2. The Gram-Positive Bacteria
Chapter 19

3. The Gram-Positive Bacilli of Medical Importance

4. Some Gram-positive soil bacteria are capable of producing endospores.
A reminder:
Some Gram-positive soil bacteria are capable of producing endospores.
Bacillus anthracis
With central endospores
Clostridium perfringens
With subterminal endospores
Clostridium tetani
With terminal endospores

5. Bacillus gram-positive, endospore-forming, motile rods mostly saprobic
aerobic & catalase positive
versatile in degrading complex macromolecules
source of antibiotics
primary habitat is soil
2 species of medical importance
Bacillus anthracis
Bacillus cereus

6. Bacillus anthracis large, block shaped rods
central spores that develop under all conditions except in the living body
virulence factors – capsule & exotoxins
3 types of anthrax
Cutaneous – spores enter through skin
black sore- eschar; least dangerous
Pulmonary –inhalation of spores
Gastrointestinal – ingested spores
treated with penicillin or tetracycline
vaccine – toxoid 6X over 1.5 years; annual boosters
dead livestock burned/chemically decontaminated before burial to prevent soil contamination

7. Bacillus anthracis Biological warfare threat. Letter attacks of 2001
Resulted in only 22 cases of anthrax and five deaths
Great disruption and concern
More than 32,000 people who may have come into contact with the letters were given prophylactic antibiotics.

8. Anthrax- Domestic Bioterrorism
Starting one week after the 9/11/2001 attack letters containing weaponized anhtrax were sent to media offices and to Democratic Senators Tom Daschle and Patrick Leahy -- the two individuals expected to resist passage of the USA PATRIOT ACT.
The attacks came in two waves of letters with Trenton, New Jersey postmarks:
the first, with postmarks of Sepmtember 18, were sent to ABC, NBC, CBS, the New York Post, and the National Equiror at American Media Inc.
the second, with postmarks of October 9, were sent to Senators Daschle and Leahy.

9. Cutaneous anthrax showing the eschar

10. Bacillus cereus common airborne & dustborne
grows in foods, spores survive cooking & reheating
ingestion of toxin-containing food causes nausea, vomiting, abdominal cramps & diarrhea; 24 hour duration
no treatment
spores abundant in the environment

11. Clostridium gram-positive, spore-forming rods
anaerobic & catalase negative
over 120 species
oval or spherical spores produced only under anaerobic conditions
synthesize organic acids & alcohols (useful for some biotechnology application) & exotoxins
cause wound & tissue infections & food intoxications

12. Clostridium perfringens
causes gas gangrene in damaged or dead tissues
2nd most common cause of food poisoning, worldwide
virulence factors
toxins – alpha toxin – causes RBC rupture, edema & tissue destruction
collagenase
hyaluronidase
DNase

13. C. perfringens infection

14. C. perfringens wound infection

15. Clostridium perfringens
treatment of gangrene –
debridement of diseased tissue
large doses of cephalosporin or penicillin
hyperbaric oxygen
Hyperbaric treatment chamber
Exposure to increased oxygen levels inhibits anaerobes and speeds healing

16. Clostridium difficile
normal resident of colon, in low numbers
causes antibiotic-associated colitis
treatment with broad-spectrum antibiotics kills the other bacteria, allowing C. difficile to overgrow
produces entertoxins that damage intestine
major cause of diarrhea in hospitals

17. C. difficile infection. Lining of the colon as seen via a sigmoidoscope
Mild
Severe pseudomembranous colitis

18. Clostridium tetani common resident of soil & GI tracts of animals
causes tetanus or lockjaw, a neuromuscular disease
spores usually enter through accidental puncture wounds, burns, umbilical stumps, frostbite, & crushed body parts
tetanospasmin – neurotoxin causes paralysis
vaccine booster needed every 10 years

19. Tetanospasmin (a potent neurotoxin)
Tetanospasmin (a potent neurotoxin). Toxin inhibits release of neurotransmitter from neurons controlling inhibition of skeletal muscle contraction

20. Patient with Tetanus
Figure 19.17

21. Neonatal tetanus

22. Clostridium botulinum
Causes 3 diseases
food poisoning – (an intoxication) spores are in soil, may contaminate vegetables; improper canning does not kill spores & they germinate in the can producing botulinum toxin
toxin causes paralysis by preventing release of acetylcholine
infant botulism – caused by ingested spores that germinate in the body & release toxin
wound botulism – spores enter wound & cause food poisoning symptoms (although GI tract is not colonized)

23. Botulism

24. Clostridium botulinum
Botox
In 2007, almost 4.6 million procedures were performed with FDA-approved BOTOX® Cosmetic.
BOTOX® Cosmetic works by temporarily reducing the contractions of the muscles that cause the persistent frown lines that have develop over time.
The benefits may last up to 4 months
Over 11 million people have used botox

25. Listeria monocytogenes
non-spore-forming gram-positive
primary reservoir is soil & water
can contaminate foods & grow during refrigeration
Most cases are from contaminated dairy products, poultry and meat
Disease is often mild/subclinical …may have fever, diarrhea
Listerosis in immunocompromised patients, fetuses & neonates affects brain & meninges
20% death rate
ampicillin & trimethoprim-sulfamethoxazole
Prevention – pasteurization & cooking

26. Listeria monocytogenes
Pathogen isolated from:
12 % of ground beef
25-30% of chicken/turkey carcasses
6% of luncheon meats
also in dairy products

27. Erysipelothrix rhusiopathiae
gram-positive rod widely distributed in animals & the environment
primary reservoir – tonsils of healthy pigs
enters through skin abrasion, multiplies to produce erysipeloid, dark red lesions
penicillin or erythromycin
vaccine for pigs

28. Erysipeloid lesion on hand of animal handler

29. Corynbacterium diptheriae
gram-positive irregular bacilli (pleiomorphic)
produce catalase
possess mycolic acids & a unique type of peptidoglycan
2 stages of disease
local infection –upper respiratory tract
diptherotoxin production & toxemia
pseudomembrane formation can cause asphyxiation

30. United States

31. pseudomembrane

32. Diptheria What can happen if you don’t vaccinate!
1990s epidemic in former Soviet Union countries. In 1993 and 1994, more than 50,000 cases were reported during a serious outbreak of diphtheria in countries of the former Soviet Union. (Contrast US had only 15 total cases between )
Reduced vaccination programs resulted in greatly reduced “herd immunity”
Multitude of susceptible persons allowed spread in the community.
Most cases are in children aged 1-10 in crowded settings.

33. Propionibacterium acnes
gram-positive rods
aerotolerant or anaerobic
nontoxigenic
common resident of sebaceous glands
Causes 85% of acne cases

34. Mycobacteria gram-positive irregular bacilli acid-fast staining
strict aerobes
produce catalase
possess mycolic acids & a unique type of peptidoglycan
do not form capsules, flagella or spores
grow slowly
Mycobacterium tuberculosis
Mycobacterium leprae

35. Mycobacterium tuberculosis
produces no exotoxins or enzymes that contribute to infectiousness
contain complex waxes & other substances that prevent destruction by lysosomes of macrophages
transmitted by airborne respiratory droplets
Estimated that 1/3 of world’s population is infected
only 5-10% infected people develop clinical disease. (still…about 2 million people die every year)

36. Primary TB infectious dose 10 cells
phagocytosed by alveolar macrophages & multiply intracellularly
after 3-4 weeks immune system attacks, forming tubercles, granulomas consisting of a central core containing bacilli surrounded by WBCs

37. Granulomatous infection by
M. tuberculosis
Tubercule formation

38. Primary Tuberculosis
Figure 19.22a

39. Extrapulmonary (disseminated) TB
Secondary TB
reactivation of bacilli
tubercles expand & drain into the bronchial tubes & upper respiratory tract
gradually patient experiences more severe symptoms
violent coughing, greenish or bloody sputum, fever, anorexia, weight loss, fatigue
untreated 60% mortality rate
Extrapulmonary (disseminated) TB
during secondary TB, bacilli disseminate to regional lymph nodes, kidneys, long bones, genital tract, brain, meninges = Bad News

40. Secondary Tuberculosis
Figure 19.22b

41. Diagnosis tuberculin testing (Mantoux test) X rays
direct identification of acid-fast bacilli in specimen
cultural isolation and biochemical testing

42. Tuberculin skin test. Tests for exposure.
Tests for hypersensitivity(type IV)
Vaccination causes a positive test result…so test is only useful in countries that do employ widespread vaccination

43. Treatment of TB
Current US treatment: combination of 4 antibiotics for 2 months.
Multi-drug resistant TB treatment: much more expensive and can go on for 2 years.
vaccine based on attenuated bacilli Calmet-Guerin (BCG) strain of M. bovis used in other countries (not U.S.)
Success rate of vaccination ??? ~80% in children and 20-50% in adults.

44. Directly Observed Therapy (DOT) …to assure that medication is actually taken

45. Mycobacterium leprae Hansen’s bacillus
strict parasite – has not been grown on artificial media or tissue culture
slowest growing of all species
multiplies within host cells in large packets called globi
causes leprosy, a chronic disease that begins in the skin & mucous membranes & progresses into nerves

46. leprosy endemic regions throughout the world
spread through direct inoculation from leprotics
2 forms
tuberculoid – superficial infection without skin disfigurement which damages nerves and causes loss of pain perception
lepromatous – a deeply nodular infection that causes severe disfigurement of the face & extremities
treatment by long-term combined therapy

47. Leprosy

48. Leprosy

49. Feather test for leprosy.
Tickling to determine sensitivity to touch

50. Actinomycetes
Genera Actinomyces & Nocardia are nonmotile filamentous bacteria related to mycobacteria
may cause chronic infection of skin and soft tissues
Actinomyces sp – responsible for diseases of the oral cavity & intestines
Nocardia brasiliensis causes pulmonary disease similar to TB

51. Periodontal infection
Periodontal infection. Infection works its way to the surface of the face to rupture and drain
Actinomycosis

52. Nocardiosis
Pulmonary infection that has disseminated to the skin

53. Lactobacillus
Ferment carbohydrates to lactic acid. The acidity inhibits many competing bacteria and creates an ecological niche where they have the advantage.
Major inhabitant of the vaginal tract. Not a pathogen.
Used in production of many foods
L. delbrueckii used in yogurt production
L. acidophilus used in acidophilus milk production

54. Gram Positive Cocci
Oral streptococci on agar plate

55. General characteristics of the Staphylococci
Spherical cells arranged in irregular clusters
Gram positive
Common inhabitant of the skin & mucous membranes
Lack spores and flagella
May have capsules
numerous species

56. Staphylococcus aureus
grows in large, round, opaque colonies
optimum temperature of 37oC
facultative anaerobe
withstands high salt, extremes in pH, & high temperatures
produces many virulence factors

57. S. aureus
SEM showing “grape-like” clusters

58. Enzymes of S. aureus
coagulase – coagulates plasma and blood; produced by 97% of human isolates; diagnostic
hyaluronidase
staphylokinase
DNase
lipases
penicillinase

59. S. aureus
Coagulase Test
Catalase Test

60. Toxins of S. aureus hemolysins – lyse RBCs; a, b, g leukocidin
enterotoxins
exfoliative toxin
toxic shock syndrome toxin

61. S. aureus 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

62. S. aureus diseases Ranges from localized to systemic
localized -abscess, folliculitis, furuncle, carbuncle, impetigo
systemic – osteomyelitis, bacteremia
toxigenic disease – food intoxication, scalded skin syndrome, toxic shock syndrome

63. S. aureus
Skin Lesions
Boil or Furuncle
Carbuncle

64. S. aureus
Osteomyelitis

65. S. aureus Staphylococcal scalded skin syndrome (SSSS)
Exfoliative toxin
Epidermal shedding/separation

67. Staphylococcus aureus
Food Poisoning
Food contaminated by handling with bare hands. Bacteria grow and produce toxin.
Refrigerate food to prevent growth. Heating doesn’t always destroy toxin.
Symptoms usually start within 1-6 hours after eating contaminated food (usually gone in a day)
Symptoms include: diarrhea, vomiting, nausea, cramps

68. Staphylococcus aureus
Toxic Shock Syndrome (TSS)
First linked to use of ultra-absorbent tampons that bound Mg ions and created a favorable environment for increased colonization and growth of vaginal S. aureus and increased TSS toxin production.
Toxin enters bloodstream and causes fever, vomiting organ damage and potential fatality.
Height of problem …15,000 affected/yr with 15% mortality.

70. SuperBug ? MRSA: Methicillin Resistant Staphylococcus aureus
A strain of staph that is highly resistant to broad-spectrum antibiotics.
Once found almost exclusively in hospital settings the bacteria is now showing up in the wider community
People can be colonized by the bacteria (skin and nose primarily) and be healthy but act as carriers to spread the microbe.
It is thought that perhaps 25-30% of people may harbor S.aureus.

71. MRSA has become a subject for some intensive public education efforts

73. MRSA-2
A CDC survey completed recently suggests that in 2005 there were ~ 94,000 cases of MRSA in the US and that perhaps 19,000 people died. (A higher rate than AIDS in the US)
85% of the cases were in hospitals, nursing homes etc. But many are concerned about the large number of infections acquired in normal community environments.
Spread may be by hands (direct contact) or from contaminated surfaces.
Infections range from an inconsequential skin lesion to a fatal septicemia or pneumonia

74. Other Staphylococci
S. epidermidis – lives on skin & mucous membranes; endocarditis, bacteremia, UTI
S. hominis – lives around apocrine sweat glands
S. capitis – live on scalp, face, external ear
All 3 may cause wound infections
S. saprophyticus – infrequently lives on skin, intestine, vagina; UTI

75. Streptococci
Gram-positive spherical/ovoid cocci arranged in long chains
Non-spore-forming, nonmotile
Can form capsules & slime layers
Facultative anaerobes
Do not form catalase !!!
Most parasitic forms are fastidious & require enriched media
Small, nonpigmented colonies
numerous species

76. Streptococcus

77. Streptococci
Lancefield classification system based on cell wall Ag – 14 groups (A,B,C,….)
Another classification system is based on hemolysis reactions
b-hemolysis – A,B,C,G & some D strains
a –hemolysis – S. pneumoniae & others collectively called viridans

79. Human streptococcal pathogens
S. pyogenes
S. agalactiae
viridans streptococci
S. pneumoniae
Enterococcus faecalis

80. b-hemolytic S. pyogenes
Group A
Most serious streptococcal pathogen
Strict parasite…Inhabits throat, nasopharynx, occasionally skin
Produces multiple virulence factors

81. S. pyogenes Humans only reservoir
Transmission – contact, droplets, food, fomites
Skin infections –pyoderma, impetigo, erysipelas
Systemic infections – strep throat, pharyngitis, scarlet fever
Sequelae -rheumatic fever, glomerulonephritis

82. Pharyngitis and Tonsilitis Strep Throat
Note white pus patches on tonsils

83. Toxic sequelae to Strep throat

84. Streptococcal skin infections
Impetigo lesions
Erysipelas

85. FLESH-EATING Bacteria Necrotizing fasciitis
Streptococcus pyogenes

86. Rheumatic fever is a possible sequelae to S. pyogenes infection.
Antibodies against the bacteria may cross-react with host tissue leading to damage of heart valves

87. Group B: S. agalactiae
Regularly resides in human vagina, pharynx & large intestine
can be transferred to infant during delivery & cause severe infection
Most prevalent cause of neonatal pneumonia, sepsis, & meningitis
15,000 infections & 5,000 deaths in US
Pregnant women often screened & treated
wound and skin infections & endocarditis in debilitated people

88. Enterococcus faecalis, & E. faecium
Normal colonists of human large intestine
Cause opportunistic urinary, wound, and skin infections, particularly in debilitated persons
Enterococcus …Genus related to streptococci and so studied together

89. Viridans group a-hemolytic Large complex group
Most numerous & widespread residents of the oral cavity & also found in nasopharynx, genital tract, skin
Not very invasive but…dental or surgical procedures facilitate entrance

90. Viridans group
Bacteremia, meningitis, abdominal infection, tooth abscesses
Most serious infection – subacute endocarditis – blood-borne bacteria settle & grow on heart lining or valves
Persons with preexisting heart disease are at high risk & may receive prophylactic antibiotics before surgery or dental procedures

91. Subacute bacterial endocarditis.
Colonization of heart valve surfaces lead to layers of fibrin and bacteria called vegetations

92. Viridans group
SEM of Plaque
S. mutans produces slime layers that adhere to teeth, basis for plaque
involved in dental caries

93. S. pneumoniae Causes 60-70% of all bacterial pneumonias
arranged in pairs and short chains
All pathogenic strains form large capsules – major virulence factor
Causes pneumonia & otitis media
Vaccine available for high risk people

94. Gram stain of sputum from pneumonia patient showing diplococci

95. S. pneumoniae 5-50% of all people carry it as normal flora in pharynx
Very delicate, does not survive long outside of its habitat
Pneumonia occurs when cells are aspirated into the lungs of susceptible individuals
Pneumococci multiply & induce an overwhelming inflammatory response
Treated with penicillin

96. S. pneumoniae

97. Middle ear infections (otitis media)

98. S. pneumoniae
Buldging eardrum (otitis media)
Normal eardrum

99. Ruptured/torn eardrum
Healthy eardrum
Ruptured/torn eardrum
Tubes
Tubes are usually left in for 8 to 18 months and most often fall out on their own