Medically Important Bacteria

Classification of Medically Important Bacteria

• Classification involves arranging organisms taxonomic groups based on similarities and relationships
• Nomenclature gives organisms names based on international rules and characteristics
• Identification uses classification for practical purposes

Identification

• Isolating and distinguishing desirable organisms from undesirable ones is key
• Confirming special properties of a culture
• Identifying the causative agent of a disease

Classification Factors

• Morphology of bacteria matters
• Biochemical characteristics
• Cell wall nature
• Staining characteristics
• Ability to grow with or without oxygen
• Ability to form spores

Cell Wall Differences

• Some bacteria have rigid, thick walls
• Others have flexible, thin walls, like spirochetes such as Treponema and Borrelia
• Mycoplasma are wall-less bacteria

Rigid Wall Living

• Free-living bacteria are extracellular
• Non-free-living bacteria are obligate intracellular parasites
• Rickettsia and Chlamydia are examples of obligate intracellular parasites

Rigid Walled Cells and Free Living Types

• Gram-positive bacteria include Cocci like Streptococcus and Staphylococcus
• Spore-forming rods include Aerobic Bacillus and Anaerobic Clostridium
• Non-spore forming rods include Nonfilamentous Listeria and Filamentous Nocardia

Rigid Walled Cells and Gram-Negative Bacteria

• Cocci such as Nesseria
• Straight Facultative Respiratory bacteria like Haemophilus and legionella
• Zoonontic bacteria such as Brucella, Yersinia spread animal to human
• Enteric bacteria such as E. coli, Shigella, Proteus

Rigid Walled Cells and Aerobic/Anaerobic Bacteria

• Facultative bacteria include Curved Champylobacter & Vibrio
• Aerobic bacteria such as Pseudomonas
• Anaerobic bacteria such as Bacteriods are often found in the digestive system, specifically the appendix

Classifying Features

• Acid-fast bacteria, like Mycobacterium

Laboratory Diagnosis Approaches

• Bacteriologic approach identifies organisms by staining and culturing
• Immunologic (serologic) approach identifies organisms by detecting antibodies in patient serum

Steps Prior to Lab Work

• Choose the right specimen
• Get it without contamination
• Transport it quickly
• Give the lab essential info

Laboratory Work Steps

• Observe the organism with a microscope after staining
• Get a pure culture by inoculating a bacteriologic medium
• Identify the organism via:
  • Biochemical reactions
  • Growth on selective media
  • DNA probes -specific antibody reactions

Diagnosing Bacterial Infections

• Get a specimen from the infected spot.
• Stain it (Gram stain or acid-fast). Look at shape, size, arrangement, and Gram reaction.
• Culture on appropriate media like blood agar. Streak for isolated colonies, and incubate with/without oxygen.
• Identify with tests like sugar fermentation, DNA probes, or antibody tests. Check for hemolysis or pigment.
• Test antibiotic susceptibility.

Bacterial Infection Diagnosis with Negative Culture

• Check for antibodies in the patient's serum
• Detect antigens in the patient's specimen with known antibodies
• Detect nucleic acids with PCR and DNA probes

Cultures

• Blood agar supports the growth of many bacteria to allow observation of the type of hemolysis
• Red blood cells lack a functioning nucleus and therefore are incapable of supporting the growth of either viruses or the obligate intracellular bacteria.
• "Cooked" blood agar or chocolate agar has a chocolate color because the blood is heated

"Selective, Differential" Media

• Selective media: Allow selective growth of bacteria via certain compounds
• Differential media: allows differentiation between two bacteria based on biochemical reaction

Bacteriologic Agars

• Blood agar can isolate various bacteria and to detect hemolysis
• Bordet-Gengou agar isolates Bordetella pertussis to increase the concentration of blood allows growth
• Charcoal-yeast extract agar allows to isolates Legionella pneumophila to increase the concentration of iron and cysteine allows growth
• Chocolate agar allows isolates Neisseria meningitidis and Neisseria gonorrhoeae from sterile sites to inactivate inhibitors of growth by heating

Culturing Haemophilus influenzae on Chocolate agar

• It needs X and V factors for growth

Culturing Clostridium perfringens on Egg yolk

• Lecithinase produce degrades egg yolk to produce insoluble precipitate

Culturing Gram-Negative Enteric Bacteria on Eosin-Methylene Blue

• Selects against gram positive bacteria and differentiates between lactose fermenters and non-fermenters

Culturing Mycobacterium tuberculosis on Löwenstein-Jensen

• Selects against gram-positive bacteria in respiratory tract flora and contains lipids required for growth

Culturing Gram-Negative Enteric Bacteria on MacConkey

• Selects against gram positive bacteria and differentiates between lactose fermenters and non-fermenters

Tellurite for Corynebacterium diphtheriae

• Tellurite metabolizes to tellurium, which has black color

Thayer-Martin Agars

• Isolate N. gonorrhoeae from nonsterile sites
• Chocolate agar has antibiotics to inhibit growth of normal flora

Culturing Gram-Negative Enteric Bacteria on Triple Sugar Iron (TSI)

• Distinguishes lactose fermenters from nonfermenters and H2S producers from nonproducers

Direct Collection

• Involves collected from normally sterile tissue (lung, liver) or body fluids (CSF, blood)
• Use needle aspiration to surgical biopsy
• +ve findings are diagnostic for the tissue
• -ve findings exclude the infection at the suspected tissue

Spinal Fluid CSF

• Involves the spinal column at L3 and L4

Indirect Samples

• Use inflammatory exudates (expectorated sputum, voided urine)
• Methods are convenient for both physician and patient

Collecting Samples

• Use sterile swab because they are convenient
• Transport to the lab soon as possible (N. gonorrhoeae needs to delivered fast in few minutes)
• Transport media with buffered fluid / semisolid media that containing minimal nutrients to prevent drying, maintain a neutral pH, minimize bacterial growth (free oxygen for obligate anaerobes)

Blood Cultures

• Blood cultures are performed in case of sepsis, endocarditis, osteomyletis, meningitis or pneumonia
• The most frequently isolated organisms from blood cultures are Gram +ve S. aureus and S. pneumoniae, and Gram -ve E. coli, K. pneumoniae
• Take three 10-ml blood samples in 24 hours.
• Check blood for turbidity and CO2 production
• If +ve growth, gram stain, subculture, and antibiotic sensitivity
• -ve result on other media

Throat Cultures

• To detect the presence of group A beta-hemolytic
• to detect streptococci (Streptococcus pyogenes) which cause pharyngitis
• To detect Candida (thrush)
• Use a throat swab and inoculate on blood agar and gram stain (not done- why)
• Check 24 incubation ẞ- hemolytic streptococcus after 24 hours (group A, non-group A)

Sputum Cultures

• To detect Pneumoniae and tuberculosis
• Use Gram stain smear to check specimen
• Culture on a blood agar
• Check if Mycoplasma appears. there will be a rise in antibody titer
• To detect Legionella pneumonia, use charcoal-yeast agar (high concentration iron sulfur)
• To detect Tuberculosis, use acid-fast stain

Spinal Fluid Cultures

• To detect Meningitis (causes)
• Use a Gram stain
• Use immunochemical tests to detect capsular antigen (latex particle agglutination, counter- immunoelectrophoresis

Stool Cultures

• To detect Enterocolitis such as Shigella, salmonella, and campylobacter
• Gram stain is not done
• Detect Salmonella & Shigella (Mackonkey, Eosin- methylene blue EMB), non lactose fermenting colonies: triple sugar iron TSI

Urine Cultures

• To detect Pyelonephritis and cystitis
• To detect the different strains of bacteria like E. coli, proteus
• Use midstream urine
• Culture samples within 1 hour (store in refrigerator at 4°C, less than 18h)

Genital Tract Cultures

• To detect Abnormal discharge and Sexually transmitted diseases
• To detect the presence of Neisseria gonorrhoeae, use microscopic examination of a Gram stained smear
• To detect Treponema pallidum, use microscopy serology

Wound & Abscess Cultures

• Depends on anatomic site
• If there is a Lung, brain abdomen abscess, detect anaerobes such as Bacteroids fragilis and gram positive cocci
• Place specimen in anaerobic collection tubes
• It is important to culture the specimen on several different media under different atmospheric condition

Immunological Methods

• Immunological tests include "serologic" tests can determine whether antibodies are present in the patient's serum as well as detect the antigens of the organism in tissues or body fluids
• The antigens of the causative organism can be detected by using specific antibody often labeled with a dye such as fluorescein (fluorescent antibody tests).
• The presence of antibody in the patient's serum can be detected using antigens derived from the organism
• Detect a significant fourfold (or greater) increase in titer to indicate a current infection

Nucleic Acid-Based Methods

• Nucleic acid amplification: PCR is used for amplification of bacteria specific DNA
• Nucleic acid probe: detect bacteria DNA or RNA directly using a labeled DNA or RNA probe
• Nucleic acid sequence: identify bacteria based on the base sequence of the organism's ribosomal RNA