Heat-injured Listeria Monocytogenes Research Articles

Evaluation of the Thin Agar Layer Method for the Recovery of Pressure-Injured and Heat-Injured Listeria monocytogenes

A sublethally injured bacterial cell has been defined as a cell that survives a stress such as heating, freezing, acid treatment, or other antimicrobial intervention but can repair the cellular damage exerted by the stressor and later regain its original ability to grow. Consequently, sublethally injured cells are not likely to be included in conventional enumeration procedures, which could result in unrealistically low counts unless efforts are made to encourage recovery of the injured cells before enumeration. The objective of this study was to evaluate the use of the thin agar layer (TAL) method for the recovery of pressure-injured and heat-injured Listeria monocytogenes in a tryptic soy broth with 0.6% yeast extract system. Pressure injury consisted of treatment of a culture of mixed L. monocytogenes strains with high hydrostatic pressure at 400 or 600 MPa for 1 s, 2 min, 4 min, or 6 min at a process temperature of 12±2 °C. Heat injury consisted of treatment of a culture of mixed L. monocytogenes strains at 60±1 °C for 3, 6, or 9 min. Growth media were tryptic soy agar (TSA) with 0.6% yeast extract, modified Oxford medium (MOX), and TAL, which consisted of a 7-ml layer of TSA overlaid onto solidified MOX. Counts of viable L. monocytogenes on TAL were higher than those on MOX in the heat-injury experiment but not in the pressure-injury experiment. Therefore, the effectiveness of the TAL method may be specific to the type of injury applied to the microorganism and should be investigated in a variety of cellular injury scenarios.

A Solid Agar Overlay Method for Recovery of Heat-Injured Listeria monocytogenes

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58°C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.

Comparison of Growth Kinetics for Healthy and Heat-Injured Listeria monocytogenes in Eight Enrichment Broths

Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels. In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths. Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical ManualListeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth. Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L. monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth. The Gompertz equation was used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells. Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents.

Detection of Heat Injury in Listeria monocytogenes Scott A

Methods of detecting live pathogens in foods that may be growth inhibited following heat treatment are essential to food safety. Among the techniques available, reverse transcription polymerase chain reaction (RT-PCR) amplification of messenger RNA from heat-injured Listeria monocytogenes Scott A is preferable to direct PCR in an attempt to avoid false positives from dead cells. The RT-PCR has a detection limit of 3 × 106 CFU/g, compared to 3 CFU/g for untreated controls, but may not be suitable for the identification of all viable cells. Physically apparent changes in cellular structures from heat injury in L. monocytogenes are expected to result. Ultrastructural analyses did depict notable heat damage as cytoplasmic clearing after 5 min at 60°C. The heat-injured survivors can be readily distinguished from total viable cells using selective media. As a result, combinations of molecular and visual methods including selective media improve detectability of heat-injured, viable L. monocytogenes Scott A.

Optimizing Detection of Heat-Injured Listeria monocytogenes in Pasteurized Milk

Optimal conditions for the detection of heat-injured cells of Listeria monocytogenes in modified Pennsylvania State University (mPSU) broth were determined using a response surface design generated by a computer program, EChip. Different combinations of incubation temperatures and lithium, magnesium, and d-serine concentrations were evaluated to determine the optimum conditions for the detection of heat-injured L. monocytogenes in filter-sterilized whole milk inoculated with selected problematic background microflora. A concentration of 212mM lithium chloride completely inhibited the growth of Enterococcus faecium while permitting recovery and detection of L. monocytogenes. A concentration of 15.8mM MgSO4 was found to be optimum for the recovery and detection of L. monocytogenes. A concentration of 140.2mM d-serine was found to completely inhibit the germination of Bacillus subtilis var. globii spores but not recovery and detection of L. monocytogenes. Under optimum concentrations of LiCl, MgSO4, and d-serine and in the absence of background microflora, the effect of incubation temperature on percentage detection was described by a second-order polynomial model, and 28°C was determined to be optimal. In the presence of background microflora, the effect of incubation temperature on percentage detection of heat-injured cells was described by a third-order polynomial model, and 30°C was found to be optimal. Optimizing the levels of highly specific and selective agents, nutrients, and incubation temperature in one recovery enrichment system dramatically increased the Listeria/background microflora ratio. This resulting medium, optimized PSU (oPSU) broth, greatly improved the detection of heat-injured and nonheat-injured L. monocytogenes by both conventional and molecular methods (Oxoid's Listeria Rapid Test, Gen-Probe's Accuprobe Listeria monocytogenes Culture Identification Test, and Qualicon's BAX for screening Listeria monocytogenes).

Influence of modified atmospheric storage, lactic acid, and NaCl on survival of sublethally heat-injured Listeria monocytogenes

The effect of package atmosphere on survival of uninjured and sublethally heat-injured Listeria monocytogenes, inoculated onto tryptose phosphate agar containing 0.85% lactic acid and 2% NaCl (TPALAS) was investigated. Inoculated TPALAS plates were packaged in air, 100% N 2 (N 2), 30% CO 2–70% N 2 (CO 2–N 2), and vacuum and stored at 4 and 20°C for up to 31 days. Recovery of L. monocytogenes from TPALAS was influenced by the injury status (i.e., injured and uninjured) of the inoculum, storage atmosphere (air, N 2, CO 2–N 2, and vacuum), storage temperature (4 and 20°C), and recovery media [tryptose phosphate agar (TPA) and modified Oxford agar (MOX)] ( P<0.05). Overall, storage at 4°C supported greater survival than storage at 20°C ( P<0.05). Uninjured L. monocytogenes stored at 4°C was recovered on TPA better than sublethally heat-injured L. monocytogenes stored at 4°C ( P<0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 4°C followed the order N 2>CO 2–N 2>air>vacuum ( P<0.05), whereas recovery of uninjured L. monocytogenes stored at 4°C followed the order N 2>CO 2–N 2>vacuum>air ( P<0.05). Air and vacuum atmospheres supported greater survival of uninjured and heat-injured L. monocytogenes than N 2 and CO 2–N 2 atmospheres at 20°C ( P<0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 20°C followed the order vacuum>air>CO 2–N 2=N 2 ( P<0.05), whereas recovery of uninjured L. monocytogenes stored at 20°C followed the order vacuum>air>CO 2–N 2>N 2 ( P<0.05). Uninjured L. monocytogenes stored under N 2 at 4°C was recovered best, whereas sublethally heat-injured L. monocytogenes stored under N 2 at 20°C was recovered poorest ( P<0.05). Factors such as package atmosphere and storage temperature, involved in the production, storage, and distribution of fermented foods must be thoroughly evaluated when determining strategies for control and detection of L. monocytogenes in such products.

Comparison of Different Enrichment Broths and Background Flora for Detection of Heat-Injured Listeria monocytogenes in Whole Milk

Various primary enrichment broths, including University of Vermont medium (UVM), Listeria enrichment broth (LEB), modified LEB, and aerobic and anaerobic L-PALCAMY, were compared with aerobic and anaerobic Pennsylvania State University (PSU) broths for the detection of severely heat-injured (62.8°C for 5, 10, or 15 min; no colony appearance after heat injury on aerobic Trypticase soy agar containing 0.6% yeast extract and modified Oxford medium) Listeria monocytogenes Scott A. Anaerobic conditions were produced by adding l-cysteine and then purging the headspace with N2. The effect of uninjured background flora (103 CFU/ml of Enterococcus faecium) on frequency of detection was examined. Anaerobic PSU broth resulted in the lowest false-positive rate and the highest frequency of detection of severely heat-injured L. monocytogenes compared with UVM, LEB, and modified LEB (P < 0.05). The presence of E. faecium significantly enhanced the detection of heat-injured (10 min at 62.8°C) L. monocytogenes in aerobic and anaerobic PSU and aerobic and anaerobic L-PALCAMY broths (P < 0.05). The highest concentration of uninjured E. faecium (>106 CFU/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). A heat-resistant, LiCl-tolerant Lactobacillus isolate from raw milk increased the rate of both false-positive and false-negative reactions.

Factors influencing resuscitation and growth of heat injured Listeria monocytogenes 13-249 in sous vide cooked beef

The growth of Listeria monocytogenes 13-249 in vacuum-packed, minced beef was investigated as a function of degree of heat injury (including no injury i.e. uncooked beef), growth phase (logarithmic and late stationary phase), pH (5.6 and 6.2), and storage temperature (3, 10 and 20°C) during a storage period of 30 days. Late logarithmic and late stationary phase cultures of L. monocytogenes 13-249 showed similar growth in refrigerated, vacuum-packed, raw minced beef with a high pH (6.2). In normal pH (5.6) beef there was no growth at 3°C while growth at 10 and 20°C was only observed for logarithmic phase cultures. Heat injured late stationary phase cultures with 95–99.9% injured cells in the surviving population (as measured by differential plating on enriched vs. selective media after sous vide cooking) did not grow or repair sublethal injuries in sous vide cooked beef at 3°C while repair and growth took place at 10 as well as at 20°C. In logarithmic phase cultures heat injury occurred very rapidly and ≥99.9% heat injury was observed in all trials in spite of much lower pasteurization values and fewer log 10 reductions compared with late stationary phase cultures. Regardless of growth phase, all cultures where a high degree of heat injury (≥99.9%) was observed, did not subsequently grow in the beef product at 3 or 10°C within 30 days. Growth of heat injured cultures preexposed to heat shock (46°C, 30 min) or slowly rising temperatures (0.3°C min −1) before heat injury was also investigated. Heat shocked or heat adapted cultures generally responded in the same manner as non-stressed cultures (no growth at 3°C) except that a longer lag phase was observed in beef processed at slowly rising temperatures and in normal pH beef at 10°C. Although processing at slowly rising temperatures may slightly increase the survival of L. monocytogenes 13-249 in cooked beef, there seem to be no indication of an increase in subsequent growth potential of the surviving cells.

Comparison of Different Reducing Agents for Enhanced Detection of Heat-Injured Listeria monocytogenes

The effect of different reducing agents (l-cysteine, Oxyrase, and Enterococcus faecium) and their combinations on the detection of heat-injured (62.8°C, 7.5 min or 10 min) Listeria monocytogenes was examined. The incorporation of l-cysteine (0.5 g/liter) yielded higher percentage detection than any of the other reducing agents (P < 0.05). The combination of Oxyrase (10 U/ml) and E. faecium (103 CFU/ml) synergistically enhanced the detection of L. monocytogenes heat-injured for 10 min at 62.8°C(P < 0.05). Simultaneous addition of l-cysteine (0.5 g/liter) and Oxyrase (10 U/ml) significantly lowered the recovery of heat-injured L. monocytogenes (P < 0.05). Higher activities of Oxyrase (50 U/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). The rates of depletion of relative percentage O2 were in the order: l-cysteine (0.5 g/liter; 6.63%/min) > Oxyrase (10 U/ml; 5.00%/min) >E. faecium (108 CFU/ml; 1.66%/min) >E. faecium (103 CFU/ml; 0.20%/min). The final levels of redox potential (Eh) achieved were −110.5 mV, −100 mV, −83.5 mV, and −25 mV for E. faecium (l08 CFU/ml), l-cysteine, Oxyrase, and E. faecium (103 CFU/ml), respectively.

Development of a Simple Recovery-Enrichment System for Enhanced Detection of Heat-Injured Listeria monocytogenes in Pasteurized Milk

A simple anaerobic recovery-enrichment system, semisolid Penn State University (ssPSU) broth, that enhances recovery of heat-injured Listeria monocytogenes, was rapidly achieved in 10-ml screw-capped tubes by adding Bacto-agar (2.5 g/liter) and L-cysteine (0.5 g/liter) to Penn State University broth. Glucose was removed from the formulation for ssPSU broth to prevent the growth of thermoduric lactobacilli. Ferric ammonium citrate was added to ssPSU broth to detect esculin hydrolysis and to indicate the presumptive presence of L. monocytogenes. Replacement of phosphate buffer with 3-[N-morpholino]propanesulfonic acid (MOPS) buffer and addition of magnesium sulfate (15 mM) enhanced recovery and detection of L. monocytogenes heat treated at 62.8°C for 20 min. D-Serine, at a concentration of 150 mM, was found to inhibit germination of Bacillus spp. spores but did not inhibit severely heat-injured L. monocytogenes. Finally, ssPSU broth was modified (to mPSU broth) to contain the following: (i) Bacto-agar, 2.5 g/liter; (ii) ferric ammonium citrate, 0.5 g/liter; (iii) MOPS buffer, pH 7.0; (iv) D-serine, 13.7 g/liter; (v) D-alanine, 11.6 g/liter; and (iv) magnesium sulfate, 1.81 g/liter. Incubation temperature significantly affected the recovery and detection of severely heat-injured L. monocytogenes. L. monocytogenes that were heat challenged in filter-sterilized whole milk at 62.8°C for 20, 25, and 30 min could not be detected at incubation temperatures ≥30°C but were consistently detected after incubation at 25°C for 174, 199, and 330 h, respectively. Heat-injured cells of L. monocytogenes that were added to various commercial brands of pasteurized whole milk were also detected using mPSU broth. When clostridial spores (104 spores per ml) were added to filter-sterilized milk containing either heat-injured or non–heat-injured L. monocytogenes, only the latter could be detected in mPSU broth. The mPSU broth system requires no purging with nitrogen gas to create anaerobic conditions and permits recovery, growth, and detection of L. monocytogenes in one vessel in the presence of thermoduric background microflora commonly found in pasteurized milk.

Thin Agar Layer Method for Recovery of Heat-Injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm–diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37°C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55°C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)—in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.

The influence of commonly used selective agents on the growth of Listeria monocytogenes

The effects, upon addition of various selective agents to a rich growth medium (BHIEM) on the growth of Listeria monocytogenes and non- Listeria were studied in microtitre wells. A combination of nalidixic acid (0.1 g/l), acriflavine (0.02 g/l) and fosfomycin (0.1 g/l) added to the medium (BHIEM NAF) was found to be the least inhibitory to Listeria while at the same time, effectively inhibitory to non- Listeria organisms. Recovery of heat injured Listeria monocytogenes in BHIEM, BHIEM NAF and the two traditional Listeria-media, UVM I and II, was compared. Recovery in BHIEM was obtained within 24 h of incubation at 30°C, whereas recovery in BHIEM NAF took up to 48 h. Growth and recovery in UVM I and II never reached the same level as in BHIEM or BHIEM NAF.

Mathematically modeling the repair of heat-injured Listeria monocytogenes as affected by temperature, pH, and salt concentration

Heat-injured cells of Listeria monocytogenes were inoculated into Listeria repair broth (LRB) adjusted to various pH levels (4.2, 5.0, 6.6, 8.0 and 9.6) and salt concentrations (0.5%, 2.5%, 5.0%, 7.5% and 10.0% w v ) at controlled temperatures (4, 10, 22, 37 and 43 °C) in a complete factorial manner (5 3). Repair of the injured microorganisms was evaluated using selective and non-selective plating media. The Gompertz parameters, which were generated by fitting the equation with the bacterial counts, were used to calculate the repair percentage as a function of time from which the repair time was estimated. All growth curves fit the Gompertz equation well (R 2 ≥ 0.972). A first-order model described the repair trend closely (R 2 = 0.989 ± 0.011). Heat-injured Listeria could fully repair in LRB only under 63 of 125 conditions tested during 21 days of incubation. Refrigeration temperature was the most effective means to prevent the repair of heat-injured Listeria. The minimum temperature required for repair increased with an increase in NaCl concentration. The pH ranges at which the repair could occur were narrower at 4 and 10 °C than those at higher temperature. The repair was observed in media containing 10% NaCl between temperatures of 22 and 43 °C at pH 6.6.

Enrichment in Fraser broth supplemented with catalase or Oxyrase ®, combined with the microcolony immunoblot technique, for detecting heat-injured Listeria monocytogenes in foods

The microcolony immunoblot technique using monoclonal antibodies to Listeria monocytogenes was evaluated for its suitability to detect heat-injured cells. Pasteurized milk and filtrates of homogenized raw ground beef slurry and cabbage were inoculated with L. monocytogenes Scott A, heated, diluted, inoculated into Fraser broth (FB) supplemented with 400 μg of catalase ml −1 or 0.01 unit of Oxyrase ® ml −1, and incubated at 30 °C for 6 h. Three inoculum populations (high, medium, and low) were used. The extent of injury was dependent on the heating menstruum. Forty percent of the cells were injured in beef slurry filtrate, whereas 79 and 94% were injured in milk and cabbage filtrate, respectively, when foods were heated at 52 °C for 20 min. Populations of viable cells were determined using the immunoblot technique and by surface plating on modified Oxford (mMOX) agar. Recovery of cells from heated foods was enhanced in FB supplemented with catalase or Oxyrase ® compared to recovery in control broth. Essentially all unheated (control) cells could be detected within about 30 h using enrichment and the immunoblot technique; 54 h were required to easily detect colonies on mMOX. In most cases, the number of cells detected in heated milk or filtrates of homogenized beef after enrichment in FB supplemented with catalase or Oxyrase ® was significantly higher than populations detected using unsupplemented FB; however, enrichment in FB supplemented with catalase or Oxyrase ® did not significantly increase cell populations in heated cabbage filtrate. Within each heat treatment and level of inoculum, cell populations detected on mMOX agar after incubating plates for 48 h or on immunoblots after 24 h were not significantly different. Results indicate that the immunoblot technique in conjunction with enrichment in FB containing either catalase or Oxyrase ® can be successfully used to detect healthy and heat-injured cells of L. monocytogenes in diverse types of foods within 34 h.

Comparison of Oxygen Scavengers for Their Ability to Enhance Resuscitation of Heat-injured Listeria monocytogenes

The recovery of heat-injured Listeria monocytogenes Scott A in Fraser broth (FB) supplemented with sodium thioglycolate, sodium pyruvate, L-(+)-cysteine hydrochloride, catalase or Oxyrase? was studied. After 3 h of incubation at 30°C, recovery was enhanced by all oxygen scavengers except sodium pyruvate. Oxyrase? (0.005 U ml−1) promoted the highest recovery (34%) compared to recovery in control broth (19%). All oxygen scavengers enhanced the recovery of injured L. monocytogenes in FB within 6 h of incubation. After 6 h at 30°C, 49 and 55% of injured cells underwent resuscitation in FB containing 2.5 mg of sodium pyruvate ml−1 and 400 μg of catalase ml−1, respectively, compared to 24% resuscitation in FB not supplemented with oxygen scavengers. The percentage recovery was increased as the incubation time was extended to 6 and 24 h. Nearly all injured cells were recovered within 24 h of incubation, regardless of supplementation of FB with oxygen scavengers. Fraser broth containing 2.5 mg of sodium pyruvate ml−1, 400 μg of catalase ml−1 or 0.01 U of Oxyrase? ml−1 were tested to determine the optimal incubation time and temperature for recovering heat-injured L. monocytogenes. Percentage recovery of injured cells increased with an increase in temperature from 25 to 30°C and from 30 to 35°C. The highest percentage of injured cells recovered was observed in FB containing 400 μg of catalase ml−1 (67%) and 0.01 U of Oxyrase? ml−1 (68%) within 6 h of incubation at 35°C. Catalase (400 μg ml−1) and Oxyrase? (0.01 U ml−1) in FB resulted in significantly higher recovery of injured cells from heated whole milk; however, recovery of injured cells from heated skim milk was not significantly higher. Enrichment in FB containing catalase or Oxyrase? has potential for recovering heat-injured L. monocytogenes cells within 6 h compared to 24 h required in conventional methods.

Enhanced Recovery of Severely Heat-Injured, Thermotolerant Listeria monocytogenes from USDA and FDA Primary Enrichment Media Using a Novel, Simple, Strictly Anaerobic Method

A novel, simple, strictly anaerobic method was developed, which dramatically enhanced the recovery of severely heat-injured Listeria monocytogenes from pasteurized milk, compared to current aerobic methods. Cells of L. monocytogenes were grown in trypticase soy broth supplemented with 0.6% yeast extract (TSB-YE) at 43°C for 18 h and then severely heat-injured at 62.8°C for 5 min in raw milk in sealed, totally submerged thermal-death-time tubes. Recovery in primary enrichment media was enhanced in all cases by purging the headspace with N2 gas. The following treatments (in order of effectiveness) significantly improved recovery compared to conventional aerobic controls: addition of filter-sterilized cysteine + N2 purging > pre-reduced Hungate media + N2 purging = Oxyrase® + lactate + N2 purging > filter-sterilized cysteine - N2 purging > Oxyrase® - lactate + N2 purging. Addition of filter-sterilized cysteine to a final concentration of 0.5 g/l with subsequent N2 purging increased recovery from 0% to 60% with the U.S. Department of Agriculture (USDA) UVM broth and from 11 to 100% with the Food and Drug Administration (FDA) LEB. Addition of either 1% pyruvate or a l0-fold higher concentration of cysteine (5.0 g/L) completely inhibited recovery. Faster and more efficient recovery of L. monocytogenes using cysteine and N2 purging was attributed to the absence of oxidative stress during primary enrichment. Potential difficulties in recovering very severely injured cells are discussed.

A novel strictly anaerobic recovery and enrichment system incorporating lithium for detection of heat-injured Listeria monocytogenes in pasteurized milk containing background microflora.

Heat-injured cells of Listeria monocytogenes were recovered from heated raw milk containing noninjured Enterococcus faecium by combining a simple method for obtaining strict anaerobiosis with a novel enrichment broth, Penn State University broth (PSU broth). Strictly anaerobic conditions were rapidly achieved by adding 0.5 g of filter-sterilized cysteine per liter to PSU broth and then purging the preparation with N2 gas. Little resuscitation or growth occurred in strictly anaerobic PSU broth without lithium chloride because of overgrowth by E. faecium. The growth of E. faecium decreased dramatically with increasing LiCl concentration; LiCl concentrations of 8 and 10 g/liter were completely bacteriostatic. The mechanism of inhibition by LiCl appeared to involve competition with the divalent cations Ca2+ and Mg2+. Heat-injured L. monocytogenes consistently recovered and grew rapidly in strictly anaerobic PSU broth containing 4, 6, or 7 g of LiCl per liter. The use of strictly anaerobic PSU broth containing 7 g of LiCl per liter permitted detection of severely heat-injured L. monocytogenes in one simple recovery-enrichment step by eliminating oxygen toxicity and inhibiting the growth of background microflora, without preventing the resuscitation and subsequent growth of heat-injured L. monocytogenes. L. monocytogenes heated in raw milk at 62.8 degrees C for 10, 15, and 20 min could be consistently recovered from strictly anaerobic PSU broth enrichment cultures at 30 degrees C after 48, 96, and 144 h, respectively, and hence, use of PSU broth may result in better recovery of both injured and noninjured cells from foods than currently used U.S. Department of Agriculture and Food and Drug Administration preenrichment procedures.

Recovery of heat-injured Listeria monocytogenes

Concern has been expressed that the ability of heat-injured Listeria monocytogenes cells to resuscitate during refrigerated storage of food may lead to underestimation of their heat resistance. The recovery of heat-injured L. monocytogenes was therefore examined as a function of incubation temperature and composition of recovery medium. Heat-injured cells exhibited a broad optimum temperature for recovery centered around 20–25°C. The best recovery medium of those tested was blood agar. Incubation of cells in broth or chicken slurry at 5°C (cold-enrichment) did not allow repair of potentially lethal injury i.e. it did not allow recovery of cells that would otherwise have died if incubated at a higher temperature. In some cases incubation of heat-injured cells at 5°C resulted in death of a proportion of the population. Repair of sublethal heat-injury, measured as the time of incubation in tryptone soya broth needed to regain the ability to grow on Listeria selective agar, was slower and less complete at 25°C than at 2°C; repair took 10–15 h at 25°C compared with 8–12 days at 5°C. Refrigeration of heat-treated foods should not therefore increase the risk that heat-injured cells will recover from the heat treatment.

Universal Preenrichment Broth for the Simultaneous Detection of Salmonella and Listeria in Foods

A medium and method for preenriching food products which allow the simultaneous recovery and detection of Salmonella and Listeria are described. To prevent the pH of the medium from rapidly dropping in the presence of extraneous microorganisms found in foods, this medium, universal preenrichment (UP) broth, is highly buffered and low in carbohydrates. The medium allows sublethally injured bacteria to resuscitate and multiply to sufficiently high numbers so that highly selective, secondary enrichment media can be employed to help select the specific bacteria in question from a mixed bacterial background culture. As few as 10 heat-injured Salmonella multiplied to at least 106/ml following a 24 h enrichment in UP, even in mixtures of high levels of known competitive microflora or from naturally occurring microflora found in chicken, hot dogs, or Brie cheese. As few as 10 heat-injured Listeria monocytogenes multiplied to at least 105/ml in these same experiments. From the UP broth, secondary selective preenrichment broths which favor the growth of Salmonella or Listeria can be inoculated, and subsequent protocols for the recovery of either Salmonella or Listeria can then be followed.

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.