Comparison of subtypes of Listeria monocytogenes isolates from naturally contaminated watershed samples using a combination of non-selective and selective enrichment methods

Authors: Gorski, Lisa; Walker, Samarpita; Liang, Anita; Nguyen, Kimberly; Govoni, Jessica; Carychao, Diana; Cooley, Michael; MANDRELL, ROBERT - Former ARS Employee

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2014
Publication Date: 3/20/2014
Citation: Gorski, L.A., Walker, S., Liang, A.S., Nguyen, K.M., Govoni, J.A., Carychao, D.K., Cooley, M.B., Mandrell, R.E. 2014. Comparison of subtypes of Listeria monocytogenes isolates from naturally contaminated watershed samples using a combination of non-selective and selective enrichment methods. PLoS One. 9(3):e92467. DOI: 10.1371/journal.pone.009246.

Interpretive Summary: Different methods to isolate bacteria from natural sources can show a bias in the types of strains that arise in the analysis. In order to test whether two different methods to enrich Listeria monocytogenes from watershed samples were revealing the same types of strains 304 isolates obtained by both methods were subtyped. Serotype is commonly used to subtype L. monocytogenes, but it is not as discriminating as molecular subtyping by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Among the 62 samples tested by both enrichment methods 304 isolates were isolated, 157 by one method and 147 by the other. The same three serotypes were detected by both methods. Serotype 1/2a is more commonly found in food and food processing and water isolates, but the vast majority of the 304 isolates in this study were serotype 4b, which is implicated more often in listeriosis outbreaks. In all 62 different MLVA types, and the most common types were found by both enrichment methods. Forty-three MLVA types were found only in isolates from one method or the other; however, 30 of these MLVA types were only isolated once. The virulence gene inlA, which encodes the Internalin A protein, was sequenced in 77 isolates. This gene is found often to be mutated in L. monocytogenes isolates from food and food processing plants. Among the inlA genes sequenced in our study 88% of them were intact. Using both enrichment methods allowed for detection of a greater diversity of isolates among the samples than the use of one method alone. This limited study provides evidence for a wide diversity of L. monocytogenes strains this watershed.

Technical Abstract: Two enrichment methods for Listeria monocytogenes using Immuno Magnetic Separation were tested to determine if they selected the same subtypes of isolates. Both methods included a non-selective enrichment and one included subculture in Fraser Broth. Naturally contaminated watershed samples from the Central California Coast were used subtype diversity was measured by serotype and Multiple Number Variable Tandem Repeat Analysis. A portion of the 304 isolates were screened to determine if they carried premature stop codons (PMSC’s) in the virulence gene inlA. Three different serotypes were detected from both methods with serotype 4b strains present in 97% of the samples, serotype 1/2a in 18%, and serotype 1/2b in 13%. Sixty-two different MLVA types were detected with the most common MLVA types found in both enrichment methods. The most common MLVA type-12 was detected in over half of the watershed samples. Forty-two types were detected from one method or the other, and thirty different MLVA types were detected only once. Twenty-four watershed samples contained MLVA types that were discovered only through the use of both methods. The inlA genes from 77 isolates were sequenced and 88% of them were intact. Four isolates contained inlA genes with PMSC’s and five isolates had inlA alleles with a 9 nucleotide deletion. Using both enrichment methods allowed for detection of a greater diversity of isolates among the samples than the use of one method alone. This limited study provides evidence for a wide diversity of L. monocytogenes strains this watershed.