Anthony D. Hitchins

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.

Assessment of alimentary exposure to Listeria monocytogenes.

Survey data on the frequency of foodborne occurrence and dietary exposure to Listeria monocytogenes were used to estimate the mininmal mean per person annual rate of exposure in the United States during the late 1980s. The estimate was restricted to ready-to-eat (RTE) foods because proper cooking was assumed to be listericidal. The mean amount of each food type per L. monocytogenes occurrence was calculated in about 100 sources, and dietary intake data were used to calculate the mean number of occurrences of L. monocytogenes consumption per person per year. The mean number of occurrences consumed annually per person was determined to be 10 to 100 for RTE food values of 2 to 20% of the total dietary intake, respectively. The frequency of foodborne listeriosis (approximately 10(-5)) was consistent with the estimated exposure rate only if the susceptible population was unexpectedly small or extremely high doses were necessary for infection. Because little evidence is available to support a high rate of unreported non-severe infections, this study was concerned only with severe listeriosis cases. Published frequencies of L. monocytogenes concentrations in food were used to convert occurrences to colony forming units (CFU). Low L. monocytogenes concentrations (approximately 1 CFU/g) were too frequent to be responsible for listeriosis in susceptible subjects, would have caused listeriosis only with extremely low probability in a one-cell threshold infection model. The probability of exposure to a higher dose (> or = 10(3) CFU) was large enough to account for the observed rate of listeriosis.

The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes

ABSTRACT 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.

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 extracts 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.

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 McBrides 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.

Discovery of Natural Atypical Nonhemolytic Listeria seeligeri Isolates

ABSTRACT 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.

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 Administrations 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.

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

ABSTRACT 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.

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.

Microbial survey of shared-use cosmetic test kits available to the public

SummarySome people like to try cosmetics before purchasing them. With repeated use by different customers, however, the tester kits provided by many retail outlets can become potential vectors of microbial pathogens. A survey was conducted to assess the health risk from bacteria found on shared-use cosmetics. A total of 3027 shared-use cosmetic product samples were collected from 171 retail establishments throughout the contiguous United States. Eye, face and lip cosmetics were tested within situ nondestructive swabbing and the use of the Transette 3R Modified Amies Charcoal Culture and Transport System. Bacteria were isolated from about 50% of the items for all three categories. Semiquantitatively-estimated mean densities were 2288, 1685 and 1088 CFU g−1 for eye, face and lip products, respectively. Ranges for all categories were 0–155 CFU g−1. About 5% of the items had bacterial counts above 5000 CFU g−1 (eye products) or 10 000 CFU g−1 (other products). More than 60% of isolates were typical of microflora from human skin; the remainder were environmental microbes. About 60% of the isolates were Gram-positive cocci:Staphylococcus spp. (especiallyS. epidermidis) andMicrococcus spp. The Gram-negative pathogenPseudomonas aeruginosa constituted 0.07% of the isolates. The survey results suggest that the preservation systems of some of the cosmetics failed under excessive use (abuse), and indicated a potential for microbiological safety problems with shared-use consmetics.