Competition of Thermally Injured Listeria monocytogenes with a Mesophilic Lactic Acid Starter Culture in Milk for Various Heat Treatments

Overnight tryptose broth cultures of three L. monocytogenes strains were combined, centrifuged, suspended in 200 ml of tryptose phosphate broth, and heated at 56°C for 20 min and at 64°C for 2 min to obtain low-heat-injured (LHI) and high-heat-injured (HHI) cells, respectively, showing >99.0% injury. Flasks containing 200 ml of raw, low-heat-treated (56°C for 20 min), high-heat-treated (64°C for 2 min), pasteurized, and ultrahigh-temperature (UHT) milk were tempered to 31.1°C and inoculated to contain 104 to 106 CFU/ml of LHI, HHI, or healthy L. monocytogenes cells and a commercial Lactococcus lactis subsp. lactis–Lactococcus lactis subsp. cremoris starter culture at levels of 0.5, 1.0, and 2.0%. Numbers of healthy and injured L. monocytogenes cells and starter organisms were determined using tryptose phosphate agar with or without 4.0% NaCl at selected intervals during 24 h of incubation at 31.1°C. The presence of L. monocytogenes did not adversely affect the growth of the starter culture at any inoculation level. Overall, L. monocytogenes survived the 24-h fermentation period and grew to some extent. In starter-free controls, 76 to 81% of LHI cells and 59 to 69% of HHI cells were repaired after 8 h of incubation, with the lowest repair rates being observed for raw rather than heat-treated or pasteurized milk. Increased injury was observed for healthy L. monocytogenes cells at the 1.0 and 2.0% starter levels, with less injury seen for LHI and HHI cells. Raw and subpasteurized milk allowed less of a decrease in the percentage of injury and also showed higher numbers of injured cells than did pasteurized and UHT milks. These findings may have important implications for the survival of Listeria spp. in certain cheeses that can be prepared from raw or heat-treated milk.

Survival and Growth of Alkali-Stressed Listeria monocytogenes on Beef Frankfurters and Thermotolerance in Frankfurter Exudates

Cells of Listeria monocytogenes exposed at 4°C to 1% solutions of two alkaline cleaners or alkali-adapted in tryptose phosphate broth (pH 10.0) at 37°C for 45 min, followed by 4°C for 48 h, were inoculated onto beef frankfurters containing high fat (16 g) and high sodium (550 mg) or low fat (8 g) and low sodium (250 mg) per 57-g serving. Frankfurters were surface inoculated (2.0 log10 CFU/g), vacuum packaged, stored at −20, 4, or 12°C, and analyzed for populations of L. monocytogenes at 2-day to 2-week intervals. Populations did not change significantly on frankfurters stored at −20°C for up to 12 weeks. After storage at 4°C for 6 weeks (1 week before the end of shelf life), populations of control cells and cells exposed to alkaline cleaners were ca. 6.0 log10 CFU/g of low fat, low sodium (LFLS) frankfurters and ca. 3.5 log10 CFU/g of high fat, high sodium (HFHS) frankfurters. Growth of alkali-adapted cells on both types of frankfurters was retarded at 4°C. Growth of L. monocytogenes on frankfurters stored at 12°C was more rapid than at 4°C, but a delay in growth of alkali-adapted cells on HFHS and LFLS frankfurters was evident during the first 9 and 6 days, respectively. Alkali-adapted cells had a significantly (P ≤ 0.05) lower logistic D59°C-value (decimal reduction time) than alkaline cleaner-exposed cells, but the D59°C-value was not different from that of control cells. Cells exposed to a nonbutyl alkaline cleaner, and then heated in LFLS frankfurter exudates, had a significantly lower D62°C-value than cells that had been exposed to some of the other treatments. Growth characteristics of L. monocytogenes inoculated onto the surface of frankfurters may be altered by previous exposure to alkaline environments. Differences in growth characteristics of L. monocytogenes on HFHS versus LFLS beef frankfurters stored at refrigeration temperatures indicate that composition influences the behavior of both alkaline-stressed and control cells.

Survival and Heat Resistance of Listeria monocytogenes after Exposure to Alkali and Chlorine

ABSTRACT A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59°C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21°C. Cells ofL. monocytogenes incubated at 37°C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56°C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 ± 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log10 CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log10 CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56°C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log10-unit reductions is not compromised in these cells.

Kinetics of Methyl t-Butyl Ether Cometabolism at Low Concentrations by Pure Cultures of Butane-Degrading Bacteria

ABSTRACT Butane-oxidizing Arthrobacter (ATCC 27778) bacteria were shown to degrade low concentrations of methylt-butyl ether (MTBE; range, 100 to 800 μg/liter) with an apparent half-saturation concentration (K s ) of 2.14 mg/liter and a maximum substrate utilization rate (kc) of 0.43 mg/mg of total suspended solids per day. Arthrobacter bacteria demonstrated MTBE degradation activity when grown on butane but not when grown on glucose, butanol, or tryptose phosphate broth. The presence of butane, tert-butyl alcohol, or acetylene had a negative impact on the MTBE degradation rate. NeitherMethylosinus trichosporium OB3b nor Streptomyces griseus was able to cometabolize MTBE.

Survival of Listeria monocytogenes on the Surface of Egg Shells and During Frying of Whole and Scrambled Eggs

Studies were done to determine the survival characteristics of Listeria monocytogenes on shell eggs and after cooking raw whole and scrambled eggs by frying. Samples were inoculated with low or high populations of a five-strain mixture of L. monocytogenes. Survival of the organism on shells of unbroken eggs was monitored over a 6-week storage period at 5 and 20°C. The presence of L. monocytogenes was determined by subjecting egg samples to primary enrichment in tryptose phosphate broth followed by plating the broth on Lee Modified Oxford agar. Enumeration was done by directly plating serially diluted wash buffer from shell eggs and diluted fried eggs directly on Lee Modified Oxford agar. Both low (102 CFU per egg) and high (104 CFU per egg) populations of L. monocytogenes on the surface of egg shells decreased to <10 CFU per egg after 6 d of storage at 5 and 20°C. Frying whole eggs “sunnyside up” until albumen was partially coagulated reduced both low (102 CFU/g) and high (105 CFU/g) populations of L. monocytogenes by only 0.4 log10 CFU/g. In contrast, frying one or three scrambled eggs to an internal temperature of 70–73°C reduced low (102 CFU/g) populations of L. monocytogenes to undetectable and <102 CFU/g, respectively. Frying three scrambled eggs containing high (105 CFU/g) populations caused a 3 log10 reduction. Frying one scrambled egg containing a high population resulted in <102 CFU/g. Both low (104 CFU/g) and high (107 CFU/g) populations of L. monocytogenes remained unchanged or decreased slightly when raw slightly beaten whole eggs were allowed to stand for up to 3 h at 20°C.

Caffeine and Theobromine

Two components of cocoa powder, caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine), were evaluated for their effect on growth of L. monocytogenes strain V7. Caffeine (0.5%) and theobromine (2.5%) were added singly or in combination to skim milk or a modified tryptose phosphate broth (MTPB), which were sterilized, inoculated to contain ca. 1 × 103 CFU L. monocytogenes/ml, and incubated at 30°C. Both compounds allowed some growth of the pathogen; however, longer lag phases occurred in samples with (6 to 9 h) rather than without (<3 h) caffeine. Generation times at 30°C ranged from 1.10 h (2.5% theobromine in broth) to 2.28 h (2.5% theobromine plus 0.5% caffeine in milk). Generation times were significantly (p<0.05) lengthened in the presence (2.17 h) rather than in the absence (1.2 h) of caffeine. Populations in samples without caffeine were more than ten times greater (8.57 log10 CFU/ml) than in samples with caffeine (7.21 log10 CFU/ml). Theobromine concentration (0 or 2.5%) and substrate (MTPB or skim milk) had only limited effects on growth of the pathogen. The change in pH of the medium was a function of the extent of growth of L. monocytogenes and the buffering capacity of the substrate. A smaller pH change (0.09 unit) occurred in the milk medium than in broth (0.48 unit) because of the minimal buffering capacity of the 0.2% tryptose solution. Samples with caffeine had a small decrease in pH (0.07 unit) because growth of L. monocytogenes was inhibited and thus acid production was minimal.

Chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses

SUMMARYThe chemical disinfection of virus-contaminated non-porous inanimate surfaces was investigated using coxsackievirus B3, adenovirus type 5, parainfluenzavirus type 3 and coronavirus 229E as representatives of important nosocomial viral pathogens. A 10 µl amount of the test virus, suspended in either faeces or mucin, was placed onto each stainless steel disk (about 1 cm in diameter) and the inoculum allowed to dry for 1 h under ambient conditions. Sixteen disinfectant formulations were selected for this study based on the findings of an earlier investigation with a human rotavirus.After 1 min exposure to 20 µl of the disinfectant, the virus from the disks was immediately eluted into tryptose phosphate broth and plaque assayed. Using an efficacy criterion of a 3 log10or greater reduction in virus infectivity titre and irrespective of the virus suspending medium, only the following five disinfectants proved to be effective against all the four viruses tested: (1) 2% glutaraldehyde normally used as an instrument soak. (2) a strongly alkaline mixture of 0·5% sodium o-benzyl-p-chlorophenate and 0·6% sodium lauryl sulphate, generally used as a domestic disinfectant cleaner for hard surfaces, (3) a 0·04% solution of a quaternary ammonium compound containing 7% hydrochloric acid, which is the basis of many toilet bowl cleaners. (4) chloraminc T at a minimum free chlorine level of 3000 p.p.m. and (5) sodium hypochlorite at a minimum free chlorine concentration of 5000 p.p.m. Of those chemicals suitable for use as topical antiseptics, 70% ethanol alone or products containing at least 70% ethanol were ineffective only against coxsackievirus B3. These results emphasize the care needed in selecting chemical disinfectants for routine use in infection control.

Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae)

SUMMARYBlastocrithidia triatomae parasitizes vectors of Chagas' disease and is very difficult to cultivate in conventional media. However, co-cultivation with a cell line of its host Triatoma infestans (TI-32; in Schneider's Drosophila medium supplemented with 20% foetal calf serum and 10% tryptose phosphate broth) led to vigorous growth at 24 or 28 °C without an adaptation phase. More than 60 primary cultures were initiated successfully without any failures. Subcultures could be started immediately at weekly intervals. The doubling time was similar in both the primary cultures (41 h) and the 33rd subculture (39 h). The importance of the reduviid cells for B. triatomae became clear after removal of the insect cells, when multiplication of epimastigotes stopped and mainly cysts were formed. Cysts produced in vitro were infective for reduviids. Scanning electron microscopy showed that B. triatomae attached to the host cells, inserted its flagellum into them and destroyed them.

Survival of human rhinovirus type 14 dried onto nonporous inanimate surfaces: effect of relative humidity and suspending medium

To study the survival of human rhinovirus 14 on environmental surfaces, each stainless steel disk (1 cm in diameter) was contaminated with 10 μL (about 105 plaque-forming units) of the virus suspended in either 1χ tryptose phosphate broth (TPB), 5 mg/mL of bovine mucin in normal saline, or undiluted human nasal discharge. The inoculum was dried in a laminar flow cabinet for 1 h under ambient conditions. The disks were then placed in a glass chamber (20 ± 1 °C) with the relative humidity at either low (20 ± 5%), medium (50 ± 5%), or high (80 ± 5%) level. At appropriate intervals, the disk to be tested was placed in 1 mL of tryptose phosphate broth and the eluate titrated in A-5 HeLa cells. When the virus was suspended in either tryptose phosphate broth, mucin, or the nasal discharge and subjected to initial drying, there was a 3.0 ± 1.0, 82.0 ± 6.7, and 89.0 ± 3.0% loss in virus infectivity, respectively. The half-life of the TPB-suspended virus was about 14 h at the high relative humidity as compared with < 2 h at the other two relative humidity levels. The half-lives for the mucin-suspended virus at the high, medium, and low relative humidity were 1.42, 0.55, and 0.24 h, respectively; the corresponding values for the nasal discharge suspended virus being 0.17, 0.25, and 0.09 h.