Microbiology

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Mr D Raj
 Micro
• Not asked commonly
• Expect this to be hard and random
• Need to know!
• Will probably not get asked lots of this
• This is for info – so don’t have to look up

Mr D Raj
 Late at night – send of knee
aspirate?
• How is gram stain done?
• Smear thin film
• Fix in bunsen burner
• Add crystal violet
• Add iodine – fixes stain in cell wall
• Add decolouriser – acetone
• Add saffronin counterstain
• Look under slide
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 Principle
• Crystal violet stains everything purple
• Iodine fixes it, but only in G+ve
• Acteone washes it off G-ve
• Saffronin stains G-ve so it can be seen

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 Classify bacteria
• G+ve/-ve
• Bacilli – what do they look like? Rods
• Cocci – what do they look like? Grapes
• Aerobic/anaerobic – lives in the presence
or absence of free oxygen

Mr D Raj
 Name G +ve cocci
• Staph
• Strep

Mr D Raj
 Name G-ve cocci
• Neisseria meningitidis
• Neisseria gonorrhoeae

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 Name G+ve bacilli
• ANAEROBIC – Clostridia
(Perfringens/Tetani/Difficile/Botulinum)
• AEROBIC – Anthrax/Listeria/Diptheria

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 Name G-ve Bacilli
• Enterobacter (gut) Food poisioning
E. Coli Cholera
Proteus Shigella
Serratia Salmonella
Klebsiella Campylobacter
Pseudomonas

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Difference between G+ve and G-ve
• Gram stain
• G+ve produce exotoxin (e.g TSS). Has
thick cell wall which block violet escape
• G-ve has lipopolysaccharide in call wall
outer membrane. This produces G-ve
sepsis effects

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 Name types of staph
• Coag +ve
• Coag –ve: Does not produce plasma free
coagulase
• Name one: Staph epidermidis

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 What is the concern with CNS?
• Attach to and infect prosthetic materials
• THR
• Lines
• Catheter UTI

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 Types of strep
• Alpha haemolytic
• Viridans (endocarditis)
• Pnemoniae – pneumonia / otitis media

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 Types of strep
• Beta-haemolytic
• Gp A: Strep pyogenes – Rh F/ Scarlet
fever / cellulitis
• Gp D: Enterococci (strep faecalis)

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 Choose an antibiotic
• How does it work?
• Penicillin – beta lactam ring
• Prevents cell wall synthesis
• Bacterocidal

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 MRSA
• What does it stand for
• How is it resistant? Produces beta
lactamase

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 MRSA
• How is spread prevented?
• ADMISSION
• Screen admissions
• Isolate high risk admissions
• WARD and theatre
• Handwash+++++
• Hygeine

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 MRSA
• Eradicate if found on swabs
• 5 days intranasal mupirocin
• Chlorhexidine baths (on day of surgery of
receiving THR)
• Synergistic effect of these 2 drugs
• Swab again, and repeat but not too many times
• Discharge screening
• New drugs? Linezolid - myelotoxicity

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 What if outbreak occurs?
• Mutlidisciplinary meeting
• Micro
• Ward nurse
• Theatre nurse

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 Open fracture
• What Abx and why?
• Giannoudis
• I = 2nd generation cef
• II and III= 2nd generation cef and aminoglycoside
– why? G+VE and G-VE
• Farm/soil? Penicillin
• No data to show to give for >48-72 hours
• A&E cultures controversial(+ve in 65%, 7% get
infected)
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 Prior to closed # ORIF. When
should antibiotics be given?
• In anaesthetic room
• Concentration needs to be at Rx doses in
tissues at knife to skin
• How decide? Need to know half life
• Ceph – 30 mins
• Evidence for 3 doses? V little good
evidence – consensus=3

Mr D Raj
 Tetanus prophylaxis
• Clean wound (clean incised/graze/scald)
• No Immunity = needs full course of
vaccination
• Last 3 course dose >10 years = Single
vaccine injection
• Last 2 course dose within 10 years = nil

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 Tetanus
• Tetanus prone wound
(soil/puncture/open#/bite)
• No immunity = 3 dose Vaccinate + Ig
• >10 years = Single dose vaccinate + Ig
• <10 years = nil. Can give Ig if
contamination +++

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 Do you use an inside knife?
• Controversial
• Studies for and against
• Relatively cheap – while undecided then
play safe and use one

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 Tell me about how theatre design
prevents infection
• V – ventilation
• I – Illumination
• T – Temp
• A – Air contamination
• L – Location
• DESIGN

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 Tell me about how theatre design
prevents infection
• Outer = hospital
• Clean = Up to theatre door
• Aseptic = Theatre
• Disposal Zone = Sluice

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 Prevent THR infection
• Ultra-clean air theatre
• Lidwell 1982 – multicentre trial
• Showed air cleanliness proportional to deep joint
sepsis – 50% reduction
• Infection reduced by a further 25% with body
exhaust suits
• Combine these two with Abx- 0.06% infection
• Charnley showed Vertical laminar flow reduced
infection from 7.5% to 0.5%
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 Laminar flow in your theatre?
• We use Howarth Exflow as laminar flow v
expensive
• Air passed through HEPA filter in ceiling
• Vertical downflow in shape of inverted
trumpet
• 300 air changes/hour
• 10 CFU/m3

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 Gowns and drapes
• Disposable non-woven (gore-tex better but
expensive)
• Elasticated neck/sleeves/waist
• Open weave is comfortable and air
circulates
• BUT
• Pore size allows bacteria through
• Gets wet and allows strike through
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 Gloves
• All get perforated
• HAT – reduce contamination 15x in
laminar flow
• MASKS – 95% effective until damp
• Controversial, makes no difference in
general surgery / one use
• BUT orthopaedics – good practice

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 Skin prep
• 2% chlorhexidine
• 70% alcohol
• Allow to dry
• Equally effective

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 What organisms cause THR
sepsis?
• Staph aureus
• Staph epidermidis / CNS (up to 50%)
• Also beta haem strep – 13%
(Webb/Bannister)
• Enterococcus/Haem Inf 5%

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Acute Osteomyelitis - pathology
• Inflammation – rise in IO pressure
• Suppuration
• Pus forms within bone at 2-3 days and
appears as sub-periosteal abscess
• Necrosis – at 7 days get dead bone
(decreased b.s. and periosteal stripping)
• New bone – 10-14 days – get sequestrum
• Resolution or chronic disease
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 X-ray changes
• Normal for 10 days
• Demineralisation 10-14 days
• Sequestrum / new bone formation after

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 Organisms
• ALL = STAPH AUREUS
• Infant = E.Coli / Gp B strep
• Young = H. Inf/ Strep pyogenes
• Sickle = Salmonella

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Cierny and Mader classification of
chronic osteomyelitis

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 TB
• Spread – inhaled – can be oral (milk)
• Get primary TB – respiratory infection
• Sub-pleural Ghon focus
• Resolves
• Can spread to
bones/lungs/joints/meninges/miliary
• Post-primary TB later in life – reactivation
or repeat exposure
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 Microscopy
• Zieh-Neelsen stain
• Appear and red acid fast organisms
• Culture in Lowenstein-Jensen method for
6 weeks
• Granuloma on histology – caseating
necrosis centrally, lymphocytes, epitheloid
macrophages and multinucleate giant cells
around
• 4 for 2 and 2 for 4
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 Draw the serum levels of Hep B
antigens and antibodies after acute
infection

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 Hep B
• Get infected – get HB surface Ag (HBsAg)
in 1st 6/12
• Get antibodies to HBsAg when immune rg
sign of previous infection or
VACCINATION
• If HBsAg persists (>6/12) then CARRIER
• If anti-e Abs present then not so bad
• If e antigen persists then very infective
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• Polio
• TB
• Hep B

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 Clostridium Difficile
• Clostridium difficile is the major cause of antibiotic-associated diarrhoea
and colitis, a healthcare associated intestinal infection that mostly
affects elderly patients with other underlying diseases.
• C. difficile is a bacterium of the family Clostridium (the family also includes
the bacteria that cause tetanus, botulism, and gas gangrene). It is an
anaerobic bacterium (i.e. it does not grow in the presence of oxygen) and
produces spores that can survive for a long time in the environment.
• Its usual habitat is the large intestine, where there is very little oxygen. It can
be found in low numbers in a small proportion (less than 5%) of the healthy
adult population. It is kept in check by the normal, 'good' bacterial population
of the intestine. It is common in the intestine of babies and infants, but does
not cause disease because its toxins (poisons) do not damage their
immature intestinal cells.

Mr D Raj
 Clostridium Difficile
• What does it cause?
• C. difficile can cause diarrhoea, ranging from a mild disturbance to a very
severe illness with ulceration and bleeding from the colon (colitis) and, at
worst, perforation of the intestine leading to peritonitis. It can be fatal.
• Generally, it is only able to do this when the normal, healthy intestinal
bacteria have been killed off by antibiotics. When not held back by the
normal bacteria, it multiplies in the intestine and produces two toxins (A and
B) that damage the cells lining the intestine. The result is diarrhoea.

Mr D Raj
 Clostridium Difficile
• How does it spread?
• Although some people can be healthy carriers of C. difficile, in most cases
the disease develops after cross infection from another patient, either
through direct patient to patient contact, via healthcare staff, or via a
contaminated environment. A patient who has C. difficile diarrhoea
excretes large numbers of the spores in their liquid faeces. These can
contaminate the general environment around the patient's bed (including
surfaces, keypads, equipment), the toilet areas, sluices, commodes, bed
pan washers, etc. They can survive for a long time and be a source of hand-
to-mouth infection for others. If these others have also been given
antibiotics, they are at risk of C. difficile disease.

Mr D Raj
 Clostridium Difficile

• How is it diagnosed?
• A sample of diarrhoeal faeces is tested for the presence of the C. difficile
toxins. This is the main diagnostic test and gives a result within a few
hours. In outbreaks, or for surveillance of the different strains circulating in
the population, C. difficile can be cultured from faeces and the isolates sent
to the Anaerobe Reference Laboratory (National Public Health Service,
Wales; Microbiology, Cardiff) for typing and testing for susceptibility to
antibiotics.

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 Clostridium Difficile
• How common is it?
• When C. difficile was first recognised to cause antibiotic-associated
diarrhoea and colitis in the late 1970s, laboratory diagnosis was difficult and
the number of cases was not monitored. Since 1990 laboratories have
reported the number of cases diagnosed to the Health Protection Agency in
a voluntary system. The number of reports increased from less than 1,000
in the early 1990s to 22,000 in 2002, 28,000 in 2003 and 44,488 in 2004.
Some of this was due to improved diagnostic tests and improved reporting
by laboratories, but there has clearly been a very significant increase in the
number of cases. Since January 2004, C. difficile has been part of the
mandatory surveillance programme for healthcare associated infections.

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 Clostridium Difficile
• What is type 027 and why is it of concern?
• The typing system analyses part of the C. difficile DNA (chromosome) in a
test called ribotyping. Over 100 types have been identified. Type 027 was
rare in the UK; the first isolate was identified in 1999 and the second in
2002. Individual isolates were identified in 2003-05.When outbreaks at
Stoke Mandeville and the Royal Devon and Exeter Hospitals were
investigated in 2004-05, Type 027 was found to predominate in their cases.
The same type has caused a large outbreak of severe disease in hospitals
in Canada (Quebec) and North-eastern USA since 2000. Type 027
produces much more of the toxins than most other types because a
mutation has knocked out the gene that normally restricts toxin production.
It causes a greater proportion of severe disease and appears to have a
higher mortality. It also seems to be very capable of spreading between
patients.

Mr D Raj
 Clostridium Difficile
Prevention and control
There are three important components to the prevention
and control of C. difficile disease:
1. prudent antibiotic prescribing to reduce the use of
broad spectrum antibiotics

2. isolation of patients with C. difficile diarrhoea and good

infection control nursing- handwashing (not relying solely
on alcohol gel as this does not kill the spores), wearing
gloves and aprons, especially when dealing with bed
pans etc
3. enhanced environmental cleaning and use of a
chlorine containing disinfectant where there are cases of
C. difficile disease to reduce environmental
contamination with the spores.
Mr D Raj
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