DIAGNOSIS OF SEPTICEMIA

Causative organisms:

(Mention those bacteria that you are well-conversant with)

Gram negative cocci – meningococci, gonococci, hemophilus
Gram negative bacilli – Salmonella typhi, EIEC, almost all causes of UTI, Infective endocarditis (including HACEK organisms), meningitis.
Gram positive organisms (rare) – Streptococcus, Staphylococcus, Pneumococcus, almost all organisms that cause osteomyelitis and pneumonia.

Material for diagnosis:

Venous blood – 3 samples at 0, 30 and 90 min intervals from admission
Urine
CSF (if features of meningeal irritation are present)
Swab from suspected skin lesion


Steps of diagnosis:
Exams and Tests

Physical examination may show:

Low blood pressure
Low body temperature or fever
Signs of associated disease (such as meningitis, epiglottitis, pneumonia, or cellulitis)

Tests that can confirm infection include:
Blood culture
Urine culture
CSF culture
Culture of any suspect skin lesion
CBC
Platelet count
Clotting studies
PT
PTT
Fibrinogen levels
Blood gases

BLOOD CULTURE
A minimum of 10 ml of blood is taken through venipuncture and injected into two or more "blood bottles" with specific media for aerobic and anaerobic organisms.
Care needs to be taken that the bottles are not contaminated with bacteria from staff members or other patients. To that end, the patient's skin is rubbed or sprayed with denaturated alcohol and betadine applied to the sampling site. Sterile gloves should be used to minimize contamination.
To maximise the diagnostic yield of blood cultures multiple sets of cultures (each set consisting of aerobic & anaerobic vials filled with 3-10 mL) may be ordered by medical staff.
Set 1= L. antecubital fossa at 0 minutes
Set 2= R. antecubital fossa at 30 minutes
Set 3= L. or R. antecubital fossa at 90 minutes
Ordering multiple sets of cultures increases the probability of discovering a pathogenic organism in the blood and reduces the probability of having a positive culture due to skin contaminants.
After inoculating the culture vials on the hospital floor, they are sent to the microbiology lab clinical pathology department. Here the cultures are entered into a blood culture machine, which keeps the samples at body temperature. The blood culture instrument reports positive blood cultures (cultures with bacteria present, thus indicating the patient is "septic") by monitoring carbon dioxide levels produced by bacteria in the vials via fluorescence detected by a light emitting diode (LED). Most cultures are monitored for 5-days, after which, if the vials are negative, they are removed.
If a vial is positive, a microbiologist will perform a Gram Stain on the blood for a rapid, general ID of the bacteria, which they will report to the attending physician of the septic patient. The blood is also subcultured or "Subed" onto agar plates to isolate the pathogenic organism for culture and suceptability testing, which takes up to 3 days time. This culture & sensitivity (C&S) process IDs the species of bacteria. Antibiotic sensitivities are then assessed on the isolate to inform clinicians on appropriate antibiotics for treatment.
Some guidelines for infective endocarditis recommend taking up to 6 sets of blood for culture (around 60 ml).

URINE EXAMINATION:
(Summarize the Laboratory diagnosis for UTI)
CSF EXAMINATION:
(Summarize the Laboratory diagnosis of Meningitis)

ROUTINE EXAMINATIONS
CBC - may show neutrophilia (enteric fever may show neutropenia)
Platelet count - may decrease following DIC
Clotting studies
PT - may rise following DIC r hepatocellular failure
PTT - may rise in DIC
Fibrinogen levels – may be low in DIC
FDP – may rise in DIC
Blood gases – may be abnormal in respiratory failure (raised pCO2 and low pO2)

CONCLUSION:
Based on the above investigations we can reach a diagnosis of septicemia.

Mock test on Bacteriology (General and Systemic)

MICROBIOLOGY (BACTERIOLOGY) MOCK TEST

Full marks 40 Time – 2 hours

Attempt all questions. Supplement your answer with charts and diagrams as needed.

1. A middle aged lady attended the skin OPD with complaints of hypopigmented patch over the right elbow with progressive loss of sensation. What is your provisional diagnosis? How would you proceed to confirm the clinical diagnosis in the laboratory? How is the efficacy of treatment evaluated in such cases? [1 + 7 + 2]

Or, A middle aged person attended the STD clinic with a solitary painless ulcer with hard base on the glans penis. What is the commonest organism that causes such a lesion? What are the methods of isolation and demonstration of the organism in the laboratory? Discuss the role of serological tests in diagnosing late stages of the disease. [1 + 4 + 5]

Or, Name the bacteria responsible for diarrheal diseases in man. How would you proceed to investigate a case of bacterial diarrhea in the laboratory if you are supplied with the fresh fecal matter of the infected individual collected in a sterile plastic bottle? [2 + 8]

2. Write short notes on any THREE: [3 x 4 = 12]

a) Acid fast stain OR Bacterial growth curve
b) Plasmids OR Bacterial flagella
c) El Tor Vibrio OR Shiga toxin producing E. coli
d) Importance of ASO titer OR Interpretation of Mantoux test

3. Comment on any THREE: [3 x 4 = 12]

a) The choice of specimen for diagnosis depends on the stage of Enteric fever.
b) Koch’s postulates have many exceptions.
c) Morphology of the colonies of motile bacteria depends on the concentration of agar agar in Nutrient agar medium.
d) Bacteriological profile of purulent meningitis varies with the age of the patient.
e) Slide and tube coagulase tests are based on different biological principles.
f) Both chromosomal and extra-chromosomal factors are involved in the origin of bacterial drug resistance.

4. Differentiate between any THREE: [3 x 2 = 6]

a) Staphylococcus aureus and S. saprophyticus
b) Autoclaving and Tyndallisation
c) Streptococcus pneumoniae and Streptococcus pyogenes
d) Chlamydia and bacteria
e) Blood agar and chocolate agar

Microbiology: An overview of Bacterial Toxins

This chapter will help you to

• Define exotoxins and Endotoxins


• Differentiate between exotoxins and endotoxins


• Classify exotoxins according to mechanism of action


• Write short notes on: Pore forming toxins, Enzymatic toxins, Superantigens, Proteolytic toxins, Toxins acting on second messenger systems


• Write short notes on: Lipid A, Lipopolysaccharide (LPS), Lipooligosaccharide (LOS), O-antigen


• Discuss the pathogenesis of endotoxic shock
  

Ťoxigenesis, or the ability to produce toxins, is a mechanism by which many bacteria cause disease. There are two main types of bacterial toxins, lipopolysaccharides, which are associated with the outer membrane of the cell wall of Gram-negative bacteria, and proteins, which are released from bacterial cells. The cell-associated toxins are referred to as endotoxins and the extracellular diffusible toxins are referred to as exotoxins.
Endotoxins are cell-associated substances that are structural components of bacteria. Chemically, endotoxin refers to the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) located in the outer membrane of Gram-negative bacteria.
Exotoxins are usually secreted by bacteria. However, in some cases, exotoxins are released by lysis of the bacterial cell. Exotoxins are usually proteins or polypeptides, that act enzymatically or through direct action on host cells and stimulate a variety of host responses.

Endotoxins and Exotoxins vary greatly in structure and function

Endotoxin is a molecular complex of lipid and polysaccharide; hence, the alternate name lipopolysaccharide. The complex is secured to the outer membrane by ionic and hydrophobic forces, and its strong negative charge is neutralized by Ca2+ and Mg2+ ions.

Members of the family Enterobacteriaceae exhibit O-specific chains of various lengths, whereas N gonorrhoeae, N meningitidis, and B pertussis contain only core polysaccharide and lipid A. Some investigators working on the latter forms of endotoxin prefer to call them lipooligosaccharides to emphasize the chemical difference from the endotoxin of the enteric bacilli. Nevertheless, the biologic activities of all endotoxin preparations are essentially the same, with some being more potent than others.

Exotoxins, unlike the lipopolysaccharide endotoxin, are protein toxins released from viable bacteria. They form a class of poisons that is among the most potent, per unit weight, of all toxic substances. Most of the higher molecular-sized exotoxin proteins are heat labile; however, numerous low molecular-sized exotoxins are heat-stable peptides. Unlike endotoxin, which is a structural component of all Gram-negative cells, exotoxins are produced by some members of both Gram-positive and Gram-negative genera. The functions of these exotoxins for the bacteria are usually unknown, and the genes for most can be deleted with no noticeable effect on bacterial growth. In contrast to the extensive systemic and immune-system effects of endotoxin on the host, the site of action of most exotoxins is more localized and is confined to particular cell types or cell receptors. Tetanus toxin, for example, affects only internuncial neurons. In general, exotoxins are excellent antigens that elicit specific antibodies called antitoxins. Not all antibodies to exotoxins are protective, but some react with important binding sites or enzymatic sites on the exotoxin, resulting in complete inhibition of the toxic activity (i.e., neutralization). Exotoxins can be made into toxoids, which can be used for active immunisation.

Bacterial Exotoxins

Bacterial exotoxins can be classified into 5 classes according to their mechanisms of action:

1. Toxins that damage membranes


2. Toxins that act as enzymes


3. Toxins that activate second messengers


4. Toxins that activate immune response


5. Toxins that act as protease

A) Toxins that damage membranes

These toxins get inserted into the membrane and form trans-membrane pores. This leads to influx of water by endosmosis, resulting in cellular swelling and lysis.

Examples:

Table 2

• Perfringiolysin O from Clostridium perfringens attach to cell membrane cholesterol and atke part in the pathogenesis of gas gangrene


• Alpha toxin from Staphylococcus aureus targets cell membrane and causes abscesses


• Pneumolysin from Streptococcus pneumoniae target cell membrane cholesterol and cause pneumonia


• Hemolysin from Escherichia coli damage renal tubular cell membrane and cause UTI


• Streptolysin O from Streptococcus pyogenes taget membrane cholesterol and is implicated in Sore throat
  
  B) Toxins that act as enzymes
  
  These toxins have an “A plus B Subunit” arrangement. Subunit A acts as the catalyst (active subunit) and subunit B helps in binding (binding subunit). Occasionally a translocation subunit (T subunit) may be present that helps “A subunit” to reach the target substrate in the cell. They ultimately catalyse some reactions in the cell, which inactivates the cellular protein synthetic apparatus.
  

Figure 1: Ribbon structure of Diphtheria toxin

Classical example of this type of toxin is the Diphtheria toxin. This toxin splits NAD into ADP-Ribose and Nicotinamide. The ADP-Ribose (ADPR) attaches to a regulatory protein called EF 2 that helps in protein synthesis, and covalently modifies it. This inactivates EF 2 and inhibits protein synthesis in the host cell, leading to cell death.

C) Toxins that activate second messengers

These toxins bind to cell surface receptors and act like hormones to activate one or more second messenger pathways in the cell. This subsequently causes changes in the cellular functions, leading to cell death.

Examples of toxins of this class are as follows:

Table 3

• E. coli secretes a heat-labile toxin that increases cellular ADP-ribosyl transferase and a heat-stable toxin that stimulates guanylate cyclase causing raised cyclic nucleotide levels in the intestinal cells. this leads to diarrhea.


• Bacillus anthracis, that causes Anthrax, secretes an edema factor which stimulates Adenylate cyclase


• Bordetella pertussis (causes whooping cough) liberates the pertussis toxin that acts via ADP-ribosyl transferase

D) Toxins that activate immune response

Several bacterial toxins can act directly on the T cells and antigen-presenting cells of the immune system, and stimulate them non-specifically. Impairment of the immunologic functions of these cells by toxin can lead to human disease. These antigens belong to a class of molecules called superantigens.


Figure 2: Schematic representation of a superantigen

Normally an antigen binds at the antigen-binding-groove (ABG) of the T cell receptor (TCR). Superantigens bind outside the ABG and causes widespread nonspecific stimulation of the T cells, leading to immunological dysfunction.

Examples of super-antigenic toxins are as follows:

Table 4

• Staph. aureus secretes enterotoxins that is implicated in Food poisoning, and exfoliative toxins that causes Staphylococcal Scalded Skin Syndrome


• toxic-shock syndrome toxin from Staphylococcus binds TCR and MHC II and causes Toxic Shock Syndrome


• Strep. pyogenes elaborates Pyrogenic exotoxins that cause Scarlet fever

E) Toxins that act as protease

These toxins cleave various regulatory proteins of the cell and impair cellular function. Examples of this group of toxins include:

Table 5

• B. anthracis secretes a Lethal factor which is a Metalloprotease


• C. botulinum secretes the Neurotoxins, A-G which are Zinc-metalloprotease in nature


• Clostridium tetani releases the tetanus toxin which act as Zinc-metalloprotease and cleaves neuronal regulatory proteins like Synaptobrevin.
  
  
  Summary of Exotoxins:
  


• Exotoxins are protein in nature


• They are actively secreted by the bacteria


• They can act by damaging membranes, Inhibiting protein synthesis, Activating second messengers, Activating immune response & as Proteases


• They can be made into toxoids
  
  
  
  Bacterial Endotoxins
  
  Bacterial endotoxins are characterized by the following features:


• Part of the outer membrane of the cell wall of Gram-negative bacteria


• Invariably associated with Gram-negative bacteria whether the organisms are pathogenic or not


• Although the term "endotoxin" is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is reserved to refer to the LPS complex
  
  
  Chemical nature of Endotoxin
  
  
  Figure 1: Structure of endotoxin

LPS consists of three components or regions: Lipid A, an R polysaccharide and an O polysaccharide.

• Region I. Lipid A is the lipid component of LPS. It contains the hydrophobic, membrane-anchoring region of LPS. Lipid A consists of a phosphorylated N-acetylglucosamine (NAG) dimer with 6 or 7 fatty acids (FA) attached. Usually 6 FA are found. All FA in Lipid A are saturated. Some FA are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present. The structure of Lipid A is highly conserved among Gram-negative bacteria. Among Enterobacteriaceae Lipid A is virtually constant.

Figure 2: Structure of Lipid A

The primary structure of Lipid A has been elucidated and Lipid A has been chemically synthesized. Its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide, the PO4 groups, the acyl chains, and also the KDO-containing inner core.

Region II. Core (R) antigen or R polysaccharide is attached to the 6 position of one NAG. The R antigen consists of a short chain of sugars. For example: KDO - Hep - Hep - Glu - Gal - Glu - GluNAc -
Two unusual sugars, heptose and 2-keto-3-deoxyoctonoic acid (KDO), are usually present, in the core polysaccharide. KDO is unique and invariably present in LPS and so it has been used as an indicator in assays for LPS (endotoxin).
With minor variations, the core polysaccharide is common to all members of a bacterial genus (e.g. Salmonella), but it is structurally distinct in other genera of Gram-negative bacteria. Salmonella, Shigella and Escherichia have similar but not identical cores.

Region III. Somatic (O) antigen or O polysaccharide is attached to the core polysaccharide. It consists of repeating oligosaccharide subunits made up of 3 - 5 sugars. The individual chains vary in length ranging up to 40 repeat units. The O polysaccharide is much longer than the core polysaccharide, and it maintains the hydrophilic domain of the LPS molecule. A major antigenic determinant (antibody-combining site) of the Gram-negative cell wall resides in the O polysaccharide.
Great variation occurs in the composition of the sugars in the O side chain between species and even strains of Gram-negative bacteria. At least 20 different sugars are known to occur and many of these sugars are characteristically unique di-deoxy-hexoses, which occur in nature only in Gram-negative cell walls. Variations in sugar content of the O polysaccharide contribute to the wide variety of antigenic types of Salmonella and E. coli and presumably other strains of Gram-negative species. Particular sugars in the structure, especially the terminal ones, confer immunological specificity of the O antigen, in addition to "smoothness" (colony morphology) of the strain. Loss of the O specific region by mutation results in the strain becoming a "rough" (colony morphology) or R strain.

LPS and virulence of Gram-negative Bacteria

Both Lipid A (the toxic component of LPS) and the polysaccharide side chains (the nontoxic but immunogenic portion of LPS) act as determinants of virulence in Gram-negative bacteria.

The O polysaccharide and virulence
Virulence, and the property of "smoothness", is associated with an intact O polysaccharide. The involvement of the polysaccharide chain in virulence is shown by the fact that small changes in the sugar sequences in the side chains of LPS result in major changes in virulence. There are several postulated mechanisms of virulence:

1. O-specific antigens could allow organisms to adhere specifically to certain tissues, especially epithelial tissues.
2. Smooth antigens probably allow resistance to phagocytes, since rough mutants are more readily engulfed and destroyed by phagocytes.
3. The hydrophilic O polysaccharides could act as water-solubilizing carriers for toxic Lipid A. It is known that the exact structure of the polysaccharide can greatly influence water binding capacity at the cell surface.
4. The O antigens could provide protection from damaging reactions with antibody and complement. Rough strains of Gram-negative bacteria derived from virulent strains are generally non virulent. Smooth strains have polysaccharide "whiskers" which bear O antigens projecting from the cell surface. The O antigens are the key targets for the action of host antibody and complement, but when the reaction takes place at the tips of the polysaccharide chains, a significant distance external to the general bacterial cell surface, complement fails to have its normal lytic effect. Such bacteria are virulent because of this resistance to immune forces of the host. If the projecting polysaccharide chains are shortened or removed, antibody reacts with antigens on the general bacterial surface, or very close to it, and complement can lyse the bacteria. This contributes to the loss of virulence in "rough" colonial strains.
5. The O-polysaccharide or O antigen is the basis of antigenic variation among many important Gram-negative pathogens including E. coli, Salmonella and Vibrio cholerae. Antigenic variation guarantees the existence of multiple serotypes of the bacterium, so that it is afforded multiple opportunities to infect its host if it can bypass the immune response against a different serotype. Furthermore, even though the O polysaccharides are strong antigens, they seldom elicit immune responses which give full protection to the host against secondary challenge with specific endotoxin.

Lipid A and Endotoxic shock
The physiological activities of LPS are mediated mainly by the Lipid A component of LPS. Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system.
Since Lipid A is embedded in the outer membrane of bacterial cells, it probably only exerts its toxic effects when released from multiplying cells in a soluble form, or when the bacteria are lysed as a result of autolysis, complement, phagocytosis, or antibiotic therapy.

The injection of living or killed Gram-negative cells or purified LPS into experimental animals causes a wide spectrum of nonspecific pathophysiological reactions, such as fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock and death. The sequence of events follows a regular pattern:

(1) Latent period
(2) Physiological distress (diarrhea, prostration, shock)
(3) Death

How soon death occurs varies on the dose of the endotoxin, route of administration, and species of animal. Animals vary in their susceptibility to endotoxin.

The mechanism is complex. In humans, LPS binds to a lipid binding protein (LBP) in the serum which transfers it to CD14 on the cell membrane, which in turn transfers it to another non-anchored protein, MD2, which associates with Toll-like receptor-4 (TLR4). This triggers the signaling cascade for macrophage/endothelial cells to secrete pro-inflammatory cytokines and nitric oxide that lead to characteristic "endotoxic shock". CD14 and TLR4 are present on several cells of the immunological system cells, including macrophages and dendritic cells.

In monocytes and macrophages, three types of events are triggered during their interaction with LPS:

1. Production of cytokines, including IL-1, IL-6, IL-8, tumor necrosis factor (TNF) and platelet-activating factor. These, in turn, stimulate production of prostaglandins and leukotrienes. These are powerful mediators of inflammation and septic shock that accompanies endotoxin toxemia. LPS activates macrophages to enhanced phagocytosis and cytotoxicity. Macrophages are stimulated to produce and release lysosomal enzymes, IL-1 ("endogenous pyrogen"), and tumor necrosis factor (TNFalpha), as well as other cytokines and mediators.

2. Activation of the complement cascade. C3a and C5a cause histamine release (leading to vasodilation) and affect neutrophil chemotaxis and accumulation. The result is inflammation.

3. Activation of the coagulation cascade. Initial activation of Hageman factor (blood-clotting Factor XII) can activate several humoral systems resulting in:
a. Coagulation: a blood clotting cascade that leads to coagulation, thrombosis, acute disseminated intravascular coagulation, which depletes platelets and various clotting factors resulting in internal bleeding.
b. Activation of the complement alternative pathway (as above, which leads to inflammation)
c. Plasmin activation which leads to fibrinolysis and hemorrhaging.
d. Kinin activation releases bradykinins and other vasoactive peptides which causes hypotension.
The net effect is to induce inflammation, intravascular coagulation, hemorrhage and shock.

LPS also acts as a B cell mitogen, stimulating the polyclonal differentiation and multiplication of B-cells and the secretion of immunoglobulins, especially IgG and IgM.



Summary of Endotoxins:

• Endotoxins are LPS in nature
• They are integral part of the cell wall of gram negative bacteria
• O polysaccharide allows organisms to adhere specifically to certain tissues, especially epithelial tissues, allows resistance to phagocytes and act as water-solubilizing carriers for toxic Lipid A. They also provide protection from damaging reactions with antibody and complement and are the basis of antigenic variation among many important Gram-negative pathogens
• LPS binds to a lipid binding protein (LBP) in the serum which transfers it to CD14 on the cell membrane. This in turn transfers it to another non-anchored protein, MD2, which associates with Toll-like receptor-4 (TLR4). This triggers the signaling cascade for macrophage/endothelial cells to secrete pro-inflammatory cytokines and nitric oxide that lead to endotoxic shock
• Endotoxin causes shock by production of cytokines, activation of the complement cascade and activation of the coagulation cascade
• LPS also acts as a B cell mitogen
  
  
  
  References:


• Mechanisms of Bacterial pathogenesis – Toder’s online textbook of bacteriology: Toder K, University of Wisconsin-Madison Department of Bacteriology


• Bacterial Toxins: Friends or Foes? – Emerging Infectious Diseases: Vol 5, No 2, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA


• Pathogenesis of bacterial infection – Medical Microbiology: Jawetz, Melnick & Adelberg

For any Further Query, please email at: tirthankar82@yahoo.co.in