LISTERIA2011

Listeria monocytogenes
• Bacterias pequeñas, Gram +, no
esporo-formadores
• Móviles cuando crecen a 20-25°C,
y no-móviles a 37°C
• Anaerobio facultativo
• Catalasa positivo
• Rumiantes mantienen Listeria spp
en el ambiente rural vía un
continuo enriquecimiento fecal-oral
• Patógeno intracelular
Listeria monocytogenes
Identificación fenotípica de las especies de Listeria
+
xilosa
+
-
L. monocytogenes
L. seeligeri
+
ribosa
+
-
L. ivanovii Subsp. ivanovii
L. ivanovii Subs. londinensis
-
xilosa
+
prueba de CAMP
Rhodococcus equi
Hemólisis
-
-
manitol
+
L. innocua
L. welshimeri
L. grayi
Sólo tres especies son hemolíticas, L. monocytogenes, L. seeligeri y L. ivanovii. Las dos primeras
producen una estrecha zona de hemólisis, a veces limitada al diámetro de la colonia; L. ivanovii muestra
una hemólisis amplia. La prueba de CAMP es positiva para L. monocytogenes y L. seeligeri en la
proximidad de una estría de Staphylococcus aureus, mientras que L. ivanovii sólo da positiva la prueba de
CAMP en presencia de una estría de Rhodococcus equi.
CAMP-positive Listeria
monocytogenes inoculated
at right angles to bhemolytic Staphylococcus
aureus. Note the arrowshaped zone of weak
enhanced hemolysis
indicating a positive CAMP
test. Gloves are worn when
working with L.
monocytogenes. (Anne
Hanson, University of
Maine)
Manifestaciones de L. monocytogenes
•
•
•
•
Síntomas gripales
Diarrea meningitis
Encefalitis
Meningoencefalitis
Mortalidad del 30-40 %
The term listeriosis encompasses a wide variety of disease symptoms that are similar in
animals and humans. Listeria monocytogenes causes listeriosis in animals and humans; L.
ivanovii causes the disease in animals only, mainly sheep. Encephalitis is the most common
form of the disease in ruminant animals. In young animals, visceral or septicemic infections
often occur. Intra-uterine infection of the fetus via the placenta frequently results in abortion in
sheep and cattle.
Brotes dados por L. monocytogenes
Location (year)
No. of cases
(no. of deaths)
No. perinatal / no.
nonperinatal
Foods
associated
No. of immunocompromised
Maritime Provinces
(1981)
41 (17)
34/7
Coleslaw
Massachusetts
(1983)
49 (14)
7/42
Pasteurize
d milk
42
California (1985)
142 (48)
93/49
Jalisco
cheese
48
Canton de Vaud,
Switzerland
(1983–1987)
122 (31)
63/59
Vacherin
Mont d’Or
cheese
–
36 (16)
4/32
Ice cream,
salami
24
9 (1)
2/7
>300 (?)
unknown
Philadelphia
(1986–1987)
Connecticut (1989)
United Kingdom
(1987–1989)
United States (22
states, 1998–1999)
>100 (21)
Shrimp
Pate
0
0
unknown
Grupos susceptibles de la población
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•
•
•
Individuos inmuno-comprometidos
Mujeres embarazadas
Neonatos
Ancianos
Previamente tiene que sobrevivir al acido estomacal y pasar la
barrera intestinal
• Puede cruzar la barrera hematoencefálica
Causa meningitis
• Puede cruzar la barrera placentaria
Causa aborto
Dosis infecciosa mínima: animales vs. humanos
mice
nonhuman
primates
human
ID50 103 – 107
109 kill
109
noticeable illness (loss of
appetite, septicemia, irritability,
occasional diarrhea)
103 – 109
(range based on
estimates from
outbreaks)
little known; dependent on host
and strain
Mecanismo de la invasión
Internalización
Lateral clustering of cadherin molecules is required to form stable cell-to-cell contacts. The intracellular catenins bind to the cytoplasmic tail of
cadherin-11. p120 catenin (p120ctn) binds the cadherin tail at the juxtamembrane domain, whereas β-catenin binds the distal domain, the βcatenin binding sequence. α-Catenin associates with β-catenin and is directly linked to the actin cytoskeleton.
Kiener and Brenner Arthritis Res Ther 2005 7:49
Liberación
Fosfolipasas: PC y PI
Jacobs et al.
Movilidad y multiplicación
Actin-based Motility
• Polimeriza actina formando una cola
– Se mueve por el citoplasma
– invade celulas adyacentes
– La polimerizacion se localiza en el
extremo de la bacteria
Polymerization of host actin at the Listeria monocytogenes surface provides a mechanism of intracellular
motility for the bacteria after they invade eukaryotic cells. The micrograph is an image of a kidney
epithelial cell from Potoroo tridactylis infected with Listeria monocytogenes. Filamentous actin is
stained red with rhodamine phalloidin. The bacteria are stained green by indirect immunofluorescence
with polyclonal antibody raised againstListeria monocytogenes. UC Berkeley
Fuente de C intracelular?
Aceptor de electrones intracelular?
Análisis de Listeria monocytogenes
Determinación de UFC por recuento en placa sembradas por extensión en
superficie
Medio PALCAM:
polimixina, acriflavina, cloruro de litio, ceftazidima, esculina, manitol,
si en caso no se usa revitalización previa se macera diluye el alimento y se
siembra en superficie. se incuba 24-48h 30oC y se cuentan colonias regulares
planas, color negro-verdoso con halo negro, sobre fondo cereza.
con revitalización
se hace lo mismo pero con ½ rico: glucosa, peptona de soja, extracto de
levadura. Se incuba 6h a temp ambiente y se agrega una capa de PALCAM
templado a 45oC y se incuba 24-48h 30oC.
La prueba P-A se hace en caldo BHI del mismo modo y se consideran + los tubos
negros y tb los débilmente negros.
Confirmación:
tomar la colonias PALCAM características, sembrar en
medio no selectivo xej tripticasa soja hacer GRAM,
catalasa
continuar con los bacilos cortos GRAM + Catalasa +
sembrar en agar sangre, agar xilosa, agar ramnosa.
se consideran +: bacilos cortos, GRAM +, Catalasa +,
móviles, Hemolíticos, xilosa negativo, ramnosa + sin
gas.
Listeria monocytogenes motility test in semisolid medium showing the typical "umbrella" growth. The left
side shows a negative control and the right side shows the positive umbrella growth.
Gini G., R. Menéndez, V. Gran, y R. Figueroa. 1986. Listeriosis en Guatemala. Reporte del Primer caso
de Listeriosis Neonatal. Rev. Guatemala Pediátrica 8:67-72.
Listeria monocytogenes, removing colonies to see the subtle hemolysis directly
beneath the colonies. (Rebecca Buxton, University of Utah)
caldo selectivo para L.
monocytogenes, Universidad
de Vermont Modificado (UVM)
(CM 863 Oxoid)
Legislación microbiológica
•
•
•
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El nivel de L. monocytogenes tolerado en los países es muy variable y va de
tolerancia 0 en 25 g en EE.UU. hasta permitir la presencia de 100 UFC/g en algunos
países de la Unión Europea.
En Mayo de 2004 numerosas asociaciones de industrias de los EE.UU., han
presentado una petición al Departamento de Alimentos y Medicamentos -FDA- para
que se eleve hasta 100 UFC/g la tolerancia de L. monocytogenes en alimentos que
no permiten el desarrollo.
El Código Alimentario Argentino exige en quesos de mediana, alta y muy alta
humedad ausencia de L. monocytogenes en 25 g de muestra.
Asimismo, el Servicio Nacional de Sanidad y Calidad Agroalimentaria -SENASAestablece la investigación de L. monocytogenes en salchichas tipo Viena y/o
chacinados y/o salazones cocidos feteados y otros productos cocidos.
Numerosos investigadores, incluyendo la ICMSF (International Commission on
Microbiological Specifications for Foods), proponen un valor objetivo de inocuidad
alimentaria no mayor de 100 UFC/g al momento del consumo.
Outbreak of Invasive Listeriosis Associated with the Consumption of Hog Head Cheese --Louisiana, 2010
Weekly
April 8, 2011 / 60(13);401-405
During January--June 2010, a total of 14 cases of laboratory-confirmed invasive listeriosis were
reported to the Louisiana Office of Public Health (OPH). Isolates of Listeria monocytogenes from
the blood samples of eight patients were identified as serotype 1/2a and had pulsed-field gel
electrophoresis (PFGE) pattern combinations that were indistinguishable from one another. The
detection of this cluster prompted an investigation in coordination with CDC, the Louisiana
Department of Agriculture and Forestry (LDAF), and the U.S. Department of Agriculture's Food
Safety and Inspection Service (USDA-FSIS). In-depth epidemiologic and environmental
investigations of the cluster were initiated on July 26, including food history interviews of four
patients. Three patients reported eating hog head cheese (a meat jelly made from swine heads
and feet); the product was purchased at two grocery stores in Louisiana. A traceback
investigation determined that a single brand of hog head cheese was common between the two
grocery stores. L. monocytogenes serotype 1/2a was cultured from one of three product samples
and from two of 16 environmental samples collected by LDAF at the processing establishment;
the product and one of the two environmental samples yielded isolates with PFGE pattern
combinations that were indistinguishable from the patient isolates. On August 14, LDAF
coordinated a voluntary recall of approximately 500,000 pounds of hog head cheese and
sausage because of possible contamination with L. monocytogenes. This is the first published
report of an invasive listeriosis outbreak associated with hog head cheese, which is a ready-toeat (RTE) meat. USDA-FSIS has a "zero tolerance" policy for L. monocytogenes
contamination of RTE food products (1), requesting recall of such products at any detectable
level of L. monocytogenes contamination. LDAF imposes and enforces equivalent requirements
in state-inspected establishments.
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6013a2.htm
Preguntas
NEW OXOID MEDIUM IMPROVES ISOLATION OF LISTERIA MONOCYTOGENES
Oxoid Limited has launched a new chromogenic medium for the improved isolation, enumeration and identification of Listeria from
food samples. In addition to providing presumptive identification of Listeria species, Oxoid Chromogenic Listeria Agar ( OCLA ) Plate
(PO5165A) is also able to differentiate pathogenic Listeria monocytogenes and Listeria ivanovii from other Listeria species.
The X- glucoside chromogen within the medium is cleaved by the enzyme b - glucosidase , common to allListeria species, resulting
in clearly visible blue/green colonies. (Other micro-organisms that possess b - glucosidase , such as enterococci , are inhibited by
selective agents, lithium chloride, nalidixic acid and polymixin B.) In addition, phospholipase in Listeria monocytogenes (and
pathogenic Listeria ivanovii )hydrolyses lecithin in the medium to produce a distinctive opaque white halo around the colony.
The selectivity of the medium is further enhanced by the addition of ceftazidime and amphotericin B, which inhibit other background
flora, such as bacilli, yeasts and moulds.
Studies have shown the OCLA Plate to have superior performance to PALCAM and Oxford medium for the isolation of Listeria
monocytogenes 1.
Thiol-activated cytolysins : structure,
function and role in pathogenesis
BILLINGTON S. J. ; JOST B. H.; SONGER J. G.
Department of Veterinary Science and Microbiology, 1117 East Lowell Street, The University of
Arizona, Tucson, AZ 85721, ETATS-UNIS
Members of the thiol-activated family of cytolysins are involved in the mechanism of pathogenesis
of a number of Gram-positive species. While they are pore-forming toxins, their major pathogenic
effects may be more subtle than simple lysis of host cells, and may include interference with
immune cell function and cytokine induction. Crystal structure, electron microscopy, mutagenesis
and antibody binding studies have led to the modeling of a novel mechanism of pore formation,
encompassing membrane-binding, membrane insertion and oligomerization. Despite their
designation as thiol-activated cytolysins, it is now clear that thiol activation is not an important
property of this group of toxins.
FEMS microbiology letters 2000, 182:197-205
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JBC 272:17395-17399. 1997
•
Thiol Activation of Endopeptidase EC 3.4.24.15
•
A NOVEL MECHANISM FOR THE REGULATION OF CATALYTIC ACTIVITY
•
Corie N. Shrimpton , Marc J. Glucksman § , Rebecca A. Lew , John W. Tullai § , Elliott H. Margulies § , James L. Roberts § and A. Ian
Smith
From the Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria 3181, Australia and the § Dr. Arthur M. Fishberg
Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029
•
•
Endopeptidase EC 3.4.24.15 (EP24.15) is a thermolysin-like metalloendopeptidase involved
in the regulated metabolism of a number of neuropeptides. Unlike other thermolysin-like
peptidases EP24.15 displays a unique thiol activation, a mechanism that is not clearly
understood. In this study we show that both recombinant and tissue-derived EP24.15 are
activated up to 8-fold by low concentrations (0.1 mM) of dithiothreitol. Additionally, under
non-reducing conditions, recombinant and native EP24.15 forms multimers that can be
returned to the monomeric form by reduction. We have also shown that competitive inhibitor
binding occurs only to the monomeric form, which indicates that catalytic site access is
restricted in the multimeric forms. Through systematic site-directed mutagenesis we have
identified that cysteine residues 246, 253, and possibly 248 are involved in the formation of
these multimers. Furthermore, both a double mutant (C246S/C253S) and a triple mutant
(C246S/C248S/C253S) are fully active in the absence of reducing agents, as measured by
both inhibitor binding and hydrolysis. The formation and disruption of disulfide bonds
involving these cysteine residues may be a mechanism by which EP24.15 activity is
regulated through changes in intra- and extracellular redox potential.
Listeria monocytogenes expresses cell-surface and secreted proteins that enable attachment to host cells, escape
from the phagocytic vacuole and locomotion in the cytosol of the invaded cell. Internalin A (InlA) and InlB mediate
the attachment of L. monocytogenes to the surface of host cells, and listeriolysin O (LLO) lyses the phagosomal
membrane. The actin-assembly-inducing protein (ActA) is expressed in a polarized manner and catalyses actin
polymerization, which propels bacteria through the cell and into neighbouring cells. To escape the secondary
vacuole in the newly invaded cell, L. monocytogenes expresses the phosphatidylcholine-specific phospholipase
PlcB, a secreted zinc metalloproteinase (Mpl) and LLO.
Triple Sugar Iron Agar Test
TSI contains glucose, lactose and sucrose as well as a pH-sensitive color indicator. It also contains an iron ingredient for detecting hydrogen sulfide
production, which blackens the medium if it occurs. Fermentation of the sugars by the test organism is interpreted by color changes in the butt
and the slant of the mediums.
Interpretation
Acid (yellow) butt – alkaline (pink) slant (K/A)
- glucose fermented
- sucrose or lactose not fermented
acid butt - acid slant (A/A)
- glucose fermented
- lactose and/or sucrose fermented
alkaline butt - alkaline slant (K/K)
- neither glucose, lactose or sucrose fermented
- organism probably not an enteric bacillus
Gas production - indicated by bubbles in the butt. With large amounts of gas production, the agar may be broken or pushed upward.
Hydrogen sulfide production - indicated by any amount of blackening in the butt.
TSI agar
Indole test
Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the
production of indole, pyruvic acid and ammonia. Indole production is an important characteristic in the identification of
many species of microorganisms being particularly useful in separating E. coli (positive) from members of the
Klebsiella-Enterobacter-Hafnia-Serratia group (mostly negative)
Voges Proskauer
The Voges-Proskauer test is used to detect the presence of
acetylmethylcarbinol, one of the end products of glucose
metabolism.
Blood-brain barrier, light micrograph
Blood-brain barrier. Confocal light micrograph of a section through a blood vessel in the brain, showing the arrangement
of cells that form the blood-brain barrier. The lumen (interior) of the vessel runs horizontally across the upper
frame. The endothelial cells that line the blood vessels of the brain are packed more tightly than elsewhere in the
body. This barrier protects the brain from many potentially harmful molecules and micro-organisms, but also
presents a challenge for the administration of drugs to the brain. Surrounding the blood vessel are glial cells
(green), which provide structural support for neurons (nerve cells, red) and supply them with nutrients and oxygen.
It is also thought that glial cells help maintain the blood-brain barrier.
C.J.GUERIN, PhD, MRC TOXICOLOGY UNIT/ SCIENCE PHOTO LIBRARY