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Listeria Monocytogenes

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Listeria Monocytogenes Listeria monocytogenes L.monocytogenes morphology Introduction: history • Murray in 1926: – Bacterium monocytogenes – blood monocytes in rabbits – human and animal pathogen • Pirie in 1940: – Listeria monocytogenes – Joseph Lister • Since 1980 – foodborn pathogen Introduction: general • Cause of listeriosis • Found in environment – water, soil, sudge, … Found in animal feaces • Taxonomy: – Kingdom: Bacteria Division: Firmicutes Class: Bacilli Order: Bacillales Family: Listeriaceae Genus: Listeria Species: L. monocytogenes Listeria monocytogenes • L.monocytogenes is the only important human pathogen among the six species: Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri and Listeria grayi. Only two species of the genus are generally considered to be pathogenic, L. monocytogenes in humans and L. ivanovii in other mammals. L.monocytogenes is a Gram- positive , facultatively anaerobic, catalase-positive, oxidase- negative, non-sporeformer. • L.monocytogenes elaborates a 58 kDa β-haemolysin, listeriolysin O, which acts synergistically with the haemolysin produced by Staphylococcus aureus Characteristics Basic features • Gram positive • Facultatively anaerobe • Catalase positive • Oxidase negative • Non-spore former • Coccoid to rod shaped • 0,4 – 0,5 µm x 0,5 – 2,0 µm • Cultured at 20 – 25°C • Petritious flagella and tumbling motility • T-range: grow 0 – 42 °C, Opt: 30 – 35°C • Min pH: 4,4 – 5,6 pH • Salt tolerated Characteristics Biochemical facts • Foodborn: The pathogen can be present inin thethe originaloriginal foodstuffoodstuf oror enterenter thethe foodchain during processing. – Lactating cows can shed L. monocytogenes inin milkmilk asas aa consequenceconsequence ofof mastitismastitis forfor aa rather long time. – Soft cheese such as Camembert can be contaminated on the surface duringduring ripening;ripening; Food items which may contain L. monocytogenes Food items which in general are free of Listeriae Sausages (salami, pate¤) All kinds of food immediately after heating Raw meat, in particular turkey and chicken Pasteurized milk, yoghurt (industrial products!) Sandwiches Hard cheese Lettuce, raw mushrooms Chocolate, marmalade, cookies Raw milk and products made from this material Raw carrots Soft cheese (Munster, Roquefort, Camembert, Brie) Raw apples Fresh cheese (ricotta, feta) Raw tomatoes Seafood (mussels, salmon) All kind of meals which are conserved after heating Characteristics Pathogenesis and clinical features The manifestations of listeriosis include: – Septicemia (blood poisoning) – Meningitis – Encephalitis (an acute inflammation ot the brain) – Intrauterine or cervical infections in pregnant women spontaneous abortion (2nd/3rd trimester) or stillbirth. Symptoms: • Influenza-like symptoms: persistent fever. • Gastrointestinal symptoms: nausea, vomiting, and diarrhea Characteristics Pathogenesis and clinical features Incubation Period: • Is variable and ranges from 3 to 70 days, with the median incubation period being three weeks. • In infections during pregnancy, the mother usually survives. Treatment: – Parenteral penicillin or ampicillin with aminogycosides. – Trimethoprim-sulfamethoxazole for patients allergic to penicillin. Prevention: • Similar to prevent from other foodborne illnesses, such as salmonellosis. L.monocytogenes • Organism grows over a wide range of temperature from 0 – 42oC with an optimum between 30 and 35oC . • Organism is ubiquitous in the environment. Isolation from fresh and salt water, soil, sewage sludge, decaying vegetation, silage. • Oportunistic pathogen – incubation periods for disease from 1 day to 90 days. Symptoms vary from a mild, flu-like illness to mengitis and meningoencephalitis. • Attack : pregnant women, children (0 – 2 years) or elderly (more 65 years) and the immunocompromised population. Introduction: properties • Gram positive, facultative anaerobe • Non-sporeformer • Rod shaped with flagella – tumbling mobility • Growth temperature: 0 – 42°C – optimum: 30 – 35°C • Min pH for growth: 4.4 – 5.6 • D60: few minutes D70: few seconds • Other properties: – catalase +, oxidase –, hemolysis Foods containing L. monocytogenes • Raw vegetables – cabbage, tomatoes, lettuce, … • Meat – delicatesses: salami, ham, paté, … – pork sausages, chicken nuggets, … • Dairy products – pasteurised milk – soft cheeses: brie, camenbert, … – even butter Listeriosis • Flu-like illness, meningitis, meningoencephalitis, sepsis, (endocarditis) • Patients – pregnant women – newborn and old people – immunocompromised • Incubation: 1 – 90 days • Infective dose: not known Listeriosis • Symptoms: – fever, headache, sometimes stomacache – pregnant women: abortion, stillbirth, premature labour – newborn: pneumonia, sepsis, abscesses, meningitis, infection of nervous system • Treatment with antibiotics – ampicillin, vancomycin, … • Mortality 20-25% – meningitis 70% – sepsis 50% – neonatal infections 80% !!! Early diagnosis is rather rare !!! Listeriosis: lifecycle Methodology Traditional Identification • Microscopic examination of plates illuminated from below at 45° blue-grey to blue-green Conformation • Sugar-fermentation test ( distinguish from other Listeria species) Methodology Modern Isolation: Two of the most widely-used culture reference methods for detection of Listeria in all foods are – FDA bacteriological and analytical method (BAM) – the International Organization of Standards (ISO) 11290 method Identification: – CAMP test ( figure) – Chromogenic substrates – Antibody-based tests • Enzyme-linked immunosorbent assay (ELISA ) • Immuno-capture – Molecular tests • DNA hybridization • Polymerase chain reaction (PCR) Methodology Modern Future directions for the detection and identification of L. monocytogenes • Tests targeting RNA – Reverse transcription (RT)-PCR – Real-time PCR – Nucleic acid sequence-based amplification (NASBA) • DNA microarrays – PCR-based microarrays – Oligonucleotide-based microarrays Detection: standard • Isolation / enrichment (48h) – buffered Listeria enrichment broth base (BLEB) – slow: enrichment at 4°C – faster: antibiotics at optimal growth temperature • Incubation 24-48h at 30°C – selective agents: lithium chloride, polyethanol, antibiotics, … – elective agents: aesculin, ferric ammonium citrate – agar: PALCAM, MOX, … Detection: standard • Identification – blue-green colonies (Henry illumination) – black halo (aesculetin + iron) – other Listeria: yellow (mannitol fermentation) • Confirmation – catalase, oxidase, nitrate reduction, CAMP tests, … – fast methods !!! Takes 5-7 days !!! Detection: new methods • DNA probes – hybridisation • Recombinant DNA – plasmid • New, faster media – Rapid'L.Mono Medium • Commercial kits – AccuProbeTM, GeneTrak Conclusion L. monocytogenes is a wide spread organism, able to grow in many foods Listeriosis dangerous difficult to recognise pregnant women, newborn and eldery people, immunocompromised Standard detection methods take a long time Listeria monocytogenes Scanning EM showing Flagella L.monocytogenes • Association with food : documented were: coleslaw salad, raw vegetable (celery, tomatoes, lettuce), dairy products as raw milk, soft cheeses, smoked salmon, pork tongue in aspic. • Ability to multiply at refrigerator temperatures • Morrtality: 20 – 40% Detection Listeria monocytogenes according to ISO 11290-1:1996 Characteristics of L. monocytogenes Gram-positive facultative anaerobic bacterium resistant to environmental conditions 0,5 – 45 °C pH from 4,3 to 9,6 high concentration of NaCl (10 – 15 %) Found in the environments (studied, not known exactly) from there it can infect food Characteristics of L. monocytogenes Intracellular parasite - causative agent of alimentary disease listeriosis High mortality (~ 30 %) in the threaten groups elder people, babies, persons with weakened or insufficiently developped immune system abortions at pregnant women http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=8082 Food safety criteria COMMISSION REGULATION (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs (Text with EEA relevance) ANNEX I Microbiological criteria for foodstuffs Chapter 1. Food safety criteria Chapter 2. Process hygiene criteria Chapter 3. Rules for sampling and preparation of test samples COMMISSION REGULATION (EC) No 1441/2007 of 5 December 2007 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs (Text with EEA relevance) COMMISSION REGULATION (EU) No 365/2010 of 28 April 2010 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs as regards Enterobacteriaceae in pasteurised milk and other pasteurised liquid dairy products and Listeria monocytogenes in food grade salt (Text with EEA relevance) Food safety criteria • Listeria monocytogenes • All ready-to-eat foods - Target: Reduction of human listeriosis • Although the disease is relatively rare, listeriosis is a significant public health concern • Clinical severity and high mortality (20-30%) http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=8082 Food safety criteria 2073/2005 Food category Microorgani Sampling Limits 2 Analytical Stage where the criterion sm/their plan 1 reference applies toxins, method 3 n c m M metabolites 1.1 Ready-to-eat foods L. 10 0 Absence EN/ISO Products intended for infants monocytoge in 25 g 11290-1 placed on the market during and ready-to-eat foods nes their shelf-life for special medical purposes4 (1) n = number of units comprising the sample; c = number of sample units
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