Clinical Microbiology: Bacteria and Infectious Disease

• Clinical Microbiology, Part 1 covers topics including introduction & bacteria review, body system infections, antibiotic susceptibility tests, bacteria, protozoa, viruses and STIs.

Organisms that Cause Infectious Diseases

• These organisms are categorized into 5 major groups: bacteria, fungi, protozoa, helminths (worms), and viruses.
• Bacteria are in their own domain.
• Fungi (yeasts and molds) are unicellular or relatively simple multicellular organisms.
• Protozoa and helminths (worms) are complex multicellular organisms.
• Viruses are noncellular.

Bacteria

• Bacteria are single-celled organisms without organelles or nuclei.
• Bacteria's DNA is tangled in “nucleoid DNA".
• Gram stain is performing in 4 steps.
• Medically-important bacteria are tested.
• Antimicrobial susceptibility testing and result interpretation is needed.

Bacterial Structure

• Almost all bacteria have an inner plasma membrane and a peptidoglycan cell wall.
• Gram+ bacteria have a much thicker peptidoglycan cell wall than gram- bacteria.
• Gram- bacteria have an additional outer membrane
• The outer membrane is an asymmetric bilayer with lipopolysaccharide (LPS) on the outside, and phospholipid on the inside.

Gram Staining

• Gram staining is the technique of differentiating bacteria according to their cell walls, through the following four steps:
• Crystal violet dye (purple) is applied and taken up into the cell.
• Mordant (iodine) is applied to set the stain, forming insoluble complexes in the cell so it is not easily removed.
• A lipid detergent, such as alcohol, is applied.
  • In gram- bacteria, the outer membrane is washed away because of its thin cell wall, making the cell colorless as the crystal violet washes away
  • Gram+ bacteria retain crystal violet in the cell because of their thicker cell wall, so they appear purple.
• Cells are counterstained with safranin (pink).
• Gram+ bacteria appear purple or blue because the pink safranin is overshadowed by the crystal violet.
• Gram- bacteria appear pink or red.

Bacteria: Gram Positive vs. Gram Negative

• Gram-positive bacteria have 1 cell membrane and a thick cell wall, and retain crystal violet dye, staining purple with gram stain.
• Gram-negative bacteria have 2 cell membranes and a thin cell wall, and do not retain crystal violet dye, staining pink with gram stain.

Gram Staining and Other Techniques

• Gram staining does not work well on all bacteria, so alternative techniques exist.
• Some bacteria have envelope components that resist decolorization by acid/ethanol-based staining ingredients, so they are identified with acid-fast staining.
• Mycobacterium (e.g., M. tuberculosis) are a notable example of acid-fast bacteria.

Gram-Negative Bacteria

• Lipopolysaccharide (LPS) in the outer membrane is a potent endotoxin.
• LPS is highly antigenic, causing a systemic inflammatory response but making it impossible to build immunity to LPS.
• Bacteria itself causes limited local tissue damage.
• The death of bacteria causes a systemic inflammatory response, which becomes a problem.
• The outer membrane of gram- bacteria makes them more resistant to many antibiotics compared to gram+ bacteria.

Gram-Positive Bacteria

• Gram Positive Bacteria are invasive and causes local tissue damage.
• Gram Positive Bacteria causes Less systemic disease than gram-negative bacteria.

Bacterial Structure and Classification

• Bacteria are classified in many ways, with classifications updated as science advances, and historically classified primarily on morphology and biochemical characteristics.
• The three basic groups based on shape are cocci, bacilli, and spirochetes.
• Cocci are round/spheres that may be arranged in pairs (diplococci), chains (streptococci), or clusters (staphylococci).
  • Gram-positive cocci are most clinically relevant.
• Bacilli are rods.
  • Gram-negative rods are most clinically relevant.
• Spirochetes are spiral shaped.
• Some bacteria are variable in shape (pleomorphic), with additional shapes like coccobacilli beyond those listed above.

Classical Categorization of Medically Important Bacteria

• The classical categorization of medically important bacteria are separated into I. Rigid, thick cell walls, II. Flexible, thin cell walls (spirochetes), III. No cell wall (mycoplasmas).
• Rigid, thick cell walls category are further subdivided into Extracellular and Obligate intracellular parasites.
• Extracellular bacteria can grow on lab medium in absence of other host cells, and subdivided according to shape, staining reaction, and oxygen/spore-forming abilities.
  • Gram+ cocci and rods
  • Gram- cocci and rods.
• Obligate intracellular parasites can only grow within host cells.

Bacterial Taxonomy

• The following taxonomy is not complete.
• It provides a framework for understanding some of the more clinically relevant bacteria, but is not all traditionally organized.

Gram+ Cocci

• Gram+ cocci are either Staph (Clusters) or Strep (Chains).
• Refer to chart for distinctions between the clusters including Staphylococcus aureus, Staph. epidermidis, and the chains including Strep. pyogenes, Strep. agalactiae and Strep. viridans.

Gram+ Rods

• Gram+ Rods are spore forming or non-spore forming
• Refer to chart for distinctions between the groups including Bacillus (aerobic), Clostridium (anaerobic), Actinomyces(filamentous) and Listeria (non-filamontous)

Gram- Cocci

• The Gram- Cocci chart lists Neisseria (such as gonorrhoeae and meningitidis) and Moraxella.
• Both Neisseria and Moraxella are typically diplococci.

Gram- Rods

• Gram - Rods chart includes the types of Respiratory organisms, the Zoonotic organisms, and the Enteric organisms.

Antibiotic Susceptibility Testing

• There are various testing methods for determining sensitivity/resistance of bacteria to antibiotics.
• Dilution methods such as broth microdilution, include different concentrations of antibiotic treatments.
• Quantitative Minimum inhibitory concentration (MIC) is the lowest concentration of a specific drug that will inhibit visible growth of an organism.
• MIC ranges are classified into interpretative categories.
• Kirby-Bauer method (disk diffusion test) is a simple, inexpensive method.
• Sample colonies are cultured then exposed to antibiotic disks, where antibiotic diffuses outward with the highest antibiotic concentration near the disk.
• The diameter of inhibited growth is measured with the zone of inhibition (ZOI).

Antibiotic Susceptibility Testing

• The Kirby-Bauer method (disk diffusion test) is qualitative.
• Prespecified breakpoints (ranges) are used for the diameter of zone of inhibition (ZOI).
  • It is used to classify activity of a drug against a specific organism into susceptible, susceptible-dose, intermediate, or resistant.
• The higher the susceptibility of the organism to the drug, the higher the ZOI and the lower MIC.
• A culture & sensitivity (C&S) test can take up to 3 days for a final report, with preliminary reports given.
• Samples taken can be from blood, sputum, wound, or urine.
• "S" stands for susceptible/sensitive, and "R" stands for resistant on test results.

Multi-Drug Resistant Organisms (MDROs)

• MDROS pose a "catastrophic threat" according to WHO.
• Resistance can be inherent to the species or acquired through mutations and non-resistant bacteria.
• The clinical role is to use antibiotics only when the body needs them to fight infection patient education.
• From the laboratory role, Suppress sensitivity results on broad-spectrum antibiotics, include reminders with C&S reports.
  • For example, "50% of long-term care residents have asymptomatic bacteriuria".

Protozoa

• Protozoa are single-celled organisms with nuclei and more complex cellular machinery than bacteria.
• Protozoa are closer to single-celled animals than to bacteria.
• Protozoa causes common Protozoal Infections.
• Can specifically relate to the four Plasmodium malaria species.

Protozoa: Entamoeba histolytica

• Entamoeba histolytica causes amebiasis/amebic liver disease, known for causing liver abscesses ("anchovy paste").
• Diagnostic studies include:
  • E. histolytica-specific stool antigen test (~90% sensitive/specific)
  • Stool microscopy is also an option but less sensitive/specific/Stool specimens show cysts or trophozoites
  • Sigmoidoscopy may supplement stool antigen/stool microscopy (mucus collected from ulcers for microscopy and biopsy samples taken from edge of ulcers, Mucosal biopsy shows flask shaped ulcers)
  • Blood serologic testing does not differentiate acute infection vs. past exposure; but if negative in someone with 7-10 days of symptoms, then E. histolytica is less likely.

Protozoa: Giardia lamblia

• Giardia lamblia causes giardiasis, which is the most common protozoal infestation in U.S.
• A typical scenario is drinking untreated water, such as while hiking/camping, where multiple family members are affected.
• Trophozoites from ingested cysts attach to the intestinal epithelium, causing mucosal inflammation and malabsorption.
• Malabsorption results in steatorrhea (fatty diarrhea).
• Diagnosis can be determined by stool microscopic examination for ova (eggs) and parasites (O&P).
• Double-nucleated flagellated organisms are seen on stool sample.

Protozoa: Toxoplasma gondii

• Toxoplasma gondii is an obligate intracellular protozoan, found worldwide in humans and in many species of mammals and birds.
• It is commonly around cats.
• It causes toxoplasmosis.
• You can diagnosis with Anti-toxoplasma IgM and IgG antibodies and serology is most important. -IgM peaks, IgG and can cause congenital infection and reactivate in immunocompromised.

Protozoa: Trichomonas vaginalis

• Trichomonas vaginalis causes trichomoniasis.
• It needs to be discovered Traditionally via microscopic "wet mount” examination of vaginal/penile discharge.
• This will show motile flagellates
• For the Pearls, treat patient and sexual partners because partners can keep infecting each other.

Protozoa: Malaria

• Malaria is contracted from a Mosquito-borne cyclical flu like illness
• It ranges from mild to fatal
• Parasitic protozoan
• Plasmodium causes Malaria.
• Transmitted from Anopheles mosquito's saliva into blood/releasing merozoites.
• Multiple Stages
• Sporozoite infects liver. -Replicates until hepatocyte bursts. -Also forms gametocytes.

Protozoa: Malaria (Diagnosis)

• Diagnosis occurs with light microscopy examination of Giemsa-stained blood smear(classic test).
• The technique, requires expertise and labor intensive and can miss cases with very low parasite loads
• Rapid antigen tests. -Detect parasite antigens, are Useful and Simpler and quicker than blood smear.
• Test-use generally limited to reference labs for research/epidemiology.

Protozoa: Malaria (Symptoms)

• Molecular can amplify parasite DNA
• Nucleic acid tests: PCR.
• Anemia (testing and diagnosis of tissue-invasive diseases must be verified.)

Viruses

• Viruses are studied by medical virology.
• They can invade host cells and replicate.
• Viruses relates to common viral infections.
• Used to inventory the indications for a Tzanck smear.

Viruses

• A virus is a genome that is a sequence of DNA or RNA, contained in a capsid composed of structural proteins that protect, transport, and deliver the viral genome.
• There are also enveloped viruses that contain their own transcription factors, and its envelope comes from host cell plasma membranes.
• The genome is usually stuffed with glycoproteins.

Viruses

• Naked viruses have only a capsid and must penetrate host cell plasma membranes.
• Enveloped viruses have hijacked part of the host cell plasma membrane, disguising the virus and enabling it to enter cells by fusion of membranes.

Viral Genome

• DNA viruses are double-stranded and replicate in nuclei
• Host cell machinery does not distinguish viral sequences from host sequences.
• Viral DNA hijacks host polymerases and reproduces.
• Most viral DNA brings its own viral polymerase.
• RNA viruses are single stranded and replicate in cytoplasm.
• They bring its own viral polymerases to replicate.

Viral Life Cycle

• General life cycle of viruses includes 1) invade and 2) release
• Viruses are not alive, and can can hijack cells.
• Viruses exist until they bump into a cell.
  • Naked viruses attach and penetrate using capsid proteins, then uncoat and release the genome.
  • Enveloped viruses Glycoproteins facilitate membrane fusion.
• From the invasion process. Genome is copied from the Host ribosomes
• They hijacked to make viral proteins used to make new capsids.
• There are Naked and Enveloped viruses for Exit and entry.
• Enveloped viruses needs to be acute or chronic or latent. Cytolytic
• Exit when its acute.

Viral Infections: Epstein-Barr Virus (EBV)

• EBV is most common cause of infectious mononucleosis (IM): fever, sore throat, lymphadenopathy, atypical lymphocytosis.
• Diagnosis (Check WBC count) due to with differential+ heterophile antibody test with Monospot
• The Gold Standard will also be EBV-specific antibody testing on.

Viral Infections: Human Papillomavirus (HPV)

• HPV are Double-stranded DNA viruses that only infect humans with >200 types.
• Low-risk HPVs mostly cause no disease
• HPV's can cause anogenital and oropharynx. The vaccine for treatment is Gardasil 9® protects against infection from nine HPV types (nonavalent.)
• 2-part series, boys and girls, starting between ages 9-12.

Viral Infections: Varicella-Zoster Virus (VZV)

• Is used to show and give viral information.
• Shingles(herpes zoster) with dermatomal pattern that is Painful .
• It can diagnosis infections and conditions.

Tzanck Smear

• Tzanck Smear is Simple, rapid, and inexpensive .

Viral Infections: Rhabdovirus

• Rhabdovirus causes rabies
• Low cases in US.
• There can be rabies from transcript infection.
• If it is wild life ,euhanthiza brains observed. Isolate pets

Viral Infections: Cytomegalovirus (CMV)

CMV: Usually, asymptomatic for the patients infection.

• CMV can cause many clinical presentations
• Can also cause congenital with infection
• Cannot be reliably diagnosed. PCR is a treatment that is preferred.

Viral Infections: Herpes Simplex Virus (HSV)

• Painful vesicular eruptions, usually in orolabial area (HSV1) or on genitals (HSV2).
• Usually diagnosed clinically but should be confirmed with lab testing with PCR ( preferred).
• Lab text is other PCR to be less and and the use of Tzanck.
• Limited test to diagnose patient.

Sexually Transmitted Infections (STIs)

• Commonly Analyze using laboratory testing.
• Has Trichomonas, HSV and HPV (certain types).

Sexually Transmitted Infections (STIs): Syphilis

• Syphilis is caused by Treponema pallidum (spirochete).
• Multistage disease beginning with painless, indurated ulcer, usually on the genitals
• Serology is the recommended approach. -The test should include the test should be non. -False-positives.

Sexually Transmitted Infections (STIs): Gonorrhea

•Caused by Neisseria gonorrhoeae. •Creamy, yellow discharge from the (Penile or cervical). •Diagnosis will be Nucleic acid amplification testing (NAAT): most accurate ("gold standard")