DEVELOPMENT OF NEW ENRICHMENT BROTHS AND PLATING AGARS FOR ISOLATION OF HEMOLYTIC SPECIES OF LISTERIA

A new basal broth medium was formulated for optimal growth of Listeria monocytogenes, which occurred at 37°C when the initial pH was 6.8. This formulation was used as the basal medium for development of a highly selective plating agar which proved suitable for direct culture of vaginal and rectal swabs for L. monocytogenes. A modification with slightly lower selectivity was necessary for recovery of hemolytic strains of Listeria ivanovii and Listeria seeligeri. The same basal medium was used as a pre-enrichment broth and for the development of a selective enrichment broth which were incorporated into a two-step enrichment procedure for the isolation of L. monocytogenes from foods. These new media were compared with several others that have been proposed by comparing recoveries of Listeria from laboratory seeded foods (100% positive), raw milk (50 samples, all negative) and comminuted meat products (75% positive) .     

Impact of dilution ratios on Listeria monocytogenes growth during University of Vermont medium enrichment of deli meats

In the U.S. Department of Agriculture (USDA) method for Listeria detection, a 25-g composite food sample is enriched in 225 ml of University of Vermont medium (UVM), giving a detection limit of 0.04 CFU/g. However, in a recent large-scale four-state deli meat survey for L. monocytogenes, 125-g samples enriched in 1,125 ml of UVM were requested to increase the detection limit to 0.008 CFU/g. To circumvent problems associated with large volumes of UVM, the impact on L. monocytogenes growth of lower dilution ratios used for enrichment and most-probable-number (MPN) detection was compared with the results obtained using the conventional 1:10 dilution. In this study, 125-g samples of cured turkey, uncured turkey, ham, and roast beef were inoculated with a six-strain L. monocytogenes cocktail to contain approximately 1 x 10(3) CFU/g. This cocktail was then diluted 1:3, 1:5, or 1:10 in UVM, homogenized, enriched at 30 degrees C, and periodically plated on modified Oxford agar to determine generation times during 24 h of incubation. The same enrichment protocol was also assessed in a three-tube MPN assay using 125-g samples inoculated with L. monocytogenes to contain approximately 1 CFU/g. The effects of two homogenization methods, stomaching and pulsifying, on Listeria growth were compared using oven-roasted turkey breast diluted 1:3, 1:5, and 1:10 in UVM. Overall, the growth rates, generation times, and MPN values for each of the four selected deli meats were similar (P > 0.05) using UVM enrichment ratios of 1:3, 1:5, and 1:10, with no significant (P > 0.05) differences in L. monocytogenes growth rate or generation time between experiments using pulsifying and stomaching. These findings indicate that lower volumes of UVM can be used in the USDA procedure when examining deli meats without compromising Listeria recovery.     

Feasibility of a defined microflora challenge method for evaluating the efficacy of foodborne Listeria monocytogenes selective enrichments

Comparison of isolation methods for microbial pathogens is complicated by the variable interference caused by the competitive microflora present in test samples such as foods. In principle, using measured amounts of a standard competitor in a defined surrogate food matrix might control the effect of variable interference. This possibility was investigated using Listeria monocytogenes and enrichment broths belonging to the acriflavine-nalidixate selective agent class. Triplicate test sample sets were prepared. Each set consisted of suspensions of variable levels of the standard competitor, Enterococcus faecium strain 111 (approximately 10 to 10(9) CFU/25 g), mixed with a low constant level (10 to 100 CFU/25 g) of L. monocytogenes. These test samples were enriched at 30 degrees C for 48 h in different selective media and streaked onto selective isolation agars. The input CFU ratio (E. faecium/L. monocytogenes) that permitted a 50% end point L. monocytogenes recovery was 2.2 x 10(6) or higher for the Food and Drug Administration one-step enrichments and 0.8 x 10(6) for the International Standards Organization (ISO) two-step enrichment. These and other results show that this evaluation method is feasible with this class of enrichments. Interestingly, L. monocytogenes could be detected in enrichment cultures at high-input E. faecium/L. monocytogenes ratios even when the enriched samples were plated onto nonselective media. The pinpoint colonies of L. monocytogenes embedded in a confluent lawn of E. faecium 111 were detectable by their contrasting coloration in Henry obliquely transmitted illumination.     

Comparison of a cold enrichment and the FDA method for isolating Listeria monocytogenes and other Listeria spp. from ready-to-eat food on retail sale in the U.K

A cold enrichment and the modified FDA selective enrichment method were compared for their ability to detect Listeria monocytogenes and other Listeria species from various ready-to-eat foods on sale in the UK. Of 57 food samples examined using cold enrichment, five yielded L. monocytogenes, and two L. innocua. The FDA enrichment method yielded three samples positive for L. monocytogenes only. Foods examined included soft cheeses, fermented meat sausages, pates and salads.     

Injury,resuscitation and detection of Listeria spp. from frozen environments

The ability of Listeria spp. to be reversibly freeze-injured was determined and methods for detection of freeze-injured Listeria were evaluated. Strains of Listeria monocytogenes and Listeria innocua tested showed no significant variation as to the extent of injury sustained during storage for 24 h at -9 to -11°C. Average 24 h injury ranged from 44-64% for L. monocytogenes strains and 41-54% for L. innocua strains. The most substantial increase in injury was seen in the first 24 h and injury remained constant or increased slightly throughout the 14 day period. Freeze injury was reversible in all Listeria strains tested when trypticase soy broth and Listeria repair broth (LRB) were used as repair media. In order to determine if nonselective pre-enrichment followed by selective enrichment using LRB, resulted in increased detection of Listeria existing naturally in processing environments, samples were obtained from two dairy and one meat processing plant. Use of nonselective pre-enrichment followed by selective enrichment using LRB did not enhance recovery of Listeria from frozen environments. This may be attributed to a limited amount of freeze-injury occurring in a naturally existing population of Listeria.     

Evaluation of enrichment procedures for recovering Listeria monocytogenes from dairy products

Six different enrichment media and five selective plating media were compared for their suitability for the recovery of Listeria monocytogenes from dairy products. These included media used to test milk products by the U.S. Food and Drug Administration (FDA) and the U.S. Centers for Disease Control (CDC), and media developed by the U.S. Department of Agriculture (USDA) for testing meat and poultry products. Test samples included naturally contaminated goat's milk, cultured milk products and ice cream manufactured with L. monocytogenes, and unpasteurized milk inoculated with heat- and freeze-injured cells of L. monocytogenes. Generally, the media and two-stage enrichment protocol developed by the USDA, with plating of samples after two consecutive 24-h incubation periods, yielded better recoveries than all other enrichment media incubated for 24 h. A modified USDA procedure, incorporating nonselective pre-enrichment of samples by omitting acriflavine and nalidixic acid from the primary USDA enrichment broth, and transfer of a larger volume of the initial culture broth to the secondary enrichment media, significantly increased recoveries of low numbers of sublethally stressed L. monocytogenes. Prolonged incubation of samples in the FDA enrichment broth, for 7 days, did not consistently improve recoveries over the initial 24-h incubation time of the medium. The selective plating medium developed by the USDA, lithium chloride-phenylethanol-moxalactam agar, was the most effective plating agar for isolation of L. monocytogenes following enrichment of samples in any broth culture, and increased recoveries of L. monocytogenes by 19-40% compared with other selective agar media tested.     

Incidence and seasonal variation of Listeria species in bulk tank goat's milk

Four hundred and fifty raw goat's milk samples obtained from the bulk tanks of 39 goat farms were analyzed for Listeria spp. over a 1-year period. Modified versions of the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) and Food and Drug Administration (FDA) protocols were used for recovery of Listeria. Overall, 35 (7.8%) samples yielded Listeria spp. with Listeria monocytogenes identified in 17 of the 35 (3.8%) Listeria-positive samples. Listeria innocua was detected in 26 (5.8%) samples. Eight milk samples contained both L. monocytogenes and L. innocua. Milk samples from 18 of the 39 (46.2%) farms were positive for Listeria at least once during this 1-year study. The modified USDA-FSIS method, which used Listeria repair broth rather than University of Vermont (UVM) broth for primary enrichment followed by a 4-h nonselective incubation period, yielded more Listeria-positive samples (77.1%) than the FDA method (51.4%). All L. monocytogenes isolates belonged to serotypes 1 (62.6%) or 4 (37.4%). Moreover, five different Listeria ribotypes were identified from 34 selected L. monocytogenes isolates, 2 of which were deemed to be of clinical importance. Listeria isolation rates were markedly higher during winter (14.3%) and spring (10.4%) as compared to autumn (5.3%) and summer (0.9%) with these trends similar to those previously reported for cow's milk.     

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 Manual Listeria 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.     

Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains

Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp. and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were respectively present in 56 and 34 of 315 samples analysed. Fraser broth incubated for 48 h gave the fewest false negative Listeria spp. results [4/56; (7.1%)], but concurrently only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes, Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp. positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes positive samples correctly and gave fewer false positive results. The overall conclusion was that more than one isolation method is needed to accurately estimate L. monocytogenes contamination rates.     

The development of an efficient and rapid enzyme linked fluorescent assay method for the detection of Listeria spp. from foods

Conventional isolation methods, including the Health Products and Food Branch (HPFB), Health Canada method used for the isolation and identification of Listeria species and Listeria monocytogenes from foods and environmental samples, can take a week or more to complete and are usually labor-intensive. This has led to the development of various rapid methods which attempt to generate results comparable to standard methods but in a reduced time-frame with less hands-on operation. Our previous work with rapid detection systems indicated that the recommended enrichment protocols failed to grow the Listeria to detectable levels in a reliable and consistent manner.In the present study, a novel enrichment protocol is described and consists of samples being pre-enriched in Palcam broth (incubated at 35 degrees C for 26 h), enriched in UVM 2 (30 degrees C for 26 h) and then plated and analysed by a rapid detection kit, with results being generated after 52 h of incubation.In total, 200 naturally contaminated samples were analysed by both the HPFB standard method and the Palcam method. The results showed that the Palcam method is comparable to the HPFB method. Further analysis involved a rapid detection system, which applies ELISA techniques and automation in an enzyme-linked fluorescent assay (ELFA) system. This system, referred to as the Vitek Immuno Diagnostic Assay System or VIDAS, can identify Listeria to the genus or species (L. monocytogenes) level.In this comparison, an additional 324 naturally contaminated samples were analysed by both the Palcam and ELFA methods. The sensitivity and specificity of the ELFA method were 98.1% and 97.0%, respectively, while the efficiency was 97.5%. False-negative and false-positive rates were 1.9% and 3.0%, respectively. These results show that the ELFA method (when using the Palcam method for pre- and secondary enrichment) was efficient and gave reliable results after 52 h of incubation, and met Health Canada's criteria for approval as a rapid method.     

Combined secondary enrichment of primary enrichment broths increases Listeria detection.

The efficacy of combining dual primary enrichment cultures into a single secondary broth was evaluated for detecting Listeria in naturally contaminated meats and environmental samples obtained from dairy processing plants. A total of 336 samples were tested using University of Vermont modified Listeria enrichment broth (UVM) and Listeria repair broth containing selective agents (LRBS) as primary enrichment media. Eighty samples (23.8%) yielded Listeria by at least one method. Neither primary enrichment broth was significantly better (P>0.05) than the other in identifying Listeria-positive samples. UVM media, when used as a primary enrichment broth, identified 66 Listeria-positive samples, while the use of LRBS as a primary enrichment broth identified 65 Listeria-positive samples. Listeria detection improved significantly (P<0.01) when two primary enrichment media were used for sample analysis. It is not clear whether this improvement was due to simply replicating the primary enrichment or to the particular pair of primary enrichment media used. The use of a dual secondary enrichment procedure was better (P<0.05) than the use of either individual primary enrichment medium alone. The overall rate of recovery increased from 81.3 to 82.5% for single secondary enrichment to 93.8% using a dual secondary enrichment technique. Analysis of results obtained when combining two independent isolation methods versus combining two primary enrichment media into one single secondary enrichment broth indicated that there was no significant difference (P>0.05) in either procedure. Inoculum size (0.1 ml versus 0.2 ml) did not have an effect on the overall rate of recovery. The procedure developed increased the sensitivity of testing while decreasing the potential workload associated with an increase in enrichment procedures.     

Nitrite-induced injury of Listeria monocytogenes and the effect of selective versus nonselective recovery procedures on its isolation from frankfurters

Sodium nitrite (NaNO2) is used as a curing agent in frankfurters. Although previous studies have documented the bacteriostatic abilities of NaNO2 toward Listeria monocytogenes, few if any studies have been conducted that consider the possibility of sublethal injury to L. monocytogenes by exposure to NaNO2. The goals of this study were to determine whether NaNO2 has the ability to injure L. monocytogenes, to determine whether nitrite injury is reversible, and to compare the recovery of L. monocytogenes from frankfurters containing nitrite with Listeria repair broth (LRB) and University of Vermont modified Listeria enrichment broth (UVM). NaNO2, when used at concentrations of 100 and 200 ppm, was found to injure L. monocytogenes. The injury was completely reversible, or growth of uninjured Listeria occurred in LRB when injury was between 98.5 and 98.7%. However, total recovery was not observed in LRB when injury exceeded 99%. UVM was unable to reverse the effects of nitrite-injured L. monocytogenes. With respect to time, inoculum, and meat type, LRB was found to be consistently superior to UVM at recovering L. monocytogenes from frankfurters. Nitrite injury might be a factor influencing detection and recovery of L. monocytogenes from frankfurters.     

Validation of NMKL method No. 136--Listeria monocytogenes, detection and enumeration in foods and feed

A collaborative study was organised to define the performance characteristics of the revised NMKL Method No.136 "Listeria monocytogenes. Detection and enumeration in foods". Chromogenic L. monocytogenes specific plating medium, Agar Listeria according to Ottaviani and Agosti (ALOA) was introduced in the revised method in order to improve the sensitivity and specificity of the method, and to shorten the analysis time. Efficacy of ALOA One Day from AES (ready-to-use agar in bottles), Listeria Chromogenic Agar (Agosti and Ottaviani Listeria agar) from Lab M (LCA) (dehydrated powder), Chromogenic Listeria Agar Plates from Oxoid (OCLA) (ready-to-use plates) and L. monocytogenes blood agar medium LMBA from Lab M (dehydrated powder) were tested. Three types of food matrices (vacuum-packed hot-smoked salmon, soft cheese and cooked ham) and one feed matrix (wheat grain) inoculated with two levels of L. monocytogenes with or without L. innocua were used in the study. A total of 24 samples were analysed both in the detection and enumeration part of the study by 18 and 17 Nordic laboratories, respectively. The sensitivities of ALOA, LCA, OCLA and LMBA in the detection of L. monocytogenes in food samples after one-step enrichment (Half-Fraser) were 94.4-96.4% and after two-step enrichment (Half-Fraser followed by Fraser) 97.7-100%. For wheat grain the respective figures were 84.7-88.9% and 90.3-93.1%, respectively. The precision characteristics were generally good for the enumeration of L. monocytogenes in the food samples with high levels of inoculation. Several poor values obtained from the food samples with low levels of inoculation probably reflect high uncertainty of measurement when less than 10 cfu/g was counted. Poor values obtained from the wheat grain samples by any of the media evaluated were due to poor precision for feed samples. According to the study, the revised NMKL Method No.136, 4th ed. showed excellent results in the detection and satisfactory results in the enumeration of L. monocytogenes in foods. The results for the detection of L.monocytogenes in wheat grain were good, but the method cannot be recommended for the enumeration of L. monocytogenes in feed-stuffs. Any one of the media evaluated can interchangeably be used as an obligatory isolation medium for the detection and enumeration of L. monocytogenes in foods, and for the detection in feed-stuffs. The L. monocytogenes specific plating media that were evaluated shorten the time of analysis and significantly reduce the work load. The detection of positive samples mostly after Half-Fraser enrichment, reduces the analysis time further, and makes it possible to skip the secondary enrichment. However, secondary enrichment cannot be totally left out, because samples with low levels of L. monocytogenes, with high levels of competing flora, and with injured L. monocytogenes, do need secondary enrichment.     

Comparison of two chromogenic media for the detection of Listeria monocytogenes with the plating media recommended by EN/DIN 11290-1

The efficacy of two selective chromogenic culture media, Agar Listeria Ottaviani and Agosti (ALOA) and RAPID' L. mono for the detection of Listeria monocytogenes in food, was compared with that of an official culture method according to the EN/DIN 11290-01 and -02 protocols [corresponding to the section 35 LMBG (German Food Act) method]. A total of 310 pre-packed ready-to-eat food samples (100 of graved and cold smoked salmon, 130 of different raw and cooked sausages and 80 of delicatessen and mixed salads) were examined. L. monocytogenes was identified in 52 investigated salmon samples. Using two chromogenic media, 50 samples were found positive for L. monocytogenes. Compared to the reference method there were no false-positive results. By the EN/DIN 11290-01 culture procedure after the selective enrichment in Fraser broth 12 out of 130 samples of sausages were positive for L. monocytogenes. These 12 samples were also positive for L. monocytogenes with the chromogenic medium RAPID' L. mono. One sample was false negative with ALOA. Three additional samples were found positive with ALOA and four with RAPID' L. mono. The standard method was inadequate to confirm these samples as positive. Listeria spp. were isolated from 7 samples of mixed salads with both methods. One, 3 and 3 samples were found to contain L. monocytogenes, L. innocua and L. seeligeri, respectively. Both chromogenic media enabled a rapid and specific detection of L. monocytogenes within 24h after enrichment. Visual detection of pathogenic L. monocytogenes and other Listeria spp. was easier on chromogenic media.     

Selective enrichment media affect the antibody-based detection of stress-exposed Listeria monocytogenes due to differential expression of antibody-reactive antigens identified by protein sequencing

Selective enrichment broths are frequently used to recover stressed Listeria cells to detectable levels, but the ability of antibodies to detect these cells from various commonly used enrichment media is unknown. In this study, a polyclonal (PAb) and monoclonal (MAb) antibody were used to examine the variation in antigen expression on healthy or stress-recovered Listeria monocytogenes cells grown in brain heart infusion broth, buffered Listeria enrichment broth (BLEB), Listeria repair broth (LRB), University of Vermont medium (UVM), and Fraser broth (FB) for immunodetection. Indirect enzyme-linked immunosorbent assay (ELISA) data showed that L. monocytogenes subjected to stresses (acid, cold, heat, and salt) and then grown in BLEB gave the highest reaction with the anti-Listeria PAb while those grown in LRB gave the highest reaction with the MAb C11E9. Cells grown in UVM and FB gave poor ELISA values with both antibodies. Western blotting with PAb revealed differential expression of surface proteins of 62, 58, 50, 43, and 30 kDa on L. monocytogenes cells, with most proteins displaying elevated expression in BLEB and LRB but reduced or no expression in UVM or FB. Similar differential expressions were noticed for C11E9. PAb-reactive proteins were identified as putative LPXTG-motif cell-wall anchor-domain protein (62 kDa; lmo0610), flavocytochrome C fumarate reductase chain A homolog protein (58 kDa; lmo0355), enolase (50 kDa; lmo2455), glyceraldehyde 3-phosphate dehydrogenase (43 kDa; lmo2459), and hypothetical phospho-sugar binding protein (30 kDa; lmo0041), respectively, and the MAb-reactive 66-kDa protein was confirmed to be N-acetylmuramidase (lmo2691). In conclusion, BLEB and LRB favorably supported increased expression of antigens and proved to be superior to UVM and FB for immunodetection of stressed L. monocytogenes cells.     

Evolution of Listeria populations in food samples undergoing enrichment culturing

The isolation of Listeria monocytogenes from food is carried out using a double enrichment. It is believed that the double enrichment can allow the overgrowth of Listeria innocua in samples where both species are present. In this study, we have evaluated the impact of overgrowth between Listeria species and strains during each step of the enrichment process. The effect of factors minimizing interactions between strains or phage inhibitory effects has also been estimated. In an artificially contaminated food undergoing enrichment, overgrowth could result from competitive interactions between Listeria spp. resulting from the production of bacteriocins and bacteriophage at high initial contamination levels (>10(4) cfu/g), but not at lower levels (50-100 cfu/g) as generally found in contaminated foods. At high levels of inoculation, the competitive effect could be reduced by solidification of the selective broths, to limit the diffusion of the inhibitors. Overgrowth resulting from differences in growth rate occurred independent of the initial contamination level. However, in naturally contaminated foods undergoing enrichment, there were no absolute correlations between growth rates or inhibitory profiles in terms of strain evolution during enrichment. In fact, Listeria strains which were predominant in the original sample in most cases remained the dominant strains at the end of the enrichment, although the relative proportion of any given strain could change significantly over the enrichment process. Additional factors which have yet to be identified impact on the evolution of Listeria in the two-step enrichment process. Analysis of strain evolution in eight naturally contaminated foods has indicated that the second enrichment step in Fraser broth can be reduced from 48 to 24 h without impacting on the recovery of L. monocytogenes. Our limited survey of naturally contaminated foods also demonstrated that maximum recovery of L. monocytogenes and other Listeria strains was found following 24 h incubation in 1/2 Fraser Broth. This finding suggests that it may be possible to shorten the current two-step isolation method further without reducing method sensitivity.     

The effect of aeration,initial inoculum and meat microflora on the growth kinetics of Listeria monocytogenes in selective enrichment broths

The effect of increased aeration, initial inoculum and the meat microflora on the growth kinetics of Listeria monocytogenes in meat samples enriched in Listeria selective broth were investigated. Growth rate and lag phase duration were calculated following linear regression analysis on the growth data. Neither aeration or variations in the initial inoculum levels altered the length of the lag phase or the growth rate of L.monocytogenes. The growth kinetics of the meat microflora was similar under all of the experimental conditions investigated. Overall the meat microflora was shown to have a faster growth rate than L. monocytogenes in selective media. Organisms growing during Listeria selective enrichment were identified as Lactobacillus plantarum, Lactobacillus delbrueckii and pseudomonads. The implications of the growth of these organisms in Listeria selective broths are discussed.     

pH and solute concentration of suspension media affect the outcome of high hydrostatic pressure treatment of Listeria monocytogenes

The effect of pH and solute concentration of suspension media on high hydrostatic pressure (HHP) induced inactivation of Listeria monocytogenes (approximate 10(8) CFU/ml) was investigated by the using treatment between 300 MPa and 600 MPa at 25 degrees C for 10 min. The suspension media used in this study represented different concentrations (0.1% to 10%) of buffered peptone water (BPW) with an adjusted pH of 4 to 7. An increase in the concentration of BPW resulted in a decreased HHP-induced inactivation of L. monocytogenes that was dependent on the pH of the medium. HHP-treatment at 300 MPa showed no bactericidal effect at neutral pH regardless of the BPW concentration. When the pH of BPW (0.1% to 5%) was reduced to 4, L. monocytogenes was completely inactivated (more than an 8 log cycle reduction) with a HHP-treatment of at least 300 MPa. HHP-treatment above 400 MPa completely inactivated L. monocytogenes in a relatively dilute BPW (0.1% and 1%) with an adjusted pH below 6. While only a 2 log cycle reduction was observed in 10% BPW at the pH ranging from 5 to 7 after treatment with 600 MPa, L. monocytogenes in 10% BPW at pH 4 was completely inactivated. Even though a significant bactericidal effect of HHP-treatment was not observed when applied with a low pressure such as 300 MPa or suspended in higher BPW at neutral pH, a reduction of the pH greatly affected the HHP-induced inactivation of L. monocytogenes. These results indicated that information concerning the pH of food or media would greatly assist an optimization of HHP-treatment for the inactivation of bacteria.     

Detection of Listeria monocytogenes in pigs and pork

In this study, we surveyed hogs (n = 300) as well as pork products (ground pork and raw chitterlings) for Listeria monocytogenes. Pig specimens collected before (tonsil swabs) and after slaughter (tonsils, lymph nodes, carcass swabs, and rectal contents) were examined for L. monocytogenes by enrichment with conventional enrichment broths followed by subculturing to selective agar. A multiplex polymerase chain reaction assay targeting the highly conserved 16S rRNA gene of the Listeria species as well as the hlyA gene unique to L. monocytogenes was used to screen aliquots of the enrichment (method I) as well as to confirm presumptive Listeria colonies from Columbia agar with 0.05% glucose supplemented with polymyxin B-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol (PALCAM; method II). Subculturing to PALCAM agar was the more sensitive of the two methods on the basis of the overall detection of Listeria. For hog tissues, method I detected L. monocytogenes (0.87% positive) and no other Listeria spp. in all samples (n = 1,849). In contrast, method II detected significantly more (P < 0.05) L. monocytogenes (2.38%) and Listeria spp. (0.38%) in these tissues. For small intestines (n = 300 raw chitterlings), L. monocytogenes was identified in 8.3% of enrichments with University of Vermont modified Listeria enrichment broth; plating to PALCAM slightly improved recovery (9%). Overall, ground pork samples (n = 340) harbored L. monocytogenes (45% positive) and other Listeria species (1.5% positive), as determined by method I. Subculturing to PALCAM significantly (P < 0.05) improved the detection of L. monocytogenes (50.2%) but not that of other Listeria species (1.7%). L. monocytogenes isolates (n = 243) were assigned to serotype 1 (53.5%), serotype 4 (25%), and serotypes other than 1 and 4 (21.4%).     

Modelling the competitive growth of Listeria monocytogenes and Listeria innocua in enrichment broths

The overgrowth of Listeria innocua in enrichment broths designed for the isolation of Listeria monocytogenes is believed to result from two factors: a selective growth advantage of L. innocua, and/or an inhibitory interspecies interaction. The generation times of 13 isolates of L. innocua and L. monocytogenes were determined in Brain Heart Infusion (BHI) and a variety of enrichment media. No significant differences were found in growth characteristics between either species in the various media, suggesting that the growth advantage of L. innocua in enrichment media was not as significant as previously described. Kinetic analysis of mixed cultures of L. monocytogenes and isolates of L. innocua producing a variety of inhibitory activities demonstrated the possibility of an inhibitory interaction between these two species resulting in the overgrowth of the enrichment culture with L. innocua. Modelling the evolution of the ratio between two populations in an enrichment process was used to analyze the impact of a selective growth advantage in L. innocua in an enrichment process for growth of L. monocytogenes. These findings support the widely held view that an overgrowth of L. innocua in the enrichment process can result from both a selective growth advantage as well as the production of inhibitory compounds. From a practical perspective, these interactions can result in an increase in false negatives.