Listeria

Prepared by: Amira Mohammad Ali Fahim Faysal

ContentsIntroductionCharacteristics of listeriaListeria monocytogenesListeria diseaseLife cycle of listeriaIsolation of listeria

Introduction

Listeria is a genus of bacteria in the firmicutes division.Listeria consists of 10 species and each of the species consists of 2 sub species.

IntroductionSpecies of ListeriaL. fleischmanniiL. grayiL. innocuaL. ivanoviiL. marthiiL. monocytogenesL. rocourtiaeL. seeligeriL. weihenstephanensisL. welshimeri

Introduction

•Listeria are Gram positive cell•They are rod shaped, 0.4-0.5 x 0.5-2.0μm•The are facultative anaerobic. This means they can survive with or without oxygen•They do not form spore.

IntroductionThe natural habitat of listeria are: domestic and wild animals birds insects Soil and waste water vegetation

Characteristics of ListeriaGrowth temperatureAir requirementAcidityWater activityFormation of biofilmsMotility

Characteristics of Listeria

Growth temperatureListeria are psychrotrophsThey survive in teperature below freezing to body temperatureTheir optimum growth temperature is 30°C-37°C

Characteristics of Listeria

Air RequirementListeria is a facultative organismCan grow under aerobic (oxygenated conditions)Can grow under reduced oxygenated conditions (anaerobic)When grown in a laboratory, aerobic condition is provided.

Characteristics of Listeria

AcidityListeria prefer to grow at pH 7-8Listeria can tolerate the range pH 5-10Recent studies have shown that Listeria may survive and grow in material with a pH as low as 4.4 When grown in laboratory the pH requirement is 5.6.

Characteristics of Listeria

Water activity (aw)Listeria grows well at low water activityTheir optimum aw is >0.95; can multiply at aw of 0.90Some listeria can survive at aw of <0.90Listeria was isolated from salami with aw of 0.79-0.86

Characteristics of Listeria

Formation of biofilmsBiofilms are assemblage of bacterial cells attached to a surface

Characteristics of ListeriaFormation of biofilmsListeria has the ability to colonize the surfaces, forming biofilmThey can remain attached to the equipments during food productionThis results in cross contamination of listeria and the food.

Characteristics of Listeria

MotilityListeria are motile via flagellaThe motility of flagella is active at 30°c and below but not at 37°c.

Listeria monocytogenesListeria monocytogenes is a gram positive, motile, rod shaped bacteriaThey have petrichous flagella. They are motile 22-28 but not at 37They are non-spore formingThe are facultative intracellular organismThey grow best at -18°c to 10°c

Listeria monocytogenesHas been recognized as a foodborne pathogen since the 1980It is the only Listeria species pathogenic to humansIntracellular pathogen, causes the food-borne illness, listeriosis, in susceptible persons (pregnant women, immunocompromised people and olderly aged people)

Listeria monocytogenesL.monocytogenes are:catalase-positive Oxidase-negativeand expresses a beta hemolysin, which causes destruction of red blood cells

Listeria monocytogenesThere are 12 serotypes of L. monocytogenes:

1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e and 7

Listeria diseaseThe infection caused by listeria species is known as listeriosis.Among other species of genus listeria, only L.monocytogenes and L.ivanovii are known to be pathogenic. L.monocytogenes is associated with human and animal illnessListeriosis infection is caused by eating contaminated food by L.monocytogenes

Listeria diseasePeople who are at risk of listeriosis

Listeria diseasePeople who are at risk of listeriaImmunocompromised individuals (HIV/AIDS infection)Pregnant womanNew born babiesPersons with low stomach acidityCancer patientsAlcoholics and drug abusersPerson with corticosteroid therapyElderly people

Listeria diseaseManifestation of listeriosisSepticemiaMeningitisEncephalitisIntrauterine and cervical infections in pregnant womenInfection in pregnant women can also lead to abortion, still birth or premature delivery.

Listeria diseaseSymptoms of listeriosisPersistent feverNauseaDiarrheaconstipitation

Listeria diseaseDiagnosis of ListeriosisListeriosis is clinically defined when the organism is isolated from blood, cerebrospinal and even placenta and foetus in case of abortion.

Listeria diseaseTreatmentAntibiotics used to treat listeriosis are: tetracyclin, erythromycin, streptomycin, cephalosporin and fosfomycinThe first choice of treatment is using ampicillin, penicillin G combined with an aminoglycoside and gentamycinThe second choice of treatment is using the association of timethoprim with sulphonamide

Listeria DiseaseAntibiotic resistance of L.monocytogenesL.monocytogenes strains are resitant to drugs like: tetracyclin, gentamicin, penicillin, ampicillin, streptomycin, erythromycin, kanamycin, sulfonamide, trimethoprim and rifampicinTetracyclin-resistant gene in L.monocytogenes codes for a protein which is responsible for active effulx of tetracyclin from the bacteria

Listeria diseasePrevention of listeriosisCook raw meat and chicken properlyHeat chilled meal properlyHeat the unpasteurized milkAvoid eating ripened soft cheese Avoid cross contaminating vegetables and meatWash hands properly

Listeria diseaseOutbreak of listeriosisIn 1971 the first outbreak of foodborne listeriosis occurred in a Boston HospitalReason: Contaminated vegetables prepared in the hospitalNumber of people affected- 23

Listeria diseaseOutbreak of listeriosisIn 1981, another case of listeriosis occurred in the maritime provinces, CanadaReason: L.monocytogenes was isolated from the colselowPeople affected: 34 pregnant women and 7 adults

Listeria diseaseOutbreak of listeriosisRecently (1998-1999) a large multistate listeriosis outbrea was recordedReason- consumption of hot dogsPeople affected- 101 individuals with 21% mortality

Listeria diseaseListeria infection is relatively uncommon.However the fatality rate can be 30% among the risk peopleIt is estimated that Listeria causes approximately 1600 cases of listeriosis annually, resulting in 400 to 500 death in US.

Life cycle of listeria

Life cycle of listeriaListeria go through an intracellular life cycle involving early escape from the phagocytice vacuoleRapid intracytoplasmic multiplication occursBacterially induced motility is triggeredThe motile cell can spread to the neighbouring cell and continue the life cycle

Isolation of listeria

Isolation of listeriaCulturing listeriaThe sample should be enriched in enrichment broth, Buffered listeria enrichment broth for 30-48°cA loop of inoculum should be subcultured in a listeria selective agar, Oxford listeria agar for 24 hours at 37°cDark brown colonies with black zones are should be subcultured on nutrient agar

Isolation of listeriaBiochemical testsGram staining- pink rod shaped cells

Isolation of listeriaBiochemical testsCulturing in blood agar- this will show a beta haemolysis

Isolation of listeriaBiochemical testsCulturing in blood agarlHaemolysis means break down of red blood cells and releasing their contents into the surrounding fluid.lThe haemolysis occurs in three ways:lBeta heamolysis- complete break down of RBClAlpha heamolysis- Partial break down of RBClGamma hemolysis- the lack of hemolysis

Isolation of listeriaBiochemical testsMotility testing at 20-25°c and at 37°c•Listeria Motility Medium is a semisolid medium recommended for testing motility of Listeria monocytogenes.•Listeria Motility media consists of- casein enzymic hydrolysate, peptic digest of animal tissue, agar and a pH of 7.3±0.2•There will be motility at 20-25°c, but not at 37°c

Isolation of listeriaBiochemical testsCatalase test•This test demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2).•Catalase test can be done in two ways:•1.tube method•2.slide method

Isolation of listeriaBiochemical testsCatalase test•1. Tube method•Pour 1-2 ml of hydrogen peroxide solution into a test tube.•Using a sterile wooden stick or a glass rod, take listeria colonies of the 18 to 24 hours test organism and immerse in the hydrogen peroxide solution.•Observe for immediate bubbling.

Isolation of listeriaBiochemical testsCatalase test•2.Slide method•Use a sterile loop to transfer a small amount of colony growth in the surface of a clean, dry glass slide.•Place a drop of 3% H2O2 in the glass slide.•Observe for the evolution of oxygen bubbles.

Isolation of listeriaBiochemical testsCatalase testBubbles will be observed.

Isolation of listeriaBiochemical testsSeroagglutination test using commercially listeria spp. Antisera- agglitunation patterns will be similar to listeria spp.

•Seroagglutination test is done by two ways:•Determination of the O-antigen•Determination of the H-antigen

Isolation of listeriaBiochemical testsSeroagglutination test using commercially listeria spp. Antisera•Determination of the O-antigen•Determination of the O-antigen is carried out with heat-inactivated bacteria using the slide agglutinationmethod. A dense bacterial antigen suspension should be prepared by suspending cells cultured on a BHI (Brain Heart Infusion) agar plate with 0.2%w/v sodium chloride to adjust the cell concentration to about 10 mg/ml, heating the suspension at 121°C for 30 minutes followed by centrifugation at 3,000 rpm for 20 minutes, and resuspending the precipitate with a small amount of 0.2%w/v sodium chloride.

Isolation of listeriaBiochemical testsSeroagglutination test using commercially listeria spp. Antisera•Determination of the O-antigen1. Place a drop of each I/II antiserum, V/VI antiserum and physiological saline (30 μl) as a control onto aclean glass slide partitioned into several parts with a glass pencil.2. Place an antigenic suspension for group O (5-10 μl) onto the serum and physiological saline on theglass slide.3. Mix the reagents by tilting the glass slide back and forth for 1 minute and the agglutination pattern isobserved. Agglutination is grossly observed with light through the slide using fluorescent light. It shouldbe first confirmed that no agglutination is found on the reaction with antigenic suspension andphysiological saline. Only strong agglutination observed within 1 minute in the reaction with each serumshould be regarded as positive. Delayed or weak agglutination is regarded as negative.4. If a specimen tests positive with I/II antiserum, perform steps 1-3 above using I and IV antiserum. If

positive with V/VI antiserum, using VI, VII , VIII and IX antiserum.

Isolation of listeriaBiochemical testsSeroagglutination test using commercially listeria spp. AntiseraB. Determination of the H-antigenDetermination of the H-antigen is carried out using the test tube method with the bacteria cultured in liquid media, In order to obtain clear test results, as L.Monocytogenes possesses only 1 – 4 flagella, it is recommended that the motility of the testing organisms should be raised by passing them through a semi-liquid agar medium.

Isolation of listeriaBiochemical testsSeroagglutination test using commercially listeria spp. AntiseraB. Determination of the H-antigen1.Organisms passed through the semi-liquid BHI media (0.2% agar) with a Craigie's tube 3 - 4times may be used for inoculation of the preparatory culture in the liquid BHI medium. Then, a cellsuspension should be prepared by culturing in the BHI medium at 30oC overnightand adding anequal amount of physiological saline containing 1%w/v formalin.2.Put two drops of each H-antisera into separate test tubes using the syringe attached to thecontainers and then add 0.5 ml of the cell suspension to each. Use one tube that does not containantisera as a control.3. After mixing thoroughly, keep the tubes in a water bath (50oC - 52oC) for one hour and observewith the naked eye as to whether agglutination occurs or not. Take care not to agitate the tubesduring observation since the agglutination tends to break up easily. The name of the antiserum thatproduced positive agglutination should be taken as the name of the H antigen possessed by thetested L. Monocytogenes.

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