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Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers.

ABSTRACT: A method for direct detection of Listeria monocytogenes in 25 ml of raw milk is presented. The detection limit can be situated between 10 and 5 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers specific for Listeria monocytogenes.

SUBMITTER: Herman LM

PROVIDER: S-EPMC167343 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers.

Herman L M LM De Block J H JH Moermans R J RJ

Applied and environmental microbiology 19950201 2

A method for direct detection of Listeria monocytogenes in 25 ml of raw milk is presented. The detection limit can be situated between 10 and 5 CFU. The detection method is based on chemical extraction of the milk components and PCR amplification with two nested pairs of primers specific for Listeria monocytogenes. ...[more]

PMID: 7574620

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Project description:The prevalence and levels of Listeria monocytogenes in retail raw foods covering most provincial capitals in China were studied with testing of 1036 samples of vegetables, edible mushrooms, raw meat, aquatic products and quick-frozen products from September 2012 to January 2014. The total prevalence of Listeria monocytogenes was 20.0% (207/1036), and the most probable number (MPN) values of 65.7% of the positive samples ranged from 0.3 to 110 MPN/g. Geographical differences were observed in this survey, and the results of both qualitative and quantitative methods indicated that the levels in the samples from North China were higher than those in the samples from South China. A total of 248 isolates were analyzed, of which approximately half belonged to molecular serogroup 1/2a-3a (45.2%), followed by 1/2b-3b-7 (30.6%), 1/2c-3c (16.1%), 4b-4d-4e (5.2%) and 4a-4c (2.8%). Most of the isolates carried hly (100%), inlB (98.8%), inlA (99.6%), inlC (98.0%) and inlJ (99.2%), and 44.8% of the isolates were llsX-positive. Seventeen epidemic clones (ECs) were detected, with 7 strains belonging to ECI (2.8%) and 10 belonging to ECIII (4.03%). Resistance to clindamycin (46.8%) was commonly observed, and 59 strains (23.8%) were susceptible to all 14 tested antibiotics, whereas 84 (33.9%) showed an intermediate level of resistance or were resistant to two or more antibiotics, including 7 multi-resistant strains that exhibited resistance to more than 10 antibiotics. The data obtained in the present study provides useful information for assessment of the possible risk posed to Chinese consumers, and this information will have a significant public health impact in China. Furthermore, the presence of virulence markers, epidemic clones, as well as the antibiotic resistance amongst the isolates strongly implies that many of these strains might be capable of causing listeriosis, and more accurate treatment of human listeriosis with effective antibiotics should be considered. This research represents a more full-scale and systematical investigation of the prevalence of L. monocytogenes in retail raw foods in China, and it provides baseline information for Chinese regulatory authorities that will aid in the formulation of a regulatory framework for controlling L. monocytogenes with the aim of improving the microbiological safety of raw foods.

Project description:The occurrence and the antibiogram signatures of Listeria monocytogenes (Lm) recovered from 65 milk samples and its products within the Eastern Cape province were examined. The EN ISO 11290:2017 procedures Parts 1 and 2 described by the International Organization for Standardization for the enumeration and isolation of Lm was adopted for the study. Lm was detected in 18.46% of all the samples examined, and the strains recovered from the samples belong to serotypes 4b and 1/2b. The virulence determinants including prfA, plcA, plcB, inlA, inlC, hly, mpl, actA, inlJ and inlB were detected in all the isolates. About 95.24% of the studied Lm isolates demonstrated potential capacity for biofilm formation. The antibiogram profile revealed high resistance against sulfamethoxazole (71.43%), trimethoprim (52.86%); erythromycin, cefotetan and oxytetracycline (42.86% respectively). About 85.71% exhibited multiple antibiotic resistance phenotypes against the test antibiotics. The resistance determinants encoding resistance against the β-lactamase antibiotics [such as the blaTEM, blaSHV, blaTEM variants (TEM-1 and TEM-2) and the blaZ], the tetracycline resistance genes (including tetA, tetD, tetG and tetM and tetK) were detected among resistant isolates. In addition, the aminoglycoside resistance gene aph (3)-IIa (aphA2)a was detected only in one isolate. Finally, the sulfonamide resistance genes including the sul2 and the sul1 genes were the most frequently observed among Lm isolates. Generally, 71.43% of all Lm isolates recovered from the samples investigated harboured one or more resistance genes encoding resistance against various antibiotics. The antibiogram signatures of Lm isolates observed in this study is an indication that empirical treatment of listeriosis may be challenging in the future as the pathogen may obliterate the success of antibiotics. We, therefore, advocate for the recognition of the One Health approach to ensuring food safety and curbing the spread of antimicrobial resistance in food.

Dataset Information

Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers.

Publications

Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers.

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