The Majority of Genotypes of the Virulence Gene inlA Are Intact among Natural Watershed Isolates of Listeria monocytogenes from the Central California Coast.

Internalin A is an essential virulence gene involved in the uptake of the foodborne pathogen Listeria monocytogenes into host cells. It is intact in clinical strains and often truncated due to Premature Stop Codons (PMSCs) in isolates from processed foods and processing facilities. Less information is known about environmental isolates. We sequenced the inlA alleles and did Multi Locus Variable Number Tandem Repeat Analysis (MLVA) on 112 L. monocytogenes isolates from a 3-year period from naturally contaminated watersheds near a leafy green growing area in Central California. The collection contained 14 serotype 1/2a, 12 serotype 1/2b, and 86 serotype 4b strains. Twenty-seven different inlA alleles were found. Twenty-three of the alleles are predicted to encode intact copies of InlA, while three contain PMSCs. Another allele has a 9-nucleotide deletion, previously described for a clinical strain, indicating that it is still functional. Intact inlA genes were found in 101 isolates, and 8 isolates contained the allele predicted to contain the 3-amino acid deletion. Both allele types were found throughout the 3-year sampling period. Three strains contained inlA alleles with PMSCs, and these were found only during the first 3 months of the study. SNP analysis of the intact alleles indicated clustering of alleles based on serotype and lineage with serotypes 1/2b and 4b (lineage I strains) clustering together, and serotype 1/2a (lineage II strains) clustering separately. The combination of serotype, MLVA types, and inlA allele types indicate that the 112 isolates reflect at least 49 different strains of L. monocytogenes. The finding that 90% of environmental L. monocytogenes isolates contain intact inlA alleles varies significantly from isolates found in processing plants. This information is important to public health labs and growers as to the varieties of L. monocytogenes that could potentially contaminate fresh produce in the field by various means

Sample and isolate information for aberrant InlA types.

<p>Sample and isolate information for aberrant InlA types.</p

Dendrogram of intact alleles of <i>inlA</i>, and associated information.

<p>Alleles labeled with “1” and letters indicate sequences that were found in more than one isolate. Alleles labeled with “RM” and a number are unique alleles. Also listed are the Lineage information and MLVA types in which the alleles are found, watershed information, sample dates, and the number of isolates of each allele. Watershed information: A, Alisal; C, Carr Lake, G, Gabilan; S, Salinas River; T, Tembladero; X1 –X3, sites not associated with watersheds.</p

The Majority of Genotypes of the Virulence Gene <i>inlA</i> Are Intact among Natural Watershed Isolates of <i>Listeria monocytogenes</i> from the Central California Coast

<div><p>Internalin A is an essential virulence gene involved in the uptake of the foodborne pathogen <i>Listeria monocytogenes</i> into host cells. It is intact in clinical strains and often truncated due to Premature Stop Codons (PMSCs) in isolates from processed foods and processing facilities. Less information is known about environmental isolates. We sequenced the <i>inlA</i> alleles and did Multi Locus Variable Number Tandem Repeat Analysis (MLVA) on 112 <i>L</i>. <i>monocytogenes</i> isolates from a 3-year period from naturally contaminated watersheds near a leafy green growing area in Central California. The collection contained 14 serotype 1/2a, 12 serotype 1/2b, and 86 serotype 4b strains. Twenty-seven different <i>inlA</i> alleles were found. Twenty-three of the alleles are predicted to encode intact copies of InlA, while three contain PMSCs. Another allele has a 9-nucleotide deletion, previously described for a clinical strain, indicating that it is still functional. Intact <i>inlA</i> genes were found in 101 isolates, and 8 isolates contained the allele predicted to contain the 3-amino acid deletion. Both allele types were found throughout the 3-year sampling period. Three strains contained <i>inlA</i> alleles with PMSCs, and these were found only during the first 3 months of the study. SNP analysis of the intact alleles indicated clustering of alleles based on serotype and lineage with serotypes 1/2b and 4b (lineage I strains) clustering together, and serotype 1/2a (lineage II strains) clustering separately. The combination of serotype, MLVA types, and <i>inlA</i> allele types indicate that the 112 isolates reflect at least 49 different strains of <i>L</i>. <i>monocytogenes</i>. The finding that 90% of environmental <i>L</i>. <i>monocytogenes</i> isolates contain intact <i>inlA</i> alleles varies significantly from isolates found in processing plants. This information is important to public health labs and growers as to the varieties of <i>L</i>. <i>monocytogenes</i> that could potentially contaminate fresh produce in the field by various means.</p></div

Alignment of Intact Alleles Showing SNP changes.

<p>The entire 2403 bp length of <i>inlA</i> is shown with the 5 regions as described in the caption to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167566#pone.0167566.g002" target="_blank">Fig 2</a>. Bases 1–87 (gray) is the signal sequence, bases 88–1071 (red) is the leucine rich repeat region, bases 1072–1386 (yellow) is the intergenic region, bases 1387–1950 (green) is the B repeat region, and bases 1388–2403 (purple) is the membrane anchor region. The boxed alleles were found only in Lineage II strains, while the unboxed alleles were found only in Lineage I strains.</p

Sampling Area.

<p>The watersheds are indicated by overlaid colors on the green map background. Sampling sites not associated with watersheds along the waterways are indicated with X1 and X2 on the map. Sampling locations were grouped into watersheds to allow statistical analysis as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167566#pone.0167566.ref026" target="_blank">26</a>]. Watershed areas are shaded labeled. Salinas River watershed is indicated with the blue line along the Salinas River; Tembladero is the yellow box; Gabilan is the pink box; and Alisal is the orange box. The Carr Lake watershed is between the Tembladero and Alisal watersheds within the city of Salinas. The base layer of the map is from the DEMIS Mapserver. (<a href="http://www.demis.nl/home/pages/wms/demiswms.htm" target="_blank">http://www.demis.nl/home/pages/wms/demiswms.htm</a>), which are public domain. DEMIS World Map Server generates maps using public domain data with no usage restrictions.</p

Lengths of predicted InlA proteins with functional regions labeled.

<p>The N and C termini are labeled. The final number of the right of each construct is the predicted lengths of the proteins. The InlA protein is divided into 5 regions with the first 29 amino acids (gray region) encoding a Signal Sequence. Amino acids 30–357 (red) encode a leucine rich repeat needed for attachment to the E-cadherin receptor. Amino acids 358–462 (yellow) encode an intergenic repeat. Amino acids 463–650 (green) encode the B repeats section, and amino acids 651–800 encode the Membrane Anchor region. An “L” on the Membrane Spanning region of the top two constructs indicate where the LPXTG motif is located. The region that is deleted in the 797-amino acid protein in relation to the full-length protein is indicated by an asterisk (*).</p

Comparison of Subtypes of <i>Listeria monocytogenes</i> Isolates from Naturally Contaminated Watershed Samples with and without a Selective Secondary Enrichment

<div><p>Two enrichment methods for <i>Listeria monocytogenes</i> using Immuno Magnetic Separation (IMS) were tested to determine if they selected the same subtypes of isolates. Both methods used a non-selective primary enrichment and one included subculture in Fraser Broth, while the other involved direct plating of IMS beads. Sixty-two naturally contaminated watershed samples from the Central California Coast were used as a source of <i>L. monocytogenes</i>, and subtype diversity was measured by serotype and Multiple Number Variable Tandem Repeat Analysis (MLVA). Three different serotypes were detected from both methods with serotype 4b strains making up 87% of the isolates, serotype 1/2a making up 8%, and serotype 1/2b making up 5%. The data suggest that serotype 1/2a strains were more likely to be isolated from the Fraser Broth culture method. Sixty-two different MLVA types were detected and the more common MLVA types were detected by both culture methods. Forty-three MLVA types were detected only from one culture method or the other, while 19 types were detected from both culture methods. The most common MLVA type-12 was detected in 33 of the 62 water samples, and represented 31% of the isolates from both culture methods. This limited study provides evidence that using both enrichment culture methods allowed for detection of a greater diversity of isolates among the samples than the use of one method alone, and that a wide diversity of <i>L. monocytogenes</i> strains exist in this watershed.</p></div

MLVA types detected from more than one water sample and enrichment type from which they were isolated.

<p>MLVA types detected from more than one water sample and enrichment type from which they were isolated.</p