ABSTRACT
ABSTRACT: Listeria monocytogenes (Lm) is one of the leading causes of death because of foodborne illness, affecting the elderly, pregnant women, neonates, and people who are immunocompromised. Serologically, Lm can be classified into 13 serotypes, although only 4 are typically linked with food contamination and illness. Since 2000, a shift in serotypes involved in listeriosis outbreaks has been observed, suggesting that tracking of serotypes could help identify emerging trends. A PCR method developed in 2004 allowed detection of the four major serotypes as molecular serogroups, corresponding to broad phylogenetic groups. In this study, a novel quantitative PCR (qPCR) method was developed that uses two multiplex qPCRs, one to confirm the Listeria genus and Lm species and the second for Lm molecular serogrouping. This method was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) method for Lm and the seroagglutination method, using a 208-strain panel. Comparison of the genus and species qPCR assay with the BAM methods found an equal or slightly higher accuracy for the qPCR method (>98%), compared with the BAM protocol (>96%), when evaluated against independent characterization data. Molecular serogrouping using the qPCR method (96.6%) was more accurate than the seroagglutination assay (75.6%). The qPCR method identified Lm 4bV strains, which could not be resolved using seroagglutination. The qPCR could not identify lineage III and IV serotype 4b strains but did correctly identify 16 of 18 lineage III and IV strains. The qPCR method performed genus identification for the Listeria species Lm, L. innocua, L. welshimeri, L. ivanovii, and L. seeligeri. In addition, the method performed species identification for Lm and classified Lm into six molecular serogroups: 2A, 2B, 2C, 4B, NT, and 4bV. This method provided a rapid and accurate confirmation of Lm and serogroup determinations; furthermore, it could help identify otherwise unlinked strains by enabling whole genome sequencing analysis based on broad phylogeny, independent of other information.
Subject(s)
Listeria monocytogenes , Listeria , Listeriosis , Aged , Female , Humans , Infant, Newborn , Listeria monocytogenes/genetics , Phylogeny , Pregnancy , Serogroup , SerotypingABSTRACT
A precise and accurate method for enumeration of low level of Listeria monocytogenes in foods is critical to a variety of studies. In this study, paired comparison of most probable number (MPN) and direct plating enumeration of L. monocytogenes was conducted on a total of 1730 outbreak-associated ice cream samples that were naturally contaminated with low level of L. monocytogenes. MPN was performed on all 1730 samples. Direct plating was performed on all samples using the RAPID'L.mono (RLM) agar (1600 samples) and agar Listeria Ottaviani and Agosti (ALOA; 130 samples). Probabilistic analysis with Bayesian inference model was used to compare paired direct plating and MPN estimates of L. monocytogenes in ice cream samples because assumptions implicit in ordinary least squares (OLS) linear regression analyses were not met for such a comparison. The probabilistic analysis revealed good agreement between the MPN and direct plating estimates, and this agreement showed that the MPN schemes and direct plating schemes using ALOA or RLM evaluated in the present study were suitable for enumerating low levels of L. monocytogenes in these ice cream samples. The statistical analysis further revealed that OLS linear regression analyses of direct plating and MPN data did introduce bias that incorrectly characterized systematic differences between estimates from the two methods.
Subject(s)
Colony Count, Microbial/methods , Food Contamination/analysis , Food Microbiology , Ice Cream/microbiology , Listeria monocytogenes/isolation & purification , Agar , Algorithms , Bayes Theorem , Culture Media , Least-Squares Analysis , Limit of Detection , Polymerase Chain Reaction , Probability , Reproducibility of ResultsABSTRACT
A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.
