Microbiology Lecture Notes: Listeria and Erysipelothrix week10pt2

Introduction to Gram-Positive Rods

• Focus Genera and Species:

  • Genus Listeria: Main species of interest is Listeria monocytogenes.

  • Genus Erysipelothrix: Main species of interest is Erysipelothrix rusiopathiae.

• Context: The lecture is held on Gadigal country.

• Overview of Topics:

  • Laboratory features of gram-positive rods.

  • Significance as animal pathogens.

  • Key virulence factors and host/environmental interactions.

  • Disease risk management and treatment strategies.

Listeria: General Characteristics

• Morphology: Gram-positive rods with a uniform shape; no pleomorphism is observed.

• Environmental Distribution: Widely distributed in nature; acts as a common saprophytic organism. It can survive and replicate easily in external environments.

• Acquisition: Commonly acquired as a commensal in the gut through ingestion.

• Temperature Tolerance:

  • It can grow in a wide range of temperatures.

  • It is classified as a psychotropic (or psychrotrophic) bacteria, meaning it can grow at temperatures between $0\,^\circ\text{C}$ and $7\,^\circ\text{C}$.

  • This allows the bacteria to survive and proliferate in refrigerated conditions, posing a significant risk in both public health (human food safety) and animal health (cold storage of feed).

• Motility: The organism is motile but possesses no flagellae. It uses internal propulsion mechanisms to move within host cells.

• Classification: It is a facultative intracellular parasite, which impacts how the host immune system manages infection.

Laboratory Features of Listeria monocytogenes

• Identification Marks: Short, regular-shaped gram-positive rods.

• Oxygen Requirements: Classified as a facultative anaerobe.

• Biochemical Reactions:

  • Catalase Test: Positive.

  • Esculin Hydrolysis: Hydrolyzes esculin, resulting in a black reaction. Selective media incorporated with esculin allows colonies to be visualized under UV light, where they fluoresce.

  • CAMP Test: Shows a positive reaction when streaked against Rhodococcus equi.

  • Hemolysis: Exhibits a narrow zone of complete hemolysis on sheep blood agar plates.

Pathogenesis and Virulence Factors of Listeria monocytogenes

• Invasion and Survival:

  • Internalin: A protein expressed by the bacteria that enables invasion into the host cell (including non-phagocytic cells).

  • Listerialysin O: An enzyme that prevents phagosome-lysosomal fusion, allowing the organism to survive and avoid lysis within the phagosome.

• Intracellular Movement:

  • Act A: Once inside the cell, the bacteria express Act A, which allows them to produce an actin "motile tail."

  • Propulsion: This tail propels the bacteria laterally through the cytoplasm.

• Intercellular Spread:

  • Lecithinase: This enzyme allows the bacteria to spread directly into neighboring cells without exiting the intracellular environment.

  • Immune Avoidance: By staying intracellular and moving directly between cells, Listeria avoids extracellular host defenses like antibody responses. Consequently, cell-mediated immune (CMI) responses are critical for clearing the infection.

Forms of Listeriosis in Animals and Humans

Visceral Form

• Pathophysiology: Occurs when the organism is ingested, enters the bloodstream, and spreads systemically.

• Clinical Presentation: Causes granulomatous foci or internal abscesses in various organs.

• Susceptible Populations:

  • Neonatal animals: Experience systemic disease and internal abscesses.

  • Pregnant hosts (Ruminants and Humans): Infection in the bloodstream leads to fetal infection and abortion. This is the primary public health risk for pregnant women consuming contaminated foods.

• Route: Exogenous ingestion of soil-contaminated feed. The bacteria enter via internalins through the intestinal route (lamina propria) into the bloodstream.

• Clinical Signs: Gastroenteritis, intestinal inflammation, liver abscesses, jaundice, and pneumonia (if lodged in lungs).

Neural Form

• Pathophysiology: Often associated with damage to oral, nasal, or oculomucosal membranes. The organism enters peripheral nerves (specifically the trigeminal nerve) and migrates to the brain.

• Host Specificity: Much more common in ruminants (cattle and sheep) than in humans.

• Clinical Sign (Circling Disease):

  • The infection causes meningoencephalitis.

  • If the bacteria infect the right side of the brain, the animal circles to the left.

  • If the bacteria infect the left side of the brain, the animal circles to the right.

• Risk Factors:

  • Young animals shedding teeth (providing an entry avenue).

  • Drought conditions: Abrasive feed causes oral and mucosal abrasions that facilitate entry from the environment.

Host and Environmental Factors for Listeria

• Asymptomatic Carriage: Animals can carry the organism in their gut as commensals, shedding it in feces and seeding the environment. Stress can trigger endogenous septicemia in these carriers.

• Immune Status: Suppression of cell-mediated immunity (due to pregnancy, immaturity, age, or drugs) increases susceptibility.

• Zoonotic Risk:

  • Direct contact with animals is rare; most human cases are urban and linked to animal-sourced food.

  • Risk Foods: Dairy products (milk, soft cheeses), raw vegetables (soiled with contaminated earth), undercooked meats, and fermented products like mayonnaise or yogurt.

  • Outbreak Case Study (Rock Melons): Approximately $8$ years ago in Australia, contaminated rock melons caused $7$ deaths and $1$ miscarriage. The jagged surface of the melons allowed soil/Listeria to accumulate. Heavy rain made harvesting muddy, and washing was insufficient. The infectious dose in compromised individuals is low (approximately $100$ cells).

  • Outbreak Case Study (Canada Nursing Home): Contaminated meat slicers in a processing plant created biofilms. Slicing meat transferred Listeria to products used for sandwiches in nursing homes. This resulted in over $200$ recalled products, over $50$ cases, and $22$ deaths.

• Silage Management: Listeria loves decaying vegetation. If silage fermentation is poor and the pH remains above $5.5$, Listeria will proliferate. The goal is to keep silage pH below $5.5$.

• Prevention:

  • Avoid soft cheeses and unpasteurized dairy during pregnancy.

  • Wash vegetables thoroughly.

  • Thoroughly cook food; pasteurization kills Listeria.

  • Maintain strict hygiene of food processing equipment (e.g., disassembling meat slicers to remove biofilms).

Erysipelothrix rusiopathiae

• General Features:

  • Gram-positive short rod; can be filamentous.

  • Non-motile.

  • Non-catalase producer.

• Culture: Produces pinpoint colonies with alpha (incomplete) hemolysis, often appearing with a greenish tinge.

  • Smooth form: Short regular rods.

  • Rough form: Filamentous; produces a "bottle brush" appearance in gelatin slabs.

• Virulence Factors:

  • Protective Antigen: A glycoprotein complex that prevents phagocytosis.

  • Adhesins: Allow adherence to intestinal epithelial cells.

  • Neuraminidases: Associated with vascular damage and thrombosis; plays a role in adhesion.

  • Capsule: Antiphagocytic.

  • Enzymes: Phospholipases and antioxidant enzymes allow survival within phagocytic cells.

Erysipelas: Clinical Manifestations

• Primary Host: Most significant in pigs, but also seen in fish, aquatic animals (dolphins, walruses), and birds.

• Septicemic Form (Acute): Caused by highly virulent strains; results in acute death.

• Chronic Forms (Less Virulent Strains):

  • Skin Disease (Diamond Skin Disease): Produces pathognomonic diamond-shaped skin lesions. This is an immune-mediated Arthus reaction (type III hypersensitivity) involving thrombosis.

  • Arthritis: Localization in the joints causing lameness.

  • Vegetative Endocarditis: Colonization of heart valves leading to congestive heart failure.

• Significance: Diamond skin lesions degrade carcass quality, leading to financial loss as the skin is sold with the meat in the pork industry.

• Zoonosis (Erysipeloid): In humans, it causes "erysipeloid" (not to be confused with human erysipelas, which is streptococcal). It presents as non-suppurative skin swelling, usually following contact with fish or pig carcasses. It typically resolves in $2$ to $3$ weeks.

Management of Erysipelas

• Transmission: Ingestion, wound contamination, or arthropod bites. It can be found in the tonsils/gut of healthy carriers. It survives well in the environment (resistant to drying/salting) but is susceptible to moist heat.

• Treatment: Penicillin is the drug of choice. Withholding periods must be observed to avoid drug residues in pork.

• Controls:

  • Isolate sick animals.

  • Minimize stress and overcrowding.

  • Heat-treat or properly dispose of infected carcasses.

  • Quarantine new animals for a $30$-day incubation period.

• Vaccination:

  • Killed vaccines are available.

  • They are effective at preventing acute septicemia and death but are less effective at preventing chronic forms (skin lesions or arthritis).

  • Vaccinating sows/gilts provides maternal antibodies to piglets.

Questions & Discussion

• Question: Are there any questions about Erysipelothrix or Listeria?

• Response: All good? Great. Tomorrow you begin Gram-negative rods with Karola Venturini (Enterobacteriaceae).

• Discussion on Practicals:

  • First practicals are Friday in the Lees Building, PCT lab.

  • Groups start at $11:00$ and $14:00$.

  • Capacity is limited ($70$ students max); students must stick to their timetables.

  • Practicals are sequential: a swab taken in week 1 is followed through in weeks 2 and 3. Attendance at all three is strongly encouraged for the best outcome.

Appendix: Comparative Summary

Introduction to Gram-Positive Rods

• Focus Genera and Species:

  • Genus Listeria: The main species of interest is Listeria monocytogenes, which is significant due to its role in listeriosis, a serious infection primarily affecting pregnant women, newborns, elderly, and immunocompromised individuals.

  • Genus Erysipelothrix: The main species of focus is Erysipelothrix rusiopathiae, known for causing swine erysipelas and other infections in various animal species.

• Context: The lecture is held on Gadigal country, acknowledging the traditional custodians of the land and their continuing connection to culture and community.

• Overview of Topics:

  • Laboratory features of gram-positive rods, highlighting their identification and biochemical characteristics.

  • The significance of these bacteria as important pathogens in veterinary and human medicine, emphasizing the diverse diseases they cause.

  • Discussion on key virulence factors, mechanisms of pathogenesis, and host/environmental interactions that facilitate their survival and infection.

  • Strategies for disease risk management and treatment to mitigate the public health impact of infections caused by these organisms.

Listeria: General Characteristics

• Morphology: Gram-positive rods exhibiting a consistent and uniform shape, with no pleomorphism observed which aids in identification under microscopic examination.

• Environmental Distribution: Listeria monocytogenes is widely distributed in various environments including soil, water, and decaying vegetation, functioning as a saprophyte. Its ability to survive in harsh conditions, including high salinity and low pH, poses significant health risks in food safety.

• Acquisition: This pathogen is commonly acquired through ingestion of contaminated foods, where it can be found as a commensal organism in the gut microbiota of animals.

• Temperature Tolerance:

  • It can grow across a wide range of temperatures, classified as a psychrotrophic bacterium, which allows it to thrive at refrigeration temperatures between $0 \, ^\text{°C}$ and $7 \, ^\text{°C}$. This adaptability is a major concern in food safety and public health as it can persist in cold storage conditions.

• Motility: The bacterium is motile through a unique cellular propulsion mechanism and does not have flagella; this motility aids its invasion of host cells.

• Classification: It is classified as a facultative intracellular pathogen, which enables it to evade host immune responses and persist within host cells.

Laboratory Features of Listeria monocytogenes

• Identification Marks: Characteristically identified as short, rod-shaped gram-positive bacteria. Laboratory tests show positive results during Gram staining.

• Oxygen Requirements: This organism is classified as a facultative anaerobe, allowing it to grow in both aerobic and anaerobic conditions, which is crucial for survival in various environments.

• Biochemical Reactions:
  
  

  • Catalase Test: Positive, indicating its ability to produce the enzyme catalase.

  • Esculin Hydrolysis: Listeria can hydrolyze esculin resulting in a blackening of the medium, which can be useful for visual identification in selective media under UV light.

  • CAMP Test: Demonstrates a positive reaction when co-streaked with Rhodococcus equi, indicating its unique interaction in a mixed culture.

  • Hemolysis: It exhibits a narrow zone of complete hemolysis on sheep blood agar, which aids in differentiating it from other species in the lab.

Pathogenesis and Virulence Factors of Listeria monocytogenes

• Invasion and Survival:

  • Internalin: This surface protein is vital for the bacterium's invasion into host cells, including non-phagocytic cells, which is key for establishing infection.

  • Listerialysin O: An important virulence factor that prevents the fusion of phagosome and lysosome, enabling the organism to survive within the phagocytic cells and evade immune responses.

• Intracellular Movement:

  • Act A: It facilitates the polymerization of actin, enabling the formation of an actin filament "tail" that propels the bacterium laterally within the cytoplasm of the host cells, aiding its spread.

• Intercellular Spread:

  • Lecithinase: This enzyme allows for direct spread into adjacent cells without leaving the intracellular environment, minimizing exposure to immune surveillance.

  • Immune Avoidance: The organism’s intracellular lifestyle effectively evades extracellular immune mechanisms such as antibody responses. Consequently, cell-mediated immune responses are essential for clearance of the infection and affected individuals may present signs of severe disease if CMI is compromised.

Forms of Listeriosis in Animals and Humans

Visceral Form

• Pathophysiology: The visceral form occurs when Listeria monocytogenes is ingested, leading to systemic spread via the bloodstream, which can result in multi-organ infection.

• Clinical Presentation: This form is associated with the development of granulomatous lesions or abscesses in internal organs, contributing to significant morbidity and mortality.

• Susceptible Populations:

  • Neonatal animals: Particularly susceptible to systemic disease and may present with severe internal abscesses due to immature immune systems.

  • Pregnant hosts (Ruminants and Humans): Fetal infection can occur leading to abortion in pregnant women and ruminant species, which has severe public health implications.

• Route of Transmission: Exogenous ingestion of contaminated feed, whereby bacteria utilize internalins to penetrate the intestinal mucosa (lamina propria) before entering the bloodstream.

• Clinical Signs: These may include gastroenteritis, abdominal pain, intestinal inflammation, liver abscesses, and respiratory distress if the infection reaches the lungs.

Neural Form

• Pathophysiology: This form often follows trauma to the oral, nasal, or ocular mucosa, allowing the bacteria to enter peripheral nerves, particularly the trigeminal nerve, and disseminate to the central nervous system.

• Host Specificity: It is seen more frequently in ruminants, specifically cattle and sheep, than in humans.

• Clinical Sign (Circling Disease):

  • Infection of either side of the brain leads to circling behavior, indicating neurotropic infection, with resultant encephalitis characterized by severe neurological symptoms.

• Risk Factors:

  • Young animals during teething may have abrasions that allow entry of the bacteria.

  • Drought conditions may exacerbate mucosal damage and provide a route for infection through abrasive feeds.

Host and Environmental Factors for Listeria

• Asymptomatic Carriage: Many animals, particularly ruminants, can carry Listeria asymptomatically in their gastrointestinal tract, shedding it in feces and contaminating the environment. Stressors can lead to activation of endogenous septicemia in these carriers.

• Immune Status: Factors such as suppressed cell-mediated immunity due to pregnancy, age, immaturity, or immunosuppressive medications significantly increase susceptibility to infections.

• Zoonotic Risk:

  • Human cases are primarily linked to the consumption of animal-derived foods rather than direct contact with animals. Thus, understanding food safety is critical for the prevention of listeriosis.

  • Risk Foods: Common sources include dairy products (especially unpasteurized milk and soft cheeses), raw vegetables (contaminated with soil), undercooked meats, and certain processed products like mayonnaise and yogurt.

  • Outbreak Case Study (Rock Melons): In a notable outbreak about $8$ years ago in Australia, contaminated rock melons were responsible for $7$ deaths and $1$ miscarriage due to Listeria. The irregular surface of the melons allowed soil contamination, particularly after heavy rains, leading to insufficient washing post-harvest.

  • Outbreak Case Study (Canada Nursing Home): Bioburden management failures in meat processing facilities led to cross-contamination. Inadequate sanitation of meat slicers resulted in recalls of over $200$ products linked to Listeria with serious consequences including $50$ reported cases and $22$ deaths.

• Silage Management: Listeria thrives in decaying organic matter. Poorly fermented silage with pH levels above $5.5$ can favor the growth of Listeria, underscoring the importance of proper fermentation techniques.

• Prevention Strategies:

  • Pregnant individuals should avoid soft cheeses and unpasteurized dairy products to reduce risk.

  • Emphasize washing fruits and vegetables thoroughly before consumption.

  • Cooking food thoroughly and following pasteurization guidelines are key to killing Listeria.

  • Regular sanitation and hygiene practices in food processing environments are crucial, including the regular disassembly of equipment to remove biofilms that harbor pathogens.

Erysipelothrix rusiopathiae

• General Features:

  • It is a gram-positive, short rod that can occasionally exhibit filamentous forms, complicating identification in some cases.

  • Notably non-motile and does not produce catalase, distinguishing it from other closely related bacteria.

• Culture Characteristics: Cultures exhibit pinpoint colonies characterized by alpha (incomplete) hemolysis, often showing a greenish hue on certain growth media.

  • The smooth form appears as short regular rods, while the rough form manifests filamentous growth leading to a characteristic "bottle brush" appearance on gelatin slabs.

• Virulence Factors:

  • Protective Antigen: A glycoprotein complex that effectively prevents phagocytosis, allowing the organism to survive in hostile environments.

  • Adhesins: Essential for attachment to intestinal epithelial cells, contributing to pathogenicity.

  • Neuraminidases: Enzymes that play a role in vascular damage leading to thrombosis and assist in adhesion to host tissues.

  • Capsule: Provides an antiphagocytic barrier that assists the bacterium in avoiding immune clearance.

  • Enzymatic Activity: Phospholipases and antioxidant enzymes help the bacterium survive immune responses within phagocytic cells.

Erysipelas: Clinical Manifestations

• Primary Host: The primary veterinary focus of Erysipelothrix rusiopathiae is pigs, although it can also infect various species including fish, marine mammals like dolphins and walruses, and avian species.

• Septicemic Form (Acute): Characterized by highly virulent strains that can lead to sudden death in affected animals due to overwhelming infection.

• Chronic Forms (Less Virulent Strains):

  • Skin Disease (Diamond Skin Disease): Notable for producing pathognomonic diamond-shaped lesions on the skin, which are indicative of immune-mediated responses and lead to significant welfare and economic impacts in the swine industry.

  • Arthritis: Localization in joints can result in lameness and secondary complications.

  • Vegetative Endocarditis: Infection of the heart valves can lead to severe heart complications and congestive heart failure.

• Significance: The presence of diamond skin lesions can severely degrade the quality of the carcasses, leading to financial losses as the pork industry relies on the quality of the meat and skin.

• Zoonosis (Erysipeloid): Erysipelothrix can cause "erysipeloid" infections in humans, distinct from erysipelas caused by streptococci. Infections typically arise from handling infected animal carcasses, leading to localized non-suppurative skin swelling, which usually resolves within $2$ to $3$ weeks without serious complications.

Management of Erysipelas

• Transmission: The bacterium is typically transmitted via ingestion, contamination of wounds, or through bites from arthropods. It can be isolated from the tonsils and intestines of healthy carrier animals, posing a constant risk for outbreaks.

• Treatment: Penicillin remains the first-line treatment for infected animals, though it is crucial to observe withholding periods to prevent drug residues in pork products and ensure food safety.

• Control Measures:

  • Isolation of infected animals and minimizing stress is essential in controlling the spread of disease.

  • It is important to heat treat or appropriately dispose of infected carcasses to reduce environmental contamination.

  • Quarantine measures should be practiced for all incoming animals for a period of at least $30$ days to observe for signs of infection.

• Vaccination:

  • Vaccination against Erysipelothrix rusiopathiae using killed vaccines can significantly reduce the incidence of acute septicemia and mortality but may be less efficacious against chronic conditions such as skin lesions or arthritis. Vaccinating sows and gilts can provide passive immunity to their piglets through maternal antibodies.

Questions & Discussion

• Question: Are there any questions regarding the pathogenesis, clinical presentations, or management of Erysipelothrix rusiopathiae or Listeria monocytogenes?

• Response: Any outstanding questions? If all is clear, we will move on to our next topic covering Gram-negative rods with guest lecturer Karola Venturini, focusing on Enterobacteriaceae.

• Discussion on Practicals:
  
  

  • Practical sessions will commence this Friday in the Lees Building's PCT lab.

  • Sessions begin at $11:00$ and $14:00$, with a maximum capacity of $70$ students; adherence to assigned timetables is necessary.

  • Each practical will build on the previous one, with a swab taken in week 1 tracked through weeks 2 and 3. Attendance at all sessions is recommended for optimal learning outcomes.

Appendix: Comparative Summary