Bacteriology- Select gram ⊕ and gram ⊝ infections

Microbiology & Infectious Disease
Scanning electron micrograph of Staphylococcus aureus bound to the surface of a human neutrophil. “Granulocytic Phagocytes,” by Frank R. DeLeo and William M. Nauseef.
Prepared and presented by
Marc Imhotep Cray, M.D.

host-agent-environment interaction model
 Fundamental relationships involved in
host-agent-environment interaction
model
 In the host, pathogenetic mechanisms
extend from level of populations
(eg, person-to-person transmission) to
level of cellular and molecular processes
(eg, genetic susceptibility)
Hammer GD and McPhee. Pathophysiology of Disease: An
Introduction to Clinical Medicine. McGraw-Hill Education, 2014.
 The environment includes vectors
(insects and other carriers that transmit
infectious agents) and zoonotic hosts
or reservoirs (animals that harbor
infectious agents and often act to
amplify the infectious agent)

Host Defenses
 Types= Host defenses include:
 Nonspecific, antigen-independent mechanisms like anatomic
and physiological barriers including skin, lysozyme in tears,
stomach acidity, etc.
 Specific antigen-dependent mechanisms involving humoral
and cell-mediated immunity
 Host defenses can become harmful and contribute to disease
process by inducing formation of autoantibodies
 (e.g. M proteins of S. pyogenes Ag-Ab complexes can damage kidneys
and lead to acute poststreptococcal glomerulonephritis)

Hierarchy of Microbes: [Smallest  Largest]
4
Viruses
Bacteria
Single-celled eukaryotes (animal-like cells):
Parasitic protozoa
Fungi/yeast
Higher eukaryotes:
Parasitic intestinal worms
Tissue worms

Distribution
5
Microbes are everywhere and in high numbers
1 gram of human feces contains about 1 trillion
bacteria

The World from the Microbe's Perspective
6
Microbes cannot
think they simply
exploit the human
environment solely for
growth and survival
MISSION: “SURVIVE AND MULTIPLY TO ENSURE
SURVIVAL OF THE SPECIES”

Common Clinical Ways of Categorizing Microbes
7
“Avirulent” or “nonpathogenic” - harmless
“Virulent” or “pathogenic” - capable of causing disease
Normal flora - microbes that colonize the body and usually do not
cause disease
Opportunistic pathogens - microbes that normally do not cause
disease, but may under certain circumstances
Frank pathogens - microbes that always cause disease
Other - diseases caused mostly by ingestion of preformed toxins
or when toxins are produced from bacteria during infection

Question
What is the term for parasitic relationships between
microorganisms and the human body in which the
human body is harmed?
a. Infectious disease
b. Mutual disease
c. Communicable disease
d. Commensal disease

Answer: a
RATIONALE: A parasitic relationship is one in which only the infecting
organism benefits from the relationship and the host either gains
nothing from the relationship or sustains injury from the interaction.
If the host sustains injury or pathologic damage in response to a
parasitic infection, the process is called an infectious disease.
Mutual and commensal relationships do not harm the human body.
Communicable diseases can be passed from one human to another;
they are not considered parasitic.

Question
Which of the following sequences accurately describes the
stages of an infectious disease?
a. Incubation, prodromal, current, recovery, and resolution
b. Subacute, prodromal, acute, postacute, and convalescent
c. Prodromal, subacute, acute, postdromal, and resolution
d. Incubation, prodromal, acute, convalescent, and
resolution.

Answer: d
RATIONALE: The course of any infectious disease can be divided into
several distinguishable stages after the point of time in which the
potential pathogen enters the host.
These stages are:
 The incubation period
 The prodromal stage
 The acute stage
 The convalescent stage, and
 The resolution stage
There are no postacute, subacute or postdromal stages to an
infectious disease.

In the figure below, label the areas that represent the course through
which an infectious disease progresses: resolution, acute phase,
convalescent phase, incubation phase, infection, and prodromal phase.
Kipp B. Study Guide for Porth’s Essentials of Pathophysiology, 3rd Ed.
Wolters Kluwer Health | Lippincott Williams & Wilkins, 2011.

Kipp B. Study Guide for Porth’s Essentials of Pathophysiology, 3rd Ed.
Wolters Kluwer Health | Lippincott Williams & Wilkins, 2011.

Question
There are two criteria that have to be met in order for a
diagnosis of an infectious disease to occur. What are these two
criteria?
a. Recovery of probable pathogen and documentation of signs
and symptoms compatible with an infectious process.
b. Propagation of a microorganism outside the body and
testing to see what destroys it.
c. Identification by microscopic appearance and Gram stain
reaction
d. Serology and an antibody titer specific to the serology

Answer: a
RATIONALE: The diagnosis of an infectious disease requires two criteria: the
recovery of a probable pathogen or evidence of its presence from the
infected sites of a diseased host, and accurate documentation of clinical signs
and symptoms compatible with an infectious process.
Culture and sensitivity (C&S) are the growing of microorganisms outside
the body and the testing to see what kills it.
Identifying a microorganism by microscopic appearance and Gram stain reaction
are not the criteria for diagnosis.
Serology, an indirect means of identifying infectious agents by measuring
serum antibodies in the diseased host, and the quantification of those antibodies,
an antibody titer, are not criteria for diagnosis.

Basic Concepts in Bacteriology

Bacterial structures
Le T and Bhushan V. Microbiology. In: First Aid for the USMLE Step 1 2016. M-H, 2016.

Bacterial structures (2)
Cell envelope
1. General structure= macromolecular layers that
surround bacterium
It includes:
 A cell membrane and a peptidoglycan layer except for
mycoplasma
 An outer membrane layer in Gram-negative bacteria
 A capsule, a glycocalyx layer, or both (sometimes)
 Antigens that frequently induce a specific antibody
response

Cell envelope
2. Cell wall
 portion of cell envelope that is external to cytoplasmic
membrane and internal to capsule or glycocalyx
 It confers osmotic protection and Gram-staining characteristics
 In Gram-positive bacteria it is composed of:
o Peptidoglycan
o Teichoic acid
o Polysaccharides
 In Gram-negative bacteria, it is composed of:
o Peptidoglycan
o Lipoprotein
o An outer phospholipid membrane that contains lipopolysaccharide

Cell envelope
3. Peptidoglycan (also called mucopeptide or murein) is unique to
prokaryotes It is found in all bacterial cell walls except
Mycoplasma
 Structure
o This complex polymer consists of a backbone composed of alternating
N-acetylglucosamine and N-acetylmuramic acid and a set of identical
tetrapeptide side chains
 Peptidoglycan is site of action of certain antibiotics such as
penicillin and cephalosporins
 In Gram-positive bacteria, it comprises up to 50% of cell wall
 In Gram-negative bacteria, it comprises only 2% to 10% of cell wall

Bacterial structures/ Gram + Cell Envelope
Johnson AG, Ziegler RJ & Hawley L. Microbiology and Immunology 5th ed. Baltimore: LLW, 2010.

Bacterial structures/Gram - Cell Envelope
Johnson AG, Ziegler RJ & Hawley L. Microbiology and Immunology 5th ed. Baltimore: LLW, 2010.

Concept of "Pathogenesis"
23
Pathogenesis - the course of the infectious process
"Virulence" factors or "pathogenicity" factors
 Microbes which can cause disease are thought to carry out
process by utilizing one or more properties called virulence
factors or pathogenicity factors
IMPORTANT TO NOTE:
 The study of microbial virulence factors is a major emphasis In
microbiology And infectious disease research.
 Understanding molecular mechanisms by which pathogens exert
their virulence properties leads to new antibiotics and vaccines.

Mechanisms of Pathogenesis
Bacteria carry a variety of
virulence factors that facilitate
pathogenesis:
These include:
 factors that help them evade
immune system
 factors that facilitate
adhesion
 factors that facilitate spread
through tissues
 invasion of host cells
 a variety of different toxins
that are toxic to host

Virulence factors
 Features may be genetically encoded on bacterial chromosome or located on
plasmids
 Structural bacterial components virulence factors include:
o Antiphagocytic surface proteins and capsules
o Adhesins that promote colonization
o Endotoxins of Gram-negative bacteria/LPS of outer membrane
o Immunoglobulin G (IgG) antibody binding surface proteins
o Antigenic switching of surface antigens due to phase variation or
antigenic variation processes
Extracellular gene products, include:
 Degradative enzymes like collagenase and hyaluronidase that facilitate
tissue invasion
 IgA antibody-degrading proteases
 Exotoxins
Growth properties=capacity for intracellular growth & ability to form biofilms

Toxins
Endotoxins consist of lipid-A component of Gram-negative bacteria
Endotoxins actions:
 Induce release of endogenous pyrogens (e.g., interleukin 1 [IL-1], tumor necrosis factor
[TNF], prostaglandins, etc.)
 Increase vascular permeability
 Initiate complement and blood coagulation cascades
 Cause fever, hypotension, disseminated intracellular coagulation, and shock
Exotoxins are secreted by Gram-positive and Gram-negative bacteria; they may
be genetically encoded in bacterial chromosome, a plasmid, or a phage
Exotoxins actions:
 five mechanisms of action:
o Alter cellular components
o Act as superantigens that cause inappropriate release of cytokines
o Inhibit protein synthesis
o Increase cAMP
o Alter nerve impulse transmission

Le T and Bhushan V. Microbiology. In: First Aid for the USMLE Step 1 2016. McGraw-Hill, 2016.
Antimicrobial therapy

Bacterial taxonomy

Bacterial taxonomy (2)

Bacteria Staining
 Bacteria may be stained by a variety of dyes, including methylene
blue, crystal violet, carbol-fuchsin (red), and safranin (red)
 The two most important methods, the Gram and
acid-fast techniques use staining, decolorization, and
counterstaining in a manner that helps to classify, as well as, stain
organism
 Gram-positive bacteria stain blue/purple with Gram stain
o This is retained when washed with ethanol and acetone
 Gram-negative bacteria do not retain Gram stain when washed
with ethanol and acetone
o Counterstain results in pink/red color

Gram and acid-fast stains
 Four bacteria and a polymorphonuclear
neutrophil are shown at each stage
 All are initially stained purple by crystal
violet and iodine of Gram stain (A1) and
red by carbol-fuchsin of acid-fast stain
(B1)
 After decolorization, Gram-positive and
acid-fast organisms retain their original
stain
o Others are unstained (A2, B2)
 Safranin of Gram counterstain stains
o Gram-negative bacteria and makes
background red (A3), and
methylene blue leaves a blue
background for contrasting red acid-
fast bacillus (B3) Ryan KJ and Ray CG Eds. Sherris Medical Microbiology, 5th Ed. New York:
McGraw-Hill, 2010.

Clinical Bacteriology
Gram-positive lab algorithm

Question
A 54-year-old man develops a pyogenic infection along the
suture line after knee surgery. The laboratory gives a preliminary
report of a beta-hemolytic, catalase-positive, coagulase-positive,
Gram-positive coccus. The most likely causative agent is
(A) Moraxella catarrhalis
(B) Staphylococcus aureus
(C) Staphylococcus epidermidis
(D) Streptococcus agalactiae
(E) Streptococcus pyogenes

Clinical Bacteriology
Gram-negative lab algorithm

Gram-positive Organisms
Staphylococcal infections
Staphylococcus aureus causes:
 Skin infections
 Osteomyelitis
 Pneumonia
 Endocarditis
 Toxic shock syndrome
Virulence factors – remember SET:
 Surface proteins for adherence
 Enzymes
 Toxins
 Grow in clusters
Staphylococcus aureus
Sputum sample from pt. with pneumonia
shows gram positive cocci in clusters

Gram-positive Organisms (2)
Streptococcal infections
Facultative or obligate anaerobes
 Streptococcus pneumoniae:
acquired pneumonia
 Enterococci: UTI and endocarditis
Virulence factors:
 Capsules resist phagocytosis
 M protein inhibits alternative
pathway of complement system
 Pneumolysin destroys
membranes of host cells
 Grow in pairs or chains
Streptococcus pneumoniae
Sputum sample from a pt. with pneumonia
shows gram positive diplococci

Diphtheria
Corynebacterium diphtheriae
 Rod shaped
 Exotoxin causes damage to heart and nerves
 Symptoms include pseudomembranous
pharyngitis (grayish-white membrane)
with lymphadenopathy, myocarditis, and
arrhythmias
 Lab diagnosis based on gram ⊕ rods with
metachromatic (blue and red) granules and
⊕ Elek test for toxin
 Toxoid vaccine prevents diphtheria

Anthrax
Bacillus anthracis
 Gram ⊕, spore-forming rod that produces
anthrax toxin Only bacterium with a polypeptide
capsule (contains d-glutamate)
Cutaneous anthrax
 Painless papule surrounded by vesicles ulcer
with black eschar (painless, necrotic)
uncommonly progresses to bacteremia and death
Pulmonary anthrax
 Inhalation of spores flu-like symptoms that
rapidly progress to fever, pulmonary hemorrhage,
mediastinitis, and shock death
 Also known as woolsorter’s disease
Bacillus cereus Gram ⊕ spore-forming rod
Causes food poisoning (Reheated rice
syndrome)
 Spores survive cooking rice
 Keeping rice warm results in germination
of spores & enterotoxin formation
 Emetic type usually seen with rice and
pasta N/V within 1–5 hr. Caused by
cereulide, a preformed toxin
 Diarrheal type causes watery, nonbloody
diarrhea and GI pain within 8–18 hrs.

Nocardia vs Actinomyces: Both are gram ⊕ and form long, branching filaments
resembling fungi
Nocardia
 Aerobe
 Acid fast (weak)
 Found in soil
 Causes pulmonary infections in
immunocompromised with CNS
involvement (can mimic TB but
with (- PPD)
 cutaneous infections after trauma in
immunocompetent
 Treat with sulfonamides (TMP-SMX)
Actinomyces
 Anaerobe
 Not acid fast
 Normal oral, reproductive, and Gl flora
 Causes oral/facial abscesses that drain
through sinus tracts, forms yellow
"sulfur granules;" can also cause PID
with IUDs
 Treat with penicillin

Listeriosis
Listeria monocytogenes
 Gram ⊕, facultative intracellular rod (bacillus) acquired by ingestion of
unpasteurized dairy products & cold deli meats, via transplacental transmission,
or by vaginal transmission during birth
 Forms “rocket tails” (red) via actin polymerization
that allow intracellular movement and cell-to- cell
spread across cell membranes, thereby avoiding antibody
 Can cause amnionitis, septicemia, and spontaneous abortion in pregnant
women, granulomatosis infantiseptica, neonatal meningitis, meningitis in
elderly and immunocompromised patients; mild, self-limited gastroenteritis in
healthy individuals
 Treatment: ampicillin in infants, immunocompromised and elderly as empirical
treatment of meningitis

Gram-negative Organisms
Neisserial infections: Gram ⊝ diplococci
Neisseria meningitides meningitis and Neisseria gonorrhoeae STI
Gonococci
 No polysaccharide capsule
 No maltose metabolized
 No vaccine due to antigenic
variation of pilus proteins
 Sexually or perinatally transmitted
 Causes gonorrhea, septic arthritis,
neonatal conjunctivitis, pelvic
inflammatory disease (PID)
 Condoms decrease sexual transmission,
erythromycin eye ointment prevents
neonatal blindness
 Treatment: ceftriaxone + (azithromycin or
doxycycline) for possible chlamydial
coinfection
Meningococci
 Polysaccharide capsule
 Maltose fermentation
 Vaccine (type B vaccine not widely
available)
 Transmitted via respiratory and oral
secretions
 Causes meningococcemia with petechial
hemorrhages and gangrene of toes
 Meningitis
 Waterhouse-Friderichsen syndrome
(adrenal insufficiency, fever, DIC shock)
Rifampin, ciprofloxacin, or ceftriaxone
 prophylaxis in close contacts
 Treatment: ceftriaxone or penicillin G

Gram-negative Organisms (2)
Pseudomonas infection
 Pseudomonas aeruginosa is an
opportunistic aerobic, motile, gram
⊝ rod (bacillus )
 Non-lactose fermenting, oxidase ⊕
 Produces pyocyanin (blue-green
pigment); has a grape-like odor
 Produces endotoxin (fever, shock)
 exotoxin A (inactivates EF-2)
 phospholipase C (degrades cell
membranes) and
 pyocyanin (generates reactive
oxygen species)
PSEUDOMONAS is associated with:
Pneumonia, pyocyanin
Sepsis
Ecthyma gangrenosum
UTIs
Diabetes, drug use
Osteomyelitis (e.g., puncture wounds)
Mucoid polysaccharide capsule
Otitis externa (swimmer’s ear)
Nosocomial infections (catheters,
equipment)
exotoxin A
Skin infections (hot tub folliculitis)
Ecthyma gangrenosum
rapidly progressive, necrotic
cutaneous lesion caused by
Pseudomonas bacteremia.
Immunocompromised pts.

Plague
 Yersinia pestis = Zoonotic bacteria (Zoonosis: infectious disease
transmitted betw. animals and humans)
Transmission & Source:
 Fleas (rats and prairie dogs are reservoirs)
Whooping cough
 Bordetella pertussis Gram ⊝, aerobic coccobacillus
 Virulence factors include pertussis toxin (disables Gi) and tracheal
cytotoxin
 Causes whooping cough
 Prevented by Tdap, DTaP vaccines
 May be mistaken as viral infection due to lymphocytic infiltrate resulting
from immune response

Haemophilus influenza
 Small gram ⊝ (coccobacillary) rod
 Aerosol transmission
 Nontypeable (unencapsulated) strains are most common cause of mucosal infections (otitis
media, conjunctivitis, bronchitis) as well as invasive infections since vaccine for capsular type
b was introduced
 Produces IgA protease
 Culture on chocolate agar
HaEMOPhilus causes
Epiglottitis (endoscopic appearance in (A) , can be “cherry red” in children; “thumbprint sign” on
x-ray (B)
Meningitis
Otitis media, and
Pneumonia
 Treatment: amoxicillin +/− clavulanate for
mucosal infections; ceftriaxone for meningitis
 Rifampin prophylaxis for close contacts
(A)
(B)

Mycoplasma pneumoniae
 Pleomorphic (Illust.)
 Classic cause of atypical “walking” pneumonia
(insidious onset, headache, nonproductive
cough, patchy or diffuse interstitial infiltrate)
 X-ray looks worse than patient
 High titer of cold agglutinins (IgM)
 Grown on Eaton agar
 Treatment: macrolides, doxycycline, or fluoroquinolone
 (N.B. penicillin ineffective since Mycoplasma have no cell wall)
o Not seen on Gram stain
 Bacterial membrane contains sterols for stability
 Mycoplasmas pneumonia is more common in patients < 30 years old
 Frequent outbreaks in military recruits and prisons

Gram ⊕, spore-forming, obligate anaerobic rods
Clostridia (with exotoxins=C tetani, C botulinum, C perfringens & C difficile )
Clostridia tetani
 Produces tetanospasmin, an exotoxin causing Tetanus
 Tetanus toxin (and botulinum toxin) are proteases that cleave SNARE
proteins for neurotransmitters
 Blocks release of inhibitory neurotransmitters, GABA and glycine,
from Renshaw cells in spinal cord
 Causes spastic paralysis, trismus (lockjaw), risus sardonicus (raised
eyebrows and open grin)
 Prevent with tetanus vaccine
 Treat with
 antitoxin +/− vaccine booster
 diazepam (for muscle spasms), and
 wound debridement

Gram ⊕, spore-forming, obligate anaerobic rods (2)
Clostridia botulinum
 Produces a heat-labile toxin that inhibits ACh release at neuromuscular
junction causing botulism
 In adults, disease is caused by ingestion of preformed toxin
 In babies, ingestion of spores (e.g., in honey) leads to disease (floppy
baby syndrome)
o Botulinum is from bad bottles of food, juice, and honey (causes a
descending flaccid paralysis)
 Treat with antitoxin
Note:
 Local botox injections used to Tx focal dystonia, achalasia, and muscle spasms
 Also used for cosmetic reduction of facial wrinkles

Clostridia perfringens
 Produces α toxin (lecithinase, a
phospholipase) that can cause myonecrosis
(gas gangrene) and hemolysis
 Spores can survive in undercooked food
when ingested, bacteria release heat-labile
enterotoxin  food poisoning
Perfringens perforates a gangrenous leg Le T and Bhushan V. Microbiology. In: First Aid for the
USMLE Step 1 2016. McGraw-Hill, 2016.

Clostridia difficile
Produces 2 toxins
 Toxin A, enterotoxin, binds to brush border of gut
 Toxin B, cytotoxin, causes cytoskeletal disruption
via actin depolymerization diarrhea
pseudomembranous colitis (Illust.)
 Difficile causes diarrhea
Often 2° to antibiotic use, especially clindamycin or
ampicillin and associated with PPI use
Diagnosed by detecting one or both toxins in stool by
PCR
 Treatment: metronidazole or oral vancomycin
 For recurrent cases, consider repeating prior regimen,
fidaxomicin, or fecal microbiota transplant
Le T and Bhushan V. Microbiology. In: First Aid for the
USMLE Step 1 2016. McGraw-Hill, 2016.

50
THE END
See next slide for further study tools and resources.

Further study:
51
eLearning (IVMS Cloud)
 Infectious Disease
 Microbial biology & Immune System
Textbooks:
 Ryan KJ and Ray CG Eds. Sherris Medical Microbiology, 5th Ed. New York:
McGraw-Hill, 2010.
 Carroll KC etal. Jawetz, Melnick, & Adelberg’s Medical Microbiology 27th Ed.
New York: McGraw-Hill, 2016.

Bacteriology- Select gram ⊕ and gram ⊝ infections

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