Med Micro

stained bacteria and other microscopic organisms

small objects (≤0.2 um) - Treponema pallidum and flagella

three-dimensional image of the organism

staining organisms with fluorescent dyes; colors vary depending on the fluorochrome selected

• magnetic coils (rather than lenses) are used to direct a beam of electrons from a tungsten filament through a specimen and onto a screen
• high magnification and resolution- individual viral particles
• Transmission EM: electrons such as light pass directly through the specimen
• Scanning EM: electrons bounce off specimen surface at an angle; three- dimensional

• Wet mount: specimen mixed with water or saline; motile bacteria, stool concentrates for eggs and parasite larvae
• Potassium hydroxide (10% KOH): detect fungal elements in sputum
• Lactophenol cotton blue: stains fungal hyphae
• India ink prep: for the detection of capsule (Cryptococcus neoformans)

• Gram stain
• Iron hematoxylin and trichrome stains (for trophozoites & cysts from stools)
• Wright-Giemsa stain (for blood parasites)

Kinyouns or Auramine-rhodamine (a fluorescent dye for Mycobacteri, Nocardi)

• Acridine orange fluorescent dye targets nucleic acid of dead and alive microorganisms
• Fluorescent antibody stains

The observable characteristics of an organism's growth on or in the media.

• allows growth of many pathogens
• Examples....
• Sheep blood agar: Staphylococcus aureus, Streptococcus pneumonia, Klebsiella sp., etc
• Chocolate agar: Haemophilus, some pathogenic Neisseria strains
• Thioglycolate broth- enrichment broth for aerobes and anaerobes

• Inhibitory substances (antibiotics) are added to suppress the growth of susceptible bacteria, but allow particular pathogen’s growth
• Modified Thayer Martin- selects for N. gonorrhoeae and inhibits most other bacteria
• Inhibitory mould agar- selects for fungi and inhibits bacteria

• selects for specific bacteria and differentiates them
• MacConkey agar: lactose, glucose, neutral red indicator, bile salts and crystal violet (inhibit GP bacteria); selects for enteric GNR and differentiates between lactose fermenters and nonlactose fermenters

• allows the growth of bacteria that require specific ingredients
• Buffered charcoal yeast extract (BCYE) for Legionella
• MacConkey sorbitol agar for recovery of Escherichia coli O157

cells grow as a single layer and can be maintained in the lab indefinitely

cells prepared from tissues/organs immediately before use

• Advantages- identify difficult or slow growing organisms, strain determination (Mycobacteria tuberculosis, Chlamydia, N. gonorrhoeae, slow growing fungi)
• Detection tools: PCR (increases the DNA/RNA required for analysis) and Probes (target specific nucleic acids in a strand of unknown DNA or RNA. Specific for genera, species, and strains)

• A rapid nucleic acid test that produces 400 results or more in 8 hrs
• Used to diagnose disease and screen blood donations
• Amplifies RNA or DNA a billion fold in 15-30 minutes Uses DNA probes tagged with chemiluminescent signal.
• Culture required for Mycobacteria tuberculosis and Group B strep
•  Patient specimen for N. gonorrhoeae, Chlamydia trachomatis, Group A strep, HIV

• PCR based testing and nucleic acid
• hybridization
• Identifies pathogens in clinical specimens: sputums and blood samples
• 48 tests/run with results in 2.5 hours.
• Cultures unnecessary

use of antibodies and antigens to determine the infecting agent, a primary or reinfection, and the course of the disease.

foreign proteins and polysaccharides; cell wall, capsule, membrane proteins; many different antigenic sites are on a bacterium or virus.

A specific region of an antigen that binds to an antibody. There can be more than one epitope per antigen, each binding to a specific antibody.

• amount of antibodies in a patient’s serum (quantitation of the antibody strength); the lowest dilution of the sample that retains a detectable activity
• Diagnostic for an infection is a four-fold rise in titer pf antibodies; from acute to convalescent phase.
• Serially dilute the serum two-fold and add a set amount of antigen; antibodies specific to the antigen will bind and precipitate; the last dilution with precipitate is the titer
• Dilutions are 1:2, 1:4, 1:8. Titers are 2,4,

During a primary infection, IgM is first produced.

When re-exposed to a previous infection, a booster.

• Direct precipitation techniques
• Direct labeling the antibody with a radioactive,
• fluorescent, or enzyme probe
• Indirectly through measurement of an antibody-directed reaction, such as complement fixation

antibody cross-links the antigen into a complex that is too large to stay in solution (forming precipitation)

• detects and quantifies an antigen
• Antibody in agar and antigen in a well and diffuses outward. A line of precipitate indicates a complex has formed. A line far from the well indicates a higher quantity of antigen than a line close to the well

• Determine the relatedness of different antigens
• A central well and surrounding wells. Precipitation lines indicate an antibody-antigen reaction

http://micro.digitalproteus.com/pics/immunoprecipitation.jpg

Detecting antigens on the cell surface or within the cell

• Legionnaires’ disease: Legionella antigen complexs with
• tagged antibodies

The flow cytometer evaluates individual cell parameters as the cells flow past a laser beam at rates of more than 5000 per sec.

• Antigen immobilized on a plastic surface, bead, or filter to capture and separate the specific antibody from other antibodies in a patient's serum.
• Anti-human antibody with a covalently linked enzyme (e.g., horseradish peroxidase, alkaline phosphatase, β- galactosidase) then detects the affixed patient antibody.
• Crytococcal meningitis, Giardiasis, Rotavirus gastroenteritis, etc.
• Can also be used to quantitate the soluble antigen in a patient's sample

• for quantitation of antibody or antigen.
• 

• Proteins of an infectious agent are separated by electrophoresis according to their molecular weight or charge are transferred (blotted) onto a filter paper (nitrocellulose, nylon).
• When exposed to a patient's serum, the immobilized proteins capture virus-specific antibody and are visualized with an enzyme-conjugated antihuman antibody.
• Western blot analysis is used to confirm ELISA results in patients suspected to be infected with the human immunodeficiency virus (HIV).

• A standard but technically difficult serologic test
• Complement is added to the Ag-Ab mixture
• Antibody-antigen complexes bind, activate, and fix (use up) the complement. The residual complement is then assayed through the lysis of red blood cells coated with antibody.
• RBC lysis indicates no antibody-antigen complex formed
• No RBC lysis indicates the complement was used for the
• antigen-antibody complex.
• Diagnosis of fungal, respiratory virus, arbovirus, and Q fever infections

• Use of the specificity of an antibody to prevent infection (neutralization) or other activity (hemagglutination inhibition), identify the strain of the infecting agent (virus), quantitate antibody responses to a specific strain of virus
• Hemagglutination inhibition is used to distinguish different strains of influenza A

• Antibodies or antigens are coupled with latex beads. Add serum or a suspension of an organism. Visible agglutination occurs or not.
• Crytococcal meningitis, Staphylococcus aureus
• Virus-specific antibody causes latex particles coated with viral antigens to clump. Antibody- coated latex particles are used to detect soluble viral antigen

1935 - protected mice against systemic streptococcal infection.

1928 - noted mold Penicillium prevented the multiplication of staphylococci.

Range of antimicrobial activity (Broad vs Narrow)

• Level of antimicrobial activity that inhibits the growth of an organism
• Minimum inhibitory concentration (MIC)
• The lowest concentration that inhibits the growth of the
• organism

• Level of antimicrobial activity that kills an organism
• Minimum bactericidal concentration (MBC)
• The lowest concentration that kills 99.9% of the population

Combinations of antibiotics enhanced bactericidal activity

One antibiotic interferes with the activity of the other

• (1) Broaden the antibacterial spectrum
• Empirical therapy
• Treatment of polymicrobial infections
• (2) Prevent the emergence of resistant organisms during therapy
• (3) Achieve a synergistic killing effect

• Disruption of Cell Wall
• Inhibition of Protein Synthesis
• Inhibition of Nucleic Acid Synthesis
• Antimetabolite

• The most common mechanism
• Beta-lactam antibiotics (e.g., penicillins, cephalosporins, cephamycins, carbapenems, monobactams, Beta-lactamase inhibitors)
• Share a common Beta-lactam ring structure

• Building of peptidoglycan chains catalyzed by specific enzymes (serine proteases) - Transpeptidases, Transglycosylases & Carboxypeptidases
• These regulatory enzymes also called penicillin- binding proteins (PBPs)

• Bound by Beta-lactam antibiotics
• Bactericidal agents
• Antibiotic binds to specific PBPs in the bacterial cell wall
• • Inhibits formation of cross-links between peptidoglycan chains
• • Activatesautolysinsthatdegradethecellwall,resultinginbacterial
• cell death

• (1) Prevention of the interaction between the antibiotic and the target PBP
• - Gram (-) outer membrane
• (2) Modification of the binding of the antibiotic to the
• PBP
• - (1) Overproduction of PBP
• - (2) Acquisition of a new PBP through gene transfer (e.g.,
• methicillin resistance in Staphylococcus aureus)
• - (3) Modification of an existing PBP through mutation:
• recombination (e.g., penicillin resistance in Streptococcus pneumoniae) or a point mutation (penicillin resistance in Enterococcus faecium).
• (3) Hydrolysis of the antibiotic by Beta-lactamases

• Enzyme hydrolyze Beta-lactam ring
• More than 200 different Beta-lactamases
• Extended-spectrum Beta-lactamases (ESBLs) encoded on plasmids that can be transferred from organism to organism
• Specific for penicillins (penicillinases), cephalosporins (cephalosporinases), and carbapenems (carbapenemases; metallo-Beta-lactamases)
• Classes A-D
• Plasmid or chromosomal

• Natural penicillins [benzylpenicillin (penicillin G), phenoxymethyl penicillin
• (penicillin V)]
• Penicillinase-resistant penicillins [methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin]
• Broad-spectrum penicillins [aminopenicillins (ampicillin, amoxicillin, carbenicillin, ticarcillin); ureidopenicillins (piperacillin)]
• Beta-Lactam with Beta-lactamase inhibitor [ampicillin-sulbactam, amoxicillin-clavulanate, ticarcillin- clavulanate, piperacillin-tazobactam]

• Cephalosporins (Beta-lactam antibiotics) derived from 7-aminocephalosporanic acid
• Beta-lactam ring is fused with a dihydrothiazine ring
• - Same mechanism of action as the penicillins
• - Wider antibacterial spectrum
• - Resistant to many Beta-lactamases
• - Improved pharmacokinetic properties: longer half-life
• Cephamycins has oxygen in place of sulfur in the dihydrothiazine ring

• Narrow spectrum (cephalexin, cephalothin, cefazolin, cephapirin, cephradine)
• Expanded-spectrum cephalosporins (cefaclor, cefuroxime)
• Expanded-spectrum cephamycins (cefotetan, cefoxitin)
• Broad spectrum (cefixime, cefotaxime, ceftriaxone, ceftazidime
• Extended spectrum (cefepime, cefpirome)

• Carbapenems (e.g., imipenem, meropenem, ertapenem)
• Broad-spectrum antibiotics
• Active against virtually all groups of organisms, with only a few exceptions
• - Resistance has been reported for all oxacillin-resistant staphylococci, selected Enterobacteriaceae and Pseudomonas, and other gram-negative rod
• Monobactams (e.g., aztreonam).
• Narrow-spectrum antibiotics
• Active only against aerobic, gram-negative bacteria

• Bacillus licheniformis
• Topically applied products (creams, ointments, sprays)
• to trea skin infections caused by Gram (+)  Gram(-) are resistant  Inhibits cell wall synthesis
• - Interfering with dephosphorylation and the recycling of the lipid carrier
• • Transportingpeptidoglycanprecursors-cytoplasmicmembrane-cell wall)
• May damage cytoplasmic membrane and inhibit transcription
• Resistance caused by failure to penetrate into the bacterial cell

• Bacillus polymyxa
• Cyclic polypeptides insert into bacterial membranes
• like detergents
• Interacting with lipopolysaccharides and the phospholipids in the outer membrane
• Increased cell permeability and eventual cell death
• Most active against gram (-) rods
• Polymyxins B and E (colistin) cause serious
• nephrotoxicity
• - External treatment of localized infections ( external otitis, eye infections, skin infections)

• Aminoglycosides (streptomycin, kanamycin, gentamicin, tobramycin, amikacin)
• Aminocyclitol (spectinomycin)
• Tetracyclines

• Inhibition of 50S
• Oxazolidinones (linezolid)
• Chloramphenicol
• Macrolides (Erythromycin)
• Lincosamides (Clindamycin)

• Amino sugars linked to an aminocyclitol ring (through glycosidic bonds)
• Free NH, and OH groups bind to specific ribosomal proteins
• Bactericidal
• Commonly used to treat
• serious G(-) infections
• Aerobic, energy-dependent penetration
• Ex: Gentamicin, Tobramycin, Amikacin

• (1) Mutation of the ribosomal binding site
• (2) Decreased uptake of the antibiotic into the bacterial cell
• (3) Increased expulsion of the antibiotic from the cell
• (4) Enzymatic modification of the antibiotic (most
• common) on the amino and hydroxyl groups
• - Phosphotransferases, Adenyltransferases, Acetyltransferases

• Synthetic chemotherapeutic agents
• Inhibit bacterial DNA topoisomerase type II (gyrase;
• G(-)) or type IV (G(+))
• Nalidixic acid: 1st used in clinical practice
• Fluoroquinolones (ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin)
• Resistance
• - Chromosomal mutations in DNA gyrase and topoisomerase
• type IV
• - Decreased drug uptake (mutations inmembrane permeability
• regulatory genes)
• - Overexpression of efflux pumps that actively eliminate the drug

• Binds to DNA-dependent RNA polymerase and inhibits the initiation of RNA synthesis
• Bactericidal for Mycobacterium tuberculosis
• Very active against aerobic G(+) cocci
• G(-) are resistant intrinsically: decreased uptake of the hydrophobic semisynthetic antibiotic

• Mammalians do not synthesize folic acid (required as a vitamin)
• Certain microbes synthesize own folic acid
• Sulfonamides compete with p-aminobenzoic acid
• Trimethoprim inhibit dihydrofolate reductase (di- to tetrahydrofolate)
• Trimethoprim + Sulfamethoxazole: Synergistic inhibition of folic acid synthesis

• Pilin (attachment)
• Por protein (protein I; porin; prevent phagolysosome
• fusion)
• Opa protein (protein II; opacity; firm attachment)
• Rmp protein (protein III; reduction-modifiable protein; protection from antibodies)
• Transferrin-binding proteins (acquisition of iron)
• Lactoferrin-binding proteins (acquisition of iron)
• Hemoglobin-binding proteins (acquisition of iron)
• LOS Lipooligosaccharide (inflammation, TNF-α)
• IgA1 protease
• Beta-Lactamase

• Humans (only natural hosts)
• The major reservoir: asymptomatically infected person asymptomatic, particularly in women
• no other known reservoir
• Second (only to chlamydia) STD in US (~360,000 new
• infections in 2006)
• Sexual contact
• 1st contact risk: 50% female, 20% male
• Peak incidence age: 15 to 24 years
• Disseminated disease: deficiencies in late components (C5-C8) of complement (6000X higher risk)

purulent discharge (urethra, cervix, epididymis, prostate, anus) after incubation period (2-5 day)

spread from genitourinary tract through blood to skin or joints; pustular rash with erythematous base and suppurative arthritis

Ophthalmia neonatorum: purulent ocular infection acquired by neonate at birth

• Genital infections
• Men
• - Urethra (purulent urethral discharge and dysuria)
• - Most (95%) have acute symptoms
• - Complications (rare): epididymitis, prostatitis, and periurethral abscesses Women
• - Cervix (endocervical columnar epithelial cells)
• - Symptoms: vaginal discharge, dysuria, and abdominal pain
• - Ascending genital infections (10% to 20%): salpingitis,
• tuboovarian abscesses, and pelvic inflammatory disease

• A generic term for inflammation of the female uterus, fallopian tubes, and/or ovaries
• Progresses to scar formation with adhesions o nearby tissues and organs
• Viral, fungal, parasitic, though most often bacterial infections
• In US, > million women/month are affected (teenagers 15-19 yr, new mothers 20-24 yr)
• Causes over 100,000 women to become infertile in the US each year
• One of the most serious complications of sexually transmitted diseases

• Disseminated infections with septicemia and infection of skin and joints
• 1% to 3% of infected women
• Fever; migratory arthralgias; suppurative arthritis in the wrists, knees, and ankles
• Pustular rash over the extremities
• A leading cause of purulent arthritis in adults

• Purulent conjunctivitis (newborns: ophthalmia neonatorum)
• Perihepatitis (Fitz- Hugh-Curtis syndrome)
• Anorectal gonorrhea (homosexual men)
• Pharyngitis

• Microscopy
• - Gram stain
• - •sensitive and specific for men with purulent urethritis
• - •insensitivity in asymptomatic men and in both symptomatic and asymptomatic women
• Culture
• - Avoiddryingandcoldtemperatures
• - Inoculated onto both selective media (modified Thayer-Martin medium) and nonselective media (chocolate blood agar)
• Identification
• - Preliminary: oxidase-positive, gram-negative diplococci; grow on chocolate blood agar or selective media
• - Definitive: Glucose positive
• Nucleic Acid Amplification

• No more Penicillin
• β-lactamase production (plasmid-mediated)
• Chromosomally mediated changes in penicillin-binding proteins and in cell wall permeability (resistance to tetracyclines, erythromycin, and aminoglycosides)
• Fluoroquinolone (ciprofloxacin)- 1993 Ceftriaxone- 2007

• No effective vaccine
• Lack of protective immunity
• - Antigenic diversity
• - Antibodies detected to pili antigens, Por proteins, and LOS
• Chemoprophylaxis- ineffective
• Except in the protection of ophthalmia neonatorum (1% silver nitrate, 1% tetracycline, or 0.5% erythromycin eye ointments)

• Gram-negative cocci
• Cause gonorrhea which can lead to pelvic
• inflammatory disease (PID) in woman and epididymitis in men
• Can be carried asymptomatically by both men and women
• Exhibit phase variation and antigenic variation
• Can cause disseminated infections (serum-resistant strains)
• Infection are treated with antibiotics (widespread penicillin- resistance)

• A previously healthy 18-year-old man who presented to a local emergency department with the acute onset of fever and headache. His temperature was elevated (40 C), he was tachycardic (pulse of 140 per minute), and hypotensive (blood pressure 70/40 mm Hg).
• Petechiae were noted over his chest.
• Neisseria meningitidis was recovered in the patient's
• blood and CSF cultures. Despite the prompt administration of antibiotics and other support measures, the patient's condition rapidly deteriorated, and he died 12 hours after arrival in the hospital. This patient illustrates the rapid progression of meningococcal disease, even in healthy young adults.

• ’Meninges' - thin membrane, or lining surrounds the brain
• Meningitis - infection of this lining
• Affects people of all ages, most common in pre-school children
• Caused by several different viruses or bacteria
• Viral meningitis, less severe and resolves without specific treatment
• Bacterial meningitis, quite severe and may result in brain damage, hearing loss, or learning disability

• High fever, headache, and stiff neck are common symptoms- over the age of 2 years (several hours- 1 to 2 days)
• Other - nausea, vomiting, discomfort looking into bright lights, confusion, and sleepiness
•  In newborns and small infants, may absent classic symptoms, only appear slow or inactive, or be irritable, have vomiting, or be feeding poorly
•  As the disease progresses, patients of any age may have seizures

• Bacterial menigitis, an infection of the membranes (meninges) and cerebrospinal fluid (CSF) surrounding the brain and spinal cord
• A major cause of death and disability world-wide
• The etiology of bacterial meningitis varies by age group and region of the world
• Beyond the perinatal period, three organisms, transmitted from person to person through the exchange of respiratory secretions, are responsible for most cases of bacterial meningitis: Neisseria meningitidis Haemophilus influenzae Streptococcus pneumoniae
• Neisseria meningitidis, a leading cause of bacterial menigitis and sepsis in children and young adults in the United States, causes both sporadic disease and outbreaks

• Gram-negative diplococci with fastidious growth requirements
• Grows best at 35° to 37°C in a humid atmosphere
• Oxidase and catalase positive; acid produced from maltose and glucose oxidatively
• Outer surface antigens include polysaccharide capsule, pili, and lipooligosaccharides (LOS)

• Pili- bind to specific pili receptors for nasopharynx colonization
• Capsule- protects bacteria from antibody- mediated phagocytosis
• Bacteria can survive intracellular killing in the absence of humoral immunity
• Endotoxin mediates most clinical manifestations

• Upon returning home to Nebraska, a 28 year old man who had vacationed in Thailand presented to the local emergency room (ER) complaining of painful urination and a purulent discharge from his penis.
• Urethral specimens were collected for detection of Neisseria gonorrhoeae and Chlamydia trachomatis using nucleic acid amplification tests (NAATs) and the patient was empirically given a single 500-mg dose of ciprofloxacin orally and a single 1-g dose of azithromycin.
• Subsequently, the NAATs were positive for N. gonorrhoeae, but negative for C. trachomatis.

• Genera of medical interest: Neisseria, Eikenella, and Kingella
• Eikenella corrodens and Kingella kingae (opportunistic pathogens) colonize the human oropharynx

• 10 species found in humans
• Neisseria gonorrhoeae and Neisseria meningitidis, strictly
• human pathogens
• The remaining Neisseria species:
• • limited virulence • compromised patients • oropharynx and nasopharynx (anogenital) mucosal membranes

• Aerobic, Gram (-) cocci (0.6 to 1.0 um)
• Diplococci resembling coffee beans (with adjacent sides flattened)
• Not motile
• No endospores
• All are oxidase positive
• Acid produced by oxidation of carbohydrates
• - N. gonorrhoeae - glucose - N. meningitidis - both glucose
• and maltose

• Nonpathogenic species of Neisseria grow on nutrient agar at 35° to 37°C.
• N. meningitidis has variable growth on nutrient agar
• N. gonorrhoeae is a fastidious organism
• - Complex media for growth
• - Sensitive to dry conditions or fatty acids-neutralized by adding soluble starch
• - All strains require cystine for growth
• - A humid atmosphere supplemented with carbon dioxide (5% CO₂)

• Typical of gram-negative bacteria
• Outer surface of N. gonorrhoeae is not covered with a true carbohydrate capsule, with a capsule- like negative charge surface
• Serogrouping based on antigenic differences in the polysaccharide capsule of N. meningitidis
• - 13 serogroups (A, B, C, D, H, I, K, L, W-135, X, Y, Z, 29E)
• - most infections caused by A, B, C, Y, and W135

• Horizontal DNA exchange
• Phase and antigenic structural variation
• Capsular switching
• Molecular mimicry
• - Polysialic acid capsule of serogroup B meningococci is identical to structures on the human neural cell adhesion molecule (N-CAM)
• -LOS (lacto-N-neotetrose) mimics the carbohydrate moieties of human glycosphigolipids
• Blebbing and autolysis
• - Frequent vesiculation (blebbing) of the outer membrane-rapid initiation of the inflammatory cascades of sepsis
• - Naturalautolysisinstationarygrowthphase-DNArelease-genetic transformation

• Humans (only natural hosts)
• Person-to-person spread (aerosolization of RT secretions)
• Highest incidence < 5 years, institutionalized people, and patients with late complement deficiencies
• Disease occurs worldwide, most commonly in the dry, cold months of the year
• - Meningitis and meningococcemia - serogroups B and C
• - Pneumonia- serogroups Y and W135
• - Serogroups A and W135 - underdeveloped countries

Purulent inflammation of meninges associated with headache, meningeal signs, and fever; high mortality rate unless promptly treated with effective antibiotics

Disseminated infection characterized by thrombosis of small blood vessels and multiorgan involvement; small, petechial skin lesions coalesce into larger hemorrhagic lesions

Milder form of meningococcal disease characterized by bronchopneumonia in patients with underlying pulmonary disease

• Gram stain of CSF is sensitive and specific
• - of limited value for blood specimens (too few organisms are generally present, except in overwhelming sepsis.
• Culture is definitive (fastidious and dies rapidly when exposed to cold or dry conditions)
• Antigen tests- insensitive and nonspecific.

• Breast-feeding infants- passive immunity (first 6 months)
• Treatment - penicillin (drug of choice), chloramphenico,
• ceftriaxone, and cefotaxime
• Chemoprophylaxis -rifampin, ciprofloxacin, or ceftriaxone
• A polyvalent polysaccharide-protein conjugate vaccine
• effective against serogroups A, C, Y, and W135 was licensed in the United States in 2005 (11-18 yrs and other high risk)
• Immunoprophylaxis- only for serogroups A, C, Y, and W135; no effective vaccine is available for serogroup B

• Gram-negative cocci
• Cause septicemia and meningitis
• Heavily encapulated
• Hemolysin for dissemination in the bloodstream, leading to disseminated intravascular coagulations
• Vaccine with capsular polysaccharide, except for group B strains

• Commensal organisms in the oropharynx
• Implicated in isolated cases of meningitis, osteomyelitis, endocarditis, bronchopulmonary infections, acute otitis media, and acute sinusitis

• Human bite wounds: traumatic (e.g., bite, fistfight injury) introduction of oral organisms into deep tissue
• Subacute endocarditis: infection of endocardium characterized by gradual onset of low grade fevers, night sweats, and chills

Subacute endocarditis: As with E. corrodens

• A 22-year-old female schoolteacher was brought to the emergency room after a 2-day history of headache and fever. On the day of admission, the patient had failed to come to school and could not be reached by telephone. When notified of this fact, the patient's mother went to her daughter's apartment, where she found her daughter in bed, confused and highly agitated. The patient was rushed to the local hospital, where she was comatose on arrival.
•  Purpuric skin lesions were present on her trunk and arms. Analysis of her CSF revealed the presence of 380 cells/mm3 (93% polymorphonuclear leukocytes), a protein concentration of 220 mg/dL, and a glucose concentration of 32 mg/dL.
•  Gram stain of CSF showed many gram-negative diplococci, and the same organisms were isolated from blood and CSF. The patient died despite prompt initiation of therapy with penicillin.

• An 18-year-old man fell on his knee while playing basketball. The knee was painful, but the overlying skin was unbroken. The knee was swollen and remained painful the next day, so he was taken to the local emergency department.
• Clear fluid was aspirated from the knee, and the physician prescribed symptomatic treatment. Two days later, the swelling returned, the pain increased, and erythema developed over the knee.
• Because the patient also felt systemically ill and had an oral temperature of 38.8°C, he returned to the emergency department.
• Aspiration of the knee yielded cloudy fluid, and cultures of the fluid and blood were positive for S.aureus.

• Spherical shape; Gram (+) reaction; No endospores
• Catalase catabolizes hydrogen peroxide into water and oxygen gas (bubbles)
• Aerobic catalase-positive genera (Staphylococcus, Micrococcus, Kocuria, Kytococcus, Alloiococcus)
• Aerobic catalase-negative genera (Streptococcus, Enterococcus)
• Anaerobic gram-positive cocci
• 0.5 to 1.5 um in diameter, nonmotile, facultatively anaerobic (grow both aerobically and anaerobically)
• Grow in high salt (10% sodium chloride)
• Grow at 18°C to 40°C  On skin and mucous membranes (humans)
• Genus currently consists of 35 species and 17
• subspecies
• Many are found on humans
• S. aureus is catalase (+) AND coagulase (+; fibrinogen- fibrin- clot)

• S. aureus; aureus, golden (golden or yellow)
• S. epidermidis; epidermidis, outer skin (of the epidermis or outer skin)
• S. lugdunensis; Lugdunum, Latin name for Lyon, France (location of first isolate)
• S. saprophyticus; sapros, putrid; phyton, plant (saprophytic or growing on dead tissues)
• S. haemolyticus; lyse RBC

• Pathology of staphylococcal infections depends on
• 1. Production of surface proteins mediating adherence
• to host tissues
• - Capsule and slime layer
• - Teichoic acid
• - Protein A
• - Bound coagulase (clumping factor)
• - Surface adhesion proteins: MSCRAMM (microbial surface components recognizing adhesive matrix molecules)
• 2. Elaboration of extracellular proteins (specific toxins and hydrolytic enzymes)

• Cytotoxins (α, β, δ, γ, leukocidin)
• Exfoliative toxins (ET A, ETB)
• - Serine proteases
• - Staphylococcal scalded skin syndrome (SSSS)
• Enterotoxins (A-E, G-I)
• - Superantigens
• - Heat-stable at 100°C for 30 minutes
• - Resistant to hydrolysis by gastric and jejunal enzymes
• Toxic shock syndrome toxin-1
• - Superantigens
• - Heat-, proteolysis-resistant, chromos. mediated exotoxin
• - Penetrate mucosal barrier

• Coagulase
• Catalase
• Hyaluronidase
• - Hydrolyzes hyaluronic acids in connective tissue,
• - Promoting the spread of staphylococci in tissue
• - Fibrinolysin (staphylokinase)
• - Lipases
• - Nucleases
• - Penicillinase

• NF on human skin and mucosal surfaces
• Survive on dry surfaces for long periods
• Person-to-person spread through direct contact or exposure to contaminated fomites
• Risk factors
• Patients at risk
• Infections found worldwide and generally with no seasonal prevalence

• Toxin-mediated diseases
• - Food poisoning, toxic shock syndrome, and scalded skin syndrome
• Pyogenic diseases
• - Impetigo, folliculitis, furuncles, carbuncles, and wound infections
• Other systemic diseases (frequently associated with bacteremia)
• - Pneumonia (typically after viral respiratory infection), empyema (complication of pneumonia or surgical intervention), septic arthritis, osteomyelitis, acute endocarditis, and catheter-related bacteremia

• Toxin-Mediated Diseases
• • Scalded skin syndrome • Food poisoning • Toxic shock
• Suppurative Infections
• • Impetigo • Folliculitis • Furuncles or boils • Carbuncles • Bacteremia and endocarditis • Pneumonia and empyema • Osteomyelitis • Septic arthritis

• Infants (scalded skin syndrome)
• Young children with poor personal hygiene (impetigo and other cutaneous infections)
• Menstruating women (toxic shock syndrome)
• Patients with intravascular catheters (bacteremia and
• endocarditis) or shunts (meningitis)
• Patients with compromised pulmonary function or an antecedent viral respiratory infection (pneumonia)

• Microscopy
• Culture: BAP
• Biochemical: 7.5% NaCl, Mannitol, coagulase, protein A
• Serology: Aby to teichoic acids
• Fluorescent in situ hybridization (FISH): detects from clinical samples
• Pulsed-field gel electrophoresis (PFGE): genomic DNA analysis
• Antibiograms, biotyping, phage typing

• Semisynthetic penicillins resistant to β-lactamase hydrolysis (e.g., methicillin, nafcillin, oxacillin, dicloxacillin)
• Currently, 30% to 50% of the strains of S. aureus and more than 50% of the coagulase-negative staphylococci are resistant to semisynthetic penicillins
• MRSA: Methicillin-resistant S. aureus
• Acquisition of a gene (mecA) that codes for a novel
• penicillin-binding protein, PBP2’
• Expression of PBP2' renders the bacteria resistant to all penicillin, cephalosporin, and carbapenem antibiotics

• Antibiotics of choice are oxacillin (or other penicillinase-resistant penicillin), or vancomycin for oxacillin-resistant strains
• The focus of infection (e.g., abscess) must be identified and drained
• Treatment is symptomatic for patients with food poisoning
• Proper cleansing of wounds and use of disinfectant help prevent infections
• Thorough hand washing and covering of exposed skin helps medical personnel prevent infection or spread to other patients

• The goal of the National MRSA Education Initiative is to help Americans better recognize and prevent MRSA skin infections.
• MRSA is methicillin-resistant Staphylococcus aureus, a potentially dangerous type of staph bacteria that is resistant to certain antibiotics and may cause skin and other infections.
• Recent data show that Americans visit the doctor approximately 12 million times each year for suspected Staph or MRSA skin infection.

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• A school teacher who can't talk and is suffering mightily with a sore throat. He was well until two nights ago. His symptoms include 102o C fever, difficulty breathing due to enlarged tonsils, enlarged cervical lymph nodes. Two school kids in his class had similar episodes. He has no cough, and is otherwise in good health.
• A look at his throat shows markedly enlarged and reddened tonsils, plus,
• a creamy exudate on their surface.
• Throat culture is requested for lab diagnosis.
•  What is the most likely source of this infection
• (pharyngitis, strep throat)?

• Gram-positive cocci typically arranged in pairs or chains
• Facultative anaerobes; some are capnophilic
• Catalase-negative
• Ferment carbohydrates
• Blood- or serum-enriched media

• (1) Serologic properties
• - Lancefield groupings (originally A to W) of cell wall carbohydrates
• (2) Hemolytic patterns
• - Complete (beta [β]) hemolysis
• - Incomplete (alpha [α]) hemolysis
• - No (gamma [γ]) hemolysis
• (3) Biochemical (physiologic) properties

• S. pyogenes - Small colonies with a large zone of hemolysis
• S. agalactiae - Large colonies with a small zone of hemolysis

• Gram-positive cocci arranged in pairs and long chains
• β-Hemolytic; more viruluent strains with capsule
• Facultative anaerobe
• Catalase negative; PYR positive; bacitracin susceptible (important identification tests)
• Group-specific carbohydrate (A antigen) and type- specific antigens (M and T proteins) in cell wall
• Produce streptolysin O and DNase B
• - Antibodies against these antigens [ASO, anti-DNase B] are clinically important
• Virulence factors

• Capsule
• Lipoteichoic acid Binds to epithelial cells
• M protein: Adhesin-mediates internalization; antiphagocytic;
• degrades C3b
• M-like proteins: Binds Ig M and G and β2-macroglobulin (protease inhibitor); antiphagocytic
• F protein: Mediates adherence to epithelial cells and internalization
• Pyrogenic exotoxins
• Streptolysin S & Streptolysin O
• Streptokinase
• DNase
• C5a peptidase

• Pharyngitis (strep throat)
• Scarlet fever
• Pyoderma
• Erysipelas
• Cellulitis
• Necrotizing fasciitis
• Streptococcal toxic shock syndrome
• Other suppurative diseases (puerperal sepsis, lymphangitis, pneumonia)

• Rheumatic fever
• Characterized by inflammatory changes of the heart (pancarditis), joints (arthralgias to arthritis), blood vessels, and subcutaneous tissues
• Specific M types (types 1, 3, 5, 6, and 18)
• Incidence (US): > 10,000 cases/yr in 1961 to 112 cases in 1994 ; Estimated 100 cases per 100,000 children per year in developing countries
• Acute glomerulonephritis
• Acute inflammation of the renal glomeruli with edema, hypertension, hematuria, and proteinuria
• Specific nephritogenic strains of group A streptococci (pharyngeal and pyodermal strains)

• Asymptomatic colonization in URT and transient colonization of skin
• Ubiquitous; Survive on dry surfaces
• Person-to-person spread
• Higher risk individuals, most children
• - 5 to 15 years old (pharyngitis) - 2 to 5 years w/ poor personal hygiene (pyoderma)
• - Children with severe streptococcal disease (rheumatic fever, glomerulonephritis)
• Seasonal incidences of specific diseases

• Microscopy
• Direct antigen tests (streptococcal pharyngitis)
• Culture is highly sensitive
• ID by negative catalase, positive PYR reaction, susceptibility to bacitracin, and presence of group A antigen
• ASO test (confirming rheumatic fever and acute glomerulonephritis)
• Anti-DNase B test (suspected acute glomerulonephritis)

• Penicillin is drug of choice (erythromycin or oral cephalosporin)
• Treat oropharyngeal carriage
• Starting antibiotic therapy within 10 days in patients with pharyngitis
• - Prevents rheumatic fever
• Antibiotic prophylaxis before procedures
• - Prevent bacteremias leading to endocarditis

• Group C Streptococcus (S. anginosus, small-colony species and S. dysgalactiae, large-
• colony species)
• Abscess formation in deep tissues (Associated with S. anginosus group)
• Pharyngitis (Associated with S. dysgalactiae, Resembles S. pyogenes infections. Complicated with acute glomerulonephritis)

• Early-onset neonatal disease
• - Within 7 days of birth; pneumonia, meningitis, and sepsis
• Late-onset neonatal disease
• - More than a week after birth; bacteremia with meningitis
• Infections in pregnant women
• - Urinary tract infections (most often)
• - Bacteremia and disseminated complications may occur
• Infections in other adult patients
• - Most common diseases include bacteremia, pneumonia, bone and joint infections, and skin and soft-tissue infections

• Isolated independently by Pasteur and Steinberg more
• than 100 years ago
• Pneumococcal disease
• - A leading cause of morbidity and mortality
• - Pneumonia • Acute onset with severe chills and sustained fever;productive cough with blood-tinged sputum; lobar consolidation
• - Meningitis • Severe infection involving the meninges with headache, fever, and
• sepsis; high mortality and severe neurologic deficits in survivors
• - Bacteremia

• A heterogeneous collection of α-hemolytic and nonhemolytic streptococci.
• Group name is derived from viridis (Latin for "green"), a reflection of the fact that many of these bacteria produce a green pigment on blood agar media.
• More than 30 species and subspecies have been identifiedinto five subgroups: Anginosus, Mitis, Mutans, Salivarius, Bovis

• Abscess formation in deep tissues (Associated with S. anginosus group)
• Septicemia in neutropenic patients (Associated with S. mitis group)
• Subacute endocarditis (Associated with S. gordonii, S. mutans, S. mitis, S. oralis, and S. sanguis)
• Dental caries (Associated with S. mutans and S. sobrinus)
• Malignancies of gastrointestinal tract (Associated with S. bovis)

• Previously classified as group D streptococci
• In 1984, reclassified into the new genus Enterococcus
• (29 species)
• Colonizes the gastrointestinal tracts of humans and animals
• The most commonly isolated, clinically important species
• - Enterococcus faecalis
• - Enterococcus faecium
• Common colonizers of the human intestinal tract
• - Enterococcus gallinarum and Enterococcus casseliflavus
• - Misidentified as vancomycin-resistant E. faecium

• Disease (Urinary tract infections, Wound infections (particularly intraabdominal and usually
• polymicrobic), Bacteremia and Endocarditis)
• Antibiotic resistance is becoming increasingly common (particularly E. faecium)
• Diagnosis (Catalase negative , PYR positive, Resistant to bile and optochin)

• A 72-year-old man was admitted to the hospital because of a fever that had risen as high as 40。 C, myalgias, and respiratory complaints. The clinical diagnosis of influenza was confirmed by the laboratory isolation of influenza virus from respiratory secretions.
• This patient's hospitalization was complicated by the development of pneumonia caused by oxacillin-resistant Staphylococcus aureus that was treated with a 2-week course of vancomycin. Declining pulmonary function necessitated the use of a ventilator, which led to the development of a secondary infection with Klebsiella pneumoniae. Ceftazidime (a cephalosporin) and gentamicin were added to the patient's treatment.
• After 4 weeks of hospitalization, the patient became septic. E. faecium resistant to vancomycin, gentamicin, and ampicillin was cultured from three blood specimens.