Listeria monocytogenes grown at 7° C shows reduced acid survival and an altered transcriptional response to acid shock compared to L. monocytogenes grown at 37° C.

Survival of the food-borne pathogen Listeria monocytogenes in acidic environments (e.g., in the human stomach) is vital to its transmission. Refrigerated, ready-to-eat foods have been sources of listeriosis outbreaks. The purpose of this study was to determine whether growth at a low temperature (i.e., 7°C) affects L. monocytogenes survival or gene transcription after exposure to a simulated gastric environment (i.e., acid shock at 37°C). L. monocytogenes cells grown at 7°C were less resistant to artificial gastric fluid (AGF) or acidified brain heart infusion broth (ABHI) than bacteria grown at higher temperatures (i.e., 30°C or 37°C). For L. monocytogenes grown at 7°C, stationary-phase cells were more resistant to ABHI than log-phase cells, indicating that both temperature and growth phase affect acid survival. Microarray transcriptomic analysis revealed that the number and functional categories of genes differentially expressed after acid shock differed according to both growth temperature and growth phase. The acid response of L. monocytogenes grown to log phase at 37°C involved stress-related transcriptional regulators (i.e., σ(B), σ(H), CtsR, and HrcA), some of which have been implicated in adaptation to the intracellular environment. In contrast, for bacteria grown at 7°C to stationary phase, acid exposure did not result in differential expression of the stress regulons examined. However, two large operons encoding bacteriophage-like proteins were induced, suggesting lysogenic prophage induction. The adaptive transcriptional response observed in 37°C-grown cells was largely absent in 7°C-grown cells, suggesting that temperatures commonly encountered during food storage and distribution affect the ability of L. monocytogenes to survive gastric passage and ultimately cause disease.

Evaluation of various selective media for the detection of Pseudomonas species in pasteurized milk.

Pseudomonas spp. are common gram-negative, post-pasteurization contaminants that contribute to spoilage of pasteurized dairy products. This study evaluated 5 common selective media for detecting Pseudomonas spp. in pasteurized milk. The performance of each selective medium for recovering 12 different Pseudomonas isolates (selected to represent a diversity of pasteurized milk isolates) was compared with that of standard plate count agar pour plates. Pseudomonas isolates showed varying abilities to produce colonies on different selective media. For 2 of 12 isolates, a 48-h incubation time was required for colony formation on any of the media tested. Violet red bile agar and coliform Petrifilm (3M, St. Paul, MN) were less effective than standard plate count agar pour plates at recovering Pseudomonas, regardless of incubation time, and MacConkey agar showed poor detection efficiency compared with SPCP after a 48-h incubation (R(2) = 0.26). Therefore, the use of violet red bile agar, MacConkey agar, or coliform Petrifilm may not be sufficient for detecting common Pseudomonas spp. in milk. The methods showing the highest detection efficiencies were crystal violet tetrazolium agar (CVTA) pour plates (R(2) = 0.95) and CVTA plates inoculated by spiral plating (R(2) = 0.89) incubated at 32 °C for 48 h. Overall, plating milk samples on CVTA followed by a 48-h incubation at 32 °C was the most effective selective method for recovering a diversity of Pseudomonas spp. from milk.

inlA premature stop codons are common among Listeria monocytogenes isolates from foods and yield virulence-attenuated strains that confer protection against fully virulent strains.

Previous studies showed that a considerable proportion of Listeria monocytogenes isolates obtained from foods carry a premature stop codon (PMSC) mutation in inlA that leads to production of a truncated and secreted InlA. To further elucidate the role these mutations play in virulence of L. monocytogenes, we created isogenic mutants, including (i) natural isolates where an inlA PMSC was reverted to a wild-type inlA allele (without a PMSC) and (ii) natural isolates where a PMSC mutation was introduced into a wild-type inlA allele; isogenic mutant sets were constructed to represent two distinct inlA PMSC mutations. Phenotypical and transcriptional analysis data showed that inlA PMSC mutations do not have a polar effect on the downstream inlB. Isogenic and natural strains carrying an inlA PMSC showed significantly reduced invasion efficiencies in Caco-2 and HepG2 cell lines as well as reduced virulence in oral guinea pig infections. Guinea pigs were also orally infected with a natural strain carrying the most common inlA PMSC mutation (vaccinated group), followed by challenge with a fully virulent L. monocytogenes strain 15 days postvaccination to probe potentially immunizing effects of exposure to L. monocytogenes with inlA PMSC mutations. Vaccinated guinea pigs showed reduced bacterial loads in internal organs and improved weight gain postchallenge, indicating reduced severity of infections in guinea pigs exposed to natural strains with inlA PMSC mutations. Our data support that (i) inlA PMSC mutations are causally associated with attenuated virulence in mammalian hosts and (ii) naturally occurring virulence-attenuated L. monocytogenes strains commonly found in food confer protective immunity.

Listeria monocytogenes F2365 carries several authentic mutations potentially leading to truncated gene products, including inlB, and demonstrates atypical phenotypic characteristics.

Searches of the genome annotation of Listeria monocytogenes F2365, an isolate from the 1985 listeriosis epidemic in California, showed that this strain carries 20 authentic mutations resulting in premature stop codons, including a nonsense mutation in inlB. Here we showed that L. monocytogenes F2365 demonstrates atypical virulence-associated characteristics, including significantly (P < 0.05) reduced invasion efficiency in Caco-2 cells as compared with a closely related lineage I serotype 4b strain as well as significantly (P < 0.05) greater variation in invasiveness when grown under different conditions compared with standard laboratory control and other lineage I serotype 4b strains. In addition, L. monocytogenes F2365 demonstrated distinct growth characteristics, including a significantly (P < 0.05) reduced exponential growth rate when compared with laboratory control and other lineage I serotype 4b outbreak-associated strains as well as a significantly (P < 0.05) longer lag phase duration time compared with another lineage I serotype 4b strain. Our results support that L. monocytogenes F2365 is characterized by genotypic and phenotypic properties that are atypical of other L. monocytogenes strains.