Transfer of antibiotic resistance from Enterococcus faecium of fermented meat origin to Listeria monocytogenes and Listeria innocua.

UNLABELLED: Listeria monocytogenes is an important foodborne pathogen that can cause infection in children, pregnant women, the immunocompromised and the elderly. Antibiotic resistance in this species would represent a significant public health problem since the organism has a high fatality/case ratio and resistance may contribute to failure of therapeutic treatment. This study was designed to explore whether the in vitro transferability of antibiotic resistance from enterococci to Listeria spp. could occur. It was found that 2/8 Listeria strains were able to acquire tetracycline resistance from Enterococcus faecium. Listeria monocytogenes GLM-2 acquired the resistance determinant tet(M) and additional streptomycin resistance through in vitro mating with Ent. faecium S27 isolated from commercial fermented dry sausage. Similarly, Listeria innocua became more resistant to tetracycline, but the genetic basis for this change was not confirmed. It has been suggested that enterococci may transfer antibiotic resistance genes via transposons to Listeria spp., and this may explain, in part, the origin of their antibiotic resistance. Thus, the presence of enterococci in food should not be ignored since they may actively contribute to enhanced antibiotic resistance of L. monocytogenes and other pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Acquisition of antibiotic resistance by pathogenic bacteria in the absence of antibiotic pressure represents an unquantified threat to human health. In the present work resistance to tetracycline and streptomycin were transferred by nonplasmid-based conjugation from Enterococcus faecium isolated from fermented sausage to Listeria monocytogenes and Listeria innocua. Thus, natural transfer of antibiotic resistance to Listeria strains may occur in the future which reinforces the concern about the safety of enterococcal strains present in foods.

Temperature-dependent effect of sublethal levels of cinnamaldehyde on viability and morphology of Escherichia coli.

AIMS: Effects of sublethal levels of cinnamaldehyde (CIN) on the viability and morphology of Escherichia coli O157:H7 and E. coli 8 WT were investigated at 6 and 37°C. METHODS AND RESULTS: The minimum inhibitory concentration of CIN against E. coli O157:H7 and E. coli 8WT was 400 mg l(-1). At 37°C and ≤300 mg l(-1), CIN delayed the multiplication of both strains, causing a ≤5 and ≤13 h lag, respectively. Delayed multiplication at ≤300 mg l(-1) was partly due to cell elongation and injury as determined by LIVE/DEAD viability, CTC vitality and bis-(1,3-dibutylbarbituric acid) trimethine oxonol staining. The greatest extent of cell elongation (87%) and greatest mean length (6.4 µm) occurred with E. coli O157:H7 at 2-h exposure to 200 mg l(-1) CIN. After initial delays in multiplication, both E. coli O157:H7 and E. coli 8WT returned to exponential growth and normal morphology before reaching the stationary phase. In contrast at 6°C, CIN at ≥100 mg l(-1) prevented cell elongation which occurred in untreated control cells. Treatment with 200 or 300 mg l(-1) CIN at 6°C was lethal to both E. coli strains. At 300 mg l(-1) , CIN caused a ≥5 log CFU ml(-1) reduction at ≤3 days and completely inactivated both of these organisms, causing ≥7 log CFU ml(-1) reduction at 7 days. CONCLUSION: Sublethal levels of CIN at 37°C delayed the multiplication of E. coli cells by causing transient cell elongation, but at 6°C ≥200 mg l(-1) CIN was lethal to E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Inhibition of cold-induced cell elongation and the enhanced lethal effect of CIN at 6°C against E. coli O157:H7 suggest that CIN may be useful for control of this pathogen at refrigeration temperatures.

Susceptibility of meat starter cultures to antimicrobials used in food animals in Canada.

Lactic acid bacteria (LAB) are extensively used in the food industry for fermentation processes. However, it is possible that these bacteria may serve as a reservoir for antibiotic resistance genes that can be transferred to pathogens, giving rise to public health concerns. Animal operations that use antimicrobials as growth promotants have been linked to the origin of resistance due to the selective effect of low levels of antimicrobial used in this management strategy. The objective of this study was to determine the antimicrobial susceptibilities and mechanisms of resistance for 30 isolates of meat starter cultures commonly used in dry sausage fermentations to 20 antimicrobial agents. Susceptibility tests were performed by broth microdilution using Iso-Sensitest broth (90%, vol/vol) and de Man Rogosa Sharpe (MRS) broth (10%, vol/vol). The results showed that all 30 isolates exhibited resistance to at least three antimicrobials regardless of antimicrobial class while 17 or 30% of strains were resistant to antibiotics in three or six different classes, respectively. The incidence of antimicrobial resistance was higher among Pediococcus pentosaceus and lower for Staphylococcus carnosus strains. Genetic determinants for the lincosamide, macrolide, and tetracycline antimicrobials were not found using PCR. Phenotypic resistance in the absence of known resistance genes found here suggests that other mechanisms or genes might have contributed to the negative results. Further studies are needed to explore the genetic mechanisms underlying the prevalence of antibiotic resistance in Pediococcus species.

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula.

AIM: To determine D- and z-values of Cronobacter species (Enterobacter sakazakii) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism. METHODS AND RESULTS: Five Cronobacter species (four C. sakazakii isolates and C. muytjensii) were heated in reconstituted milk or feeding formula pre-equilibrated at 52-58 degrees C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60-100 degrees C. The D-values of Cronobacter at 52-58 degrees C were significantly higher in whole milk (22.10-0.68 min) than in low fat (15.87-0.62 min) or skim milk (15.30-0.51 min) and significantly higher in lactose-free formula (19.57-0.66 min) than in soy protein formula (17.22-0.63 min). The z-values of Cronobacter in reconstituted milk or feeding formula ranged from 4.01 degrees C to 4.39 degrees C. Water heated to > or =70 degrees C and added to powdered milk and formula resulted in a > 4 log(10) reduction of Cronobacter. CONCLUSIONS: The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (> or =70 degrees C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.

Predicting survival of Escherichia coli O157:H7 in dry fermented sausage using artificial neural networks.

The Canadian Food Inspection Agency required the meat industry to ensure Escherichia coli O157:H7 does not survive (experiences > or = 5 log CFU/g reduction) in dry fermented sausage (salami) during processing after a series of foodborne illness outbreaks resulting from this pathogenic bacterium occurred. The industry is in need of an effective technique like predictive modeling for estimating bacterial viability, because traditional microbiological enumeration is a time-consuming and laborious method. The accuracy and speed of artificial neural networks (ANNs) for this purpose is an attractive alternative (developed from predictive microbiology), especially for on-line processing in industry. Data from a study of interactive effects of different levels of pH, water activity, and the concentrations of allyl isothiocyanate at various times during sausage manufacture in reducing numbers of E. coli O157:H7 were collected. Data were used to develop predictive models using a general regression neural network (GRNN), a form of ANN, and a statistical linear polynomial regression technique. Both models were compared for their predictive error, using various statistical indices. GRNN predictions for training and test data sets had less serious errors when compared with the statistical model predictions. GRNN models were better and slightly better for training and test sets, respectively, than was the statistical model. Also, GRNN accurately predicted the level of allyl isothiocyanate required, ensuring a 5-log reduction, when an appropriate production set was created by interpolation. Because they are simple to generate, fast, and accurate, ANN models may be of value for industrial use in dry fermented sausage manufacture to reduce the hazard associated with E. coli O157:H7 in fresh beef and permit production of consistently safe products from this raw material.

Occurrence and characterization of Salmonella from chicken nuggets, strips, and pelleted broiler feed.

Raw, frozen chicken nuggets and strips have been identified as a significant risk factor in contracting foodborne salmonellosis. Cases of salmonellosis as a result of consuming partly cooked chicken nuggets may be due in part to Salmonella strains originating in broiler feed. This study was undertaken to determine the occurrence and characterize the strains of Salmonella contaminating chicken nuggets, strips, and pelleted feeds, in an attempt to demonstrate whether the same Salmonella strains present in broiler feed could be isolated from raw, frozen chicken nuggets and strips available for human consumption. Salmonellae were recovered using the Health Canada MFHPB-20 method for the isolation and identification of Salmonella from foods. Strains were characterized by serotyping, phage typing, antimicrobial resistance typing (R-typing), and by pulsed-field gel electrophoresis (PFGE). Salmonellae were isolated from 25-g samples in 27% (n=92) of nugget and strip samples, 95% (n=20) of chicken nugget meat samples, and from 9% (n=111) of pelleted feed samples. Salmonella Heidelberg, Salmonella Enteritidis, and Salmonella Orion were the most commonly isolated serovars from chicken nuggets and strips, nugget and strip meat, and pelleted broiler feeds, respectively. Salmonella Enteritidis phage type (PT) 13a with PFGE pattern SENXAI.0006 and R-type sensitive as well as Salmonella Enteritidis PT13a with PFGE pattern SENXAI.0068 and R-type sensitive were isolated from pelleted feed, and chicken nugget and strip meat in two separate instances. Data showed that Salmonella strains isolated from broiler feed were indistinguishable from strains isolated from packaged raw, frozen chicken nuggets and strips. However, results did not rule out the possibility that breeding stock or contamination during processing may have contributed to chicken meat contamination by Salmonella.

Suitability of pea starch and calcium alginate as antimicrobial coatings on chicken skin.

The effect of incorporating trisodium phosphate (TSP) in pea starch (PS) and acidified sodium chlorite (ASC) in calcium alginate upon the antimicrobial activity of TSP and ASC was studied against a 3-strain cocktail of Salmonella inoculated on chicken skin. The influence of polymer coating concentration on skin pH, coating-skin adhesion, and coating absorption upon antimicrobial performance were investigated. Aqueous solutions of 0.5 to 4.8% (wt/vol) PS were prepared with 10% (wt/vol) TSP (PS + TSP coating), and alginate + ASC coatings contained 1% (wt/vol) calcium chloride in 1,200 ppm of ASC mixed with an aqueous solution of 0.5, 1.0, or 1.5% (wt/vol) sodium alginate. Coating drops (10 microL) were placed on chicken skin thighs, and the angle formed by the tangent of the liquid surface at the skin interface (contact angle) was measured using a digital camera to assess coating-skin adhesion. Excised skins were mounted in a ring holder, and 5 mL of the coatings was applied to the skin. Weight changes in the skins that were related to coating absorptiveness were recorded. The TSP dissolved in 3.5% PS and ASC in 1% alginate reduced Salmonella by 1.6 log cfu/g and 1.4 log cfu/g, respectively, within 24 h. These reductions were significantly greater than those caused by TSP or ASC alone in water for up to 120 h. In coatings, TSP and ASC caused significant elevation or reduction of skin surface pH for up to 120 h, respectively. The TSP destabilized PS with 88% of the coating having dripped from the skin 1 h later. Coatings with 0.5% PS were absorbed quickly by the skin and had high skin adhesion, whereas those with >3.5% PS had low skin adhesion and slow absorption. Alginate coatings with or without ASC were stable, and about 50% of the coating weight was retained at 120 h. The latter coatings appeared to have low absorptiveness because the skin gained approximately 1.0% of its weight within 60 min following application. These findings indicate that effects of the agents in coatings on skin pH, the extent of coating adhesion, and absorption may contribute to overall antimicrobial behaviors.

Inhibition of membrane bound ATPases of Escherichia coli and Listeria monocytogenes by plant oil aromatics.

Previous studies have reported that the mechanism of bactericidal action of the plant oil aromatics, eugenol, carvacrol and cinnamaldehyde involves inhibition of adenosine triphosphate generation and membrane disruption. In this study the capacity of the aromatics to inhibit the membrane bound ATPase activity of Escherichia coli and Listeria monocytogenes was investigated by experiments on isolated membranes. Inhibition of the ATPase activity of E. coli membranes was observed with 5 mM or 10 mM eugenol or carvacrol. Progressively greater inhibition by cinnamaldehyde was observed as concentration increased from 0.1 to 10 mM. L. monocytogenes ATPase activity was significantly inhibited by eugenol (5 or 10 mM), carvacrol (10 mM) and cinnamaldehyde (10 mM). Lactobacillus sakei is highly resistant to cinnamaldehyde compared to E. coli and L. monocytogenes. To determine whether this resistance was related to the relative hydrophobicity of the cell surface and hence the ability of the cell to take up the aromatics, the percentage of the three organisms partitioning in dodecane was compared. No significant difference was found between the partitioning percentage of L. monocytogenes (17.2%) and L. sakei (13.8%), indicating that surface hydrophobicity does not explain the differing sensitivity to cinnamaldehyde of these two organism. The percent partitioning of E. coli was significantly greater than both other organisms (23.3%) and may explain the greater sensitivity of E. coli to all three aromatics.

Disruption of Escherichia coli, Listeria monocytogenes and Lactobacillus sakei cellular membranes by plant oil aromatics.

The role of membrane disruption in the bactericidal activity of the plant oil aromatic compounds eugenol, carvacrol and cinnamaldehyde was investigated using confocal laser scanning microscopy, changes in ATP levels and cell viability. In 25 mM HEPES buffer pH 7 at 20 degrees C, 10 mM eugenol or carvacrol increased uptake of propidium iodide by Escherichia coli, Listeria monocytogenes and Lactobacillus sakei over a 10-min period. The same treatments resulted in lowered viability, rapid depletion of cellular ATP and release of ATP, with the exception of Lb. sakei treated with carvacrol. Eugenol or carvacrol at 5 mM to 10 mM inhibited E. coli and L. monocytogenes motility. Lb. sakei was resistant to cinnamaldehyde. Thus, its effects were only studied on E. coli and L. monocytogenes. At 10 mM, cinnamaldehyde caused a slight but statistically significant increase in propidium iodide staining of E. coli, but had no effect on L. monocytogenes. Cinnamaldehyde treatment of E. coli at 10 mM and L. monocytogenes at 40 mM resulted in decreased cellular ATP, but there was no concomitant release of ATP. Cinnamaldehyde at 5 and 10 mM inhibited E. coli and L. monocytogenes motility. Results for eugenol and carvacrol are consistent with non-specific permeabilization of the cytoplasmic membrane. Evidence for increased membrane permeability by cinnamaldehyde is less conclusive. The release of ATP from eugenol and carvacrol-treated cells and absence of release from cinnamaldehyde-treated cells could indicate that eugenol and carvacrol possess ATPase inhibiting activity. Secondary effects would also be consistent with membrane disruption.

Use of mustard flour to inactivate Escherichia coli O157:H7 in ground beef under nitrogen flushed packaging.

This study was undertaken to determine whether the glucosinolates naturally present in non-deheated mustard flour could serve as a source of allyl and other isothiocyanates in sufficient quantity to kill Escherichia coli O157:H7 inoculated in ground beef at three different levels, during refrigerated storage of the meat under nitrogen. Mustard flour was mixed at 5%, 10% or 20% (w/w) with freshly ground beef, then the beef was inoculated with a cocktail of five strains of E. coli O157:H7 at either 3, 6 or < or =1.6 log10 cfu/g. The ground beef was formed into 100 g patties and each was placed in a bag of Nylon/EVOH/PE, which was back-flushed with 100% N2, heat-sealed and stored at 4 degrees C for < or =21 days. During storage, the allyl isothiocyanate (AIT) levels in package headspaces were determined by gas liquid chromatography. By 21 days, the levels present in treatments were not significantly different. After 21 days storage, there were 0.5, 3 and 5.4 log10 decreases in numbers of E. coli O157:H7 from the initial levels of 6 log10 cfu/g in meat containing 5%, 10% and 20% mustard flour, respectively. When inoculated at 3 log10 cfu/g, E. coli O157:H7 was reduced to undetectable levels after 18, 12 and 3 days with 5%, 10% and 20% mustard flour, respectively. When immunomagnetic separation (IMS) was used for E. coli recovery following its inoculation at < or =1.6 log10 cfu/g, 5% mustard did not completely eliminate the pathogen from ground beef stored for 6 days. The natural microflora of the ground beef which developed in vacuum packages was unaffected by the addition of 5% mustard flour but some inhibition was found at higher concentrations. Sensory evaluation of the cooked ground beef showed that there were no significant differences in the acceptability of meat treated with 5 or 10% mustard flour. However, panelists could distinguish untreated controls from mustard treatments, but considered the mustard-treated meat to be acceptable. These results showed that it is possible to use mustard flour at levels of >5-10% to eliminate E. coli O157:H7 from fresh ground beef.

Inactivation of Escherichia coli O157:H7 in packaged ground beef by allyl isothiocyanate.

Commercial allyl isothiocyanate (AIT) was examined for its ability to reduce numbers of Escherichia coli O157:H7 inoculated in fresh ground beef packaged under nitrogen and stored refrigerated or frozen. A five-strain cocktail of E. coli O157:H7 containing 3 or 6 log10 cfu/g was inoculated into 100 g ground beef and formed into 10x1-cm patties. A 10-cm diameter filter paper disk treated with AIT suspended in sterile corn oil was placed on top of a single patty. One patty and paper disk were placed in a bag of Nylon/EVOH/PE with O2 permeability of 2.3 cm3 m(-2) 24 h atm at 23 degrees C. The bags were back-flushed with 100% nitrogen, heat-sealed and stored at 10, 4 and -18 degrees C for 8, 21 or 35 days, respectively. During storage, the AIT levels in the package headspaces were determined by gas liquid chromatography, and mesophilic bacteria and E. coli O157:H7 were counted. The mesophilic aerobic bacteria in ground beef patties were largely unaffected by the addition of AIT. At an initial population of 3 log10 cfu/g, E. coli O157:H7 was reduced by AIT to undetectable levels after 18 days at 4 degrees C or 10 days at -18 degrees C. In samples inoculated with 6 log10 cfu/g, a >3 log10 reduction of E. coli O157:H7 was observed after 21 days at 4 degrees C, while a 1 log10 reduction was observed after 8 and 35 days at 10 and -18 degrees C, respectively. The final AIT concentrations in the headspaces after storage at 10, 4, and -18 degrees C were 444, 456, and 112 microg/ml at 8, 21, and 35 days, respectively. Results showed that AIT can substantially reduce numbers of E. coli O157:H7 in fresh ground beef during refrigerated or frozen storage.

Use of MRSD medium and the hydrophobic grid membrane filter technique to differentiate between pediococci and lactobacilli in fermented meat and starter cultures.

Modifications of MRS medium were made by incorporation of 0.1 M L-arginine-HCl, 0.0025% phenol red, 100 IU polymyxin B sulfate, by deletion of meat extract, use of only 1.2% (w/v) glucose and increase of Mn2+ to 1000 ppm. In addition, adoption of the hydrophobic grid membrane filter (HGMF) system with 0.025% Fast Green FCF dye and adjustment of the agar medium to pH 5.5 gave MRSD (differential) medium. Incubation at 25 degrees C anaerobically under N2 or CO2 followed by a post-growth staining procedure involving use of 0.4% (w/v) bromocresol purple yielded conditions under which pediococci colonies were blue whereas homo- and heterofermentative lactobacilli were green in color. Under these conditions, 7 pediococci, 16 lactobacilli, and 18 commercial meat starter cultures were successfully analyzed by plate count to yield a differential assessment of the lactobacilli and pediococci present without interference from the 9 other genera tested. Streptococcus lactis and Leuconostoc spp. produced blue and green colonies, respectively, at 25 degrees C which might interference but these organisms are not present in significant numbers in fermented meats. Pediococcus parvulus and Streptococcus faecalis produced green and blue colonies, respectively, but their very poor growth at 25 degrees C prevented their interference. Use of the developed MRSD medium was described for enumeration of both pediococci and lactobacilli in starter cultures and in fermenting dry sausages to enable documentation of starter culture performance.

Influence of unsliced delicatessen meat freshness upon bacterial growth in subsequently prepared vacuum packed slices.

Unsliced beef pastrami, reformulated ham and bologna held at 6 degrees C were sliced 21, 17, 12 or 7 days before or at the assigned manufacturer's best before date and vacuum packaged. Packages of sliced meats were held at 6 degrees C for another 7, 12, 17 or 21 days, opened and analyses made for total bacteria, lactic acid bacteria, Enterobacteriaceae and Brochothrix thermosphacta. The maximum storage interval was 42 days; half this period unsliced, the remainder as repacked slices. Numbers of bacteria on pastrami were significantly greater than on ham and bologna (pastrami > ham > bologna) with the lactic acid bacteria dominating in all products. As unsliced meats approached their best before date, insignificant increases were generally noted for numbers of lactic bacteria, Enterobacteriaceae and B. thermosphacta. During subsequent storage of slices under vacuum, numbers of total and lactic bacteria increased exponentially at the same rate while B. thermosphacta growth was significantly slower. Numbers of Enterobacteriaceae remained low and were essentially unchanged during sliced meat storage. Within the context of study storage parameters, shelf-life appeared to be determined by length of time after slicing and repackaging rather than by best before date of the unsliced meat. Packages of sliced meat prepared from wholesale unsliced meats with 21 days left until their best before date or from unsliced meats with 12 days left until the best before date showed similar bacterial levels 21 days later. It was probable that the localization of bacterial growth at the meat surface-packaging film interface of the unsliced meats yielded slices with initially lowered bacterial content when repackaged and sampled from the uppermost slice. When Enterobacteriaceae and B. thermosphacta were absent from unsliced meats, extension of sliced meat package shelf-life beyond the best before date of the parent meat was possible. However, these bacterial groups were not always undetected.

Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms.

Interactions of monolaurin, eugenol (phenolic compound) and sodium citrate (chelator) on the growth of six organisms including common meat spoilage (Lactobacillus curvatus, Lactobacillus sake, Leuconostoc mesenteroides, Brochothrix thermosphacta) and pathogenic (Escherichia coli O157:H7 and Listeria monocytogenes) organisms were investigated. The combinations of 100 to 250 ppm monolaurin with 500 and 1000 ppm eugenol, and 0.2 and 0.4% sodium citrate were more effective than each component separately. More than one combination prevented detectable growth of each organism. Lactic acid bacteria (LAB) and E. coli O157:H7 were most resistant and L. monocytogenes and B. thermosphacta most sensitive to control by the chosen combinations. The presence of sodium citrate was necessary to yield potent inhibition of Lb. curvatus and Lb. sake growth by the monolaurin and eugenol combinations.

Microbiological safety of traditional and starter-mediated processes for the manufacture of Italian dry sausage.

Microbiological changes occurring during the commercial manufacture of Italian dry sausages (Genoa and salametti) were studied in two urban Canadian centres over a 5 month period. A comparison was made between 6 plants which used bacterial starter cultures and 4 plants where more traditional processes (without starters) were used. A total of 600 samples of raw, fermented and finished products were tested for the presence of coliforms, salmonellae, staphylococci, streptococci, the rate of pH reduction and final water activity (aW). Numbers of total bacteria peaked earlier and were significantly higher in sausages at the fermentation stage produced with starter cultures than in those traditionally manufactured. This corresponded with a more rapid drop in pH of the starter-inoculated products. Staphylococci and streptococci were significantly higher in starter-fermented Genoa sausages at the fermentation stage, but no significant differences were seen in the microbiological content or aW of mature finished sausages manufactured by the two different techniques. Of 128 randomly chosen isolates of coagulase-positive staphylococci, 34.4% were enterotoxin producers and 80% of these produced type A toxin. Enterotoxigenic staphylococci were found in 2 different samples of finished salametti and one sample of finished Genoa made with starter cultures and in one sample of finished Genoa made without added culture. Total numbers of staphylococci in these samples were not greater than 500/g. No correlation between the method of manufacture and presence of enterotoxigenic staphylococci could be made. Five subsamples from one lot of raw Genoa were the only samples positive for Salmonella during this study. Results indicated that low temperature traditional fermentations can yield products which are as safe as those produced by the higher temperature starter-controlled process. One of the most important elements in the traditional process was believed to be the selection and use of raw materials of the highest possible quality.

Occurrence and significance of streptococci in fermented Italian type dry sausage.

Commercial cultures used in Canada for the manufacture of Italian dry sausage were examined to determine their microbial composition and suitability for low temperature (less than or equal to 20 degrees C) meat fermentations. Temperature optima in both laboratory media and commercial meat mixtures were generally too high to allow these cultures to be of substantial advantage in this application. In addition, media used currently for the enumeration of streptococci and related organisms from fermented meat products were found to be inadequately specific and often required confirmatory inspection of colonies by conventional phase contrast microscopy. Streptococci were isolated from Italian dry sausage manufactured commercially with and without added starter cultures. Streptococci persisted in sausages produced by both techniques with slightly higher numbers present in starter-acidulated sausages. About 55.5% of the 312 streptococci studied were enterococci (Lancefield's Group D). Streptococci were found in several samples of commercial starter cultures but it was felt that elevated ripening temperatures used for sausage manufactured by the starter-mediated process and meat handling practices were more important factors influencing streptococci recovery from sausage material.

Cresol red thallium acetate sucrose inulin (CTSI) agar for the selective recovery of Carnobacterium spp.

Carnobacterium spp. are commonly isolated from a variety of foods, especially from meats stored under anaerobic atmospheres at refrigeration temperatures, but the role of these organisms in the spoilage of meat and meat products is yet to be determined. Cresol Red Thallium Acetate Sucrose (CTAS) agar was developed as a selective medium for enumeration of carnobacteria, however problems such as poor recovery of Carnobacterium spp. and interference by other microorganisms have precluded its general use. The aim of this study was to improve CTAS agar by broadening its spectrum of selective recovery for carnobacteria while restricting the ability of interfering species to grow. Ten Carnobacterium spp. (five ATCC cultures and five isolates from fresh pork) and 20 other genera were used in testing the agar. A wider range of Carnobacterium spp. recovery was obtained by modifying concentrations of sucrose, manganese sulphate and thallium acetate. Additions of inulin and thiamine hydrochloride also improved growth response. The additions of vancomycin and Chrisin (nisin) eliminated interference from other microorganisms. A two-temperature incubation procedure was included to improve the characteristic growth of Carnobacterium spp. on the modified medium, identified as Cresol Red Thallium Sucrose Inulin (CTSI) agar. Lactic acid bacteria and Enterobacteriaceae were unable to grow on CTSI incubated aerobically. Growth of Carnobacterium spp. on CTSI yielded pink colonies, except for Cb. mobile, which formed gray colonies. In some instances, a red precipitate formed in the center of the colony. Yellowing and clearing of the growth medium was also frequently observed. Recovery of carnobacteria using CTSI was identical to that obtained with All Purpose Tween (APT) agar.

Evaluation of antilisterial action of cilantro oil on vacuum packed ham.

Cilantro oil is an essential oil preparation extracted from the plant Coriandrum sativium. A series of experiments were conducted to evaluate the ability of cilantro oil to control the growth of Listeria monocytogenes on vacuum-packed ham. The in vitro minimal inhibitory concentration for five strains of L. monocytogenes was found to vary from 0.074% to 0.018% depending on strain. Cilantro oil treatments were then tested on ham disks inoculated with a cocktail of the five L. monocytogenes strains. The treatments studied were 0.1%, 0.5%, and 6% cilantro oil diluted in sterile canola oil or incorporated into a gelatin gel in which lecithin was used to enhance incorporation of the cilantro oil. Gelatin gel treatments were also conducted with 1.4% monolaurin with or without 6% cilantro oil to determine if an interaction between the antimicrobials could increase inhibition of L. monocytogenes. Treated ham was then vacuum-packed and stored at 10 degrees C for up to 4 weeks. The only cilantro oil treatment which inhibited growth of L. monocytogenes on the ham samples was 6% cilantro oil gel. Samples receiving this treatment had populations of L. monocytogenes 1.3 log CFU/ml lower than controls at week 1 of storage, though there was no difference between treatments from week 2 onward. It appears that immobilization of the antimicrobial in a gel enhanced the effect of treatments. Cilantro oil does not appear to be a suitable agent for the control of L. monocytogenes on ham. The possible reasons for reduced effectiveness of cilantro oil against L. monocytogenes on ham are discussed.

Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan.

A study was undertaken to evaluate the feasibility of using antimicrobial films, designed to slowly release bacterial inhibitors, to improve the preservation of vacuum-packaged processed meats during refrigerated storage. The antimicrobial films were prepared by incorporating acetic or propionic acid into a chitosan matrix, with or without addition of lauric acid or cinnamaldehyde, and were applied onto bologna, regular cooked ham, or pastrami. At various times during storage, packages were opened and the amounts of antimicrobial agents remaining in the chitosan matrix were measured. Regardless of film composition or meat product type, propionic acid was nearly completely released from the chitosan matrix within 48 h of application, whereas release of acetic acid was more limited, with 2-22% of the acid remaining in chitosan after 168 h of storage. Addition of lauric acid, but not cinnamaldehyde, to the chitosan matrix generally reduced the release of acetic acid significantly (P < or = 0.05) and the release was more limited onto bologna than onto ham or pastrami. In addition, the efficacies of the various films for inhibiting bacterial growth were tested against indigenous lactic acid bacteria and Enterobacteriaceae, and against Lactobacillus sakei or Serratia liqueficiens, surface-inoculated onto the meat products. Whereas lactic acid bacteria were not affected by the antimicrobial films under study, the growth of Enterobacteriaceae and S. liquefaciens was delayed or completely inhibited as a result of film application. Strongest inhibition was observed on drier surfaces (bologna), onto which acid release was slower, and with films containing cinnamaldehyde, as a result of its greater antimicrobial activity under these conditions.

Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms.

The antibacterial activity of selected fatty acids and essential oils was examined against two gram-negative (Pseudomonas fluorescens and Serratia liquefaciens) and four gram-positive (Brochothrix thermosphacta, Carnobacterium piscicola, Lactobacillus curvatus, and Lactobacillus sake) bacteria involved in meat spoilage. Various amounts of each preservative were added to brain heart infusion or MRS (deMan, Rogosa and Sharpe) agars, and the minimum inhibitory concentration was determined for each organism. Essential oils were analysed by gas-liquid chromatography to determine the concentration of selected components commonly found in spices. B. thermosphacta, P. fluorescens and S. liquefaciens were not affected by fatty acids, and generally overcame the inhibitory effect of essential oils after 24 h of exposure. Among the fatty acids, lauric and palmitoleic acids exhibited the greatest inhibitory effect with minimum inhibitory concentrations of 250 to 500 micrograms/ml, while myristic, palmitic, stearic and oleic acids were completely ineffective. For essential oils, clove, cinnamon, pimento, and rosemary were found to be the most active. The 1/100 dilution of those oils inhibited at least five of the six tested organisms. A relationship was found between the inhibitory effect of essential oils and the presence of eugenol and cinnamaldehyde.