 positive; STRIPS, 6 of 12 positive; FILLETS, 0 of 12 positive; and PARTS, 2 of 12 positive. Isolates were tested by PCR for presence of the toxin-encoding genes bceT, nheABC, hblACD, and cytK. Results indicate that B. cereus organisms were present on four of the five retail poultry products tested in this study, with the highest rates reported for the three fully cooked items, especially the two breaded products. All strains isolated contained the gene(s) for at least one of the toxins, although none of the strains contained the cytK gene.     

Influence of chloride and metals on silver bioavailability to Atlantic salmon (Salmo salar) and Rainbow trout (Oncorhynchus mykiss) yolk-sac fry

The effects of differing water chloride concentrations (0-10 mM) or competing metals [Cu(II), Cd(II), Zn(II), Pb(II), Co(II) (1-10,000 nM)] on Ag(I) uptake in yolk-sac fry of two salmonid species, the Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), were studied. None of the metals tested were strong competitors of Atlantic salmon yolk-sac fry whole body Ag(I) influx. Inhibition of Ag(I) influx was only seen with a 100-fold excess of Cu(II) or Cd(II) or a 1000-fold excess of Pb(II) or Co(II). At these concentrations, the degree of competition appears to be directly proportional to the conditional stability constant of the competing metal to the gill (metal-gill log K). The range of [Cl-] allowed an assessment of Ag+, AgCl(aq), and AgCl2- bioavailability. The pattern of Ag(I) uptake was similar for each fish species. At <1 mM Cl-, where the [Ag+] dominates, the Ag(I) accumulation rate was constant. Above 1 mM Cl-, where the [AgCl(aq)] is dominant and the [AgCl2-] increases, there was a decline in Ag(I) uptake rate. However, even when very little Ag+ was present (i.e., at 10 mM Cl-) Ag(I) accumulated, albeit at a lower rate. This was suggestive of passive influx by AgCl(aq) and indicated little or no entry of negatively charged silver chloride complexes. The decline in Ag(I) uptake above 1 mM Cl- demonstrated that, if Ag(I) was present as both Ag+ and AgCl(aq), salmonid Ag(I) accumulation was dominated by Ag+ uptake. Therefore, the order of bioavailability of the Ag(I) species was determined as Ag+ > AgCl(aq) > AgCl2-.     

Development of rapid real-time PCR and most-probable-number real-time PCR assays to quantify enterotoxigenic strains of the species in the Bacillus cereus group

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (10(2) to 10(7) CFU/g for cooked rice and chicken, 10(3) to 10(7) CFU/ml for milk, and 10(4) to 10(7) CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 10(0) CFU/ml. Both assays were tested with real food samples and shown to beconsiderably appropriate for B. cereus group detection and quantification.