Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

Abstract

Listeria monocytogenes is a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for two L. monocytogenes strains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate using in silico analyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistent L. monocytogenes strain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene for inlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774, lmo2775, and the 3=-terminal part of lmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype. Listeria monocytogenes is a Gram-positive, food-borne, humanpathogenic bacterium that can cause listeriosis in humans. It affects predominantly immunocompromised individuals, the elderly, young babies, and fetuses in utero (1). Although listeriosis represents only 7.4% of all reported food-borne infections, the fatality rate (17%) and hospitalization rates (92.6%) are high (2).The bacterium is common in food products and poses a special risk in ready-to-eat products that allow proliferation of the pathogen. It is not only a safety issue but also an economic concern, because 61% of food products recalled by the U.S. FDA between 1994 and 1998 were due to L. monocytogenes contamination (3). The bacterium is an intracellular human pathogen, and it also has a saprophytic life-style and can therefore be isolated fr...

Triclosan-Induced Aminoglycoside-Tolerant Listeria monocytogenes Isolates Can Appear as Small-Colony Variants

“…We sequenced the genomic DNA from the wild type and the 15 mutants to determine if this would reveal their mechanism for tolerance to gentamicin and explain the differences in colony morphology and phenotype. Genomic DNA was isolated from L. monocytogenes using the FastDNA kit (MP Biomedicals; 116540-400) as described by Holch et al (16). Samples were RNase treated (Qiagen; 158922) before quantification, and quality was analyzed using 1% agarose gel electrophoresis, a NanoDrop spectrophotometer (Saveen Werner, Sweden), and a Qubit 2.0 analyzer (Invitrogen, United Kingdom).…”

The Connection between Persistent, Disinfectant-Resistant Listeria monocytogenes Strains from Two Geographically Separate Iberian Pork Processing Plants: Evidence from Comparative Genome Analysis

“…The MLST database of the Pasteur Institute in Paris, France (updated on 3 March 2015), includes only 10 ST31 strains and 69 ST121 strains (most are of the 1/2a serotype). The ST121 type has been isolated as a persistent type in food and food-associated environments in different countries (16,42,65,66). ST121 is among the six L. monocytogenes STs most prevalent worldwide (67) and seems to be well adapted to the colonization of meat-associated environments (67,68).…”

“…monocytogenes is capable of colonizing food production plants with certain subtypes that are found only in specific sections of the plants (13,14). Furthermore, some of these subtypes may persist in food processing environments for years (15)(16)(17). Persistent strains have been identified to be major postprocessing contaminants of RTE foods, and in many cases, listeriosis outbreaks have been associated with cases of persistent environmental contamination of processing plants (18)(19)(20)(21).…”

Reservoirs of Listeria Species in Three Environmental Ecosystems

“…The presence and potential persistence of Listeria spp. in food processing facilities are often caused by environmental recontamination at the farm or plant level (14,15,16). In order to unravel the transfer of L. monocytogenes between niches, molecular subtyping is essential both in outbreak clarification and in the management of contamination events in food business operations (17,18).…”