Growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce

The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z(+), plantaricin C(+), lacticin 481(+), coagulin(+) or pediocin PA-1(+)) to grow in a lettuce extract at 4 degrees C, 10 degrees C and 32 degrees C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 degrees C. Addition of bacteriocinogenic supernatants (nisin(+), coagulin(+) and a nisin-coagulin(+) cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1-1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 degrees C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 degrees C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2-1.6 log units immediately after treatment, but, during storage at 4 degrees C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.     

Development of a physically-based model for transport of Cryptosporidium parvum in overland flow

Surface runoff from animal production facilities contains numerous microbial pathogens which pose a direct health hazard to both humans and animals. In order to preserve safe water resources and sustainable agriculture by reducing runoff-mediated contamination of agricultural watersheds, microbial transport processes need to be properly understood and quantified. Cryptosporidium parvum is a protozoan parasite responsible for the largest outbreak of waterborne disease ever recorded in US history. Infected mammals can pass as many as 10 billion Cryptosporidium oocysts per gram of faeces, hence only a few infected animals can potentially contaminate a large watershed. Little information is available on the environmental or physicochemical factors governing the transport of microbial pathogen in surface and near-surface runoff, in particular the zoonotic pathogen Cryptosporidium. The objective of this study is to develop a physically-based model for simulating transport of C. parvum oocysts in overland flow, and to compare the model results with experimental observations. Transport of oocysts in overland flow can be simulated mathematically by including terms for the concentration of the oocysts in the liquid phase (in suspension or free-floating) and the solid phase (adsorbed to the fine solid particles like clay and silt). Oocysts adsorption and decay processes are considered. These processes are solved using numerical techniques to predict spatial and temporal changes in oocyst concentrations in solid and liquid phases. The model results are also compared with experimental results to validate the model. The model output reproduced the recovery kinetics satisfactorily, but under-predicted the total recovery in a few cases when multiple peaks were observed during experiments. Similarly, the calibrated model produced a good agreement between observed and modeled total oocysts recovery. With future modification, this model may provide a promising tool for developing effective management practices for controlling microbial pathogens in surface runoff.     

Influence of biotic and abiotic factors on human pathogens in a finished compost

The role of indigenous microflora of a finished compost, defined NK12, on the growth suppression of pathogens under different moisture and temperature storages was investigated. Total count of mesophilic and thermophilic bacteria was evaluated by the most probable number method and growth of seeded Salmonella arizonae 3924 serogroup B and enteropathogenic Escherichia coli 84 M in NK12 at different moisture temperature conditions was monitored. Results on sterile and non-sterile NK12 were compared. In all tested experimental conditions, the NK12 indigenous microflora was stable and biologically active. S. arizonae 3924 and E. coli 84 M grew rapidly in sterilized NK12 at different moistures and storage temperatures, and their growth was suppressed in non-sterilized NK12. Pathogens inactivation was lower when compost was stored at 40% and 80% humidity and at 37 degrees C. Our results show that the major role in the pathogens suppression was played by the indigenous microflora of the finished compost, although physical factors too influenced the growth phenomenon.     

Coupled factors influencing the transport and retention of Cryptosporidium parvum oocysts in saturated porous media

The coupled role of solution ionic strength (IS), hydrodynamic force, and pore structure on the transport and retention of viable Cryptosporidium parvum oocyst was investigated via batch, packed-bed column, and micromodel systems. The experiments were conducted over a wide range of IS (0.1-100 mM), at two Darcy velocities (0.2 and 0.5 cm/min), and in two sands (median diameters of 275 and 710 μm). Overall, the results suggested that oocyst retention was a complex process that was very sensitive to the solution IS, the Darcy velocity, and the grain size. Increasing IS led to enhanced retention of oocysts in the column, which is qualitatively consistent with predictions of Derjaguin-Landau-Verwey-Overbeek theory. Conversely, increasing velocity and grain size resulted in less retention of oocysts in the column due to the difference in the fluid drag force and the rates of mass transfer from the liquid to the solid phase and from high to low velocity regions. Oocyst retention was controlled by a combined role of low velocity regions, weak attractive interactions, and/or steric repulsion. The contribution of each mechanism highly depended on the solution IS. In particular, micromodel observations indicated that enhanced oocyst retention occurred in low velocity regions near grain-grain contacts under highly unfavorable conditions (IS = 0.1 mM). Oocyst retention was also found to be influenced by weak attractive interactions (induced by the secondary energy minimum, surface roughness, and/or nanoscale chemical heterogeneity) when the IS = 1 mM. Reversible retention of oocysts to the sand in batch and column studies under favorable attachment conditions (IS = 100 mM) was attributed to steric repulsion between the oocysts and the sand surface due to the presence of oocyst surface macromolecules. Comparison of experimental observations and theoretical predictions from classic filtration theory further supported the presence of this weak interaction due to steric repulsion.     

The development of protein chip using protein G for the simultaneous detection of various pathogens

A protein chip using protein G for the simultaneous detection of various pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Yersinia enterocolitica, and Legionella pneumophila was developed. In order to endow the orientation of antibody molecules on solid surface, protein G was introduced. The protein G on gold (Au) surface modified with 11-mercaptoundecanoic acid (MUA) was arrayed and then four different kinds of monoclonal antibodies (Mabs) against pathogens (E. coli O157:H7, S. typhimurium, Y. enterocolitica, and L. pneumophila) on protein G spots were selectively arrayed using a microarrayer, and its spatial density was over 2400 spots cm². Using the constructed protein chip, the various pathogens such as E. coli O157:H7, S. typhimurium, Y. enterocolitica, and L. pneumophila could be detected by a sandwich method and its lowest detection limit for E. coli O157:H7 was 10² CFU/ml. The proposed fabrication technique of protein chip for the detection of various pathogens could be applied to construct other protein chips with a high efficiency.     

山东花生茎腐病病原菌研究

从山东省多地采集的花生茎腐病株上分离到15个茎腐病菌株,选取6个典型菌株,对病原菌进行形态特征、致病性、生物学特性研究。核糖体DNA-ITS序列分析表明,6个菌株的核糖体DNA-ITS序列同源性高达99％。根据病原菌的形态特征、核糖体DNA-ITS序列同源性比较,确认供试6个菌株为同一种病原菌,与Gen-Bank中可可毛色二孢属（Lasiodiplodia）（Diplodia的同物异名）的同源性高达99％以上。根据6个菌株的形态特征、致病性测定结果及生物学特性,并结合核糖体DNA-ITS序列分析,将山东花生茎腐病的病原鉴定为棉色二孢菌（Diplodia gossypina）。     

Application of caffeine, 1,3,7-trimethylxanthine,to control Escherichia coli O157:H7

The objective of this research was to determine the effectiveness of caffeine on inactivation of Escherichia coli O157:H7 in brain heart infusion (BHI) broth. Overnight samples of five E. coli O157:H7 strains of (E0019, F4546, H1730, 944 and Cider) were used in this study. These strains were individually inoculated at an initial inoculum level of 2 log CFU/ml into BHI broth containing caffeine at different concentrations (0.00%, 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, and 2.00%). Samples were then incubated at 37 °C for 24 h. Bacterial growth was monitored at different time intervals by measuring turbidity at 610 nm using a spectrophotometer. Results revealed that the addition of caffeine inhibited the growth of E. coli O157:H7. Significant growth inhibition was observed with concentration levels of 0.50% and higher. These results indicate that caffeine has potential as an antimicrobial agent for the treatment of E. coli O157:H7 infection and should be investigated further as a food additive to increase biosafety of consumable food products.     

Preparation of antibody-conjugated gold nanoparticles

We here describe a method for the simple synthesis of gold nanoparticles (~ 10 nm in diameter) conjugated with antibody to E. coli O157:H7. Gold nanoparticles with pendant carboxylic acid and alcohol functional groups were synthesized and characterized using transmission electron microscopy (TEM) and infrared spectroscopy. These nanoparticles were then reacted with anti-E. coli O157:H7, using EDC coupling chemistry, and the product was characterized with X-ray photoelectron spectroscopy. Furthermore, binding of the antibody-gold conjugates to E. coli O157:H7 was demonstrated using transmission electron microscopy.     

2004-2007年肇庆市食品中食源性致病菌监测与分析

目的了解肇庆市食品中食源性致病菌的污染状况和污染水平,初步确定高危食品的种类,为食源性疾病的监测提供科学的依据。方法按全国食品污染物监测网的工作手册,2004-2007年采集4个监测点的5大类食品(生肉、熟肉、水产品、生吃水产品和生吃蔬菜)共计325份,对沙门菌、单核细胞增生李斯特菌、大肠杆菌O157∶H7和副溶血性弧菌4种食源性致病菌进行监测分析。结果325份食品样品中,检出致病菌27株(8.31%)。其中沙门菌16株(4.92%),单核细胞增生李斯特菌10株(3.08%),大肠杆菌O157∶H7未检出,80份水产品中检出副溶血性弧菌1株(1.25%)。生肉的污染情况最为严重,检出率为11.35%,其次是熟肉,检出率为9.68%;水产品的检出率为5.45%,生食水产品的检出率为4.00%,生食蔬菜的检出率为2.56%。结论沙门菌和单核细胞增生李斯特菌对肇庆市食品的污染普遍存在。生肉、熟肉的污染尤为严重,是重要的高危食品。     

Viability of multi-strain mixtures of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 inoculated into the batter or onto the surface of a soudjouk-style fermented semi-dry sausage

The fate of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on soudjouk. Fermentation and drying alone reduced numbers of L. monocytogenes by 0.07 and 0.74 log₁₀ CFU/g for sausages fermented to pH 5.3 and 4.8, respectively, whereas numbers of S. typhimurium and E. coli O157:H7 were reduced by 1.52 and 3.51 log₁₀ CFU/g and 0.03 and 1.11 log₁₀ CFU/g, respectively. When sausages fermented to pH 5.3 or 4.8 were stored at 4, 10, or 21 °C, numbers of L. monocytogenes, S. typhimurium, and E. coli O157:H7 decreased by an additional 0.08–1.80, 0.88–3.74, and 0.68–3.17 log₁₀ CFU/g, respectively, within 30 days. Storage for 90 days of commercially manufactured soudjouk that was sliced and then surface inoculated with L. monocytogenes, S. typhimurium, and E. coli O157:H7 generated average D-values of ca. 10.1, 7.6, and 5.9 days at 4 °C; 6.4, 4.3, and 2.9 days at 10 °C; 1.4, 0.9, and 1.6 days at 21 °C; and 0.9, 1.4, and 0.25 days at 30 °C. Overall, fermentation to pH 4.8 and storage at 21 °C was the most effective treatment for reducing numbers of L. monocytogenes (2.54 log₁₀ CFU/g reduction), S. typhimurium (5.23 log₁₀ CFU/g reduction), and E. coli O157:H7 (3.48 log₁₀ CFU/g reduction). In summary, soudjouk-style sausage does not provide a favorable environment for outgrowth/survival of these three pathogens.