Chocolate agar: Haemophilus, some pathogenic Neisseria strainsThioglycolate 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
Selective and Differential media
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
Cell Monolayer Culture
cells grow as a single layer and can be maintained in the lab indefinitely
Primary Cell Culture
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)
DNA Probe Analysis of Virus Infected Cell
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
Roche Molecular Diagnostics
PCR based testing and nucleic acid
Identifies pathogens in clinical specimens: sputums and blood samples
48 tests/run with results in 2.5 hours.
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.
Detection Ag-Ab Complex
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)
Single radial immunodiffusion
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
Ouchterlony immuno-double-diffusion technique
Determine the relatedness of different antigens
A central well and surrounding wells. Precipitation lines indicate an antibody-antigen reaction
Analysis of Antigens and Antibodies by Immunoprecipitation
Detecting antigens on the cell surface or within the cell
Legionnaires’ disease: Legionella antigen complexs with
The flow cytometer evaluates individual cell parameters as the cells flow past a laser beam at rates of more than 5000 per sec.
Enzyme-linked immunosorbent assays (ELISA)
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.
Western Blot Analysis
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).
Western Blot Analysis
Complement Fixation Test
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
Diagnosis of fungal, respiratory virus, arbovirus, and Q fever infections
Antibody Inhibition Assay
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
Latex Agglutination Assay
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
Red Azo Dye Protosil
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
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
Treatment of polymicrobial infections
(2) Prevent the emergence of resistant organisms during therapy
- Antibodies detected to pili antigens, Por proteins, and LOS
Except in the protection of ophthalmia neonatorum (1% silver nitrate, 1% tetracycline, or 0.5% erythromycin eye ointments)
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 Meningococcal Case
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
Signs and Symptoms of Meningitis
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
Physiology and Structure
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)
Virulence Neisseria meningitidis
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
Clinical Case N. gonorrhoeae
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
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
Neisseria meningitidis (the meningococcus)
Cause septicemia and meningitis
Hemolysin for dissemination in the bloodstream, leading to disseminated intravascular coagulations
Vaccine with capsular polysaccharide, except for group B strains
Neisseria sicca and Neisseria mucosa
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
Many are found on humans
S. aureus is catalase (+) AND coagulase (+; fibrinogen- fibrin- clot)
Types of Staph
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
S. aureus Virulence Factors
Pathology of staphylococcal infections depends on
1. Production of surface proteins mediating adherence
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
National MRSA Education Initiative
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.
pharyngitis? strep throat?
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
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
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 of Streptococcus pyogenes
Lipoteichoic acid Binds to epithelial cells
M protein: Adhesin-mediates internalization; antiphagocytic;
M-like proteins: Binds Ig M and G and β2-macroglobulin (protease inhibitor); antiphagocytic
F protein: Mediates adherence to epithelial cells and internalization
Streptolysin S & Streptolysin O
S. pyogenes (group A) Diseases
Pharyngitis (strep throat)
Streptococcal toxic shock syndrome
Other suppurative diseases (puerperal sepsis, lymphangitis, pneumonia)
S. pyogenes (group A) Diseases
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 glomerulonephritisAcute 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
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
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)
Treatment, Control, and Prevention
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
Other β Hemolytic Streptococci Disease
Group C Streptococcus (S. anginosus, small-colony species and S. dysgalactiae, large-
Abscess formation in deep tissues (Associated with S. anginosus group)
Pharyngitis (Associated with S. dysgalactiae, Resembles S. pyogenes infections. Complicated with acute glomerulonephritis)
Streptococcus agalactiae (Group B) Diseases
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
Streptococcus pneumoniae Disease
Isolated independently by Pasteur and Steinberg more
than 100 years ago
- 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
Viridans Group of Streptococci
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
Viridans Streptococci Diseases
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)
Enterococcus and Other Gram- Positive Cocci
Previously classified as group D streptococci
In 1984, reclassified into the new genus Enterococcus
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.