Most probable number method for isolation and enumeration of Staphylococcus aureus in foods: collaborative study

Enumeration of Staphylococcus aureus in foods was collaboratively studied by comparing the present AOAC final action method, 46.062, which uses trypticase soy broth with 10% NaCl to a proposed replacement method which uses the same broth with 1% sodium pyruvate added. Fifteen collaborators analyzed uninoculated samples of milk, tuna salad, and ground turkey, as well as samples inoculated with low (10(2) cells/g), middle (10(4) cells/g), and high (10(6) cells/g) levels of S. aureus. The samples were frozen immediately to maintain the inoculated level of S. aureus in the food. A different strain of S. aureus was used for each food; heat-stressed S. aureus cells were used to inoculate the milk samples. The pyruvate-amended broth significantly (alpha = 0.05) increased enumeration of low, middle, and high levels of S. aureus from milk and ground turkey, and from tuna salad at middle and high levels. The pyruvate-amended media method has been adopted official first action to replace method 46.062.     

Quantitative evaluation of bacteria washed from stainless steel penicylinders during AOAC use-dilution method

Stainless steel penicylinders inoculated separately with test bacteria (Salmonella choleraesuis, Pseudomonas aeruginosa, or Staphylococcus aureus) are used in the AOAC use-dilution method (UDM) for disinfectant efficacy testing. Numbers of bacteria remaining on penicylinders were quantitatively assessed to determine if cells are washed from the penicylinders after a 10 min exposure to phosphate buffer dilution water (PBDW). Inoculated penicylinders were also examined by scanning electron microscopy (SEM) to determine the presence of cells remaining attached to the penicylinders after a 10 min exposure to a quaternary ammonium disinfectant and separately to PBDW. The percentage of cells washed from inoculated penicylinders exposed to PBDW was 89.9 for Salmonella choleraesuis, 48.8 for Pseudomonas aeruginosa, and 38.8 for Staphylococcus aureus. Qualitative examination of penicylinders by scanning electron microscopy confirmed the attachment of S. aureus and P. aeruginosa cells to penicylinders exposed separately to PBDW and a quaternary ammonium disinfectant. Few S. choleraesuis cells were observed on penicylinders exposed to PBDW and no cells were observed after disinfectant exposure. The variability of the numbers of viable cells entering the recovery media among the 3 UDM test bacteria due to cell detachment could be a significant factor in the recognized variability of the use-dilution method.     

Standardization of bacterial numbers of penicylinders used in disinfectant testing: interlaboratory study

An interlaboratory study was conducted to evaluate a method of standardizing bacterial numbers on penicylinders used in the AOAC use-dilution method (4.007-4.015) of disinfectant testing. Eight participating laboratories followed a broth adjustment method using their media and stock cultures of Staphylococcus aureus ATCC 6538, Salmonella choleraesuis ATCC 10708, and Pseudomonas aeruginosa ATCC 15442. The culture broths that were used to inoculate the penicylinders were incubated for 48 h at 37 degrees C after several (4-6) 24 h passages. McFarland turbidity standards of 1.0 and 0.5 were used to adjust visually the cultures of S. aureus and P. aeruginosa, respectively. S. choleraesuis was used undiluted. The results showed significant variability in numbers of test bacteria which adhered to the penicylinders, with mean values of 1.6 X 10(6) for S. choleraesuis, 3.5 X 10(6) for S. aureus, and 8.2 X 10(6) for P. aeruginosa. The results from collaborating laboratories attempting standardization of bacterial numbers on penicylinders demonstrated significant interlaboratory and cylinder variation for all 3 test organisms.     

Evaluation of enrichment and plating media for isolating Listeria monocytogenes

We compared selective enrichment broths used by the U.S. Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service, for their efficiency in the quantitative recovery of Listeria monocytogenes from a naturally contaminated Brie cheese that was obtained as part of an epidemic investigation. Quantitative recovery of Listeria in FDA broth (greater than 2.4 x 10(5) colony forming units/mL) was significantly better than recovery in USDA broth (9.3 x 10(3) colony forming units/mL). When USDA broth was supplemented with D-glucose and Phytone (papaic digest of soy protein), its recovery efficiency improved but did not equal that of FDA broth for isolating L. monocytogenes from Brie cheese. A comparison of 4 selective plating media [modified McBride's agar, gum base nalidixic acid agar, lithium chloride-phenylethanol-moxalactam agar (LPM), and acriflavine-ceftazidime agar (AC)] showed that 3 L. monocytogenes strains belonging to serotype 1/2a were partially or completely inhibited on LPM and AC agars. One strain of serotype 1/2a formed microcolonies on modified McBride's agar after 48 h of incubation.     

Historical perspectives on methodology to detect Listeria monocytogenes

While recognized as a causative agent of illness in animals and humans for some time, the foodborne role of Listeria monocytogenes is a new and emerging one. This review briefly summarizes the historical developments in methodology used to detect the presence of L. monocytogenes. Although clinical procedures exist, these procedures do not consider isolation of Listeria from heavily contaminated environments. Federal agencies such as the U.S. Food and Drug Administration and the U.S. Department of Agriculture have defined protocols for the isolation of Listeria from dairy and meat products, respectively. Each of these protocols, and current problems common to all methods for the isolation of Listeria from food products, are discussed. Finally, future challenges with respect to improvement in our abilities to recognize, isolate, and rapidly identify Listeria in foods are presented.     

Rapid sedimentological and biological assessment of hydrocarbon contaminated sediments

It is possible to rapidly detect the presence of high concentrations of sediment associated hydrocarbons using a sediment profile camera and simultaneously evaluate the general sedimentological and biological character of a contaminated area. In sediments that were heavily contaminated with hydrocarbons from spills and chronic long-term additions the presence of hydrocarbons was seen about 50% of the time in the sediment profile images as unique features, ‘H spots’. The presence of these features was related to the concentration of hydrocarbons in the sediment. In highly contaminated muddy sediments ‘H spots’ were found in images collected at stations that had from 270 to 610 ppt total hydrocarbons. Sedimentological and biological information obtained from the sediment profile images confirmed the impacted nature of Elizabeth River sediments. Sediment profile imaging provide a means of obtaining an overall evaluation of the quality of a habitat and impacts on that habitat from pollution related environmental disturbances. While qualitative, an advantage of sediment profile image data is that they can be evaluated in less than a day and used to quickly locate inclusions of hydrocarbons in the sediments for further quantitative chemical or biological sampling, or mapping of heavily contaminated areas.     

Renewable Resource-Based Magnetic Nanocomposites for Removal and Recovery of Phosphorous from Contaminated Waters

Water pollution by P (phosphorous) is a serious water-related issue being faced by mankind during the past two decades. Several technologies already exist to remove P from contaminated water, but each has its own drawbacks. The present paper discusses a novel microwave-assisted synthesis of reduced iron oxide containing renewable-resource-based media for P removal from contaminated waters. It provides a fast, easy, and economical way to produce reduced iron oxide nanocomposites without requiring the need for hydrogen or inert gas during the transformation. A wide range of other metal/ carbon nanocomposites can also be synthesized using this technology and therefore holds tremendous economic promise. The prepared media were highly effective and efficient in removing P. A 100?% P removal efficiency was attained using a 1?mg/L standard P stock solution and a maximum capacity of 43.7?mg P/g of composite was achieved using 500?mg/L standard P stock solution. The present technology is highly economical as the carbon source employed was a renewable resource media with a high regeneration capacity. The present technology may also be used for arsenic removal from similarly contaminated water. The method of preparation of media, treatment methodology, and characterization methods are also discussed.     

Essential oil constituents and in vitro antimicrobial activity of Decalepis hamiltonii roots against foodborne pathogens

Hydrodistillation of Decalepis hamiltonii roots yielded an essential oil (0.33% v/w) that contained 2-hydroxy-4-methoxybenzaldehyde (37.45%), 2-hydroxybenzaldehyde (31.01%), 4-O-methylresorcylaldehyde (9.12%), benzyl alcohol (3.16%), and alpha-atlantone (2.06%) as major constituents, with aromatic aldehydes constituting the main fraction of this root's essential oil. The oil was tested for its antimicrobial activity against foodborne pathogens responsible for food spoilage and human pathologies using standard antimicrobial assays. It exhibited strong antimicrobial activity against Bacillus cereus, Bacillus megaterium, Candida albicans, Escherichia coli, Micrococcus luteus, Micrococcus roseus, and Staphylococcus aureus at a concentration range of 1:0 with inhibitory activities of 27, 23, 16, 19, 22, 19, and 23 mm, respectively, which are comparable to those of the standards. The roots of D. hamiltonii, therefore, may be considered as an inexpensive source of an essential oil rich in antimicrobial compounds against foodborne pathogens.     

土壤重金属生物淋滤修复过程中廉价碳源的筛选研究

在室内模拟实验条件下,重点研究了一步和二步生物淋滤过程中黑曲霉利用各种廉价碳源替代蔗糖产酸修复重金属污染土壤的效果。结果发现,杨树叶、桃树叶、土豆皮去除重金属效果较好。其中杨树叶对冶炼厂土壤中重金属去除率分别为Cu63．5％、Cd100％、Pb16．8％和Zn27％;桃树叶去除效果分别为Cu61．8％、Cd100％、Pb14．6％和Zn28．5％;土豆皮去除效果分别为Cu61％、Cd100％、Pb10．6％和Zn34％。廉价碳源的使用可降低污染土壤生物淋滤修复成本。     

Recovery of Salmonella from shell eggs.

A preenrichment procedure and a direct selective enrichment procedure were compared for recovery of Salmonella artificially inoculated into liquid whole egg, egg yolk, and egg albumen. For liquid whole egg and egg yolk, the 2 procedures were comparable. With egg albumen, however, preenrichment in lactose broth gave significantly higher recoveries than did direct selective enrichment in either selenite cystine or tetrathlonate broths. The lactose preenrichment procedure was used to determine the survival of S. enteritidis in egg yolk and egg albumen over a period of 7 days. As shown by most probably number determinations, counts of S. enteritidis inoculated into egg albumen decreased by 3 log units, whereas those in egg yolk did not change significantly. It is recommended, therefore, that only the egg yolk be examined for this pathogen. In a comparison of 5 different preenrichment media (lactose broth, brain heart infusion broth, trypticase soy broth, buffered peptone water, and nutrient broth), lactose broth was somewhat less productive than the other 4 media for the recovery of Salmonella from egg yolks. Trypticase soy broth gave the highest recovery.     

Development of a sensitive enzyme-linked immunosorbent assay for the determination of domoic Acid in shellfish

Polyclonal antibodies for domoic acid were generated from rabbits after the animals had been immunized with either domoic acid-keyhole limpet hemocyanin (KLH) or domoic acid-bovine serum albumin (BSA). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of domoic acid in blue mussels and clams. The antibody titers in the serum of rabbits immunized with domoic acid-KLH were considerably higher than those in rabbits immunized with domoic acid-BSA. The antibodies from the rabbits immunized with domoic acid-KLH were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of domoic acid-horseradish peroxidase to the antibodies by domoic acid and a domoic acid analogue, kainic acid, were found to be 0.75 and 200 ng/mL, respectively. In the presence of blue mussel matrix, the detection limit of domoic acid was <25 ng/g. The overall analytical recovery of domoic acid (25-500 ng/g) added to the blue mussels and then extracted with 50% aqueous methanol in the cdELISA was found to be 81.1%. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method. Analysis of domoic acid in shellfish samples showed that 10 of the 15 shellfish examined were contaminated with domoic acid at levels of <50 ng/g.     

Plant extracts, spices, and essential oils inactivate Escherichia coli O157:H7 and reduce formation of potentially carcinogenic heterocyclic amines in cooked beef patties

Meats need to be heated to inactivate foodborne pathogens such as Escherichia coli O157:H7. High-temperature treatment used to prepare well-done meats increases the formation of carcinogenic heterocyclic amines (HCAs). We evaluated the ability of plant extracts, spices, and essential oils to simultaneously inactivate E. coli O157:H7 and suppress HCA formation in heated hamburger patties. Ground beef with added antimicrobials was inoculated with E. coli O157:H7 (10(7) CFU/g). Patties were cooked to reach 45 °C at the geometric center, flipped, and cooked for 5 min. Samples were then taken for microbiological and mass spectrometry analysis of HCAs. Some compounds were inhibitory only against E. coli or HCA formation, while some others inhibited both. Addition of 5% olive or apple skin extracts reduced E. coli O157:H7 populations to below the detection limit and by 1.6 log CFU/g, respectively. Similarly, 1% lemongrass oil reduced E. coli O157:H7 to below detection limits, while clove bud oil reduced the pathogen by 1.6 log CFU/g. The major heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were concurrently reduced with the addition of olive extract by 79.5% and 84.3% and with apple extract by 76.1% and 82.1%, respectively. Similar results were observed with clove bud oil: MeIQx and PhIP were reduced by 35% and 52.1%, respectively. Addition of onion powder decreased formation of PhIP by 94.3%. These results suggest that edible natural plant compounds have the potential to prevent foodborne infections as well as carcinogenesis in humans consuming heat-processed meat products.     

Enzyme-linked immunosorbent assay for deoxynivalenol in corn and wheat

The availability of antibody against deoxynivalenol (DON) triacetate (Tri-Ac-DON) has enabled development of a direct enzyme-linked immunosorbent assay (ELISA) and an indirect ELISA for DON in corn and wheat. In both assays, DON is extracted from the sample with acetonitrile-water, reacted with acetic anhydride to form Tri-Ac-DON, and diluted in phosphate buffer for analysis. Direct ELISA was found to be the more sensitive procedure. Fewer interferences are evidenced, and the assay is less time consuming than is indirect ELISA. For direct ELISA, recovery of 10-1000 ppb DON added to corn and wheat was 100% (SD 15, CV 15%) and 102.1% (SD 12.2, CV 11.9%), respectively. For indirect ELISA, overall recovery of 10-1000 ppb DON added to wheat was 121.5% (SD 39.5, CV 32.5%); in the higher concentration range (500-1000 ppb), recovery was 105% (SD 18, CV 17%). The minimal detection level for DON was around 10 ppb. Analysis of 7 naturally contaminated samples for DON showed that the ELISA results agreed well with those obtained by radioimmunoassay and thin-layer chromatography.     

comparison of sulphur and heavy metal contents and their regional distribution in humus and moss samples from the vicinity of Nikel and Zapoljarnij,Kola Peninsula,Russia

Terrestrial moss and humus (the O-horizon) are often used separately for determining and monitoring airborne heavy metal pollution. Here, we directly compare the results of analyses of moss and humus samples taken at a density of one site per 300 km² in a 12 000 km² area (45 samples) around the nickel smelter in Nikel, the nickel ore roasting plant in Zapoljarnij, both in Russia, and the iron ore mine and mill near Kirkenes in Norway. The samples were air dried, digested in conc. HNO₃ and analysed for more than 30 elements by ICP-MS and ICP-AES at the laboratory of the Geological Survey of Finland (GTK). For most elements, observed levels and variations are considerably greater in soil than in moss. The main contaminants, Ni and Cu, reach equally high median levels in the moss and soil, but maximum values are far higher in soil. Both media show comparable regional distribution patterns for the heavy metals, but not for sulphur. Cu and Ni can be used to delineate the limits of contamination in the survey area. Both media show the same picture, with a generally very steep gradient from east to west and background levels being reached 30–50 km from the nickel smelter. When moss is used, Cu/Ni, Cu/S and Ni/S ratios can be used to separate input from the smelting and roasting proccesses in Russia. Both media are well suited to use separately to detect airborne pollution in this heavily contaminated area. The moss data are generally easier to interpret, but moss is not available at the most polluted sites. Levels for many elements other than Cu and Ni are close to the detection levels in moss samples, but not in humus samples. Information gathered from both media thus complement each other in a regional multi-element survey.     

Fungal flora and ochratoxin a production in grapes and musts from france

Eleven samples of grapes and musts used in red table wines were investigated for the occurrence of potential ochratoxin A (OTA)-producing molds. From these samples, 59 filamentous fungi and 2 yeasts were isolated. Among the 30 genera isolated, Deuteromycetes were the most frequent (70%) followed by Ascomycetes (10%). Six of the eleven grapes samples were contaminated by potentially ochratoxinogenic strains (Penicillium chrysogenum and Aspergillus carbonarius). When cultivated in vitro on solid complex media, the 14 strains of A. carbonarius produced OTA. No other species produced OTA under the same conditions. Among must samples, eight of eleven were found to be contaminated by OTA (concentrations from <10 to 461 ng/L). There is a strong correlation between the presence of ochratoxin-producing strains on grapes and OTA in musts. These findings should be connected with the OTA contamination of human blood in these areas and in France.     

Rhamnolipid Foam Enhanced Remediation of Cadmium and Nickel Contaminated Soil

Column experiments were conducted to evaluate the feasibility of using a rhamnolipid foam to remove heavy metals (Cd and Ni) from a sandy soil contaminated with Cd (1706 ppm) and Ni (2010 ppm). Best results were obtained from the foam generated by a 0.5% rhamnolipid solution with an initial pH value of 10.0 after flushing with 20-pore-volume of solution. These conditions removed 73.2% of the Cd and 68.1% of the Ni. Removal efficiencies by foam generated by a chemical surfactant, Triton X-100, were investigated as a comparison. It removed 65.5% of the Cd and 57.3% of the Ni under the same conditions. After a 20-pore-volume liquid solution flushing, 0.5% rhamnolipid (initial pH 10.0) without foam generation removed 61.7% of the Cd and 51.0% of the Ni, whereas 0.5% Triton X-100 (initial pH 10.0) removed 52.8% of the Cd and 45.2% of the Ni. Distilled water with adjusted pH only was also used to flush through the contaminated soil column as a control. It removed 17.8% of the Cd and 18.7% of the Ni. This study shows that rhamnolipid foam technology can be an effective means for the remediation of cadmium and nickel contaminated soil.     

Effects of PAH-Contaminated Soil on Rhizosphere Microbial Communities

Bacterial associations with plant roots are thought to contribute to the success of phytoremediation. We tested the effect of addition of a polycyclic aromatic hydrocarbon contaminated soil on the structure of the rhizosphere microbial communities of wheat (Triticum aestivum), lettuce (Lactuca sativa var. Tango), zucchini (Cucurbita pepo spp. pepo var. Black Beauty), and pumpkin (C. pepo spp. pepo var. Howden) 16S rDNA terminal restriction fragment length polymorphism (T-RFLP) profiles of rhizosphere microbial communities from different soil/plant combinations were compared with a pairwise Pearson correlation coefficient. Rhizosphere microbial communities of zucchini and pumpkin grown in the media amended with highest degree of contaminated soil clustered separately, whereas communities of these plants grown in unamended or amended with lower concentrations of contaminated soil, grouped in a second cluster. Lettuce communities grouped similarly to cucurbits communities, whereas wheat communities did not display an obvious clustering. The variability of 16S rDNA T-RFLP profiles among the different plant/soil treatments were mostly due to the difference in relative abundance rather than presence/absence of T-RFLP fragments. Our results suggest that in highly contaminated soils, the rhizosphere microbial community structure is governed more by the degree of contamination rather than the plant host type.     

奶牛乳腺炎金黄色葡萄球菌临床分离株生化特性与菌体蛋白免疫原性分析

摘要：病原学调查发现金黄色葡萄球菌是奶牛乳腺炎的最主要致病菌之一。从53株金黄色葡萄球菌乳样分离株中挑选了培养和形态典型的六株进行生化试验和传代稳定性试验，进一步研究这六株菌的凝固酶、溶血素等致病性因子和耐药性。对菌体裂解产物进行蛋白电泳图谱分析发现菌体蛋白条带具有很高的相似性,TQ-1株电泳蛋白条带密度较其它几株高。用其中四株生化典型、传代稳定、致病因子较多的菌株免疫兔，制备高免血清，分析全菌抗原的蛋白转印图谱，发现XG-2株在52KD处有特殊保护性抗原条带。综合本文各试验结果，确定以TQ-1和XG-2株为疫苗候选株。     

Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomum burmannii): activity against foodborne pathogenic bacteria

Cinnamomum burmannii Blume (cinnamon stick) from Indonesia is a little-investigated spice. In this study, the antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of cinnamon stick extract were evaluated against five common foodborne pathogenic bacteria (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella anatum). Cinnamon stick extract exhibited significant antibacterial properties. Major compounds in cinnamon stick were tentatively identified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC-MS) as a predominant volatile oil component ((E)-cinnamaldehyde) and several polyphenols (mainly proanthocyanidins and (epi)catechins). Both (E)-cinnamaldehyde and proanthocyanidins significantly contributed to the antibacterial properties. Additionally, scanning electron microscopy was used to observe morphological changes of bacteria treated with the crude extract of cinnamon stick and its major components. This study suggests that cinnamon stick and its bioactive components have potential for application as natural food preservatives.     

多氯联苯污染土壤的微生物生态效应研究

以多氯联苯（Polychlorinated biphenyls,PCBs）自然污染的农田土壤为材料,分析土壤中微生物区系组成、生物量C、N、土壤基础呼吸以及微生物群落功能多样性的变化。研究结果表明,在以4-氯、5-氯同系物为主的PCBs污染土壤中,污染程度对土壤细菌、放线菌的数量影响不明显,而真菌的数量除与土壤污染程度有关,可能还受到土壤pH等性状的影响;土壤微生物C、N与土壤基础呼吸随污染程度的加剧呈下降趋势,但微生物C／N基本没有变化;Biolog分析显示,土壤微生物代谢剖面（AWCD）及Simpson指数在污染程度相差较大的两组土壤样品中差异均达到了显著性水平,表明PCBs污染引起了土壤微生物群落功能多样性下降,降低了微生物对不同单一碳源底物的利用能力。