Lectures 3-4

Question 1

Bacteria require the following for replication:

Answer 1

• Warmth
• Moisture
• Nutrient
• Oxygen and/or carbon dioxide
• The human body can provide these conditions
and thus bacterial infections are common

Question 2

In order to cause infections in the human

body, bacteria must:

Answer 2

• Gain access to the skin or other organ systems
• Gain access to food sources in the body
• Escape eradication by immune and other host
defenses

Question 3

• Virulence =

Answer 3

the degree of pathogenicity of an

organism

Question 4

Mechanisms of virulence:

Answer 4

• Adherence
• Invasion
• Metabolic byproducts (gas, acids)
• Toxins
• Degradative enzymes
• Cytotoxins / endotoxins
• Induction of excessive inflammation
• Stimulation of exaggerated immunologic
response
• Resistance to antibiotics

Question 5

Bacteria may induce disease through the following

mechanisms:

Answer 5

• Direct tissue destruction
• Release of toxins
• Stimulate immunologic processes that damage the host

Question 6

Normal flora =

Answer 6

the bacteria that normally colonize a
human host
• Normal flora have positive and negative outcomes on the
host

Question 7

 Pathogenic actions of bacteria

• Tissue destruction

Answer 7

• Fermentation and metabolism may produce acids and
gases that are damaging to tissue
• Some bacteria release degradative enzymes that
degrade tissues

Question 8

 Pathogenic actions of bacteria

• Toxins

Answer 8

• Bacterial constituents that harm tissue or trigger
disruptive physiologic processes
• Some bacteria create pre-formed toxins in foods that
induce disease soon after ingestion (S. aureus, Bacillus
ceresus, Clostridium botulinum)

Question 9

 Pathogenic actions of bacteria

• Exotoxins

Answer 9

• Produced by both Gm + and Gm – bacteria
• Toxins that are secreted by bacteria that degrade cells
or negatively affect cell function
• Ex) Botulinum toxin
• Superantigens activate T-cells but do not require the
presence of an antigen
− Activate large numbers of T cells, release large amoungs
of interleukins (cytokine storm) causing significant
autoimmune responses

Question 10

 Pathogenic actions of bacteria

• Endotoxin

Answer 10

• May be found in Gm – cell walls
• Lipopolysaccharide (LPS) serves as an endotoxin and
induces significant inflammatory reactions
− Causes release of cytokines
– Interleukin 1
– TNF
– Prostaglandins
− Induces septic shock which leads to pulmonary and
cardiovascular collapse (vasodilation, capillary leakage,
alterations of systemic vascular resistance (SVR), etc.
− Hypotension, shock, DIC and death may ensue

Question 11

When can normal flora cause disease?

Answer 11

Normal flora may induce disease when they
enter body tissues or fluids that they
normally do not occupy

Question 12

Factors to consider when evaluating

infectious process:

Answer 12

• Bacterial strain involved
• Virulence of the organism
• Size of the inoculum
• Immunologic status of the host
• Location of the infection
• Duration of the infectious process

Question 13

Sites of entry into the human body:

• Oropharyngeal, upper GI and lower GI infection

Answer 13

• Salmonella
• Shigella
• Yersinia
• Escherichia
• Vibrio
• Campylobacter
• Clostridium
• Bacillus
• Listeria

Question 14

Sites of entry into the human body:

• Inhalation through URT / LRT

Answer 14

• Mycobacterium
• Nocardia
• Mycoplasma
• Legionella
• Bordetella
• Chlamydia
• Streptococcus
• Hemophilus

Question 15

 Sites of entry into the human body:

• Skin

Answer 15

• Staphylococcus aureus

* Staphylococcus epidermidis

Question 16

 Sites of entry into the human body:

• Urogenital

Answer 16

• Neisseria
• Treponema
• Chlamydia

Question 17

 Sites of entry into the human body:

• Trauma

Answer 17

• Clostridium
• Staphylococcus
• Pseudomonas

Question 18

 Sites of entry into the human body:

• Arthropod bite

Answer 18

• Rickettsia
• Coxiella
• Francisella
• Borrelia
• Yersinia

Question 19

Sites of entry into the human body: systemic spread

Answer 19

• Bacteria entering through various portals may
enter the blood
• Once in the blood, bacteria may travel to other
parts of the body via hematogenous spread
• Bacteria may also infect the lymphatic system
and be spread throughout the body

Question 20

 Colonization

Answer 20

• Once bacteria enter the body, the bacteria may
begin to grow and colonize in that body part
• Bacteria colonize many parts of the human body
• Colonization implies that the bacteria are
sequestering and reproducing in that body part
but are not causing tissue invasion or destruction
• If cellular or physiological damage is being done,
this is the basis of infection

Question 21

 Adherence

Answer 21

• Bacteria use different methods to adhere to
various body tissues
• Adhesin proteins
• Lipoteichoic acid
• Biofilm
• Fimbriae
• Once anchored, it is more difficult for bacteria to
be “washed away” from the tissue

Question 22

 Escaping host defenses

Answer 22

• Natural selection favors bacteria that can evade
the defenses of a host organism
• Evasion of defenses allows the bacteria to grow
at an exponential rate and proliferate its DNA
• Through mutation, bacteria have developed
some methods of escaping detection and
eradication by the host:

Question 23

Mechanisms for escaping host defenses:
• Capsules and slime layers protect bacteria from
immune and phagocytic responses

Answer 23

• Capsules can be slippery which makes it difficult for
phagocytes to adhere to
• Capsules prevent destruction of bacteria even after the
bacteria has been engulfed by a phagocyte
• Biofilms can prevent antibodies and complement from
adhering to the bacteria

Question 24

 Mechanisms for escaping host defenses:

• Bacteria can evade antibody responses by:

Answer 24

• Antigenic variation
− Some bacteria (N. gonorrhoeae) can vary the structure of
surface antigens to evade an antibody response
• Inactivation of antibodies
− Some bacteria (S. aureus) may produce enzymes like
proteases that prevents an antibody from activating
complement and thus masks the bacteria from being
tagged
• Growing intracellularly
− Some bacteria (M. tuberculosis) can grow intracellularly,
even within a macrophage

Question 25

• Gram stain procedure

Answer 25

• Apply crystal violet solution • Apply iodine solution (mordant) • Decolorization with alcohol or acetone − Gram positive cells with thick cell walls hold purple iodine color − Gram negative cells will decolorize and become clear • Counterstain with safranin (pink) • Result = Gram-positive organisms will stain purple and Gram-negative organisms will stain pink

Question 26

* Not all bacteria can be gram stained | * Exceptions include:

Answer 26

• Bacteria that are old or stationary-phase cultures • Bacteria exposed to antibiotics may not gram stain • Mycobacteria − Have waxy outer shell, may be identified with acid-fast staining • Mycoplasma − Have no peptidoglycan • Peptidoglycan layer is too thin

Question 27

Atypical bacteria

Answer 27

``` • A number of bacteria do not fall into traditional categorization and are called atypical bacteria • Considered “incomplete bacteria” • Mycoplasma • Ureaplasma − Smaller than normal bacteria − Lack a true cell wall − Pleiomorphic (shape can vary) • Rickettsia − Smaller than normal − May be pleiomorphic − Obligate intracellular parasites ```

Question 28

Metabolic distinctions of bacteria

Answer 28

Bacteria may also be classified on their metabolic needs • Aerobic or anaerobic • Requirement for specific nutrients • Production of certain metabolic compounds • Presence of specific enzymes • Automated procedures have been developed to rapidly identify various bacteria

Question 29

bacteria testing- culture

Answer 29

• Using a variety of techniques, bacteria can be grown in vitro • Aerobic • Obligate anaerobes • Facultative anaerobes • Using different growth medium and knowledge of the different morphologic characteristics of each bacteria, the genus and species of the bacteria can be identified

Question 30

There are 2 main methods of determining | sensitivity:

Answer 30

1) Microserial broth dilutions − Involves growth of the microorganism in a number of different test tubes at a certain inoculum − Serial dilutions are made with each antibiotic such that the concentrations in the adjacent tube are 1/2 of the preceding tube − The minimum inhibitory concentration is the first tube that is clear (no bacterial growth) − This is the concentration of antibiotic required to inhibit the growth of the bacteria 2) Disk diffusion (Kirby-Bauer) − This method involves growing bacteria on an agar plate − Small paper disks impregnated with a standard antibiotic concentration are placed on the agar − Multiple different antibiotics are tested − The antibiotic diffuses from the disk to the surrounding agar medium − If the antibiotic is effective in inhibiting microbial growth, a clear “ring” is visible around the paper disk (zone of inhibition) − Susceptibility or resistance is determined by the size of the zone of inhibition

Question 31

Specimens used in lab Dx |  Blood

Answer 31

• One of the most important sources of bacteria • Although bacteremia is not always present, bacteria in a specific foci may enter the blood stream and travel hematogenously to other sites • Even if bacteremia is present, the bacteria may not be continuously present in blood samples • Important factors to consider: • Volume of blood sample • Number of samples taken for each culture • Location site of each sample

Question 32

Specimens used in lab Dx |  Cerebrospinal fluid (CSF)

Answer 32

• Bacterial meningitis is a fulminant disease associated with high morbidity and mortality • Antimicrobial treatment is done for bacterial meningitis (but not fungal or aseptic) • The common organisms are labile and need to be processed quickly to avoid false negatives • S. pneumoniae • N. meningitidis • Identification of the causative pathogen is important in successful treatment • Samples are obtained via lumbar puncture

Question 33

Specimens used in lab Dx |  Sterile bodily fluids

Answer 33

• A number of fluids that are normally sterile may harbor bacteria: • Peritoneal fluid • Pleural fluid • Synovial fluid • Pericardial fluid • It is often difficult to get such fluids as the volumes may be small and require invasive methods

Question 34

Specimens used in lab Dx |  URT specimens

Answer 34

• It can be difficult to obtain specimens from these areas • Contamination with oropharyngeal flora may also occur • Swabs are often used to collect nasopharyngeal specimens • Direct aspiration of sinuses is possible

Question 35

Specimens used in lab Dx |  LRT specimens

Answer 35

• Sputum expectoration • Saline induction • Direct aspiration • Bronchoscopy • BAL • The presence of squamous epithelial cells indicate the specimen is probably contaminated with mouth flora • Anaerobic bacteria may cause abscesses which require needle or surgical aspiration to culture • Some bacteria require special Cx methods (mycobacteria)

Question 36

Specimens used in lab Dx |  Ear and Eye

Answer 36

• Tympanocentesis involves aspiration of fluid from the middle ear • Invasive, not typically done in clinical practice • Complications include: − Iatrogenic introduction of bacteria into middle ear space − Scarring − Meningitis • Empiric therapy covering common organisms is common practice • Specimens obtained from eyes are usually very small • A small number of organisms may be present, making detection and identification difficult • Intraocular specimens may be obtained through direct aspiration of the eye

Question 37

Specimens used in lab Dx |  Wound tissue

Answer 37

• Specimens can be taken directly from a wound • It is important to differentiate between surface microorganisms versus those infecting a wound • Swabs are often ineffective and can be contaminated with skin flora • Abscess aspirates can be taken from the abscess itself • Drainage of fluid from wounds may help identify bacteria

Question 38

Specimens used in lab Dx |  Urine

Answer 38

• Often utilized in evaluating urinary tract infections • Cystitis • Pyelonephritis • Urine is normally sterile • If urine samples are obtained to evaluate the presence of bacteria, the first part of the urine stream should not be used • Pathogenic bacteria may colonize the urethra • Urine can be evaluated for microorganisms, tissue proteins, RBCs and WBCs

Question 39

Specimens used in lab Dx |  Genital lesions

Answer 39

• Bacteria may be isolated directly from: • Cutaneous lesions • Fluid obtained from the lesions • Pus • Blood cultures may not be useful, although serologic evaluations may be effective • Identification of the agent that causes syphilis (T. pallidum) cannot be performed using normal bacterial culture methods • Fluorescent darkfield examination • Treponemal and non-treponemal serology tests

Question 40

Specimens used in lab Dx |  Fecal specimens

Answer 40

• A large number of bacteria colonize the intestinal tract • Many bacteria may cause gastroenteritis and can be cultured • Requires a stool sample • Quantity of the sample needs to be adequate to culture out the potential pathogen • Culturing these bacteria may require different culture methods or media • Because culture may take > 2-3 days, treatment is usually initiated empirically