Clinical Microbiology and Immunology

microbiota

• microorganisms that inhabit the body

• some cause no harm → commensal

• some are helpful → mutualistic

  • help digest carbohydrates in lower GI tract

  • keep pathogenic bacteria in check

• some cause disease → pathogenic

normal flora

commensal or mutualistic microorganisms that inhabit the body

colonization

• process of becoming a normal part of our body systems

• normal flora colonize certain parts of the body → GI tract, vaginal tract, skin, upper respiratory tract

normal flora: skin

• gram positive cocci:

  • Staphylococcus spp.

    • CoNS (ex: S. epidermidis)

  • Streptococcus spp.

• gram positive rods:

  • Corynebacterium spp.

  • Cutibacterium acnes

• Candida spp.

normal flora: upper respiratory tract

• gram positive cocci:

  • Viridians group Streptococcus

• gram negatives:

  • Neisseria spp.

  • Haemophilus spp.

• oral anaerobes:

  • Peptostreptococcus

normal flora: GI tract

• gram negatives:

  • Enterobacterales

    • Escherichia coli

    • Klebsiella spp.

    • Enterobacter spp.

• gram positives:

  • Streptococcus spp.

  • Enterococcus spp.

• anaerobes:

  • Bacteroides spp.

• yeast:

  • Candida spp.

normal flora: vaginal tract

• group B Streptococcus

• Lactobacillus spp.

• Corynebacterium spp.

• Candida spp.

sterile sites

• blood

• cerebrospinal fluid

• lower respiratory tract

• upper GI tract

• urinary tract

• bone

• synovial fluid

• vitreal fluid

lower respiratory tract

colonization can occur in patients on ventilators or with permanent tracheostomies

upper GI tract

chronic use of PPIs is associated with colonization

urinary tract

• outer urinary tract may be colonized but immune system should keep bacteria from migrating up the tract

• patients with chronic foley catheters will become colonized (nearly 100%)

infection

• occurs when immune system is unable to keep normal flora in check

  • ex: E. coli which is normal gut flora evades normal urethral defenses and travels to the bladder or kidneys to cause infection

• could also occur when body is invaded by external microorganisms

  • ex: influenza, COVID-19, syphilis, Lyme disease

• results in local and/or systemic S/S

local infection

• remains isolated to one site and does not travel throughout the rest of the body

• redness, swelling/inflammation, tenderness, purulence

• ex: finger with infected splinter → redness and pus at site

systemic infection

• elicits a whole-body response

• fever/chills, hypothermia, hemodynamic instability, malaise/fatigue, aches, high WBC count, elevated CRP and ESR

• ex: patient with bacteremia exhibits fever, chills, and elevated WBC

infection

bacteria present with host inflammatory response

colonization

bacteria present without host inflammatory response

contamination

• presence of bacteria in specimen taken from a sterile site without host inflammatory response

• may be due to poor collection technique

“clean catch”

sample from a urinalysis contains <10 squamous epithelial cells

blood cultures

• two vials drawn from different sites

• if S. aureus or gram negative organism is isolated in any sample → infection

• if both vials are growing same organism → likely infection (check for S/S)

• if one of the vials is growing normal skin flora (ex: CoNS staph) → likely contamination (check for S/S)

empiric therapy

• initially, we often don’t have definitive information on the pathogen or what antibiotics will be effective

• choice of antibiotics is driven by our “best guess” based on…

  • site of infection → which organisms are most likely (ex: majority of UTIs are caused by E. coli or other enteric gram negatives)

  • patient history → risk factors and/or prior cultures

  • antibiograms

targeted therapy

• if we are able to get information from gram stain, culture, and sensitivity we can get a better idea of which antibiotics will be most appropriate

• in general, we want to pick the narrowest spectrum agent that we know will cover the pathogen

treatment considerations

• site of infection:

  • normal flora/common pathogens? (ex: UTI → E. coli)

  • will the drug reach the target?

• presentation:

  • purulent vs. non-purulent (ex: staph infections → purulent, strep infections → non-purulent)

• patient characteristics:

  • allergies

  • kidney/liver function

  • pregnancy

  • age

  • comorbidities

  • concomitant medications

antibiogram

• collection of culture and sensitivity data for a given location (ex: hospital, unit, long-term care facility)

• shows general sensitivity patterns for the organisms in that location

• different locations will have different sensitivity patterns

  • geographic (ex: Binghamton vs. Tokyo)

  • within a hospital (ex: general inpatient floors vs. ICU)

• reported as percent susceptibility to each antibiotic on the list

• can be used to guide empiric therapy

  • in general, we try to avoid using an agent if the resistance rate is >20%

  • also can be used to identify need for multidrug-resistant organism (MDRO) coverage for hospital-acquired infections

cultures

• abscess fluid (pus) → superficial wound cultures are likely to be contaminated with normal flora

• sputum → may contain normal oropharyngeal flora

• urine

• blood → ideally two bottles

• CSF

• synovial fluid

• surgical

• should never culture sinus drainage, ear drainage, or saliva

  • known to be colonized with normal flora

  • culture will grow multiple organisms (cannot tell which one is causing infection)

non-culture labs

• some tests are not cultures but identify specific organisms

• rapid antigen tests:

  • often used as point-of-care (POC) tests

  • ex: strep throat, COVID-19, influenza

• urinary antigen tests:

  • can identify S. pnenumoniae and Legionella as cause of pneumonia

• polymerase chain reaction (PCR) tests:

  • C. difficile

  • genetic markers for resistance (ex: mecA, ESBL)

  • MRSA nares (nasal swab)

gram stain

• sample is centrifuged, placed on microscopic slide, and dyed

  • only bacteria with a cell wall will react (not atypical like Mycoplasma or Chlamydia)

• interpret gram stain

  • gram positive → purple

  • gram negative → pink

• interpret morphology

• helps us identify possible bacteria

bacterial culture

• samples are placed on blood agar plates

• plates are incubated for 24 hours and read at 24-hour intervals

• if there is growth, the organism is identified

  • via gram staining, morphology, and other chemical tests (ex: oxidase, lactose fermentation, coagulase tests)

• much of this process is now automated (ex: Vitek, Microscan)

bacterial sensitivity

• determined by comparing the MIC of an organism to standard breakpoints

• reported as…

  • S → sensitive

  • I → intermediate (dose-dependent)

  • R → resistant

Kirby-Bauer

• antibiotic impregnated discs

• multiple antibiotics per dish

• susceptibility determined by “zone of inhibition” → must be certain size to be considered susceptible

E-test

• strip impregnated with only one antibiotic but in increasing concentration across the length of the strip

• placed in the middle of agar plate inoculated with bacteria

• read at the point where the triangular end crosses the strip (this is the MIC)

minimum inhibitory concentration (MIC)

• the lowest concentration of antibiotic that will stop the growth of bacteria

• unique to the patient

breakpoint

• the highest concentration of an antibiotic that can safely be achieved in a patient to define susceptibility of an organism (MIC values that predict the probability of treatment success)

• standardized for each antibiotic tested

bactericidal

antibiotic that kills the organism

bacteriostatic

• antibiotic that halts growth but does not kill the organism

• the immune system is needed to clear the organism

immunocompromised

• decreased ability of the immune system to resist infection

• disease state related (ex: HIV/AIDS)

• drug-induced (ex: high dose steroids, chemotherapy, anti-rejection medications)

autoimmune disorders

• dysregulation of one or more components of the immune system

• often characterized by…

  • production of autoantibodes against host cells (ex: rheumatoid arthritis)

  • loss of self-tolerance to healthy tissues

  • loss of tolerance to a ubiquitous antigen (ex: gluten)

systemic lupus erythematosus (SLE)

widespread inflammation and tissue damage

rheumatoid arthritis

immune system attacks joint tissues leading to inflammation

scleroderma

autoimmune attack on connective tissue leading to increased collagen production resulting in thickening of skin and disruption in organ function

Hashimoto thyroiditis

• immune system attacks thyroid

• can cause goiter and lead to hypothyroidism

drug-induced lupus erythematosus (DILE)

can be caused by hydralazine, sulfadiazine, procainamide, quinidine, etc.

TNF-alpha antagonist-induced lupus syndrome (TAILS)

can be caused by infliximab, etanercept, adalimumab, etc.

autoimmune disorders: symptoms

• immune dysregulation leads to inflammation

• most common S/S → malaise, myalgia, rash, fever

• these are non-specific symptoms associated with most autoimmune disorders

• diagnosis requires assessing complete clinical picture rather than relying on specific lab tests or symptoms

acute phase reactants

• CRP and ESR

• change quickly in response to inflammation

• not used as sole diagnostic tests for autoimmune diseases or other inflammatory conditions

• primarily used as monitoring parameters

C-reactive protein (CRP)

• non-specific indicator of inflammation

• produced by the liver, normally in small quantities

  • production is increased in the presence of inflammation

• normal ranges:

  • adults → 0.08-3.1 mg/L

  • adults (high sensitivity) → 0.02-8 mg/L

erythrocyte sedimentation rate (ESR)

• indirect measurement of acute phase response

• normally, erythrocytes sink slowly to the bottom of a test tube filled with plasma

• in the presence of inflammation associated proteins, erythrocytes and proteins aggregate and settle to the bottom of test tube more rapidly

• inflammation → increased ESR

• normal ranges:

  • adult males → 0-17 mm/hr

  • adult females → 1-25 mm/hr

  • children → 0-10 mm/hr