Examination of food chain-derived Listeria monocytogenes strains of different serotypes reveals considerable diversity in inlA genotypes, mutability, and adaptation to cold temperatures

Abstract

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.

Fig 1

Mutability of different L. monocytogenes inlA genotypes (A) and serotypes (B), assessed by the number of rifampin-resistant colonies after 48 h of growth at 35°C in the presence of 100 μg/ml rifampin. Mutability of each isolate was assayed in triplicate in each experiment, and two independent experiments were performed. Bars show mean numbers of colonies, and error bars indicate standard errors of the mean. Different letters above the bars represent significant differences (P < 0.05) between geno- and serotype groups, determined using the Mann-Whitney test (A) or the Kruskal-Wallis test followed by Dunn's multiple-comparisons test (B). Serotype 1/2b was excluded from statistical analysis, as only one isolate was recovered.

Fig 2

Invasion efficiencies (% of bacteria recovered relative to the initial inoculum) of L. monocytogenes isolates possessing inlA PMSC mutations (types 1, 3, 4, and 11) or a 3-codon deletion at amino acid positions 738 to 740 (Δ738-740) compared to those of wild-type clinical isolates (08-5578 and EGD-SmR) and a Tn1545-induced noninvasive inlA mutant of EGD-SmR (BUG5). Assays for each isolate were carried out in triplicate and repeated two times. Bars show mean invasion efficiencies, and error bars indicate standard errors of the mean. Different symbols above the bars indicate significantly higher invasion efficiencies (P < 0.05; t test) compared to those of the controls 08-5578 (●), EGD-SmR (◆), and BUG5 (■).

Fig 3

Cold growth adaptation of L. monocytogenes isolates recovered from food-processing environments (FPE), raw unprocessed foods (RUF), and ready-to-eat (RTE) foods when grown at 4°C. Differences were not statistically significant (P > 0.05; chi-square test).

Fig 4

Lag phase durations (A) and exponential growth rates (B) of 33 L. monocytogenes isolates recovered from food and food-processing environments following a downshift from 37°C to 4°C in BHI. Each isolate was assayed in duplicate, and two independent growth assays were performed. Scatter plots show mean values with standard deviations.

Fig 5

Distribution of inlA genotypes (i.e., PMSCs versus no PMSCs) (A) and identification of cold-adaptive groups (i.e., fast, intermediate, or slow) (B) observed at 4°C following a downshift from 37°C in BHI. Differences in cold growth adaptation between fast and intermediate L. monocytogenes inlA genotypes were significant (Fisher's exact test; P = 0.04). Percentages of isolates within respective inlA genotypes (A) and cold-adaptive groups (B) are indicated above bars.