Comparison of Listeria monocytogenes recoveries from spiked mung bean sprouts by the enrichment methods of three regulatory agencies.

Three selective enrichment methods, the United States Food and Drug Administration's (FDA method), the United States Department of Agriculture Food Safety Inspection Service's (USDA method), and the EN ISO 11290-1 standard method, were assessed for their suitability for recovery of Listeria monocytogenes from spiked mung bean sprouts. Three parameters were evaluated; the enrichment L. monocytogenes population from singly-spiked sprouts, the enrichment L. monocytogenes population from doubly-spiked (L. monocytogenes and Listeria innocua) sprouts, and the population differential resulting from the enrichment of doubly-spiked sprouts. Considerable L. monocytogenes inter-strain variation was observed. The mean enrichment L. monocytogenes populations for singly-spiked sprouts were 6.1 ± 1.2, 4.9 ± 1.2, and 6.9 ± 2.3 log CFU/mL for the FDA, USDA, and EN ISO 11290-1 methods, respectively. The mean L. monocytogenes populations for doubly-spiked sprouts were 4.7 ± 1.1, 5.5 ± 1.3, and 4.6 ± 1.4 log CFU/mL for the FDA, USDA, and ISO 11290-1 enrichment methods, respectively. The corresponding mean population differentials were 2.8 ± 1.1, 3.3 ± 1.3, and 3.6 ± 1.4 Δlog CFU/mL for the same three enrichment methods, respectively. The presence of L. innocua and resident microorganisms on the sprouts negatively impacted final levels of L. monocytogenes with all three enrichment methods.

Effect of Listeria seeligeri or Listeria welshimeri on Listeria monocytogenes detection in and recovery from buffered Listeria enrichment broth.

The presence of multiple species of Listeria in regulated food products is not uncommon and can complicate the recovery of Listeria monocytogenes particularly on a non-differentiating medium. The potential complications of Listeria seeligeri and Listeria welshimeri on the recovery of L. monocytogenes from inoculated food test samples using the U.S. Food and Drug Administration's (FDA) selective enrichment procedure was investigated. Post-enrichment enumeration, in the absence of food product, indicates that some L. seeligeri and L. monocytogenes pairings may have population differentials as great as 2.7 ± 0.1 logs with L. seeligeri being the predominant species. A similar observation was noted for L. welshimeri and L. monocytogenes pairings which resulted in population differentials as large as 3.7 ± 0.2 logs with L. welshimeri being the predominant species. Select strain pairings were used to inoculate guacamole, crab meat, broccoli, and cheese with subsequent recovery by the FDA Bacteriological Analytical Manual (BAM) method with 10 colonies per sample selected for confirmation. The presence of L. seeligeri had little effect on the recovery of L. monocytogenes. The presence of L. welshimeri resulted in the failure to recover L. monocytogenes in three out of the four food matrices. This work extends the observation that non-pathogenic species of Listeria can complicate the recovery of L. monocytogenes and that competition during selective enrichment is not limited to the presence of just Listeria innocua.

Heavy metal and disinfectant resistance of Listeria monocytogenes from foods and food processing plants.

The persistence of Listeria monocytogenes in food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1, cadA2, and cadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated: cadA1 was more common in isolates of serotypes 1/2a and 1/2b than 4b, while cadA2 was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups of L. monocytogenes, including exposures to heavy metals and disinfectants.

Conservation of genomic localization and sequence content of Sau3AI-like restriction-modification gene cassettes among Listeria monocytogenes epidemic clone I and selected strains of serotype 1/2a.

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.

Real-time PCR detection of 16S rRNA genes speeds most-probable-number enumeration of foodborne Listeria monocytogenes.

Quantifying foodborne pathogens at concentrations of 0.1 to 1,000 CFU/g of food generally involves most-probable-number (MPN) enumeration, which takes at least 4 days. A real-time PCR assay (RTi-PCR) was developed to accelerate MPN enumeration of foodborne Listeria monocytogenes. Foods were spiked from 70 to 110 CFU/g, and triplicate subportions from 0.0001 to 1 g were selectively enriched for 48 h at 30 degrees C. For standard MPN enumeration, the enrichments were subcultured on Oxford agar (48 h at 35 degrees C) to isolate Listeria. For RTi-PCR MPN, the L. monocytogenes cells from the same enrichments were washed and resuspended in 2 ml of sterile water. DNA was extracted by boiling for 10 min. The DNA in the extract's supernatant was targeted with published oligonucleotide primers for amplifying an Lmo-specific sequence of 16S rRNA genes. Amplification was continuously monitored with SYBR Green. The resulting amplicon was characterized by its melting temperature. The L. monocytogenes specificity of the primers was confirmed by testing L. monocytogenes (15 strains), Listeria innocua (11 strains), and Listeria welshimeri, Listeria seeligeri, Listeria ivanovii, and Listeria grayi (1 strain each). Quantitatively spiked milk, lettuce, smoked salmon, Brie cheese, ice cream, pork pâté, salami, ready-to-eat shrimp, raw ground beef, and fresh soft cheese were enumerated by both the standard and the PCR MPN method. The paired results from the two MPN methods agreed well, except for the fresh cheese. For some foods, l-g samples required a decimal dilution for a positive test result, suggesting concentration-dependent food ingredient interference with the RTi-PCR. This RTi-PCR method reduced the time necessary for the MPN enumeration of foodborne L. monocytogenes from 4 to 2 days.

Improved DNA probe detection of Listeria monocytogenes in enrichment culture after physical-chemical fractionation.

Bacterial detection in foods by nucleic acid probes is limited by microflora competition during selective enrichment. Probe target concentration by extraction and fractionation of enrichments may diminish this limitation. The 1-h AccuProbe chemiluminescent culture identification test for Listeria monocytogenes was used as a model. Its high detection threshold provides a stringent challenge for evaluating enrichment work-up protocols. Detection of L. monocytogenes, at 1-4 colony-forming units/g food, was not consistently possible in 48 h enrichment cultures using AccuProbe. Concentration by cell sedimentation was occasionally helpful but the volume of co-sedimented food limited concentration to about 10-fold. To improve concentration, enrichment sediments were sonicated or enzymatically lysed to release the probe's target, r-RNA. The RNA was separated from non-RNA material by extraction with phenol and precipitation with ethanol. Enrichments (250 mL) were concentrated 2500-fold, and the limitation was food RNA volume. A strongly competitive Enterococcus faecium food isolate was used to demonstrate the effect of artificial competition on the kit's ability to detect L. monocytogenes in enrichments. High competitor concentrations repressed the level of the target below the detection threshold, but concentration of r-RNA enabled detection of L. monocytogenes. The effectiveness of this enrichment sample work-up was demonstrated with naturally contaminated hummus.

Contribution of Selective Conditions to Microbial Competition in Four Listeria Selective Enrichment Formulations.

Microbial competition during selective enrichment negatively affects Listeria monocytogenes populations and may hinder the subsequent detection or recovery of this organism. Competition assays among 10 selected strains of Listeria and Citrobacter braakii were performed in buffered Listeria enrichment broth, 3-(N-morpholino)propanesulfonic acid-buffered Listeria enrichment broth, University of Vermont medium-modified Listeria enrichment broth, and Fraser broth. The individual contributions of each selective agent in these media were also assessed, as well as the contribution of incubation temperature. Acriflavine hydrochloride and sodium nalidixate were ineffective at preventing the overgrowth of C. braakii ; this resulted in substantially lower populations of Listeria than when the competitor was absent. At the higher levels, both of these selective agents were detrimental to Listeria populations. The highest enrichment populations of Listeria were observed when either NaCl or LiCl was present. In the absence of selective agents, the final populations of Listeria following competitive growth with C. braakii were not substantially affected by temperature; however, in the presence of selective agents, the Listeria populations were statistically higher at the higher incubation temperature. There are a limited number of selective agents available for use in Listeria -specific enrichment media, resulting in formulations that are only somewhat selective for this species. The optimization of current formulations may help researchers to improve Listeria recovery, particularly from products with a high microbial load. The understanding of the behavior and interactions between target and nontarget microorganisms in the presence of these available selective agents is a necessary step in the optimization of Listeria selective enrichment formulations.

Postenrichment population differentials using buffered Listeria enrichment broth: implications of the presence of Listeria innocua on Listeria monocytogenes in food test samples.

The recovery of low levels of Listeria monocytogenes from foods is complicated by the presence of competing microorganisms. Nonpathogenic species of Listeria pose a particular problem because variation in growth rate during the enrichment step can produce more colonies of these nontarget cells on selective and/or differential media, resulting in a preferential recovery of nonpathogens, especially Listeria innocua. To gauge the extent of this statistical barrier to pathogen recovery, 10 isolates each of L. monocytogenes and L. innocua were propagated together from approximately equal initial levels using the current U. S. Food and Drug Administration's enrichment procedure. In the 100 isolate pairs, an average 1.3-log decrease was found in the 48-h enrichment L. monocytogenes population when L. innocua was present. In 98 of the 100 isolate pairs, L. innocua reached higher levels at 48 h than did L. monocytogenes, with a difference of 0.2 to 2.4 log CFU/ml. The significance of these population differences was apparent by an increase in the difficulty of isolating L. monocytogenes by the streak plating method. L. monocytogenes went completely undetected in 18 of 30 enrichment cultures even after colony isolation was attempted on Oxoid chromogenic Listeria agar. This finding suggests that although both Listeria species were present on the plate, the population differential between them restricted L. monocytogenes to areas of the plate with confluent growth and that isolated individual colonies were only L. innocua.

The presence of the internalin gene in natural atypically hemolytic Listeria innocua strains suggests descent from L. monocytogenes.

The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenes-specific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5'-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3'-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.

Discovery of natural atypical nonhemolytic Listeria seeligeri isolates.

We found seven Listeria isolates, initially identified as isolates with the Xyl(+) Rha(-) biotype of Listeria welshimeri by phenotypic tests, which exhibited discrepant genotypic properties in a well-validated Listeria species identification oligonucleotide microarray. The microarray gives results of these seven isolates being atypical hly-negative L. seeligeri isolates, not L. welshimeri isolates. The aberrant L. seeligeri isolates were d-xylose fermentation positive, l-rhamnose fermentation negative (Xyl(+) Rha(-)), and nonhemolytic on blood agar and in the CAMP test with both Staphylococcus aureus (S(-) reaction) and Rhodococcus equi (R(-) reaction). All genes of the prfA cluster of L. seeligeri, located in the prs-ldh region, including the orfA2, orfD, prfA, orfE, plcA, hly, orfK, mpl, actA, dplcB, plcB, orfH, orfX, orfI, orfP, orfB, and orfA genes, were checked by PCR and direct sequencing for evidence of their presence in the atypical isolates. The prs-prfA cluster-ldh region of the L. seeligeri isolates was approximately threefold shorter due to the loss of orfD, prfA, orfE, plcA, hly, orfK, mpl, actA, dplcB, plcB, orfH, orfX, and orfI. The genetic map order of the cluster genes of all the atypical L. seeligeri isolates was prs-orfA2-orfP-orfB-orfA-ldh, which was comparable to the similar region in L. welshimeri, with the exception of the presence of orfA2. DNA sequencing and phylogenetic analysis of 17 housekeeping genes indicated an L. seeligeri genomic background in all seven of the atypical hly-negative L. seeligeri isolates. Thus, the novel biotype of Xyl(+) Rha(-) Hly(-) L. seeligeri strains can only be distinguished from Xyl(+) Rha(-) L. welshimeri strains genotypically, not phenotypically. In contrast, the Rha(+) Xyl(+) biotype of L. welshimeri would not present an identification issue.

Quantitative Comparison of Two Enrichment Methods for Isolating Listeria monocytogenes from Inoculated Ice Cream.

Two enrichment methods for isolating Listeria monocytogenes (the Lovett method, formerly used by the U.S. Food and Drug Administration, and the revised method for the U. S. Department of Agriculture) were compared using 25 g test portions of spiked vanilla ice cream. Both methods were found to be equally sensitive. Prolonging the enrichments to 7 d and the use of alkali pretreatment had no significant effect on the results. Reduction of the test portion size from 25 to 1 g in the revised USDA method decreased the level of sensitivity by an order of magnitude, as expected.

Initial Cell Concentration and Selective Media Effects on the Isolation of Listeria monocytogenes from Enrichment Cultures of Inoculated Foods.

To facilitate rapid quantitation of Listeria monocytogenes in artificially inoculated dairy products, seafoods, and other foods, the recently revised U.S. Food and Drug Administration methodology for Listeria in foods was further abbreviated. The ability to isolate L. monocytogenes was measured in terms of the number of inoculated colony forming units (cfu)/g of food. Isolation values for the foods tested, using modified McBride's agar (MMA), were <1, 1-10, and >11 inoculated cfu/g in 30, 44, and 26% of test samples, respectively. The corresponding percentages with lithium chloride-phenylethanol-moxalactam (LPM) agar were 41, 51, and 8%, respectively. In eight (21%) instances, isolation values on LPM agar were significantly superior to those on MMA. The superiority of LPM was most dramatic when MMA isolation values were >10 initial cfu/g. However, LPM was not an infallible remedy for MMA deficiencies even at >10 cfu/g. Ease of isolation was apparently not related to food type. For 10 different test portions of Brie cheese, isolation values ranged from 0.3 to > 10,000 of inoculated cfu/g with MMA and 0.3 to 2500 of inoculated cfu/g with LPM agar.

Evaluation of a Selective Enrichment Most Probable Number Enumeration Method for Viable Listeria spp. in Dairy Products.

A most probable number (MPN) method for enumerating low numbers of Listeria spp. in dairy foods was developed by adapting the U.S. Food and Drug Administration (FDA) Listeria isolation methodology. Milk, cheese, and other milk products were diluted and homogenized in enrichment broth (1 g/10 ml). Homogenates were inoculated with L. monocytogenes Lm82, a streptomycin-resistant variant of strain Scott A, at <1 to 320 CFU/g and further diluted in FDA enrichment broth to give 0.1, 0.01, and 0.001 g of food sample per 10 ml. Dilution aliquots (10 ml) in triplicate or quintuplicate were incubated at 30°C for 48 h before being subcultured on Oxford agar at 35°C. Esculin-hydrolyzing colonies on Oxford agar were confirmed as the inoculum strain by their ability to grow on Trypticase soy agar containing streptomycin. Differences between inoculum and MPN values were evaluated by using tabulated 95% confidence limits. The calculated MPNs agreed with the inoculum levels in 91% (58 of 64) of noncheese dairy foods and in 49% (56 of 112) of 15 varieties of ripened cheeses. Competitive microflora affected by cheese age and the kind of milk used may account for the suboptimal performance of the MPN method with the cheeses.

Pooling of Noncollaborative Multilaboratory Data for Evaluation of the Use of DNA Probe Test Kits in Identifying Listeria monocytogenes Strains.

The Accuprobe test kit, a DNA probe culture confirmation test kit for identifying bacterial isolates as Listeria monocytogenes was evaluated with 148 food-borne Listeria isolates. The identities of the isolates were confirmed by conventional tests. A subset of the Listeria isolates was also used to evaluate the Gene-Trak Listeria monocytogenes isolation kit. The data obtained with each kit were pooled with data for that kit from independent published studies. Interlaboratory performances were then estimated statistically, to achieve the main features of orthodox interlaboratory collaborative studies. The large number of strains required in this noncollaborative evaluation method advantageously provided a more representative sampling of the potential isolates that may be found in practice. Exact comparability between the two kit studies was not possible nor is it a feature of orthodox collaborative identification kit studies. However, the kits apparently performed equivalently given the different statistical confidences dictated by the study designs.

Virulence of Listeria monocytogenes , Listeria seeligeri , and Listeria innocua Assayed with In Vitro Murine Macrophagocytosis.

The survival of virulent and avirulent Listeria species internalized in cells of a murine macrophage-like cell line, RAW264.7, was monitored. Mouse macrophage cells (ca. 5 × 105/ml) suspended in fresh RPMI medium 1640 containing fetal bovine serum were mixed with 5 × 107 to 5 × 108 Listeria cells per ml and incubated 1 h at 37°C with CO2-enriched air. Gentamicin (10 µg/ml) was added to kill bacteria not internalized by the cells. At 2, 4, and 6 h postinfection, 10-µl amounts of the suspensions were lysed in microtiter plate wells during serial decimal dilution in water. Triplicate dilutions (10-µl each) were plated on trypticase soy agar, and colonies were counted after 48 h incubation at 35°C. About 0.1 to 1% of the added hemolytic pathogen L. monocytogenes Scott A and the avirulent nonhemolytic L. innocua were internalized at 2 h. The number of internal L. monocytogenes cells increased significantly by 6 h, but L. innocua cells showed no significant change. A strain of the hemolytic species L. seeligeri behaved like the nonhemolytic L. innocua . This distinction between the intracellular behavior of pathogenic and nonpathogenic species, if a general phenomenon, may be useful as an in vitro virulence assessment parameter.

Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods.

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.

Fate of Listeria monocytogenes in Shredded Cabbage Stored at 5 and 25°C under a Modified Atmosphere.

Shredded cabbage was inoculated with Listeria monocytogenes Scott A cells and stored in normal air or a modified (70% carbon dioxide and 30% nitrogen) atmosphere at 5 and 25°C. Under the normal atmosphere at 25°C, colony counts increased by 2 logs within 2 d of storage but then decreased to undetectable levels within 6 d of storage. In the modified atmosphere at 25°C, numbers also decreased to undetectable levels within 6 d, but with a less marked initial increase and a decline that was more rapid than in the unmodified atmosphere. In the cold (5°C), the counts increased gradually, but only by about 1 log, in both atmospheres. In the normal atmosphere at 5°C, however, colony counts decreased sharply after 13 d of storage. Reductions in colony counts coincided with decreases in cabbage pH and development of spoilage. The increased level of carbon dioxide was ineffective in controlling L. monocytogenes at 5°C. At 25°C cabbage spoilage was rapid and colony counts declined under both atmospheres of storage.