internal,medicine,RA,biotechnoogy,sepsis

1. Sepsis: principles of
pathophysiology and animal models
Corso di Medicina Interna
Dott. Luigi Mario Castello

2. EPIDEMIOLOGY
The incidences of severe sepsis and septic shock are rising worldwide due to:
• aging of population
• survival increase of patients with chronic conditions
• immunosuppressive therapy
• implantation of vascular catheters, PM, defibrillators, prosthetic devices
• HIV infection and malnutrition (especially in poor countries)
Sepsis causes more than 200.000 deaths per year in US
In about 2/3 of cases sepsis affects subjects with chronic diseases
Advanced age and the presence of pre-existing conditions adversely affect the
prognosis.

3. INFECTION
Penetration and proliferation of microorganisms in a tissue.
The presence of microorganisms can be confirmed with a culture of
a biological fluid sample (urine, sputum, wound exudate)
BACTEREMIA
Presence of bacteria in the bloodstream.
Bacteremia can be confirmed performing a blood culture which
allows to identify the bacterial strain and to perform antibiogram.

4. EUCAST OBJECTIVES
www.eucast.org
• To form in EUCAST, under the auspices of the European Society of Clinical
Microbiology and Infectious Diseases (ESCMID) and the European Centre for
Disease Prevention and Control (ECDC), a network of established experts in the
determination of antimicrobial breakpoints and in antimicrobial susceptibility testing.
• To determine, review and revise European clinical breakpoints and epidemiological
cut-off values for surveillance of antimicrobial resistance in close collaboration with
the European Medicines Agency (EMA) and ECDC.
• To promote the development and standardization of in-vitro antimicrobial
susceptibility testing methods used in Europe.
• To promote quality assurance of in-vitro antimicrobial susceptibility testing.
• To promote education and training in antimicrobial susceptibility testing.
• To advise ECDC and other European Union health agencies on issues related to
antimicrobial susceptibility testing and detection of resistance determinants relevant to
public health.
• To collaborate with international groups, ECDC and other European Union health
agencies involved in antimicrobial susceptibility testing and/or the epidemiology of
antimicrobial resistance in human pathogens.
• To work towards international consensus and harmonization of clinical breakpoints and
antimicrobial susceptibility testing.

5. ANTIBIOGRAM
Definition: in vitro antimicrobial susceptibility test
Semi-quantitative method: based on
diffusion (Kirby-Bauer method); small
discs containing different antibiotics, are
dropped in different zones of the culture
on an agar plate. The antibiotic will
diffuse in the area surrounding each
tablet, and a disc of bacterial lysis will
become visible. Since the concentration
of the antibiotic was the highest at the
centre, and the lowest at the edge of this
zone, the diameter is suggestive for the
Minimum Inhibitory Concentration, or
MIC, (conversion of the diameter in
millimeter to the MIC, in µg/ml, is
based on known linear regression
curves).

6. Quantitative method: is based on dilution; a dilution series of
antibiotics is established (this is a series of reaction vials with
progressively lower concentrations of the tested antibiotic). The
last vial in which no bacteria grow contains the antibiotic at the
Minimal Inhibiting Concentration (MIC).
This is the reference method for antimicrobial susceptibility
testing.
It rely on incremental dilution of the antimicrobial agents to
determine the minimum inhibitory concentrations (MICs)
ANTIBIOGRAM

7. ANTIBIOGRAM: DILUTION METHOD

8. Epidemiological
cut-off
Clinical
breakpoints
%
microorganism

9. Epidemiological
cut-off
EUCAST: EPIDEMIOLOGICAL CUT-OFF

10. EUCAST: CLINICAL BREAKPOINTS

11. MICs are expressed in mg/L or mg/mL
Epidemiological cut-off: a micro-organism is defined as wild type (WT) for a
species by the absence of acquired and mutational resistance mechanisms to the
drug in question
MICs values allows to define the isolated bacterial strain as sensitive, intermediate
or resistant to the tested antibiotic
Resistant: poor or no probability to successfully treat the infection with the tested
antibiotic
Intermediate: uncertain probability to successfully treat the infection with the
tested antibiotic
Sensitive: good probability to successfully treat the infection with the tested
antibiotic
CLINICAL USE OF ANTIBIOGRAM
RESULTS

12. • SIRS (systemic inflammatory response syndrome) is
characterized by at least two of the following:
– fever (T > 38 °C) or hypothermia (T < 36 °C)
– tachypnea (RR > 24 min-1 or PaCO2 < 32 mm Hg)
– tachycardia (HR > 90 min-1)
– Leukocytosis (WBC > 12,000 mL-1) or leukopenia
(WBC < 4.000 mL-1)
• Sepsis: SIRS with an infectious cause
DEFINITIONS

14. • Severe sepsis: sepsis with signs of organ
damage:
– Hypotension
– Oliguria (urine output < 500 mL/24 hours)
– Respiratory failure
– Low platlets count
– Severe metabolic acidosis (mainly due to lactic
acid production)
DEFINITIONS

15. Septic shock:
Sepsis with hypotension requiring fluid resuscitascion and
vasopressor administration (dopamine, epinephrine or
norepinephrine).
Multiple organ dysfunction syndrome (MODS):
Loss of function of at least two organs in an acutely ill
patient requiring a prompt medical intervention to achieve
homeostasis.
DEFINITIONS

16. RISK STRATIFICATION
Severe
sepsis
Localized
infection
Death
Fever, increase of HR and RR
Systemic
response
Organ damage
Hypotension and Shock
Septic
shockCLINICAL
DETERIORATION

17. INFECTIONS
PATHOGEN IMMUNE SYSTEM
CLINICAL MANIFESTATION
Toxines Cytokines
Other inflammatory
mediators

18. EARLY PHASE
LATE PHASE
PHASES OF SEPSIS

19. EARLY PHASE
Hyperdynamic phase:
• Low systemic vascular resistance
• Initial increase in cardiac output
• Sepsis progression leads to a gradual reduction
of CO without changes of peripheral vascular
resistance
• Haemodynamic derangement

20. LATE PHASE
HYPODINAMIC PHASE
• Low Cardiac Output (CO)
• Low Vascular Resistences
MULTI ORGAN DISFUNCTION
SYNDROME
(due to hypoperfusion with low oxygen delivery to tissues)

21. Imuune response to the lipid A
moiety of lipopolysaccharide
(LPS, also called endotoxin).
A host protein (LPS-binding protein)
binds lipid A and transfers the LPS
to CD14 on the surfaces of
monocytes, macrophages, and
neutrophils. LPS then is passed to
MD-2, that is bound to toll-like
receptor (TLR) 4 to form a
molecular complex that transduces
the LPS recognition signal into the
cell.
This signal rapidly triggers the
production and release of mediators,
such as TNF, that amplify the LPS
signal and transmit it to other cells
and tissues.
Bacterial peptidoglycan and
lipopeptides interact with different
TLRs. 11 different TLR-based
receptor complexes allows animals
to recognize many conserved
microbial molecules.

22. Imbalance between:
• Proinflammatory agents (prevailing)
• Anti-inflammatory agents (decreasing)
This imbalance leads to the onset of the systemic
inflammatory response syndrome (SIRS) while
the compensatory anti inflammatory response
(CARS) is inhibited.
PHATOPHYSIOLOGY OF SEPSIS

24. Recognition of microbial
molecules by tissue
phagocytes
cytokines,
chemokines,
prostanoids,
leukotrienes
increase blood
flow to the
infected tissue
enhance the
permeability of local
blood vessels
recruit neutrophils
to the site of
infection
elicit pain
Systemic responses
are activated by neural
and/or humoral
communication with
the hypothalamus and
brainstem
enhance local defenses by increasing blood
flow to the infected area
Increase the number of circulating neutrophils
elevating blood levels of numerous molecules

25. TNF-  stimulates leukocytes and vascular
endothelial cells
Cell-surface molecules that
enhance neutrophil-
endothelial adhesion at sites
of infection
increase prostaglandin and
leukotriene production
IL-8 and IL-17 attract circulating neutrophils to the infection site
TNF- , IL-1β, IFN  ,
IL-12, IL-17
interact synergistically with one another and
with additional mediators
Endothelial cells and
leukocytes
nitric oxide (NO)
by
activated VEC
vasodilation
prostaglandin I2
thromboxane A2

26. ANIMAL MODELS FOR THE STUDY OF
OF SEPSIS

27. STRATEGIES
• TOXIN ADMINISTRATION (LPS)
• INOCULUM OF BACTERIA
• ALTERATION OF A PROTECTIVE BARRIER
(translocation of faecal bacteria from the colon)

28. TOXIN ADMINISTRATION
• The administration of Toll Like Receptor (TLR)
agonists induces septic shock in mice.
• The model in simple.
• Low dose LPS administration is the most commonly
used approach

29. LIMITS
• The evolution of the shock is very quick
• Some phases observed in humans are lacking
• LPS injection may induce the hypodynamic phase of
the shock immediately; the hyperdynamic phase is
skipped.
• A continuous infusion of low dose LPS can be used
to induce the hyperdynamic phase but this approach
is difficult to manage in mice.

30. EXOGENOUS ADMINISTRATION OF
INFECTIOUS AGENTS
Monomicrobial or polymircobial infections
Sites for the inoculum:
• Bloodstream (intreavenous)
• Peritoneum
• Airways

31. LIMITS AND CONFOUNDING FACTORS
• The administration of high doses of bacteria often
lead to a model of intoxication, characterized by a
rapid complement mediated lysis, rather than to a
reliable model of infection (toxicosis response)
• Different bacterial loads can induce different
responses
• The bacteria virulence can affect the results
• Different host response depending on the injection
site
• Variability linked to the infusion time of the bacteria

33. ALTERATION OF A PROTECTIVE
BARRIER

34. CAECAL LIGATION AND PUNCTURE
(CLP)
The CLP model consists of the perforation of the cecum allowing the release of fecal
material into the peritoneal cavity to generate an exacerbated immune response
induced by polymicrobial infection.
This model reproduces the most important clinical conditions observed in humans. As
in humans, mice that undergo CLP with fluid resuscitation show the first (early)
hyperdynamic phase that progresses to the second (late) hypodynamic phase.
The cytokine profile is similar to that seen in human sepsis where there is increased
lymphocyte apoptosis.
Due to the multiple and overlapping mechanisms involved in sepsis, researchers need
a suitable sepsis model of controlled severity in order to obtain consistent and
reproducible results.

35. METHOD
• Anesthetize the mouse, shave the abdomen and disinfect area
• Under aseptic conditions, practice a 1 to 2 cm midline laparotomy and expose the cecum with
adjoining intestine
• The cecum is tightly ligated with a silk suture at its base below the ileo-cecal valve, and is
perforated once or twice with a 19-gauge needle. A distance of >1cm produces high grade sepsis
while a distance of ≤1 cm produces mid-to-low grade sepsis.
• The cecum is then gently squeezed to extrude a small amount of feces from the perforation sites.
The cecum is returned to the peritoneal cavity and the peritoneum and the skin are closed with 6.0
silk sutures.
• Resuscitate mice by injecting subcutaneously 1 ml of pre-warmed 0.9% saline solution using a 25G
needle. This fluid resuscitation measure will induce the hyperdynamic phase of sepsis.
• Inject subcutaneously buprenorphine (0.05mg/ kg body weight) or tramadol (20mg/kg body
weight) for post operative analgesia.
• The animals are placed temporarily on a heating pad or alternatively returned immediately to a
cage with exposure to an infrared heating lamp of 150W until they recover from the anesthesia. The
recovery time is from 30 min to 1 hour.
• Provide free access to food and water (hydrogel) placed on the bottom of the cage.
• Mice are monitored every 12 hours for survival for one to two weeks or euthanized at different
time points for analysis of different parameters.

38. MAIN ADVANTAGES OF CLP
The technique is relatively simple and cheap
The model reproduces reliably the different phases of sepsis
The degree of severity may be regulated (length of ligated cecum, diameter
of the needle used and number of holes)
The model can be used for different experimental settings (pathophysiology,
biomarkers, drugs efficacy, survival studies).
The sacrifice of mice may be performed after a few hours up to a maximum
of 28 days
Blood and different tissue samples may be obtained to study proteins, gene
activation, signal transduction, organ damage…

39. LIMITS OF CLP
In some cases the infection may be confined with the formation
of pericecal abscesses
The model is characterized by a certain degree of variability that
is not easy to control (quantity of feces extruded from the
perforation site)
A large number of variables are involved (anesthesia, fluid
resuscitation, surgical complications)

40. COLON ASCENDENS
STENT PERITONITIS
CASP model shows some differences with respect to CLP
The main difference is the insertion of a stent with defined diameter into the
ascending colon.
This lead to a constant passage of feces from the colon to peritoneal cavity
avoiding abscess formation (the infection can’t be limited).

41. METHOD
The ascending colon is exposed and a catheter (16- gauge) is
inserted through the antimesenteric wall into the lumen of the
ascending colon and is fixed with two sutures (7/0 Ethilon
thread).
The inner needle of the stent was removed and the stent was
cut 2 mm above the puncture site.
To ensure proper intraluminal positioning of the stent, stool
was milked from the cecum into the stent until a small
amount appeared.
Stent is then removed 3-9 hours after the implant

43. CASP VS CLP
The CASP model mimics closely the clinical course of
diffuse peritonitis with early and steadily increasing systemic
infection and inflammation (systemic inflammatory response
syndrome).
CLP reveals a model of intra-abdominal abscess formation
with sustained and minor signs of systemic inflammation.