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...

Listeria monocytogenes is an important food-borne bacterial pathogen and listeriosis can result in abortions in pregnant women. The bacterium can colonize food-processing environments, where specific molecular subtypes can persist for years. The purpose of this study was to determine the virulence potential of a group of food-processing persistent L. monocytogenes strains encoding a premature stop codon in inlA (encoding internalin A) by using two orally dosed models, pregnant mice and pregnant guinea pigs. A food-processing persistent strain of L. monocytogenes invaded placentas (n558; 10 % positive) and fetuses (3 % positive) of pregnant mice (n59 animals per strain), similar to a genetically manipulated murinized strain, EGD-e InlA m* (n561; 3 and 2 %, respectively). In pregnant guinea pigs (n59 animals per bacterial strain), a maternofetal strain (from a human fetal clinical fatal case) was isolated from 34 % of placenta samples (n550), whereas both food-processing persistent strains were found in 5 % of placenta samples (n536 or 37). One of the food-processing persistent strains, N53-1, was found in up to 8 % of guinea pig fetal liver and brain samples, whereas the maternofetal control was found in 6 % of fetal tissue samples. As the food-processing persistent strains carry a premature stop codon in inlA but are invasive in orally dosed pregnant mice and guinea pigs, we hypothesize that listerial crossing of the placental barrier can occur by a mechanism that is independent of an interaction between Ecadherin and InlA.

Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/β-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32×MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32×MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16–32×) to the selection agent. Five transfers (∼35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32×MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ∼30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct mechanisms. Interestingly, this type of resistance result in a concomitant high susceptibility towards plasma, and therefore the present study does not infer additional concern for peptidomimetics as future therapeutics.

We determined mammalian cell invasion and virulence gene (inlA, inlB, and actA) sequences of Listeria monocytogenes strains belonging to a molecular subtype (RAPD 9) that often persists in Danish fish-processing plants. These strains invaded human placental trophoblasts less efficiently than other L. monocytogenes strains, including clinical strains, and they carry a premature stop codon in inlA. Eight of 15 strains, including the RAPD 9 and maternofetal strains, had a 105-nucleotide deletion in actA that did not affect cell-to-cell spread in mouse fibroblasts. The RAPD 9 strains may still be regarded as of low virulence with respect to human listeriosis.Listeria monocytogenes is a Gram-positive pathogenic bacterium that can cause foodborne listeriosis, which affects immunocompromised individuals, causing septicemia and meningitis, and pregnant women, causing preterm delivery, miscarriage, or stillbirth. It is a ubiquitous environmental bacterium, and it is therefore continuously introduced to food-processing plants, where some molecular subtypes are able to persist despite thorough cleaning and disinfection procedures (1,27,32,42). Such persistent strains are likely to contaminate the food products and may be the cause of foodborne infections (30).We have shown that specific molecular subtypes of L. monocytogenes can persist for years in the seafood-processing environment (45), and strains representing a particularly prevalent, persistent molecular subtype, RAPD type 9 ([RAPD 9] random amplified polymorphic DNA), had a lower virulence potential than clinical strains in simple eukaryotic models (12). However, in a more complex biological model using oral dosing of pregnant guinea pigs, the tested RAPD 9 strain (strain La111) surprisingly infected the placentas and fetuses just as efficiently as a clinical strain (13). We therefore hypothesized that this specific subtype may have an altered (enhanced) ability to invade placental cells (e.g., trophoblasts) or an enhanced ability to spread intracellularly. (29), the United States (25), and Japan (9), and these mutations lead to attenuation in the invasion of intestinal epithelial cells (25,28,33), but it is not known if invasion into trophoblasts is affected. ActA is important for cell-to-cell spread (5) and is involved in invasion of epithelial cells (39), and ActA-mediated cell-to-cell spread plays a major role in crossing the fetoplacental barrier in both a guinea pig and a mouse model (3,22).The purpose of this study was to determine if the high level of prevalence of a RAPD 9 strain in guinea pig fetuses after oral dosing could be explained by increased invasion into and spread between trophoblastic and fibroblastic cells, respectively. Subsequently, we sequenced selected virulence genes to determine if strain variations in cell invasion and spread could be explained by differences in sequences.Strains, culture conditions, and characterization. Fifteen L. monocytogenes strains representing different origins, RAPD types, serotypes, and lineages were used (Tabl...

Aims: Antibiotics can act as signal molecules and affect bacterial gene expression, physiology and virulence. The purpose of this study was to determine whether subinhibitory antibiotic concentrations alter gene expression and physiology of Listeria monocytogenes. Methods and Results: Using an agar-based screening assay with promoter fusions, 14 of 16 antibiotics induced or repressed expression of one or more stress and/or virulence genes. Despite ampicillin-induced up-regulation of PinlA-lacZ expression, Caco-2 cell invasion was not affected. Subinhibitory concentrations of ampicillin and tetracycline caused up-and down-regulation of stress response genes, respectively, but both antibiotics caused increased sensitivity to acid stress. Six combinations of gene-antibiotic were quantified in broth cultures and five of the six resulted in the same expression pattern as the agar-based assay. Conclusions: Antibiotics affect virulence and/or stress gene expression; however, altered expression could not predict changes in phenotypic behaviour. Subinhibitory concentrations of antibiotics led to increased acid sensitivity, and we speculate that this is attributed to changes in cell envelope or reduced r B -dependent gene expression. Significance and Impact of the Study: Although subinhibitory concentrations of antibiotics affect gene expression in L. monocytogenes, the changes did not increase virulence but did enhance the acid sensitivity.