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COMMONWEALTH OF AUSTRALIA

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Opportunistic Infections

Objectives

• To appreciate the nature of opportunistic infections by understanding (1) the nature of opportunistic pathogens, (2) the contribution of host factors to infection, and (3) the reasons for the increased susceptibility of hospitalised patients to infection

• To illustrate some principles of bacterial pathogenesis and molecular epidemiology, using the opportunistic pathogen, Pseudomonas aeruginosa, as an example.

Microbial pathogenicity vs . Host resistance

colonisation

penetration

multiplication

tissue damage

physical and chemical barriers

inflammatory response phagocytosis

adaptive immune response

disease or protection

Pathogen-Host Relationships

disease threshold

Microbial Virulence

Host Resistance

How Microbial Virulence and Host Resistance Affect Disease Outcome

• E. coli • Staphylococcus aureus • Klebsiella pneumoniae • Enterococcus spp. • Pseudomonas aeruginosa • Enterobacter spp. • Serratia spp. • Proteus spp. • Clostridium difficile

Frequently Encountered Opportunistic Pathogens: “The Seed”

* may cause nosocomial epidemics

* * * * * *

• Local • anatomical defects

• surgical and other wounds

• burns

• catheterisation (bladder, IV)

• foreign bodies in general

Host Factors: “The Soil”

• Systemic • extremes of age • leucopenia • malignancy • malnutrition • diabetes • liver disease • certain infections • treatment with antimicrobials • primary (congenital) immunodeficiency

Host Factors (cont.)

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• wound infection

• UTI

• intra-abdominal infection

• pneumonia

• septicaemia (sepsis)

• meningitis (esp. neonates)

Types of Infection

• endogenous (own microbiota)

• exogenous

Sources

Culture appropriate specimen: • wound swab

• pus

• urine

• sputum

• blood

• CSF

• etc.

Diagnosis

• Depends on antibiotic susceptibility

• nosocomial strains are often multiresistant

• may need potent bactericidal agents

Treatment

• Aseptic technique (esp. hand hygiene)

Prevention

Education

Pseudomonas

• G− rod

• Motile

• Aerobes or facultative anaerobes

• Non-fermenting, non-sporing

• Some species, incl. Ps. aeruginosa, produce pigments

• Widespread

• Low nutritional requirements

Morphology of Pseudomonas aeruginosa

Colonies on nutrient agar Gram stain of culture

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Classification

• Divided into species according to:

• Biochemical tests

• Ps. aeruginosa subtyped according to:

• Serotype, biotype (seldom used)

For epidemiological studies, generally use

• RFLP or MLST (multilocus sequence typing)

Pulsed-Field Gel Electrophoresis of Genomic DNA of Ps. aeruginosa Isolates from Children with Cystic Fibrosis

1 2 3 4 5 6 7 8 9 10 11 12 kb

500-

400-

300-

200-

100-

50-

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Species of Pseudomonas and Related Genera

• Ps. aeruginosa* : ubiquitous saprophyte, important opportunistic pathogen

• Burkholderia cepacia* : opportunistic pathogen

• Stenotrophomonas maltophilia* : opportunistic pathogen

• Burkholderia pseudomallei : causes melioidosis

* These species often colonise the respiratory tract of patients with cystic fibrosis, and are intrinsically resistant to many commonly-used antibiotics

Pseudomonas aeruginosa : overview

• Epidemiology

• Diseases

• Pathogenesis (incl. biofilms and quorum sensing)

• Principles of management

Ps. aeruginosa : epidemiology

• Widespread in nature, esp. in moist environments

• Transiently colonises skin, mucous membranes, git

• A leading nosocomial pathogen:

• previously assoc. with burns, febrile neutropenia

• now mainly pneumonia, UTI, sepsis

• Major problem in patients with cystic fibrosis

Ps. aeruginosa : epidemiology (cont.)

• Infections mostly acquired from environment

• Spread in hospital on hands and fomites

• Some strains spread between patients with CF

• All strains are intrinsically resistant to many antibiotics and weak disinfectants

• Resistance also readily acquired from other bacteria

Ps. aeruginosa : diseases

• Superficial infections: • Skin: wound infection, otitis externa, folliculitis

• Eye: keratitis, corneal ulcer, (also deep infection)

• Deep and systemic infections: • Pulmonary: nosocomial pneumonia, chronic

infection in patients with cystic fibrosis

• Other: UTI, endocarditis, osteomyelitis, septicaemia, etc. in immunocompromised patients

Ps. aeruginosa Pathogenesis (1): Adhesion and Invasion

• Does not invade intact skin unless very large numbers

• Adheres weakly to intact epithelium via flagella, pili, LPS

• As with other bacteria, LPS and flagellin bind to Toll-like receptors: affect cytokine production

• LPS core binds to CFTR (CF transmembr. conductance reg.)

• Once adherent, bacteria tend to produce biofilm

• Capsule assists adherence and biofilm formation

Properties of Ps. aeruginosa in Biofilms

• Non-motile

• More capsule material - mucoid phenotype

• More adherent

• Less invasive

• Shorter LPS (no O-antigen)

• Slowed growth

• Increased resistance to antibiotics

Much of this results from the activation of genes that are regulated by quorum sensing

response to environmental signals

Bacterial “cross-talk” (= quorum sensing)

Stages in Biofilm Formation

primary-plus.com

An Example of How Quorum Sensing Works

Broth culture of light- emitting bacteria at

high cell density

B. Bassler

An Example of How Quorum Sensing Works

low cell

density

autoinducer

LuxR LuxR

LuxI

luxR lux

box luxI luxC D A B E

autoinducer

high cell

density

autoinducer

LuxR LuxR

LuxI

luxR lux box luxI luxC D A B E

autoinducer

LuxR

Light

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Properties of Ps. aeruginosa in Biofilms

• Non-motile

• More capsule material - mucoid phenotype

• More adherent

• Less invasive

• Shorter LPS (no O-antigen)

• Slowed growth

• Increased resistance to antibiotics

Ps. aeruginosa Pathogenesis (2): Spread and Multiplication

Spread through tissues and body facilitated by:

• Reduced PMNs

• Flagella

• Exoezymes: proteases, haemolysins, phospholipases, elastase. Act on pulmonary tissue and surfactant

• Exotoxins: exotoxin A; exoenzymes S and U (EXoS, ExoU), etc. Inhibit phagocytosis

• LPS-CFTR-mediated invasion: aggravates corneal damage

What Happens in Patients with Cystic Fibrosis?

• Defect in CFTR → abnormal ion transport, thickened mucus and impaired mucociliary function

• Pseudomonas and Staph not inhibited by high salt concs.

• Bacteria in biofilms resist mechanical removal and are less visible to the innate immune system

• DNA secreted by bacteria and released from dying cells → thickened mucus

• Type of host antibody response may affect outcome.

Ps. aeruginosa Pathogenesis (3): Tissue Damage

Type III secreted proteins • Exoenzymes S, T, U and Y (=ExoS, ExoT, ExoU, ExoY):

act on various host cell targets to interfere with phagocytosis and enhance cytokine production

Other Exotoxins and Enzymes • Exotoxin A: blocks protein synthesis (≡ diphtheria toxin)

• Elastase: damages connective tissue

• Phospholipase: damages cell membranes, degrades surfactant

Ps. aeruginosa : Control

• Reduce risk of susceptible patients by suitable management of burns, neutropenia, catheters, ventilators

• Be on alert for infection and treat early

• Practice high levels of hand hygiene

• Use contact lenses and solutions appropriately

• Lifelong monitoring and treatment of CF patients

Pseudomonas cannot be eradicated from the environment. Therefore management is directed towards prevention.

Ps. aeruginosa : Summary

• Ps. aeruginosa is widespread in the environment

• Its low nutritional requirements and genetic versatility allow it to occupy many different environments

• It is a prototypical opportunistic pathogen and a leading cause of hospital-acquired infections

• In humans, it shows a preference for situations where innate immunity is reduced

• Its intrinsic and acquired resistance to antimicrobials provide a therapeutic challenge

• Prevention is the key to controlling infection