Download 19. BG_7.20 GRAM PO..

Gram Positive Stains and Bacterium
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PRESENTED BY: BRYANNA GRAY
CONTACT INFORMATION:
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BRYANNA.GRAY@YAHOO.COM
4 Steps to Gram Staining
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① Pour on crystal violet stain (a blue dye) and wait
60 seconds.
② Wash off with water and flood with iodine solution.
Wait 60 seconds.
③ Wash off with water and then "decolorize" with
95% alcohol.
④ Finally, counter-stain with safranin (a red dye).
Wait 30 seconds and wash off with water.
The Result of Gram Staining
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Gram Positive Stains:
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•Crystal violet gets trapped in the latticework
•Iodine acts as mordant (binding agent)
•Grams Alcohol shrinks latticework trapping stain &
iodine
•Safranin (Red) - no space for it, hence purple stain
outermost layer
peptidoglycan layer
Phospholipid Bilayer
Cytoplasm
Gram Positive Cell Wall Properties:
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Cell Wall – Peptidoglycan, NAG and NAM
① Peptidoglycan: a polymer that is composed of polysaccharide and
peptide chains and is found especially in bacterial cell walls —called
also mucopeptide, murein
② NAG(N-acetylglucosamine):the acetyl derivative of glucosamine; it
is a component of structural glycosaminoglycans, glycolipids, and
membrane glycoproteins
③ NAM:N-Acetylmuramic acid, or MurNAc, is the ether
acetylglucosamine . It is part of a biopolymer in the bacterial cell wall,
built from alternating units of N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc), cross-linked with oligopeptides at the
lactic acid residue of MurNAc. This layered structure is called
peptidoglycan.
Gram Positive Slide
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POP QUIZ: (complete the chart)
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Gram Positive
Gram Negative
Ex. Peptidoglycan
Yes
Yes
Ex. NAG
Yes
No
NAM
Lipopolysaccharide
Cytoplasm
Red Stain
Purple Stain
Enterococcus faecalis
Corynebacterium
diptheriae
Neiserria gonorrhoeae
S. pneumoniae
Staphylococcus aureus
Shigella
Salmonella
Gram Positive Bacteria:
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① S.Aureus: Skin infection
②
Staphylococcus
aureus
Gram
Positive
Clostridium
S. pneumoniae:
pneumonia an infection
that inflames the air sacs in
one or both lungs. S.
pneumoniae can also cause
bacterial meningitis.
① Clostridium:most notably
S. Pneumoniae
the causative agent of
botulism. They are obligate
anaerobes capable of
producing endospores.
Gram Positive Infections
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STAPHYLOCOCCUS
GENUS
Characteristics of Staphylococci
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 Common inhabitant of the skin and mucous
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membranes
Spherical cells arranged in irregular clusters
Gram-positive
Lack spores and flagella
May have capsules
`
31 species
Facultative anaerobes
Staphylococcal Disease can range from localized to
systemic
S. aureus Epidemiology and Pathology
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 Present in most environments frequented by humans
 Readily isolated from fomites
 Carriage rate for healthy adults is 20-60%.
 Carriage is mostly in anterior nares, skin, nasopharynx,
intestine.
 Predisposition to infection include: poor hygiene and
nutrition, tissue injury, preexisting primary infection,
diabetes, immunodeficiency.
 Increase in community acquired methicillin resistance MRSA
Local Staphylococcal Disease
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Localized cutaneous infections–invade skin through
wounds, follicles, or glands.
 folliculitis–superficial inflammation of hair follicle;
usually resolved with no complications but can progress
 furuncle–boil; inflammation of hair follicle or
sebaceous gland progresses into abscess or pustule
 carbuncle–larger and deeper lesion created by
aggregation and interconnection of a cluster of furuncles
 impetigo –bubble-like swellings that can break and peel
away; most common in newborns
Systemic Staphylococcal infection
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 osteomyelitis–infection is established in the
metaphysis; abscess forms
 bacteremia -primary origin is bacteria from
another infected site or medical devices; endocarditis
possible
Staphylococcal Toxigenic Disease
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Toxigenic disease
 food intoxication–ingestion of heat stable
enterotoxins; gastrointestinal distress
 staphylococcal scalded skin syndrome–toxin
induces bright red flush, blisters, then desquamation
of the epidermis
 toxic shock syndrome–toxemia leading to shock
and organ failure
Staphylococcal Identification
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 Frequently isolated from pus, tissue exudates,
sputum, urine, and blood
 •Cultivation, catalase, biochemical testing, coagulase
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Clinical Concerns and Treatment
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 95% have penicillinase and are resistant to penicillin
and ampicillin.
 •MRSA –methicillin-resistant S. aureus–carry
multiple resistance
 •Abscesses have to be surgically perforated.
 •Systemic infections require intensive lengthy
therapy
Prevention of Staphylococcal Infection
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 Universal precautions by healthcare providers to prevent nosocomial infections
 •Hygiene and cleansing:
 Wash your hands. Careful hand washing remains your best defense against
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germs. Scrub hands briskly for at least 15 seconds, then dry them with a disposable
towel and use another towel to turn off the faucet. Carry a small bottle of hand
sanitizer containing at least 62 percent alcohol for times when you don't have access
to soap and water.
Keep wounds covered. Keep cuts and abrasions clean and covered with sterile,
dry bandages until they heal. The pus from infected sores may contain MRSA, and
keeping wounds covered will help prevent the bacteria from spreading.
Keep personal items personal. Avoid sharing personal items such as towels,
sheets, razors, clothing and athletic equipment. MRSA spreads on contaminated
objects as well as through direct contact.
Shower after athletic games or practices. Shower immediately after each
game or practice. Use soap and water. Don't share towels.
Sanitize linens. If you have a cut or sore, wash towels and bed linens in a washing
machine set to the hottest water setting (with added bleach, if possible) and dry
them in a hot dryer. Wash gym and athletic clothes after each wearing.
Staphylococcus infections of Interest
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 Of significance to humans are various strains of the
species S. aureus and S. epidermidis.
Significance to Humans S. aureus
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Staphylococcus aureus
Strains of S. aureus are major agents of wound
infections, boil, and other human skin infections and
are one of the most common causes of food poisoning.
S. aureus also causes meningitis, pneumonia, urinary
tract infections, and infection of the breast in women
or of the udder in domestic animals.
S. aureus
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 Grows in large, round, opaque colonies
 •Optimum temperature of 37C
 •Facultative anaerobe
 •Withstands high salt, extremes in pH, and high
temperatures
 •Carried in nasopharynx and skin
 •Produces many virulence factors
Virulence Factors of S.aureus
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 Coagulase –coagulates plasma and blood; produced
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by 97% of human isolates; diagnostic
Hyaluronidase –digests connective tissue
Staphylokinase –digests blood clots
DNase –digests DNA
Lipases –digest oils; enhances colonization on skin
Penicillinase –inactivates penicillin
Virulence Factors of S.aureus (continued)
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Toxins
 Hemolysins(α, β, γ, δ) –lyse red blood cells
 Leukocidin–lyses neutrophils and macrophages
 Enterotoxin–induce gastrointestinal distress
 Exfoliative toxin–separates the epidermis from
the dermis
 Toxic shock syndrome toxin(TSST) -induces
fever, vomiting, shock, systemic organ damage
Methicillin-resistant Staphylococcus aureus
(MRSA)
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 Methicillin-resistant Staphylococcus aureus (MRSA)
infection is caused by a type of staph bacteria that's
become resistant to many of the antibiotics used to treat
ordinary staph infections.
 Most MRSA infections occur in people who've been in
hospitals or other health care settings, such as nursing
homes and dialysis centers. When it occurs in these
settings, it's known as health care-associated MRSA (HAMRSA). HA-MRSA infections typically are associated
with invasive procedures or devices, such as surgeries,
intravenous tubing or artificial joints.
Methicillin-resistant Staphylococcus aureus
(MRSA)..continued
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 Another type of MRSA infection has occurred in the
wider community — among healthy people. This
form, community-associated MRSA (CA-MRSA),
often begins as a painful skin boil. It's spread by
skin-to-skin contact. At-risk populations include
groups such as high school wrestlers, child care
workers and people who live in crowded conditions.
Symptoms of MRSA
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Staph skin infections, including MRSA, generally start as
swollen, painful red bumps that might resemble pimples or
spider bites. The affected area might be:
 Warm to the touch
 Full of pus or other drainage
 Accompanied by a fever
These can quickly turn into deep, painful abscesses that
require surgical draining. Sometimes the bacteria remain
confined to the skin. But they can also burrow deep into
the body, causing potentially life-threatening infections in
bones, joints, surgical wounds, the bloodstream, heart
valves and lungs
MRSA Treatment
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Drug Treatment:
Vancomycin (Vancocin)
Vancomycin can cause two types of hypersensitivity
reactions, the red man syndrome and anaphylaxis
Non- Drug treatment:
 Both health care-associated and communityassociated strains of MRSA still respond to certain
antibiotics. In some cases, antibiotics may not be
necessary. For example, doctors may drain a
superficial abscess caused by MRSA rather than treat
the infection with drugs
Red Man Syndrome
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Gram Positive Infections
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STREPTOCOCCUS
GENUS
Streptococcus
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 Streptococcus is a genus of coccus (spherical) gram-
positive bacteria belonging to the phylum
Firmicutes[3] and the order Lactobacillales (lactic
acid bacteria). Cell division in this genus occurs
along a single axis in these bacteria, thus they grow
in chains or pairs, hence the name—from Greek
στρεπτός streptos, meaning easily bent or twisted,
like a chain (twisted chain). (Contrast this with
staphylococci, which divide along multiple axes and
generate grape-like clusters of cells.)
General Characteristic of Streptococci
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 Gram-positive spherical/ovoid cocci arranged in long
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chains; commonly in pairs
Non-spore-forming, nonmotile
Can form capsules and slime layers
Facultative anaerobes
Do not form catalase, but have a peroxidase system
Most parasitic forms are fastidious and require enriched
media.
Small, nonpigmented colonies
Sensitive to drying, heat and disinfectants
25 species
Streptococci Classifications
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 Lancefield classification system based on cell wall Ag
–17 groups (A,B,C,….)
 Another classification system is based on hemolysis
reactions.
 b-hemolysis –A,B,C,G and some D strains
 a–hemolysis –S. pneumoniae and others collectively
called viridans
Streptococci Classifications (continued)
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Human Streptococcal Pathogens
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 S. pyogenes
 S. agalactiae
 Viridans streptococci
 S. pneumoniae
 Enterococcus faecalis
Streptococcus pyogenes
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 Streptococcus pyogenes is a species of bacteria. Like
most other streptococci, it is clinically important in
human illness. It is an infrequent, but usually
pathogenic, part of the skin flora. It is the sole species of
Lancefield group A and is often called group A
streptococcus (GAS), because it displays streptococcal
group A antigen on its cell wall. Group A streptococcal
infection can cause illness, which typically produces
small zones of beta-hemolysis, a complete destruction of
red blood cells. (A zone size of 2–3 mm is typical). It is
thus also called group A (beta-hemolytic) streptococcus
(GABHS).
Beta-Hemolytic S. pyogenes
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 •Most serious streptococcal pathogen
 •Strict parasite
 •Inhabits throat, nasopharynx, occasionally skin
Virulence Factors of Beta-hemolytic S. Pyogenes
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 Produces surface antigens:
 C-carbohydrates–protect against lysozyme
 Fimbriae-adherence
 M-protein–contributes to resistance to
phagocytosis
 Hyaluronic acid capsule –provokes no immune
response
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Virulence Factors of β-hemolytic S.
pyogenes
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 Extracellular toxins:
 streptolysins–hemolysins; streptolysin O (SLO)
and streptolysin S (SLS) –both cause cell and tissue
injury
 pyogenic toxin (erythrogenic)–induces fever
and typical red rash
 superantigens–strong monocyte and lymphocyte
stimulants; cause the release of tissue necrotic factor
Virulence Factors of β-hemolytic S.
pyogenes
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 Extracellular enzymes
 streptokinase –digests fibrin clots
 hyaluronidase –breaks down connective tissue
 DNase –hydrolyzes DNA
β-hemolytic S. pyogenes
Epidemiology and Pathogenesis
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 Humans only reservoir
 In apparent carriers
 Transmission –contact, droplets, food, fomites
 Portal of entry generally skin or pharynx
 Children predominant group affected for cutaneous
and throat infections
 Systemic infections and progressive sequelae
possible if untreated
Scope of Clinical Disease
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Skin infections
 Impetigo (pyoderma)–superficial lesions that break
and form highly contagious crust; often occurs in
epidemics in school children; also associated with insect
bites, poor hygiene, and crowded living conditions
 Erysipelas–pathogen enters through a break in the skin
and eventually spreads to the dermis and subcutaneous
tissues; can remain superficial or become systemic
Throat infections
 Streptococcal pharyngitis–strep throat
Scope of Clinical Disease (continued)
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 Systemic infections
 Scarlet fever–strain of S. pyogenes carrying a
prophage that codes for pyrogenic toxin can lead
to sequelae
 Septicemia
 Pneumonia
 Streptococcal toxic shock syndrome
Long- Term Complications of Group A infections
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 •Rheumatic fever–follows overt or subclinical
pharyngitis in children; carditis with extensive valve
damage possible, arthritis, chorea, fever
 •Acute glomerulonephritis–nephritis, increased
blood pressure, occasionally heart failure; can
become chronic leading to kidney failure
Group B: Streptococcus agalactiae
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 Regularly resides in human vagina, pharynx and
large intestine
 Can be transferred to infant during delivery and
cause severe infection
–most prevalent cause of neonatal pneumonia, sepsis,
and meningitis
–15,000 infections and 5,000 deaths in US
–Pregnant women should be screened and treated.
 Wound and skin infections and endocarditis in
debilitated people
Group D Enterococci and Groups C and G
Streptococci
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Group D:
 Enterococcus faecalis, E. faecium, E. durans
 normal colonists of human large intestine
 cause opportunistic urinary, wound, and skin
infections, particularly in debilitated persons
Groups C and G:
 common animal flora, frequently isolated from upper
respiratory;
Identification
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 Cultivation and diagnosis ensure proper treatment to
prevent possible complications.
 Rapid diagnostic tests based on monoclonal
antibodies that react with C-carbohydrates
 Culture using bacitracin disc test, CAMP test
Identification (continued)
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Identification (continued)
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Treatment and Prevention
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Good News
Bad News
 Group A and B are
 NO VACCINES
treated with penicillin
 Sensitivity testing
needed for enterococci
AVAILABLE !!!!!!!!
Can you guess?
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 Which of the below infections has the largest
and most complex group?
A. S. pyogenes
B. S. agalactiae
C. Viridans streptococci
D. S. pneumoniae
E. Enterococcus faecalis
α-Hemolytic Streptococci: Viridans Group
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Large complex group
–Streptococcus mutans, S. oralis, S. salivarus,
 S. sanguis, S. milleri, S. mitis
 Most numerous and widespread residents of the
gums and teeth, oral cavity and also found in
nasopharynx, genital tract, skin
 Not very invasive; dental or surgical procedures
facilitate entrance
Viridans Group
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 Bacteremia, meningitis, abdominal infection, tooth
abscesses
 Most serious infection –subacute endocarditis–
blood-borne bacteria settle and grow on heart lining
or valves
 Persons with preexisting heart disease are at high
risk.
 Colonization of heart by forming biofilms
Can you guess?
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 How do dentist interact with the Viridans
group?
A. Dentist help by removing infected teeth.
B. Dentist help prevent cavities
C. Dentist help remove plaque
D. Both B and C
E. Dentist do not interact with the Viridans group.
S. mutans
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 Produce slime layers that adhere to teeth, basis for
plaque.
 Involved in dental caries
 Persons with preexisting heart conditions should
receive prophylactic antibiotics before surgery or
dental procedures.
Streptococcus pneumoniae: The Pneumococcus
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 Causes 60-70% of all bacterial pneumonias
 Small, lancet-shaped cells arranged in pairs and
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short chains
Culture requires blood or chocolate agar.
Growth improved by 5-10% CO2
Lack catalase and peroxidases –cultures die in O2
Causes pneumonia and otitis media
Epidemiology and Pathophysiology
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 5-50% of all people carry it as normal flora in the
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nasopharynx; infections are usually endogenous.
Very delicate, does not survive long outside of its habitat
Young children, elderly, immune compromised, those
with other lung diseases or viral infections, persons living
in close quarters are predisposed to pneumonia
Pneumonia occurs when cells are aspirated into the lungs
of susceptible individuals.
Pneumococci multiply and induce an overwhelming
inflammatory response.
Gains access to middle ear by way of eustachian tube
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Cultivation and Diagnosis
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 •Gram stain of specimen –presumptive identification
 •α-hemolytic; optochin sensitivity
 •Quellung test or capsular swelling reaction
Treatment and Prevention
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 Traditionally treated with penicillin G or V
 Increased drug resistance
 Two vaccines available for high risk individuals:–-
capsular antigen vaccine for older adults and other
high risk individuals
- effective 5 years
– conjugate vaccine for children 2 to 23 months
Gram Negative
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CLOSTRIDIUM
GENUS
Clostridium
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 Gram-positive, spore-forming rods
 •Anaerobic and catalase negative
 •120 species
 •Oval or spherical spores produced only under
anaerobic conditions
 •Synthesize organic acids, alcohols, and exotoxins
 •Cause wound infections, tissue infections, and food
intoxications
Tetanus (lockjaw)
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 Clostridium tetani
 Common resident of soil and GI tracts of animals
 Causes tetanus or lockjaw, a neuromuscular disease
 Most commonly among geriatric patients and IV
drug abusers; neonates in developing countries
Tetanus (continued)
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 A serious bacterial infection that causes painful
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muscle spasms and can lead to death.
Extremely rare
Fewer than 1,000 US cases per year
Spreads by contact with a contaminated object or
surface
Requires a medical diagnosis
Lab tests or imaging not required
Medium-term: resolves within months
Tetanus Pathology
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 Spores of the bacteria that cause tetanus,
Clostridium tetani, are found in soil, dust and animal
feces. When they enter a deep flesh wound, spores
grow into bacteria that can produce a powerful toxin,
tetanospasmin, which impairs the nerves that
control your muscles (motor neurons). The toxin can
cause muscle stiffness and spasms — the major signs
of tetanus.
 Tetanospasmin–neurotoxin causes paralysis by
binding to motor nerve endings; blocking the release
of neurotransmitter for muscular contraction
inhibition; muscles contract uncontrollably.
 You can't catch tetanus from a person who has it.
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Treatment and Prevention
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 Treatment aimed at deterring degree of toxemia and
infection and maintaining homeostasis
 Antitoxin therapy with human tetanus immune
globulin; inactivates circulating toxin but does not
counteract that which is already bound
 Control infection with penicillin or tetracycline; and
muscle relaxants
 Vaccine available; booster needed every 10 years
Clostridial Food Poisoning
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 •Clostridium botulinum –rare but severe
intoxication usually from home canned food
 •Clostridium perfringens–mild intestinal illness;
second most common form of food poisoning
worldwide
Botulism
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 Foodborne botulism. The harmful bacteria thrive
and produce the toxin in environments with little
oxygen, such as in canned food.
 Wound botulism. If these bacteria get into a cut,
they can cause a dangerous infection that produces
the toxin.
 Infant botulism. This most common form of
botulism begins after Clostridium botulinum
bacterial spores grow in a baby's intestinal tract. It
typically occurs between the ages of 2 months and 8
months.
Food Bourne botulism Symptoms
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 Signs and symptoms of foodborne botulism typically begin
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between 12 and 36 hours after the toxin gets into your body.
But, the start of symptoms can range from a few hours to
several days, depending on the amount of toxin ingested.
Signs and symptoms of foodborne botulism include:
Difficulty swallowing or speaking
Dry mouth
Facial weakness on both sides of the face
Blurred or double vision
Drooping eyelids
Trouble breathing
Nausea, vomiting and abdominal cramps
Paralysis
Wound botulism Symptoms
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 Most people who develop wound botulism inject drugs
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several times a day, so it's difficult to know for sure how
long it takes for signs and symptoms to develop after the
toxin enters the body. Wound botulism signs and
symptoms include:
Difficulty swallowing or speaking
Facial weakness on both sides of the face
Blurred or double vision
Drooping eyelids
Trouble breathing
Paralysis
Infant botulism
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 If infant botulism is related to food, such as honey, problems
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generally begin within 18 to 36 hours after the toxin enters the
baby's body. Signs and symptoms include:
Constipation (often the first sign)
Floppy movements due to muscle weakness and trouble
controlling the head
Weak cry
Irritability
Drooling
Drooping eyelids
Tiredness
Difficulty sucking or feeding
Paralysis
Botulism Treatment and Prevention
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 Determine presence of toxin in food, intestinal
contents or feces
 Administer antitoxin; cardiac and respiratory
support
 Infectious botulism treated with penicillin
 Practice proper methods of preserving and handling
canned foods; addition of preservatives.
Check Your Knowledge:
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① What does Vancomycin treat and what
②
③
④
⑤
hypersensitivity is associated with it?
What s the pathology of all infections discussed?
Can you differentiate the difference between
gram negative and gram positive?
What type of infection is S. Aureus and what is its
treatment?
What type of infection is MRSA and how is it
acquired?
ANY QUESTIONS FOR ME?!?!?!?!?!
References:
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① http://www.ppdictionary.com/gnbac.htm
② Parija SC (Jan 1, 2009). Textbook of Microbiology &
③
④
⑤
⑥
Immunology. India: Elsevier. ISBN 8131221636.
Kulp A, Kuehn MJ (2010). "Biological functions and
biogenesis of secreted bacterial outer membrane vesicles".
Annu. Rev. Microbiol. 64: 163–84.
doi:10.1146/annurev.micro.091208.073413. PMC 3525469.
PMID 20825345.
http://study.com/academy/lesson/bacterial-cell-walls-andthe-gram-stain-test.html
Dr. Kulkarn “Antibiotics Lecture” September 18,2015
Menilik Tadesse “Gram Positive Infections” August 13, 2013