Lecture 14: Osteomyelitis

Question 1

What are the routes of infection that can lead to osteomyelitis?

Answer 1

Route Of Infection

• Trauma (e.g., deep tissue injury, artificial joint replacement, root canal treatment)
• Spreading from local infection (e.g. diabetic ulcer, skin and soft tissue infections, cellulitis, septic arthritis)
• Hematogenous route (bacteremia) (bacteria in blood)

Question 2

Define osteomyelitis

Answer 2

Osteomyelitis means infection and inflammation of bone or bone marrow (Greek).

Bones have little pores, which are penetrated by bacteria. This causes infections such as osteomyelitis.

Question 3

Describe the pathogenesis of Osteomyelitis

Answer 3

Pathogenesis

Osteomyelitis can affect all bones, but often affects bones with good blood supply (e.g. tibia, femur).

• The bacteria infect bone, where they colonise and proliferate.
• In response, leukocytes infiltrate site of infection and fight the bacteria by generating t_oxic substances_ (radical oxygen species, enzymes) that cause inflammation and generation of pus.
• This results in devascularisation, d_ead bone (sequestrum)_ and abscess formation.

Some bacteria might also invade bone cells and evade immune responses and drugs, where they are difficult to eradicate (chronic osteomyelitis).

• Staphylococcus aureus can form biofilms, which are difficult to treat with antimicrobials.

Bacteria might spread to joint (septic arthritis).

Question 4

Often bacteria fro the bone spreads to joints which is called……

Answer 4

septic arthritis

Question 5

Who are at risk?

What are the risk factors for Osteomyelitis?

Answer 5

Risk Groups

The disease affects mainly children and young adults.

Risk factors include:

• Diabetics with foot ulcers
• Intravenous drug users
  
  • _​_Use of needles
  • Use of unsterile needles
  • Drugs - immunocompromised
• Patients with infections following trauma, bone, surgery, j_oint replacement_
• Root canal treatment
• Patients with s_kin and soft tissue infections_

Children with chicken pox infection (infrequent!)

Question 6

Describe the pathogens associated with Osteomyelitis

(what causes this disease)

Answer 6

Pathogens

• Staphylococcus aureus is most common cause of osteomyelitis (~80% osteomyelitis cases in children and young adults).
• Group B streptococcus is associated with osteomyelitis in infants.
  
  • Often found in the vagina of women- so can infect the infant as soon as he/she is born.
• Other causes include Streptococcus pyogenes (Group A streptococcus), _in adult_s. Coagulase-negative staphylococci, Haemophilus influenza, Pseudomonas aeruginosa and Enterococcus spp.

Question 7

What bones are affected by osteomyelitis in adults and in children?

Answer 7

Adults:

1) Prostatif joint infection
2) Diabetic foot infection
3) Post-traumatic infection
4) Vetebral osteomyelitis (rare)

Children

1) Femur (common)
2) Tibia (common)
3) Humerus
4) Pelvis

Question 8

~80% osteomyelitis cases in children and young adults are caused by ________

Answer 8

(~80% osteomyelitis cases in children and young adults are caused by Staphyloccoccus aureus)

Question 9

How do you diagnose osteomyelitis?

Answer 9

Diagnosis Of Disease And Identification Of Causative Bacteria

Diagnosis

1) Symptoms

• Pain/weakness of specific bone
• Redness
• Fever

2) Blood sample

• High WBC count
• Bacteria present (if associated with bacteremima)

3) Diagnosis is mainly radiology (X-ray, MRI).

• CT/X-ray
  
  • But not very sensitive, and is moderately specific
  • Radiology would not be able to pick up damage in the early stages
• MRI
  
  • Used to confirm diagnosis

2) Bacterial samples for lab diagnostic are from:

• Bone biopsy, (highly invasive)
• Blood sample (however, osteomyelitis is not always associated with bacteremia)

Question 10

Describe the Lab diagnostic for staphlococcus aureus

Answer 10

Lab Diagnostic: Gram-Staining

1) The bacterial isolates would initially be analysed using Gram-stain method. It is based on fixation, crystal violet, iodine treatment, decolorisation, counter stain with safranin.

• Staphylococcus spp, Streptococcus spp. and Enterococcus spp. would appear as Gram-positive spherical cells (cocci) (GPC) (thick peptidoglycan with one membrane), thus stained purple.
• Note that Gram-negative cells have thin peptidoglycan between two membranes, thus stained pink.

2) Staphylococci can be distinguished from other GPC using the catalase test, which would be positive for Staphylococcus spp (formation of oxygen bubbles after addition of hydrogen peroxide).
3) A coagulase test can then be used to identify S. aureus, which is the only coagulase-positive staphylococcus

Question 11

How can you differentiate between Staphylococcus and Streptococcus?

Answer 11

Both are gram-positive

In the catalase test, Staphyloccocus will be positive (presence of bubbles as the staph converts the H202 into H20 and O2)

Streptococcus will be _negative (_the coagulase in the staph aureus will convert the fibrinogen into fibrin- positive is S,aureus)

Question 12

Describe the

1) Habitat
2) Transmission
3) Source of infection
4) Diseases caused

by Staphlococcus aureus

Answer 12

Habitat:

• Anterior nares (20% of people are asymptomatic carriers)
• Transient carriage on skin

Transmission:

• Human to human

Source of infection:

• Community and hospital (one of the most common nosocomial infections)

Diseases

• Skin and soft tissue infections
• Invasive diseases
• Toxic shock
• MORE

Question 13

How can S.aureus cause skin infection?

Answer 13

If you don’t wash your hands

1) Crack in skin
2) Splinter (allow Staph sitting on the skin to move in)
3) Hair (openings)

Question 14

What are the Virulence Mechanisms of S.Aureus

Answer 14

Virulence Factors

Virulence factors are gene products that enable the bacteria to colonise host, damage host tissue, spread from the site of the initial infection and evade the immune response (harmful effects). This includes:

• Adhesins
  
  • ​(for binding to host tissue, colonisation)
• Immune evasion factors
  
  • (neutralise certain parts of immune response)
• Spreading factors
  
  • (allow bacteria to spread from local infection into deeper tissue or blood (bacteremia)
  • e.g. proteases, DNAses, hyaluronidase)
• Superantigens (SAgs)?

Question 15

Describe the Adhesins (one of the virulence mechanisms)

Answer 15

Adhesins

MSCRAMMS (microbial surface components recognizing adhesive matrix molecules) are _cell wall-attached protein_s found in most Gram-positive bacteria.

• They specifically bind to host extracellular matrix proteins, such as fibronectin, elastin, laminin, vitronectin and collagen.
• They are important for colonization of host tissue (e.g. fibronectin binding protein, collagen binding protein).

Question 16

What are the spreading factors of Staphloccocus? (one of the virulence mechanisms)

Answer 16

Spreading Factors

These are proteins that facilitate spreading from a localized infection (e.g. abscess) to surrounding tissue or blood (causing bacteremia). They include:

These basically destroy tissue and allow them to spread.

• Staphylokinase (fibrinolysin)
  
  • ​causes fibrinolysis (dissolves fibrin clots)
• Lipases
  
  • ​hydrolyse lipids, for invasion of cutaneous tissues
• Nucleases (DNases)
  
  • ​hydrolyses DNA, decrease viscosity of pus
• Proteases
  
  • include serine protease, aureolysin (metallo-protease)
• Cytolysins
  
  • ​are cell damaging proteins produced by S. aureus, and they destroy many cell types:
    
    • Leukocytes (leucocidin for immune evasion) (thus also immune evasion factor)
    • Erythrocytes (haemolysin gaining access to iron)
    • Tissue cells including epithelial cells (bacterial spreading).

Question 17

Describe the Immune evasion factors of Staphlocococcus (virulence mechanism)

Answer 17

Immune Evasion Factors

1) Cytolysins

• (also a spreading factor)
• e.g. hemolysin, leukocidin
  
  • Kills erythrocytes, leukocytes, tissue cells

2) Capsule

• Capsule is a thick layer made of polysaccharide outside cell wall, which prevents opsonisation with Ig antibodies and C3b complement (inhibits phagocytosis).
• It also contains water, which protects bacteria against desiccation.

3) Biofilms

Biofilms (i.e. slime layer, extracellular polysaccharide (EPS)) are microbial community with bacteria, attached to a substrate or interface or to each other, embedded in a matrix of extracellular polymeric substance (loose-bound water-soluble film made from secreted polysaccharides, proteins, DNA).

• They prevent antibiotics and immune components (e.g. Ig) reaching the bacteria.
• They are involved in immune evasion and antibiotic tolerance.
  
  • ​prevent immune cells from getting to the bacteria

4) Protein A

• Protein A is found on surface of most S. aureus strains.
• It binds IgG/antibodies (except IgG₃) in “wrong orientation” (via Fc region), thereby preventing opsonisation and phagocytosis (Fc receptors on phagocytes are unable to recognise IgG).

5) Cell Bound Coagulase (Clumping Factor ClF-A)

Cell-bound coagulase binds prothrombin and i_nduces fibrin polymerisation._

• Fibrin deposition on cell surface prevents opsonisation and phagocytosis.
• CIF-A also promotes cell aggregation (clumping).

Coagulase test is used to identify S. aureus, as it is not produced by any other staphylococci or by streptococci.

Question 18

Describe Superantigens

Answer 18

Superantigens are family of h_eat-resistant secreted proteins_ (>20 members), produced by Staph. aureus and Strep. pyogenes.

They are non-specific, highly potent _T cell mitogens (_stimulate lots of different T cells).

• It triggers massive release of pro-inflammatory cytokines (TNF- α, IFN-γ) and over-stimulation of host immune response.
• It results in s_ystemic inflammation_, with tissue destruction, vascular leakage, multiorgan failure, t_oxic shock._
• Some of the staphylococcal SAgs can also cause food poisoning (synergistic effect with staphylococcal enterotoxin (LPS)).*
• Toxic shock syndrome toxin (TSST) by S. aureus is the only SAg that can penetrate mucosal surfaces and is linked to menstrual toxic shock after the usage of high-absorbent tampons.*

Question 19

Superantigens are family of ___________ proteins (>20 members), produced by ________ and __________ They are non-specific, highly potent T cell mitogens (stimulate lots of different T cells).

Answer 19

Superantigens are family of heat-resistant secreted proteins (>20 members), produced by _Staph. aureu_s and Strep. pyogenes. They are non-specific, highly potent T cell mitogens (stimulate lots of different T cells).

• It triggers massive release of pro-inflammatory cytokines (TNF- α, IFN-γ) and over-stimulation of host immune response.
• It results in systemic inflammation, with tissue destruction, vascular leakage, multiorgan failure, toxic shock.

Some of the staphylococcal SAgs can also cause food poisoning (synergistic effect with staphylococcal enterotoxin (LPS)).

Toxic shock syndrome toxin (TSST) by S. aureus is the only SAg that can penetrate mucosal surfaces and is linked to menstrual toxic shock after the usage of high-absorbent tampons.

Question 20

How do you treat S.Aureus Infections?

Answer 20

Treatment Regime

Prolonged antibiotics treatment (weeks to months), such as PIC line, empirical treatment until organism is known:

• ~90% of S. aureus are resistant to penicillin (carry β-lactamase gene).
• ~30% of S. aureus are methicillin-resistant (MRSA) (methicillin is beta-lactamase-resistant penicillin, not used for treatment), and increasing resistance to other antibiotics
• Emergence of S. aureus that are vancomycin-resistant (VRSA)

Therefore, treatment usually requires the use of a beta-lactamase-resistant derivative of penicillin or clindamycin. In severe cases, combination with other antibiotics (e.g. gentamycin) is used.

In addition, _surgical debridemen_t can be considered for treatment.

Beta-Lactam Antibiotics

Beta-lactam antibiotics includes penicillins, cephalosporins and carbapenems.

• Pencillin works only against gram-positive bacteria. Extended spectrum penicillin includes amoxicillin.
• They irreversibly inhibit transpeptidase (i.e. penicillin binding protein PBP)

Resistance to Beta-Lactam Antibiotics

Beta-lactamases are plasmid encoded enzymes that can be transferred between bacterial strains and species.

• They include penicillinase, cephalosporinase, carbapenemase.
• They cleave beta-lactam ring, therefore inactivate antibiotics.

Beta-Lactam-Resistant Penicillins

Beta-lactam resistant penicillin prototype is methicillin (not for treatment as it is toxic). Clinical use includes oxacillin, nafcillin and flucloxacillin. They cannot be destroyed by beta-lactamase.

Or we can use penicillin combined with beta-lactamase inhibitor, e.g. augmentin (amoxicillin and clavulanic acid)

Resistance to Beta-Lactam-Resistant Penicillins

Since 1960s, there has been an increase of methicillin-resistant S. aureus strains (MRSA).

• Resistance is based on acquisition of a gene cassette that encodes for an alternative penicillin-binding protein (PBP-2a).
• _PBP-2a has low affinity for beta-lactam_s, e.g. penicillin and derivatives such as beta-lactamase-resistant penicillins. Therefore, it can still function to cross-link cell walls.
  
  • Not recognised by penicillin
  • so even flucloxacillin won’t work here.

Question 21

Name 3 B-lactam-resistent penicillins that are used clinically

Answer 21

Clinical use includes oxacillin, nafcillin and flucloxacillin.

They cannot be destroyed by beta-lactamase.

Question 22

What is the name of the drug which includes penecillin + B-lactamase inhibitor?

Answer 22

Or we can use penicillin combined with beta-lactamase inhibitor, e.g. augmentin (amoxicillin and clavulanic acid)

Question 23

What can you use on bacteria that are resistent to B-lactam antibiotics e.g. penecillin, cephalosporin, carbepenems

Answer 23

Beta-lactam resistant penicillin prototype is methicillin (not for treatment). Clinical use includes oxacillin, nafcillin and flucloxacillin. They cannot be destroyed by beta-lactamase.

Or we can use penicillin combined with beta-lactamase inhibitor, e.g. augmentin (amoxicillin and clavulanic acid)

Question 24

What are some diseases caused by Staphlococcus Aureus?

Answer 24

1) Osteomyletis
2) Impetigo (also can be caused by group A strep)
3) Folliculitis
4) Furuncle (boil)
5) Carbuncle
6) Cellulitis (often around an absess)
7) Septic arthritis
8) Acute infectious endocarditis
9) Bacteria pneumonia
10) Necrotising fascitis (flesh-eating disease)
11) Toxic Shock Syndrome (s.aureus can cause a different type of TSS)