Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Population Structure of Listeria monocytogenes Serotype 4b Isolates from Sporadic Human Listeriosis Cases in the United States from 2003 to 2008

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis.     

A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.     

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Three Transporters Mediate Uptake of Glycine Betaine and Carnitine by Listeria monocytogenes in Response to Hyperosmotic Stress

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.     

Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

The C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. The Listeria monocytogenes Ply118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure of Listeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding using L. monocytogenes serovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundant tagO homologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of either tagO1 (EGDe lmo0959) or tagO2 (EGDe lmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficient Listeria cells. Substitution of tagO from an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from other Listeria phage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.     

Identification of a new molecular target of class IIa bacteriocins in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> EGDe

It was shown recently (Calvez et al. 2007) that glpQ and pde genes are involved in intermediate resistance of Enterococcus faecalis JH2-2 to DvnV41, a class IIa bacteriocin produced by Carnobacterium divergens V41. The listerial orthologs of lpQ and pde genes might be lmo0052 and lmo1292 genes, respectively. Here, the role of lmo0052 and lmo1292 genes in resistance of Listeria monocytogenes EGDe to DvnV41 and MesY105 was investigated. L. monocytogenes EGDe was inactivated in lmo0052 and/or lmo1292 by homologous recombination. Listerial mutant strain EGDSC02 (inactivated in the putative glpQ gene), was slightly resistant to DvnV41. The listerial mutant strain EGDSC01 (inactivated in the putative pde gene) remained, as the wild-type strain, sensitive to DvnV41, but was affected in growth parameters.     

Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis

The cell type-specific expression of the major nuclear lamina polypeptides ("lamins") during development of Xenopus was studied using two monoclonal antibodies (L(0)46F7: specific for LIII, the single lamin of oocytes; PKB8: specific for LI and LII of some somatic cells). In the oocyte, LIII localizes in the nuclear polymer, but upon nuclear envelope breakdown it is solubilized to a form sedimenting at 9 S. In early embryos, LIII contributes to nuclear lamina formation until its depletion. Correspondingly, LI and LII begin to be expressed at a specific point in embryogenesis and appear to be integrated with LIII into a common lamina structure. Later in development, LIII reappears as a prominent nuclear lamina protein but only in certain cells (neurons, muscle cells, and diplotene oocytes). We conclude that amphibian lamins represent a family of proteins expressed in relation to certain programs of cell differentiation.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Bias in the Listeria monocytogenes Enrichment Procedure: Lineage 2 Strains Outcompete Lineage 1 Strains in University of Vermont Selective Enrichments

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.     

Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4 Listeria monocytogenes and Identify Four Distinct Lineages in Serotype 4b

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.     

The number of independent layers at the stem apex in potatoes

Cuttings from colchicine-treated cuttings of the triploid (2n=36) cultivated potato,Solanum × juzepczukii, were investigated by measuring stomata, observing the percentage of good pollen, and counting the chromosome numbers of root tips. Some cuttings of the constitution 3x∶6x∶3x+6x were found, but it was impossible to isolate clones which were 3x∶6x∶3x, the final progenies being all 3x∶6x∶6x. It is suggested that in potatoes the growing points of axillary buds trace back to two layers only, LI and LII, of the main stem and that stratified layers internal to LII in the axillary buds are derived from LII and not from LIII of the main stem. Leaflet shape is apparently determined by the constitution of LII, the constitutions of LI and LIII having little or no effect.     

Multiple forms of α-galactosidase in mature leaves of Cucurbita pepo

α-D-Galactosidase has been purified from mature leaves of Cucurbita pepo using pH and ammonium sulphate fractionation, Sephadex gel filtration and DEAE Sephadex gel chromatography. Gel filtration produced one peak of α-galactosidase activity from which three distinct enzyme forms were resolved on DEAE Sephadex and designated LI, LII and LIII. Purirications obtained were ca 75, 120 and 30 fold for LI, Lll and LIII respectively. Ll was slightly contaminated with β-galactosidase and LII with β-fructosidase activity. All forms hydrolysed the α-galactosyl linkages of raffinose and stachyose. Differences between each form were found in their pH optima, reactivity toward metal ions, thermal stability and K_m values using either p-nitrophenyl-α-D-galactoside (NPG) or raffinose as substrates. All forms were inhibited by NPG at high concentrations and by α-D-galactose. It is proposed that α-galactosidases may be components of a lysosomal system in plant cells.     

Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that causes gastroenteritis, meningitis, encephalitis and maternofetal infections. 20–30% of eubacterial ORFs are predicted to encode membrane proteins. The bacterial cytoplasmic membrane is a macromolecular structure, which plays a key role for the pathogenesis. Despite this, little knowledge exists regarding the function of cytoplasmic membrane proteins of Listeria during infection. Here, we investigated a predicted membrane protein of the pathogen L. monocytogenes, Lmo0412, of unknown function. Lmo0412 is only present in the Listeria genus and low conserved in the non-pathogenic species L. innocua. Bacterial fractionation and western blot analyses showed that Lmo0412 was only detectable in the membrane of L. monocytogenes EGDe during logarithmic growth phase. lmo0412 expression in L. monocytogenes was down-regulated during in vitro infection of JEG-3 epithelial cells. An L. monocytogenes mutant deficient in this membrane protein showed increased invasion of Caco-2 and NRK-49F host cells using in vitro infection models. Moreover, the lack of Lmo0412 in this deletion mutant increased the viable bacteria counts in the spleen and liver of mice compared to the wild type strain. Taken together, these data suggest a selective advantage conferred by the absence of Lmo0412 for the virulence of L. monocytogenes.     

Tolerance of Listeria monocytogenes to Quaternary Ammonium Sanitizers Is Mediated by a Novel Efflux Pump Encoded by emrE

A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrE_Lm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 μg/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4°C, 37°C, and 52°C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 μg/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ΔemrE_Lm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrE_Lm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments.     

Induction and origin of adventitious shoots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The chimeras between tuber mustard (Brassica juncea) and red cabbage (B. oleracea) were artificially synthesized in our previous study. Adventitious shoots were induced from nodal segments and leaf discs of TCC (LI-LII-LIII, LI -the outmost layer of shoot apical meristem; LII -the middle layer; LIII -the innermost layer. T = Tuber mustard, C = Red cabbage) chimeras. The origin of the shoots was analyzed by histology and molecular biology. As a result, the frequency of adventitious shoot induction rose with the increase of BA in MS medium in the area of the nodes. However, there was no different induction frequency of adventitious shoots from nodal segment bases in media with different BA concentrations. Most adventitious shoots (clustered shoots) arising from the node area were TTT (Tuber mustard- Tuber mustard- Tuber mustard) and only 4 shoots were chimeras, which indicated that more shoots originated from LI than from LII and LIII. All shoots from nodal segment bases were CCC (Red cabbage-Red cabbage- Red cabbage), indicating that the shoots originated from LII or LII and LIII. There were significant differences in the regeneration rate in the margin of the leaf discs among the three combinations of BA and NAA. Most adventitious shoots from the margin of leaf discs were CCC but 2 out of 70 were chimeras, which indicated that more shoots originated from LII or LII and LIII than from LI. All chimeras obtained by regeneration were different from the original explant donor in type in the present study. The origin of the adventitious shoots varied with the site of origin on the donor plant, and could be multicellular and multihistogenic.     

Characterization of Listeria monocytogenes Strains Involved in Invasive and Noninvasive Listeriosis Outbreaks by PCR-Based Fingerprinting Techniques

A total of 32 Listeria monocytogenes strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated L. monocytogenes food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.