Autofluorescence and green fluorescent protein-derived fluorescence in Listeria innocua

Fluorescent L. innocua M1 was generated by transformation with plasmid SB2019 carrying gfp3. The transformed organism exhibited 2 h longer lag phase than the parental strain. The transformation and gfp3 expression did not affect the growth rates (0.49 ± 0.02 and 0.47 ± 0.05 h⁻¹) and the maximum optical densities (1.02 ± 0.01 and 0.90 ± 0.06) of the parental or the transformed strains. The transformation of L. innocua M1 with pSB2019 resulted in cell-concentration related fluorescence which was detectable from washed cells but not in growth media. Statistical discrimination between GFP3-driven and background fluorescence signals of transformed and parental strains occurred at cell optical densities of 0.1 and above which was partially due to a relatively high endogenous autofluorescence. Although the gfp-transformed L. innocua M1 developed in this study has the potential to be a marker organism for monitoring Listeria spp. responses in mixed cultures, more work is needed to optimize the GFP-based fluorescent signal.     

The effects of carbon dioxide addition to cheese milk on the microbiological properties of Turkish White brined cheese

This study invest0igated the effect of CO₂ added to achieve three pH levels: pH 6.1, pH 6.2 and pH 6.3 for treatments X, Y, Z, respectively, on some microbiological properties of Turkish White (TW) brined cheese. For each pH, four batches of cheese were produced from: raw milk with no added carbon dioxide (UR), raw milk with carbon dioxide (TR), pasteurised milk with no carbon dioxide addition (UP) and pasteurised milk with carbon dioxide addition (TP). The microbiological analysis of TW brined cheeses was carried out for 90 days of maturation period. Total aerobic mesophilic bacteria, mesophilic lactic acid bacteria, yeasts and moulds and coliform group were determined in control and CO₂ treatment groups. Mesophilic bacteria count was determined as 5.14, 5.29, 5.67 log cfu/g for pH 6.1, 6.2 and 6.3, respectively, in CO₂‐treated raw milk cheeses. Yeasts and moulds reduction increased significantly by applying CO₂ (P < 0.01). For TW cheese samples, the most significant microbial inactivation was detected at sample groups of pH 6.1.     

Transfer of Listeria innocua from contaminated compost and irrigation water to lettuce leaves

Many foodborne outbreaks of some pathogens such as Escherichia coli O157:H7, Salmonella or Listeria have been associated with the consumption of contaminated vegetables. Contaminated manure and polluted irrigation water are probable vehicles for the pathogens. The aim of this study was to determine the potential transfer of Listeria innocua from soil fertilized with contaminated compost or irrigated with contaminated water to the edible parts of lettuce grown on these soils together with its survival in lettuce and in soil under field conditions during two different seasons. Moreover, its survival on lettuce sprinkled with contaminated irrigation water was evaluated. L. innocua survived in soil samples for 9 weeks at high concentrations, 10⁵ cfu gdw⁻¹ in fall and 10³ cfu gdw⁻¹ in spring. Pathogen survived better in fall, indicating an important influence of temperature and humidity. L. innocua population in lettuce leaves was very high on lettuce leaves after sprinkling, but decreased to undetectable levels at field conditions. There was also transfer of L. innocua from soil contaminated with compost or irrigated with contaminated water to lettuce leaves, mainly to the outer ones. Survival profiles of L. innocua on lettuce and soil samples contaminated either by application of contaminated compost or surface irrigation water was similar. Our results indicated that contaminated compost and contaminated irrigation water can play an important role in the presence of foodborne pathogens on vegetables.     

Validation of a model for growth of Lactococcus lactis and Listeria innocua in a structured gel system: effect of monopotassium phosphate

The effect of monopotassium phosphate (KH(2)PO(4)) on the chemical environment and on growth of Listeria innocua and Lactococcus lactis in coculture were investigated in a liquid and in a gelled microbiological medium at 12 degrees C and an initial pH of 6.2. As expected, addition of KH(2)PO(4) to both the liquid and gelled media resulted in an increase in buffering capacity. This effect on buffering capacity changed the profiles of lactic acid dissociation and pH evolution. At all gelatin concentrations studied, addition of KH(2)PO(4) increased the growth rate and the stationary cell concentration of L. lactis. In addition, the growth rate of L. innocua slightly increased but, in contrast, the stationary cell concentration remained unchanged. A new class of predictive models developed previously in our research team to quantify the effect of food model gel structure on microbial growth [Antwi, M., Bernaerts, K., Van Impe, J. F., Geeraerd, A. H., 2007. Modelling the combined effect of food model system and lactic acid on L. innocua and L. lactis growth in mono- and coculture. International Journal of Food Microbiology 120, 71-84] was applied. Our analysis indicate that KH(2)PO(4) influenced the parameters of the chemical and microbiological subprocesses of the model. Nonetheless, the growth model satisfactorily predicted the stationary cell concentration when (i) the undissociated lactic acid concentrations at which L. innocua and L. lactis growth cease were chosen as previously reported, and (ii) all other parameters of the chemical and microbiological subprocesses were computed for each medium. This confirms that the undissociated lactic acid concentrations at which growth ceases is a unique property of a bacterium and does not, within our case study, depend on growth medium. The study indicates that microbial growth depends on the interplay between the individual food components which affect the physicochemical properties of the food, such as the buffering capacity. Towards future research, it can be concluded that mathematical models which embody the effect of buffering capacity are needed for accurate predictions of microbial growth in food systems.     

Cold atmospheric gas plasma disinfection of chicken meat and chicken skin contaminated with Listeria innocua

Gas plasmas generated at atmospheric pressure and ambient temperatures offer a possible decontamination method for poultry products. The efficacy of cold atmospheric gas plasmas for decontaminating chicken skin and muscle inoculated with Listeria innocua was examined. Optimization of operating conditions for maximal bacterial inactivation was first achieved using membrane filters on which L. innocua had been deposited. Higher values of AC voltage, excitation frequency and the presence of oxygen in the carrier gas resulted in the greatest inactivation efficiency, and this was confirmed with further studies on chicken muscle and skin. Under optimal conditions, a 10 s treatment gave > 3 log reductions of L. innocua on membrane filters, an 8 min treatment gave 1 log reduction on skin, and a 4 min treatment gave > 3 log reductions on muscle. These results show that the efficacy of gas plasma treatment is greatly affected by surface topography. Scanning electron microscopy (SEM) images of chicken muscle and skin revealed surface features wherein bacteria could effectively be protected from the chemical species generated within the gas plasma. The developments in gas plasma technology necessary for its commercial application to foods are discussed.     

Effectiveness of High Intensity Light Pulses (HILP) treatments for the control of Escherichia coli and Listeria innocua in apple juice, orange juice and milk

High Intensity Light Pulses (HILP) represent an emerging processing technology which uses short (100-400 μs) light pulses (200-1100 nm) for product decontamination. In this study, model and real foods of differing transparencies (maximum recovery diluent (MRD), apple and orange juices and milk) were exposed to HILP in a batch system for 0, 2, 4 or 8 s at a frequency of 3 Hz. After treatment, inactivation of Escherichia coli or Listeria innocua was evaluated in pre-inoculated samples. Sensory and other quality attributes (colour, pH, Brix, titratable acidity, non-enzymatic browning, total phenols and antioxidant capacity (TEAC)) were assessed in apple juice. Microbial kill decreased with decreasing transparency of the medium. In apple juice (the most transparent beverage) E. coli decreased by 2.65 and 4.5 after exposure times of 2 or 4 s, respectively. No cell recovery was observed after 48 h storage at 4 °C. No significant differences were observed in quality parameters, excepting TEAC and flavour score, where 8 s exposure caused a significant decrease (p < 0.05). Based on these results, HILP with short exposure times could represent a potential alternative to thermal processing to eliminate undesirable microorganisms, while maintaining product quality, in transparent fruit juices.     

Quantitative Evaluation of Thermal Inactivation Kinetics of Free-Floating Versus Surface-Attached Listeria innocua Cells

Surface pasteurization is one of the decontamination treatments that can contribute to better preservation of meat products retaining most of their quality characteristics relatively intact if compared with the raw products. The current research compares the kinetics of free-floating and surface attached Listeria innocua cells by using integrated microbial and heat transfer modelling approaches. Surface pasteurization treatments are applied on a (abiotic) Teflon® model system in a novel steam surface decontamination rig. The experimental set-up prevented following four technological aspects to occur, (1) cold purge migration to the surface during the heating process, (2) inactivation kinetics of a cocktail of microbes, (3) protective effect of food components, and (4) physical distribution of bacteria throughout the depth of the product skin. Microbial load predictions are performed based on the inactivation parameters obtained during free-floating cell experiments. These predictions, when compared with the microbial data of the surface treatments, prove that the surface attached cells were much more heat resistant, despite the experimental set-up preventing the aforementioned (technological) events to occur. Indeed, surface attached cells can have different physiological/phenotypical/genetical characteristics, such as cell aggregations, colony formations, presence of flagella. In a final step, three techniques are implemented to evaluate mathematically the kinetics of the surface attached cells. Overall, this research’s significance is lying in the quantitative assessment of microbial heat resistance. The technological reasons underlying the increased microbial heat resistance on biotic and abiotic surfaces should be reevaluated, taking into account possible physiological/phenotypical/genetical characteristics.     

Behaviour of Listeria monocytogenes inoculated into Minas Frescal cheese made by direct acidification or lactic culture during refrigerated storage

The behaviour of Listeria monocytogenes Scott A artificially inoculated into Brazilian Minas Frescal cheese manufactured with the addition of a lactic culture or by direct acidification with lactic acid was evaluated at 5 and 10°C for 25 days. In cheese produced with a lactic culture, the counts remained almost unchanged during storage, whereas cheeses produced by direct acidification presented an increase in the counts of 2–3 log cycles. The profiles of the lactic bacterial counts, associated with those of L. monocytogenes, show a strong influence of the competitive starter culture on the survival of L. monocytogenes . This suggests that the addition of starter culture is an efficient way to control the growth of L. monocytogenes in these products.     

Occurrence and Antimicrobial Susceptibility of Listeria monocytogenes Isolated from Brined White Cheese in Jordan

Listeria monocytogenes is a serious foodborne pathogen that has been isolated from different dairy food products. Several foodborne outbreaks of listeriosis have been associated with consumption of cheese. The aims of this study were to determine the occurrence of L. monocytogenes and Listeria spp. in brined white cheese (BWC) sold in Jordan, and to determine the susceptibility of isolated L. monocytogenes to antimicrobials. Three hundred and fifty samples of 5 different types of BWC (akkawi, boiled, halloumi, pasteurized, and shellal) were collected from a local market in Jordan. The ISO (11290-1) procedure was followed for isolation and identification of Listeria spp. from cheese samples and a polymerase chain reaction (PCR) technique was used for confirmation of L. monocytogenes isolates. The VITEK2 automated system was used for testing antimicrobial susceptibility of L. monocytogenes isolates. The overall prevalence of Listeria spp. in cheese sample was 27.1%. L. monocytogenes was isolated from 39 (11.1%) samples. Other isolated species were L. grayi (6.9%), L. innocua (2%), L. ivanovii (4%), L. seeligeri (2%), and L. welshimeri (0.3%). The pH values and salt concentrations of L. monocytogenes positive cheese samples ranged from 5.10 to 6.32 and 5.64 to 13.16, respectively. L. monocytogenes isolates were sensitive or intermediate susceptible to imipenem, gentamicin, linezolid, teicoplanin, vancomycin, fusidic acid, trimethoprim/sulfamethoxazole, benzylpenicillin, ciprofloxacin, erythromycin, tetracycline, and rifampicin, but resistant to fosfomycin, oxacillin, and clindamycin.     

Survial of Listeria monocytogenes during the manufacture and ripening of Turkish White cheese

Listeria monocytogenes was enumerated during the manufacture and ripening of Turkish White cheese with particular reference to a) pasteurized milk, b) cheese milk after inoculation with L. monocytogenes (0 h( c) after curd formation (2 h( d) curd after pressing (6 h( e) curd after pH was reduced (17 h( f) curd after salting (32 h( and g) cheeses during ripening. Cheeses were also examined periodically for total solids, moisture and salt contents, pH values and aerobic plate count. An increase in the number ofL. monocytogenes was observed during manufacture. Following salting and throughout the storage period, numbers of L. monocytogenes decreased at a rate depending on the salt concentration, starter activity and storage time. The initial microbial number had a significant (P > 0.01) effect on the survival of L. monocytogenes during the storage period.     

Sensitization of Listeria innocua to inorganic and organic acids by natural antimicrobials

Acid tolerance of two strains of Listeria innocua as single strain culture and co-culture, were evaluated in liquid cheese whey after exposure to nisin, Microgard™ and green tea. Inorganic and organic acids were applied after natural antimicrobials treatments and microbial counts were made to analyze the bacterial response. The results have demonstrated that natural antimicrobials like nisin, Microgard™ and green tea, present in the liquid cheese whey, did not produce any cross-protection effects. On the contrary, in most of the cases, the antimicrobial treatment increased the susceptibility of L. innocua to acid stress, particularly in the treatment with nisin or green tea extract and organic acids. These results were corroborated by different techniques, including transmission electron microscopy.     

Listeria innocua and aerobic mesophiles during chill storage of inoculated mechanically recovered poultry meat treated with high hydrostatic pressure

Mechanically recovered poultry meat (MRPM) was inoculated with Listeria innocua 910 CECT at a level of approximately 10⁸ CFU g⁻¹. Vacuum-packaged samples were treated by combinations of pressure (350, 400, 450 and 500 MPa), time (5, 10, 15 and 30 min) and temperature (2, 10 and 20°C) and later stored at 2°C for 2 months. Counts of L. innocua and aerobic mesophilic bacteria were determined 1, 4, 7, 15, 30 and 60 days after pressurisation. For mesophiles, in most treatments, pressurization at 2°C gave the significantly best results. High pressure caused a marked bactericidal effect on L. innocua: reductions higher than 7.5 log units were achieved in several cases. Some cells were just sublethally injured by pressure. Samples treated at 500 MPa for 30 min at 2°C had counts of only 2.3 log units after 60 days of chill storage. Noninoculated pressurised MRPM did not show Listeria growth throughout storage. These results suggest that high pressure processing can enhance the microbiological quality of MRPM.     

Combined effect of packaging atmosphere and storage temperature on growth of Listeria monocytogenes on ready-to-eat shrimp

Cooked, peeled, and deveined shrimp were inoculated with a 5 strain mixture of Listeria monocytogenes and packaged in air, vacuum, and a 100% carbon dioxide modified atmosphere. The packaged shrimp were then stored at 3, 7, and 12 degrees C for 15 days to monitor the growth of L. monocytogenes and psychrotrophic bacteria. Uninoculated shrimp were also subjected to sensory evaluation by a trained panel to measure odor and appearance over the storage period. Results demonstrated that shrimp packaged in CO(2) and stored at 3 degrees C did not permit growth of L. monocytogenes during the 15-day storage period, while all other packaging/temperature combinations allowed for multiplication of the bacterium. Carbon dioxide packaging also resulted in the slowest growth of psychrotrophic bacteria and resulted in shrimp having acceptable sensory odor and appearance scores at the end of storage. When strict temperature control is difficult, such as during processing, transportation, retail display, or home use, additional antimicrobial hurdles may be necessary to ensure safety.     

Enumeration and growth of naturally occurring Listeria spp. in unpackaged ham

A total of 301 unpackaged retail ham samples were tested for the presence and number of Listeria spp. after 7 days at 5 degrees C to simulate domestic storage. Thirteen samples (4.3%) contained Listeria monocytogenes, with the highest count being 1.6 x 10(3)cfu g(-1). Thirteen samples contained other Listeria spp. Genotyping showed that only one L. monocytogenes isolate from the 14 tested was of a type previously identified in New Zealand human cases. Listeria-contaminated batches were incubated at 5 degrees C over approximately 3 weeks to assess the growth rate of natural contaminants. None contained L. monocytogenes, but growth occurred in one sample containing Listeria welshimeri and four containing Listeria innocua. Growth was usually slow at 0.002-0.004 log h(-1). In one sample, L. innocua grew at 0.02 log h(-1) although the maximum number reached was only 4.0-5.0 x 10(3)cfu g(-1). In five other samples little growth, if any, occurred. Growth of naturally occurring Listeria spp. at 5 degrees C was therefore generally slower than predicted by the Pathogen Modelling Programme (PMP) or did not occur.     

食源性单增李斯特菌与英诺克李斯特菌基因组特征比较

目的 通过全基因组测序对甘肃省市售食品中分离的单增李斯特菌和英诺克李斯特菌基因组特征进行比较分析。方法 收集2021—2022年甘肃省市售食品中分离的25株单增李斯特菌和7株英诺克李斯特菌作为研究对象,对菌株进行全基因组测序,分析其系统发育谱系、克隆复合群（CC）、序列型（ST）、毒力基因、抗性基因及泛基因组。结果 32株李斯特菌分属单增李斯特菌谱系Ⅰ和Ⅱ及英诺克李斯特菌3个群,单增李斯特菌分为10个亚群,英诺克李斯特菌分为5个亚群,与CC型保持一致,核心基因组多位点序列分型能将各谱系中不同CC型的菌株明显分开,谱系Ⅰ与英诺克李斯特菌的进化关系更近。25株单增李斯特菌均携带李斯特菌毒力岛LIPI-1和内化素基因,不携带LIPI-3,有2株ST87型菌株携带LIPI-4;7株英诺克李斯特菌均不携带LIPI-1和内化素基因,均携带LIPI-4,有5株菌携带LIPI-3。单增李斯特菌有16株携带SSI-1、3株携带SSI-2,7株英诺克李斯特菌均不携带SSI-1,有6株携带SSI-2。李斯特菌的泛基因组大小随着测序基因组数目的增加呈现线性增多,25株单增李斯特菌当菌株数量达到15后核心基因数目稳定在2 272个,占泛基因组基因数目的46.2%,25株单增李斯特菌和7株英诺克李斯特菌共同的核心基因1 487个,当菌株数量达到10后数目趋于稳定。结论 核心基因组多位点序列分型可将不同谱系不同克隆复合群的李斯特菌进行区分,英诺克李斯特菌与单增李斯特菌生化特性相似与其亲缘关系相近有关,致病性差异与英诺克李斯特菌缺失单增李斯特菌特有的毒力基因相关。     

Characteristics of Milk,Cheddar-Like and White Cheese Obtained from Three Goat Phenotypes during Three Lactation Periods

Goat's milk from three phenotypes: Shami, Local, and Mixed was collected during three lactation periods in 2002. The milk was analyzed for moisture, protein, fat, ash, and lactose content. Also milk was examined for yeast, mold and bacterial content. Cheddar and white cheese were manufactured from goat's bulk milk and examined for chemical composition and evaluated for color, taste and flavor, appearance, and texture and aroma. Results showed that the chemical composition of goat's milk varied significantly during the whole year and also among the three goat phenotypes. Local goats produced milk at the first lactation period with highest (44.6%) fat content: the lowest fat content was 29.8% for the Mixed color phenotype at the end of the year. Protein showed the same trend. Shami goat produced milk lower in lactose and ash content than the mixed color breed, whereas the Local (black) produced milk having intermediate content of lactose and ash. Cheddar and white cheese were highly acceptable. Their composition varied—cheddar cheese was higher in protein and NSF but slightly lower in fat content. White cheese retained more lactose. Goat's milk of Shami, Local (black) and Mixed color phenotypes were found to be highly contaminated with bacteria and mold due to lack of hygiene. Milk contained low number of pathogenic bacteria. Using a pasteurization temperature at 73°C for 16 seconds was found to eliminate them.     

Efficiency of pulse pressure treatment for inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Listeria innocua</Emphasis> in whole milk

Escherichia coli ATCC 11775 and Listeria innocua ATCC 33090 in whole milk were inactivated by single- and multi-pulsed (up to 10 pulses) high hydrostatic pressure (HHP) treatments. Both bacteria showed similar resistance to single- and multi-pulsed HHP. The efficiency of pulsed pressure treatment depends on the combination of holding time of each pulse and number of pulses. It was observed that multi-pulsed pressure treatment instead of traditional single-pulsed HHP could be used to pasteurize milk at a lower pressure level. Nevertheless, an optimization is necessary between the pulse holding time, number of pulses, and pressure levels to reach the desirable log-reduction of microorganisms compatible with industrial application. This study was partly presented in Joint 21st AIRAPT and 45th EHPRG International Conference on High Pressure Science and Technology held September 17–21, 2007 in Catania (Italy).     

Effect of combining nisin and/or lysozyme with in-package pasteurization for control of Listeria monocytogenes in ready-to-eat turkey bologna during refrigerated storage

This study investigated the efficacy of in-package pasteurization combined with pre-surface application of nisin and/or lysozyme to reduce and prevent the subsequent recovery and growth of Listeria monocytogenes during refrigerated storage on the surface of low-fat turkey bologna. Sterile bologna samples were treated with solutions of nisin (2 mg/ml=5000 AU/ml), lysozyme (10 mg/ml=80 AU/ml) and a mixture of nisin and lysozyme (2 mg nisin+10mg lysozyme/ml) before in-package pasteurization at 65 degrees C for 32s. In-package pasteurization resulted in an immediate 3.5-4.2 log CFU/cm(2) reduction in L. monocytogenes population for all treatments. All pasteurized treatments also resulted in a significant reduction of L. monocytogenes by 12 weeks compared to un-pasteurized bologna. In-package pasteurization in combination with nisin or nisin-lysozyme treatments was effective in reducing the population below detectable levels by 2-3 weeks of storage. Results from this study could have a significant impact for the industry since a reduction in bacterial population was achieved by a relatively short pasteurization time and antimicrobials reduced populations further during refrigerated storage.     

Lactosylation of milk proteins during the manufacture and storage of skim milk powders

Extensive lactosylation of milk proteins in standard skim milk powder dried against air between 185 and 90°C (inlet and outlet temperatures of the air) was detected by capillary electrophoresis. Optimisation of the drying conditions included keeping the outlet temperature low (preferably <80°C), since this was the parameter which most affected the extent of lactosylation of milk proteins. The inlet temperature was set in order to obtain the best compromise between a low extent of lactosylation and a high drying rate (170–175°C). These conditions allowed the manufacture of low-lactosylated skim milk powder. Storage of freeze-dried and control low-lactosylated skim milk powder at different temperatures showed that both temperature and moisture content affected the progress of lactosylation during storage. Further drying to less than 2.5% moisture content (w/w) and storage at low temperatures were required to prevent the development of lactosylation in the low-lactosylated skim milk powder.     

Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production

The aim of this study was to survey the presence of Staphylococcus aureus and Listeria monocytogenes during the cheese making process in small-scale raw milk cheese production in Norway.The prevalence of S. aureus in bovine and caprine raw milk samples was 47.3% and 98.8%, respectively. An increase in contamination during the first 2-3 h resulted in a 73.6% prevalence of contamination in the bovine curd, and 23 out of 38 S. aureus-negative bovine milk samples gave rise to S. aureus-positive curds. The highest contamination levels of S. aureus were reached in both caprine and bovine cheese after 5-6 h (after the first pressing). There was no contamination of L. monocytogenes in caprine cheeses and only one (1.4%) contaminated bovine cheese.This work has increased our knowledge about S. aureus and L. monocytogenes contamination during the process of raw milk cheese production and gives an account of the hygiene status during the manufacture of Norwegian raw milk cheeses.