Discrimination of intact and injured Listeria monocytogenes by Fourier transform infrared spectroscopy and principal component analysis

Fourier transform infrared spectroscopy (FT-IR, 4000-600 cm(-)(1)) was used to discriminate between intact and sonication-injured Listeria monocytogenes ATCC 19114 and to distinguish this strain from other selected Listeria strains (L. innocua ATCC 51742, L. innocua ATCC 33090, and L. monocytogenes ATCC 7644). FT-IR vibrational overtone and combination bands from mid-IR active components of intact and injured bacterial cells produced distinctive "fingerprints" at wavenumbers between 1500 and 800 cm(-)(1). Spectral data were analyzed by principal component analysis. Clear segregations of different intact and injured strains of Listeria were observed, suggesting that FT-IR can detect biochemical differences between intact and injured bacterial cells. This technique may provide a tool for the rapid assessment of cell viability and thereby the control of foodborne pathogens.     

Identification and enumeration of beta-hemolytic Listeria monocytogenes in naturally contaminated dairy products

A DNA probe was used to identify hemolytic Listeria monocytogenes in naturally contaminated dairy products: unpasteurized milk, ricotta cheese, and imported semisoft cheeses. Of 34 milk samples, 12 were suspected to contain hemolytic L. monocytogenes; 1 contained greater than 6000 viable organisms/g. The ricotta cheese, although temperature-abused, had a titer of 3.6 x 10(6) beta-hemolytic L. monocytogenes cells/g, whereas the semisoft cheeses reached a maximum of 5.6 x 10(6) cells/g. Pure cultures of L. monocytogenes isolated from both types of cheese were found positive by the CAMP test and the DNA probe.     

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.     

Direct plating technique for enumeration of Listeria monocytogenes in foods

The advantages and disadvantages of various techniques for detecting and enumerating Listeria monocytogenes in foods are reviewed, and results from a study designed to compare 14 direct plating media for their suitability to recover uninjured cells of L. monocytogenes from 4 foods are summarized. McBride Listeria agar (MLA), gum base nalidixic acid tryptone soy agar (GBNTSA), modified Despierres agar (MDA), and modified MLA (MMLA) performed best for recovering all inoculum populations from milk and ice cream mix. For Brie cheese, MLA, MDA, MMLA, and Dominguez Rodriguez isolation agar were superior for recovering L. monocytogenes; GBNTSA, MDA, MMLA, and Donnelly's Listeria enrichment agar were best for recovering the organism from cabbage. Direct plating procedures without prior enrichment can be utilized successfully for recovering L. monocytogenes from foods such as pasteurized milk and ice cream mix, which contain low populations of background microflora. However, recovery of L. monocytogenes from foods such as raw cabbage and Brie cheese, which contain high populations of other microorganisms, was not satisfactory using direct plating procedures.     

Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry

A method was developed specifically to detect naturally occurring Listeria monocytogenes in meat because the traditional cold enrichment procedure was extremely slow and other procedures were ineffective. This method could identify beta-hemolytic Listeria colonies in 3-4 days. The use of a 2-stage enrichment, highly selective LPM agar, and a thin-layer horse blood agar plate for the detection of beta-hemolytic Listeria isolates are the important steps of this method. L. monocytogenes was recovered from 20 of 41 samples of frozen ground beef, 12 of 23 samples of pork sausage, and 7 of 22 samples of poultry. These results indicate that L. monocytogenes is common in raw meat and that this method is effective for its recovery.     

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.     

Spray-dried zein capsules with coencapsulated nisin and thymol as antimicrobial delivery system for enhanced antilisterial properties

Food grade antimicrobial delivery systems were studied in this work to enhance the effectiveness of antimicrobials inhibiting the growth of Listeria monocytogenes during storage. Corn zein was used as a carrier biopolymer and nisin and thymol as antimicrobials. Capsules produced by spray drying demonstrated different microstructures and release characteristics of nisin at different usage levels of thymol. Better release profiles were achieved when glycerol was additionally used to prepare capsules. Capsules showing sustained release of significant amounts of both antimicrobials effectively inhibited the growth of L. monocytogenes at pH 6.0 and 30 °C in the growth medium. Capsules were also more effective than free antimicrobials in inhibiting the growth of L. monocytogenes in 2% reduced fat milk at 25 °C. Our work showed that engineered delivery systems have promise to fulfill the antimicrobial effectiveness during shelf life storage of foods to ensure microbiological safety.     

Radioresistance of Salmonella species and Listeria monocytogenes on minimally processed arugula (Eruca sativa Mill.): effect of irradiation on flavonoid content and acceptability of irradiated produce

This work studied the radiation resistance of Listeria monocytogenes and Salmonella species and the effect of irradiation on leaf flavonoid content and sensory acceptability of minimally processed arugula. Immersion in ozone-treated water reduced the analyzed microorganisms by 1 log. L. monocytogenes and Salmonella were not isolated from samples. Samples of this vegetable were inoculated with a cocktail of Salmonella spp. and L. monocytogenes and exposed to gamma irradiation. D10 values for Salmonella ranged from 0.16 to 0.19 kGy and for L. monocytogenes from 0.37 to 0.48 kGy. Kaempferol glycoside levels were 4 and ca. 3 times higher in samples exposed to 1 and 2 kGy, respectively, than in control samples. An increase in quercetin glycoside was also observed mainly in samples exposed to 1 kGy. In sensory evaluation, arugula had good acceptability, even after exposure to 2 and 4 kGy. These results indicate that irradiation has potential as a practical processing step to improve the safety of arugula.     

Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence

The simultaneous detection of Staphylococcus aureus, Listeria monocytogenes, and Salmonella spp. has been approached by a new multiplex PCR-based procedure followed by capillary gel electrophoresis with laser-induced fluorescence detection (multiplex-PCR-CGE-LIF). As compared to slab gel electrophoresis, the use of CGE-LIF improved from 10- to 1000-fold the sensitivity of the multiplex PCR analysis, allowing the detection of 2.6 x 10(3) cfu mL(-1) of S. aureus, 570 cfu mL(-1) of L. monocytogenes, and 790 cfu mL(-1) of Salmonella in artificially inoculated food, without enrichment. Following 6 h of enrichment, as low as 260, 79, and 57 cfu mL(-1) of S. aureus, L. monocytogenes, and Salmonella, respectively, were detected. The CGE-LIF method is shown to be reproducible, providing relative standard deviation (RSD) values lower than 0.8% for analysis time and lower than 5.8% for peak areas. The multiplex-PCR-CGE-LIF proved a powerful analytical tool to detect various food pathogens simultaneously in a fast, reproducible, and sensitive way.     

Application of hexanal,E-2-hexenal,and hexyl acetate to improve the safety of fresh-sliced apples

The aims of this work were to evaluate the effects of different concentrations of hexanal, (E)-2-hexenal, hexyl acetate, and their mixtures on the fate of pathogenic species such as Escherichia coli, Salmonella enteritidis, and Listeria monocytogenes inoculated in model systems as well as the antimicrobial activity against the target species of the chosen molecules when added to the packaging atmosphere of inoculated fresh-sliced apples. The result obtained in this work pointed out the potential use of compounds such as hexanal, (E)-2-hexenal, and hexyl acetate for both the extension of shelf life and an improvement of hygienic safety of "minimally processed foods". In fact, hexanal, (E)-2-hexenal, and hexyl acetate had a significant inhibitory effect against pathogen microorganisms frequently isolated from raw materials (E. coli, S. enteritidis, and L. monocytogenes) when inoculated in both model and real systems. In this last condition, these compounds, at the levels used (150, 150, and 20 ppm for hexanal, hexyl acetate, and (E)-2-hexenal, respectively), displayed a bactericide effect on L. monocytogenes and they exhibited significant extensions of lag phase of E. coli and S. enteritidis inoculated at levels of 10(4)-10(5) CFU/g.     

Listeria monocytogenes--a current dilemma

Since 1985, Listeria monocytogenes has gone from being an organism known to only a handful of microbiologists to being a fully recognized food-borne pathogen of concern. With nearly unprecedented speed and vigor, food processors, particularly in the dairy industry, and regulatory agencies have reacted in concert to resolve unanswered questions about Listeria so as to assure the safety of the food supply. This report summarizes what has been learned and what is being done.     

辐照对冷鲜熟制水饺微生物安全质量的影响

本试验主要研究了辐照对冷鲜熟制水饺中可能存在的致病微生物肠炎沙门氏菌、金黄色葡萄球菌和李斯特菌存活状况的影响。研究发现,肠炎沙门氏菌、金黄色葡萄球菌、李斯特菌的D10值分别为0.31、0.44和0.45kGy。随着辐照剂量的增大和贮存期的延长,不同致病菌数量变化趋势存在差异。4kGy辐照可保障冷鲜熟制水饺中3种致病菌达到不得检出的要求。     

Antibacterial and antioxidant activities of essential oils isolated from Thymbra capitata L. (Cav.) andOriganum vulgare L

Antilisterial activities of Thymbra capitata and Origanum vulgare essential oils were tested against 41 strains of Listeria monocytogenes. The oil of T. capitata was mainly constituted by one component, carvacrol (79%), whereas for O. vulgare three components constituted 70% of the oil, namely, thymol (33%), gamma-terpinene (26%), and p-cymene (11%). T. capitata essential oil had a significantly higher antilisterial activity in comparison to O. vulgare oil and chloramphenicol. No significant differences in L. monocytogenes susceptibilities to the essential oils tested were registered. The minimum inhibitory concentration values of T. capitata essential oil and of carvacrol were quite similar, ranging between 0.05 and 0.2 microL/mL. Antioxidant activity was also tested, the essential oil of T. capitata showing significantly higher antioxidant activity than that of O. vulgare. Use of T. capitata and O. vulgare essential oils can constitute a powerful tool in the control of L. monocytogenes in food and other industries.     

New bioactive biomaterials based on quaternized chitosan

Chitosan was chemically modified to produce quaternary ammonium salts in order to improve its antimicrobial activity and physicochemical properties. Quaternization of N-alkyl chitosan derivatives was carried out using alkyl iodide to elaborate water-soluble cationic polyelectrolytes ( N, N, N-trimethylchitosan, TMC). TMC was characterized by (1)H NMR spectroscopy; the quaternization degree was determined from (1)H NMR spectra and by titration of iodide ion. The antibacterial activity of hydroxypropylcellulose (HPC) films or coatings associated with chitosan or TMC as biocide was evaluated against the growth of Listeria monocytogenes and Salmonella typhimurium. The HPC-chitosan and HPC-TMC coatings exhibited a total inhibition on solid medium of both bacterial strains. Experiments conducted in liquid medium showed that the inhibitory activity against the growth of Listeria innocua was improved after chemical modification. Moreover, physicochemical properties of films were evaluated to determine their potential for food applications. The addition of the antibacterial agents showed a significant impact on the moisture barrier and mechanical properties of HPC films.     

Efficiency of two commercial ELISA kits compared with the BAM culture method for detecting Listeria in naturally contaminated foods.

The efficiency of 2 commercial enzyme-linked immunosorbent assay (ELISA) kits (Listeria-Tek and Tecra) for detecting Listeria in naturally contaminated foods was evaluated and compared with that of the culture method described in the Bacteriological Analytical Manual (BAM). Both ELISAs use modified University of Vermont (UVM-1) medium as a primary enrichment; the BAM method uses Listeria enrichment broth. Secondary enrichments for Listeria-Tek and Tecra, respectively, were Fraser broth and UVM-2, which contains additional acriflavin-HCl. When ELISA test results differed, secondary enrichments were tested against the other ELISA; Fraser broth was used to determine recovery rates because of its superiority over UVM-2. Of the 178 food samples examined, the presence of Listeria was detected and culturally confirmed in 38, 37, and 40 samples by the BAM, Listeria-Tek, and Tecra methods, respectively. Differences in results of the ELISAs compared with those of the BAM method were not statistically significant; however, differences between results of the 2 ELISA methods were significant. It was concluded that as rapid screening methods, the Listeria-Tek and the Tecra kits qualify as alternative methods to the BAM cultural method.     

Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity

The antimicrobial activity of different edible vegetable oils was studied. In vitro results revealed that the oils from olive fruits had a strong bactericidal action against a broad spectrum of microorganisms, this effect being higher in general against Gram-positive than Gram-negative bacteria. Thus, olive oils showed bactericidal activity not only against harmful bacteria of the intestinal microbiota (Clostridium perfringens and Escherichia coli) also against beneficial microorganisms such as Lactobacillus acidophilus and Bifidobacterium bifidum. Otherwise, most of the foodborne pathogens tested (Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, Yersinia sp., and Shigella sonnei) did not survive after 1 h of contact with olive oils. The dialdehydic form of decarboxymethyl oleuropein and ligstroside aglycons, hydroxytyrosol and tyrosol, were the phenolic compounds that statistically correlated with bacterial survival. These findings were confirmed by testing each individual phenolic compound, isolated by HPLC, against L. monocytogenes. In particular, the dialdehydic form of decarboxymethyl ligstroside aglycon showed a potent antimicrobial activity. These results indicate that not all oils classified as "olive oil" had similar bactericidal effects and that this bioactivity depended on their content of certain phenolic compounds.     

Intrinsic Tween 20 improves release and antilisterial properties of co-encapsulated nisin and thymol

Antimicrobial delivery systems have been proposed as potential solutions to improve effectiveness of antimicrobials in food matrixes by shielding antimicrobials from contacting food matrix components and releasing them continuously. In this work, spray-dried capsules were produced from zein solutions with the same concentrations of nisin and thymol but with varying Tween 20 contents for characterization of release kinetics of antimicrobials and antilisterial properties. At intermediate levels of Tween 20, sustained and more complete release of antimicrobials was observed at pH 6.0 and 8.0. Most capsule samples were more effective than free antimicrobials against Listeria monocytogenes in 2% reduced fat milk, and the best capsule treatment reduced the bacterial population by 2 log CFU/mL more than comparable free antimicrobials after 4 h incubation at 25 °C. Our work demonstrated that nonionic surfactant can be conveniently used to modulate characteristics of delivery systems to effectively improve antimicrobial functions in food systems.     

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.     

Kombucha fermentation and its antimicrobial activity

Kombucha was prepared in a tea broth (0.5% w/v) supplemented with sucrose (10% w/v) by using a commercially available starter culture. The pH decreased steadily from 5 to 2.5 during the fermentation while the weight of the "tea fungus" and the OD of the tea broth increased through 4 days of the fermentation and remained fairly constant thereafter. The counts of acetic acid-producing bacteria and yeasts in the broth increased up to 4 days of fermentation and decreased afterward. The antimicrobial activity of Kombucha was investigated against a number of pathogenic microorganisms. Staphylococcus aureus, Shigella sonnei, Escherichia coli, Aeromonas hydrophila, Yersinia enterolitica, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus epidermis, Campylobacter jejuni, Salmonella enteritidis, Salmonella typhimurium, Bacillus cereus, Helicobacterpylori, and Listeria monocytogenes were found to be sensitive to Kombucha. According to the literature on Kombucha, acetic acid is considered to be responsible for the inhibitory effect toward a number of microbes tested, and this is also valid in the present study. However, in this study, Kombucha proved to exert antimicrobial activities against E. coli, Sh. sonnei, Sal. typhimurium, Sal. enteritidis, and Cm. jejuni, even at neutral pH and after thermal denaturation. This finding suggests the presence of antimicrobial compounds other than acetic acid and large proteins in Kombucha.     

Symposium on microbiology update: old friends and new enemies. Listeria monocytogenes

Listeria monocytogenes, one of the "new enemies" in food microbiology, is a human and animal pathogen that is widespread in nature. The organism is a transient constituent of the intestinal flora excreted by 1-10% of healthy humans. It is an extremely hardy organism and can survive for many years in the cold in naturally infected sources. L. monocytogenes has been isolated from a wide variety of foods, including dairy products, meats, and fish. Although most of the foodborne listeriosis outbreaks have been linked to the consumption of dairy products, recent sporadic cases have been associated with meats, as well as other foods. It is now recognized that listeriolysin 0, a 60-kilodalton protein, is one of the major virulence factors of the organism. All strains of L. monocytogenes are pathogenic by definition although some appear to be more virulent than others. There have been recent reports of hemolytic isolates of L. monocytogenes, which are nonpathogenic for mice. Attachment to and penetration of cells also appear to be prerequisites for human infection. Cultural methodology for isolating the organism from foods has been in a state of flux since 1985. Rapid methods using both ELISA and DNA technology have been developed. Because of the widespread nature of the organism, it is nearly impossible to eliminate it from the food supply. However, by using a hazard analysis-critical control point approach, the health hazard associated with this organism can be reduced to a minimum.