Microbiology High Yield Topics

1) Principles and Interpretation of bacteriologic lab tests

Cultures

Type	Purpose	Methods/Principles	Further Tests/Results
Blood Cultures	To determine whether sepsis, endocarditis, osteomyletis, meningitis, or pneumonia are present	· Obtain at least three samples in a 24 hr period because the # of organisms is small and their presence intermittent. · Use 2% iodine to prevent contamination by S. epidermis	· gram stain · subculture · antibiotic sensitivity
Throat Cultures	To detect the presence of group A beta-hemolytic strep (S. pyogenes), diphtheria, gonococcal pharyngitis, or thrush.	Make sure swab touches posterior pharynx, both tonsils and tonsilar fossa	If beta-hemolytic strep are found after 24 hrs of incubation, a bacitracin disk is placed on a plate. If growth is inhibited around the disk, then it is group A strep.
Sputum Cultures	To diagnose pneumonia, TB or lung abscess	Specimen must be from sputum NOT saliva. Sputum = > 25 leukocytes & < 10 epithelial cells per 100x field	· serologic tests · biochemical tests · increase in Ab titer (Mycoplasma) · acid-fast stain (TB) · anaerobic culture (aspiration pneumonia an lung abcesses)
Spinal Fluid Cultures	To diagnose meningitis		· quellung test or immunofluorescence (N. meningitidis, S. pneumoniae, H. influenzae) · acid-fast stain (M. tuberculosis) · India ink stain (C. neoformans) · Immunologic tests for capsular antigen
Stool Cultures	To diagnose enterocolitis	· McKonkey agar for Salmonella and Shigella (selective for gram-negative rods; differential properties baesd on fact that Salmonella and Shigella do not ferment lactose whereas many other enteric gram-negative rods do) · Antibiotic containing media at 5% O2 and 10% CO2 for Campylobacter	TSI agar or agglutination test w/ antisera to organism’s O antigen
Urine Cultures	To diagnose pyelonephritis or cystitis	· Midstream specimen collected after watching the external orifice · Calibrated loop, dilutions, or agar-covered paddles for quantitative culturess\	Need > 100,000/ml to conclude that bacteriuria is present, but as few as 100/ml may be significant in asymptomatic patients
Genital Tract Cultures	To examine abnormal discharge/diagnose STD	· Thayer-Martin chocolate agar plate or transport medium (N. gonorrhoeae) · Culture of human cells/yolk sacs (C. trachomatis)	T. pallidum canNOT be cultured so look for motile spirochetes on darkfield microscopy or use serologic tests
Wound & Abscess Cultures	To determine organisms in wounds & abscesses	· Anaerobic collection tubes · Culture using several different media under several different conditions

Immunologic Methods

Test	Purpose	Principle
Capsular Swelling (quellung)	To identify S. pneumoniae, H. influenzae type B, and N. meningitidis groups A and C	Capsule swells in presence of homologous antiserum
Slide Agglutination	To identify Salmonella and Shigella	Antisera against the cell wall O antigens or the flagellar H antigens cause clumping
Latex Agglutination	To identify H. influenzae, N. meningitidis, strep, C. neoformans	Latex beads coated w/ specific Ab are agglutinated in the presence of the homologous bacteria/antigen
Counter-immuoelectrophoresis	To identify H. influenzae, N. meningitidis, S. pneuomoniae, group B strep	The unknown bacterial antigen and a known specific Ab move toward each other in an electric field. If they are homologous, a precipitate forms. Only negative antigens can be assayed
Enzyme-Linked Immunosorbent Assay	To identify many, many bacteria, fungi and viruses	A specific Ab to which an easily assayed enzyme has been linked is used to detect the presence of the homologous antigen
Fluorescent-Antibody Test	To identify many bacteria	Expose bacteria to known antibody labeled w/ fluorescent dye. Detect w/ UV micoscope
Slide/Tube Agglutination	To diagnose typhoid fever, brucellosis, tularemia, plague, leptospirosis and ricketssial diseases	Dilutions of a sample of the patient’s serum are mixed w. standard bacterial suspensions. The highest dlution capable of agglutinating the bacteria is the titer of the antibody. At least a 4-fold rise in titer between the early and late samples must be demonstrated for a diagnosis to be made
Serologic Tests for Syphilis	To identify T. pallidum	Non-treponemal tests (VDRL, RPR): use cardilipin-lecithin mix as the antigen. Clumping of the cardiolipin occurs in the presence of Ab to T. pallidum
		Treponemal tests: · FTA-ABS: patient’s serum is reacted w/ nonviable T. pallidum on a slide. Flurescein-labeled Ab against human IgG is used to detect whether IgG against T pallidum is bound · MHA-TP: patient’s sample is reacted w/ sheep erythrocytes coated w/ antigens of T. pallidum. If Ab is present, hemagglutination occurs
Cold Agglutinin Test	To detect Mycoplasma	Patients w/ Mycoplasma infections develop autoimmun Abs that agglutinate human RBCs at 4 C but not at 37 C.

2) Dermatologic Manifestations of Bacterial and Viral Infections

Infection	Dermatologic Manifestation
Bacterial
S. pyogenes	· folliculitis, cellulitis, impetigo, necrotizing fascitis · scarlet red rash (scarlet fever) · erthema marginatum (rheumatic fever)
S. aureus	· Erythematus rash (toxic shock syndrome) · Scalded skin syndrome · Skin infection
B. anthracis	Painless black vesicles (anthrax)
C. perfringens	Cellulitis/wound infection
N. meningitidis	Petechial rash (meningitis or meningiococcemia)
S. typhi	Rose spots on abdomen (enteric fever)
P. aeruginosa	Burn wound infections
H. ducreyi	Chancroid: painful genital ulcer, releasing pus
Y. enterocolitica	Rash
F. tularensis	Ulcer at site of tick bite or direct contact w. contaminated rabbit (tularemia)
P. multocida	Wound infections
R. rickettsii	Rash on wrists, ankles, soles and palms initially, becomes more generalized later (Rocky Mountain Spotted Fever)
Rickettsia (in general)	Rash in all cases except Brill-Zinsser Disease and Q fever
T. pallidum (Syphilis)	Primary stage: painless chancre (skin ulcer) Secondary stage: rash on palms and soles, condyloma latum (painless, wartlike lesion which occurs in warm places) Tertiary stage: gummas of skin and bone
T. pallidum subspecies endemicum (Bejel)	Primary and secondary lesions: in oral mucosa Tertiary lesions: gummas of skin and bone
T. pertenue (Yaws)	Primary and secondary lesions: ulcerative skin lesions near initial site of infection – often looks like condyloma lata Tertiary lesios: gummas of skin and bone
T. carateum (Pinta)	Flat red or blue lesions which do NOT ulcerate
B. burgdorferia (Lyme Disease)	Erythema chronicum migricans
M. leprae (Leprosy)	Multiple skin lumps and bumps
M. marinum	Skin granulomas (called swimming pool or fish tank granulomas) which occur at site of abrasions
M. ulrans	Chronic skin ulcers with necrotic centers
M. fortuitum M. chelonei	Skin abscess at site of trauma
Viral
Measles (Rubeola)	Koplik’s spots: small, red-based blue-white centered lesions in the mouth Rash: from head, then to neck and torso, then to feet. As the rash spreads, it coalesces
Rubella (Togavirus)	Rash: from forehead to face to torso to extremities (last 3 days)
Herpes Simplex virus-1	* Gingivostomatitis: painful group of vesicles on lips and mouth, which ulcerate and heal, usually without leaving a scar * Reactivation of gingivostomatitis when individuals are stressed out. Similar eruption of vesicles as with primary gingivostomatitis but less painful and lasting fewer days.
Herpes Simplex virus-2	* Genital herpes: painful group of focal vesicles on the cervix or external genitalia * Reactivation of genital herpes: similar eruption of vesicles, but less painful and vesicles last for fewer days * Neonatal herpes
Varicella-zoster virus	* Varicella (Chickenpox): Vesicles first erupt on trunk and face and spread to entire body. Vesicles rupture and scab over. Vesicles erupt in crops, so one crop forms as another crop scabs over. Patients are infectious until all of their lesions scab over. * Zoster (Shingles): Painful eruption of vesicles isolated to a single dermatome distribution. The vesicles dry up and form crusts, which disappear in ~3 weeks. * Herpes zoster ophthalmicus: vesicles on one side of the forehead and on the tip of the nose (the dermatomal distribution of the first division of CN V); may be associated w/ severe corneal involvement.
Human Herpesvirus 6	Roseola: rash, located mostly on the trunk, which lasts just a day or two
Human Herpesvirus 8	Kaposi’s Sarcoma (due to herpes virus): red to purple plaques or nodules all over the body
Poxviridae	Smallpox Molluscum contagiosum: small white bumps with a central dimple. Often found in the genital region.
Papovaviridae	HPV: warts
Parvoviridae	Erythema infectiosum (Fifth Disease): “slapped cheek” rash
Cocksackie A Echovirus	Rashes, “cold”

3) Common STDs

Disease	Microbiology	Clinical Features	Virulence/Transmission	Treatment
Gonorrhea	Neisseria gonorhoeae Strict human pathogen Gram negative diplococci Oxidase-positive Fastidious growth, labile Thayer-Martin media	Acute urethritis in males Cervicitis, urethritis, vaginitis, PID in women Pharyngitis, proctitis, conjunctivitis Disseminated gonococcol infection (arthritis/dermatitis, deficiency in terminal components of complement) Fitz-Hugh-Curtis Syndrome (infection of liver capsule w/ symptoms of RUQ pain) Ophthalmia neonatorum	Pilus colonization factor (antigenic variation) Opa proteins (phase variation) Lipooligosaccharide (antigenc variation) IgA1 protease Transferrin.lactoferrin binding proteins	Diagnosis (gram stain, culture) Infection does NOT equal immunity Ceftriaxone, fluoroquinolones, spectinomycin Prophylactic erythromycin eye drops for infant
Chlamydia	Chlamydia trachomatis Obligate intracellular bacteria Complex life cycle (iInfectious particle is the elementary body (EB). Once w/in an endosome, the EB inhibits phagosome-lysosome fusion & is not destroyed. It transforms into an initial body.	Urethritis Epididymitis, proctitis Mucopurulent cervicitis PID Inclusion conjunctivitis Trachoma (leading cause of preventable blindness in the world) LGV (L1, L2, L3) Reiter’s Syndrome (inflammatory arthritis of large joints) Fitz-Hugh-Curtis Syndrome (infection of liver capsule w/ symptoms of RUQ pain)	Chronic infection leads to delayed hypersensitivty (type IV) response	Diagnosis (culture, antigen, nucleic acid) Diagnosis of inclusion conjunctivitis by intracytoplasmic inclusion bodies Antibiotics (tetracycline/doxycycline, fluorquinolones, 1 dose of azithromycin) Prophylactic erythromycin eye drops for infant
Chancroid	Haemophilus ducreyi Gram negative coccobacilli Fastidious, labile	Genital ulcer Painful, soft chancre Inguinal lymphadenopathy Strong association w/ prostitution		Clinical diagnosis by exclusion of other agents of genital ulcer disease Treatment: ceftriaxone + one dose of azithromycin or erythromycin or trimethoprim/sulfameth.
Syphilis	Treponema pallidum Spirochete – fastidious, labile spiral bacterium w/ axial filaments Man is only recognized host Unculturable (propagated in rabbit testes) Gram-negative like, no LPS or recognized toxins	* Primary syphilis (3-6 wks): classical chancre, generally painless; highly contagious, rapid dissemination * Secondary syphilis (6+ wks): rash, lesions, generalized lymphadenopathy, condyloma lata, eye lesions, mild fever, malaise; infectious state * Tertiary syphilis (6-40 yrs): cardiovascular problems (aneurysm), neurosyphilis (asymptomatic, subacute meningitis, meningovascular syphilis, tabes dorsalis), gummas, general paresis, not infectious * Congenital syphilis: transplacentally acquired; novel among congenital infections in that transmission can occur in 2^nd & 3^rd trimester; high rate of fetal loss; snuffles, frontal bossing, malformations, saber shins, deafness, eye disease, Hutchinson’s teeth, Mulberry molars, neurosyphilis	Motile	Diagnosis: darkfield/ direct fluorescence microscopy to visualize chancre; nontreponemal and treponemal Abs Treatment: penicillin, erythromycin, doxycycline If secondary syphilis is untreated, 1/3 undergo spontaneous resolution, 1/3 remain infected w/o clinical manifestations, 1/3 progress to tertiary syphilis
Genital Herpes	Herpes simplex virus-2 Double-stranded linear DNA Enveloped Icosahedral symmetry	* Genitral Herpes: painful group of focal vesicles on the cervix, or on the external genitalia. Often associated w/ fever and viral symptoms. These vesicles usually do not scar * Reactivation: similar eruption of vesicles, but less painful and vesicles last for fewer days * Neonatal Herpes: acquired during passage through an infected birth canal. The risk of transmission is highest when a primary genital infection is present during delivery · Disseminated · CNS · Skin · Eye	1. Direct contact of mucous membranes 2. Sexually transmitted 3. Herpes virus travels up sensory nerve fibers to the sensory nerve ganglia, where it replicates, then returns along the sensory nerve fibers to produce skin lesions	Diagnosis: 1) Tzanck prep: reveals multinucleated giant cells and intranuclear inclusion bodies 2) Viral culture 3) PCR 4) Direct fluorescent Abs: ulcer base scrapings may be tested w/ Abs Treatment: Acyclovir
HIV	Core: 1) 2 identical SS RNA pieces · Two LTRs (long terminal repeat sequences) · gag, pol & env · Regulatory genes (tat, rev, nef) 2) Proteins: nucleocapsid proteins, protease, reverse transcriptase & integrase Capsid shell: p24 Matrix proteins Surface glycoproteins: gp 120 & gp 41	1) Acute viral illness: similar to mono (fever, malaise, lymphadenopathy); develops ~ 1 month after exposure. High levels of blood-borne HIV (viremia). 2) Clinical latency: follows for a median of 8 yrs. HIV continues to replicate & there is a steady destruction of CD4 T-lymphocyte (helper) cells. Towards the end of this period: constitutional symptoms (fever, weight loss, night sweats, adenopathy), bacterial (mycobacterium tuberculosis) & skin infections (athlete’s foot, oral thrush, herpes zoster) 3) AIDS: CD4 T-cell < 200 and/or Candida esophagitis, Pneumocystis carinii pneumonia, Kaposi’s sarcoma (HHV-8). AIDS patients also suffer from neurologic disease, B-cell lymphoma, mycobacterium tuberculosis, mycobacterium avium-intracellulare (MAC), crypotcoccus neoformans, histoplasma capsulatum, coccidioides imitis, herpes zoster, Epstein-Barr (oral hairy leukoplakia), Herpes simplex, CMV, toxoplasma gondii (mass lesions in the brain), cryptosporidium and isospora blli	Genome heterogeneity: · hyerpvariable regions lie within the env gene · gene encoding reverse transcriptase has high mutation rate HIV is transmitted from cell to cell (due to syncytial giant-cell formation). The virus can thu excape the Ab-mediated (humoral) immune system. Protection against HIV requires cell-mediated immunity	Diagnosis: 1) ELISA 2) Western Blot Treatment: 1) Zidovudine (AZT) 2) Didanosine, zalcitabine, lamivudine, stavudine, nevirapine, delavirdine 3) Protease inhibitors 4) Treat opportunistic infections Vaccine attempts: 1) Attenuated virus 2) Recombinant envelope glycoprotein 3) Live recombinant organisms 4) Direct intramuscular injecton of HIV genes 5) Soluble CD4 receptors

4) Viral Gastroenteritis in the pediatric and adult populations

Organism	Virus	Epidemiology	Symptoms & Signs	Diagnosis	Treatment & Outcome
Norwalk	RNA	Most common cause of infectious diarrhea in older children & adults Food & water borne; fecal-oral spread Occurs year round Short incubation period Shellfish amplify contamination b/c of a pumping mechanism	Watery diarrhea Nausea/vomiting (+/-) Fever (+/-) Dehydration (+/-)	Clinical	Supportive, usually resolves in 1-4 days
Rotavirus	dsRNA; acid stable	Most common cause of infectious diarhea in children 6 mos to 2 yrs Food & water borne; person-to-person spread Wintertime disease Short incubation period	Secretory diarrhea, possibly preceded by vomiting Fever (+/-) Easily dehydrated (decreased bp, increased pulse, poor turgor, sunken eyes, dry tongue)	Rotazyme (ELISA assay) Detection of antigen in stool	Supportive and treat w/ fluids
Adenovirus	Non-enveloped dsDNA	Transmission by: aerosal droplet, fecal-oral, and direct inoculation of conjunctivas	Non-bloody diarrhea (usually in children < 2 yrs)		Resolve spontaneously

5) Common Causes of Community Acquired Pneumonia

S. pneumoniae

S. aureus

S. pyogenes

H. influenzae

Legionella

Mixed aerobic and anaerobic flora

Common Causes of Nosocomial Pneumonia

Pseudomonas aeruginosa

Serratia

Klebsiella

Enterobacter

References:

1) Micro Made Ridiculously Simple

2) Medical Microbiology and Immunology Examination and Board Review (Lange)

3) Class Notes from Infectious Disease