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1. Gram-Positive Bacilli
Bacillus spp., Corynebacterium diphteria,
Listeria spp., Erysiphelothrix,
Arcanobacterium haemolyticum, and
Nocardia.
1
Medical Laboratory sciences.
Diagnostic Microbiology.
0202302
Chapter 16
Instructor: Dr. Hatem Eideh
Course: Diagnostic Microbiology
Reference: Bailey and Scott's
Diagnostic Microbiology 13th
edition. 2014.

2. Objectives
• Compare the general characteristics of the aerobic gram-positive bacilli
• Compare the clinical significance of the aerobic gram-positive bacilli and
explain how aerobic gram-positive bacilli infections are acquired.
• Describe the types of clinical specimens that are likely to contain the
aerobic gram-positive bacilli.
• Describe the microscopic morphology and colony appearance of the
aerobic gram-positive bacilli.
• Describe the clinical infections associated with Corynebacterium
diphtheriae.
• Discuss several nondiphtheria Corynebacterium spp. that are capable of
causing clinical infection in humans.
• Compare the culture and identifying characteristics of Listeria
monocytogenes with Streptococcus agalactiae (group B Streptococcus).
• Differentiate Listeria monocytogenes from other non-spore-forming gram
positive bacilli and streptococci.

3. Objectives (Cont.)
• Differentiate Erysipelothrix rhusiopathiae from other non-sporeforming
gram-positive bacilli.
• Differentiate Arcanobacterium haemolyticum from other non-
sporeforming gram-positive bacilli and Beta-hemolytic streptococci.
• Differentiate Gardnerella vaginalis from other non-spore-forming gram
positive bacilli on basis of pathogenesis and laboratory identification.
• Describe the clinical infections associated with Nocardia, Actinomadura,
and Streptomyces.
• Differentiate infections caused by Nocardia, Actinomadura,
Streptomyces, Gordonia, and Rhodococcus from infections caused by
fungal agents.
• Describe Tropheryma whipplei and Whipple disease.
• Compare the appearance of bacterial spores when visualized with Gram
and spore stains.
• Describe the clinical infections associated with Bacillus anthracis.

4. Objectives (Cont.)
• Compare the relationship among the three exotoxin proteins of
Bacillus anthracis.
• Describe the differential tests used to identify Bacillus anthracis.
• Discuss the clinical significance of Bacillus spp. other than Bacillus
anthracis.
• Describe the clinical significance of Bacillus cereus.
• Differentiate Bacillus anthracis from other Bacillus spp.

6. Bacillus spp.
• After coagulase-negative staphylococci and diphtheroids, members
of the genus Bacillus are the third most common skin contaminant
found in clinical specimens.
• On Gram stain they are large, wide, Gram-positive rods, often
occurring singly or in pairs, which can produce endospores.
• They may be confused with clostridia on direct stains
• Bacillus can form spores that can survive adverse conditions for
prolonged periods of time and are frequent contaminants of
laboratory cultures and they can be central or terminally located.

7. Bacillus spp.
• Usually they are readily distinguished from lactobacilli which
tend to occur as long, narrow Gram-positive rods which often
chain. Also, Bacillus spp. are catalase-positive, while lactobacilli
are not
• Colony morphology of Bacillus spp. is highly variable, often
growing as large to very large gray-white colonies that may be
dry in appearance, or which may produce a contiguous mat of
wet, blistery colonies.
• The majority of the species will grow on SBA and phenylethyl
alcohol (PEA) agar.

8. Bacillus spp.
• A genus of Gram-positive bacilli which are commonly
found in nature
– soil,
– water,
– airborne dust.
• Members of natural flora in the human intestines.
• Most species of Bacillus are harmless
• Two species are considered medically significant:
– B. anthracis (anthrax)
– B. cereus (food poisoning and wounds)

9. B. anthracis
• Causes anthrax in cows, sheep, and sometimes humans.
• Anthrax is transmitted to humans via
– direct contact with animal products
– inhalation of endospores.
– ingestion
• Sources of infection are usually industrial or agricultural
and the infection is classified as one of three types:
– Cutaneous infectıon (95% of human cases)
characterized by necrotic skin lesions called black
eschars
– Inhalatıon anthrax (rare) : Bioterrorism agent +
"Wool-sorter's disease," spread by inhalation of spores
from sheep's wool
– Gastroıntestinal anthrax (very rare!)

10. Diagnosis
Microscopy:
• Samples: Exudate, Blood, sputum.
• Direct smear: Large rods in chains. Spores not seen
in smears of exudate.
• Gram positive from culture centrally located spore
forming bacilli
Culture:
Blood agar
• Bacillus produces large, spreading, gray-white
colonies with irregular margins: Medusa head
• A unique characteristic of this bacterium is its
ability to produce endospores when environmental
conditions are stressful.
The only other known medically important spore-
producing bacterium is Clostridium

11. Diagnosis
• Typical skin lesion: Dark centered necrotic lesion
• Microscopy from the skin lesion: (Specimen should be taken at the margin of
healthy and diseased tissue)
– Under the microscope, B. anthracis cells appear to have square ends and
seem to be attached by a joint to other cells.
– The spores are best observed when the bacterium is cultured on artificial
media.
Culture
• Blood agar: Medusa head-like colonies
• B. anthracis typically does not grow on
PEA agar at 24 hours.
• Laboratory ındıcatıons:
• Nonhemolytic (sheep blood agar)
• Non-pigmented
• Non-motile
• Gel hydrolysis -ve
• Catalase + ve

12. anthrax eschar in a 4-year-old boy. The lesion when first seen (day 0). Note the
arm swollen

13. The pathogenicity of B. anthracis depends on two virulence factors:
•Antiphagocytic capsule.
•Exotoxins that mediate cell and tissue destruction.
• This toxin consists of three proteins:
• Protective antigen
• Lethal factor
• Edema factor

14. B. cereus
• Unlike B. anthracis, B. cereus is a motile bacterium
• Cause toxin-mediated food poisoning.
• B. cereus may also cause opportunistic eye, bone, and brain
infections.
• It is known to inhabit many kinds of food
– rice
– cereal,
– milk .
• The two toxins released by the vegetative form of the bacilli
causes
– vomiting
– diarrhea, (symptoms similar to those of Staphylococcus
food poisoning).
– Symptoms usually 1-5 hours after ingestion

15. Bacillus cereus
Laboratory Identification
a. B. cereus is normal stool flora,
to diagnose food poisoning
must culture suspected food
NOT stool
b. Gram stain – large, gram-
positive rods with spores, can
stain gram-variable or gram-
negative
c. Colony morphology –large,
feathery, spreading on SBA
d. Preliminary Identification
I. Beta-hemolytic
II. Motile
III. Penicillin – Resistant

16. Bacillus subtilis
• Known also as the hay bacillus or grass bacillus, is a gram positive, rod that
can be found growing in soil as well as the gastrointestinal tract of humans.
• B. subtilis tested positive for catalase, lipase, and amylase.
• Uses citrate as its sole carbon source also positive for carbohydrate
fermentation.
• B. Subtilis is considered non-pathogenic, It has been implicated in food
poisoning caused by poor quality bakery products among others.
• B. subtilis food poisoning has a rapid onset and with light vomiting,
commonly follow by diarrhoea.
• B. subtilis spores can survive the great amounts of heat during cooking.
• It can be found in the gastrointestinal tract of humans but this is very rare.

17. Large Gram positive (variable) rods.
Spore position: central, paracentral or
sometimes subterminal

18. Laboratory Identification: Bacillus anthracis
Characteristics B. anthracis B.cereus
Hemolysis on
BAP
= +
Motility = +
String of pearls + =
Growth on PEA = +
Gelatin hydrolysis = +
Susceptibility to
Penicillin (10U/ml)
Susceptible Resistant
B. Anthracis (string of pearls)

19. Lactobacillus
• Normal vaginal flora
• Produce lactic acid
• Reduce vaginal pH
• Inhibits grow of other organisms
• Rarely causes disease
• Lactobacillus acidophilus is widely recognised to
have probiotic effects and is one of the most
commonly suggested organism for dietary use

20. Lactobacillus
• Non-spore forming GPR
• Medium to long rods
• Aerotolerant anaerobes
• Catalase negative
• Non-motile
• Tiny alpha colonies on BAP

21. • Pleomorphic coccobacilli
• Cell wall with GP characteristics, stains Gram variable
• Non-motile
• Facultative anaerobe
• Normal Flora in 50-70% women
• Linked to bacterial vaginosis, UTIs, Pelvic
inflammatory disease (PID), and postpartum sepsis and
may infect the newborn.
• G. vaginalis probably does not cause BV, but its
presence is indicative of the condition.
41
Gardnerella vaginalis

22. 42
Bacterial Vaginosis (BV)
• Change in NF (lactobacillus)
• increase in pH allows growth of G. vaginalis
• Foul-smelling discharge – amine odor
• Untreated can lead to PID, UTIs
• Amsel and Nugent scoring systems are used to diagnose BV.
– For Amsel's criteria, clinical diagnosis and a few simple laboratory
tests are used, whereas Nugent's criteria involves assessment of
normal flora in the Gram-stained smear of vaginal discharge
• Cultures alone are too sensitive. Approximately 50-60% of
women who do not meet the criteria for BV are positive for G.
vaginalis.
• Perform wet prep or gram stain
• Clue cells squamous cells with bacteria clustered at edges

23. Clue Cells
Clue cells are certain cells in the vagina (vaginal epithelial cells) that appear
fuzzy without sharp edges under a microscope. Clue cells change to this
fuzzy look when they are coated with bacteria. If clue cells are seen, it means
bacterial vaginosis is present

24. 46
Cultures
• Appropriate for extravaginal sites
• Grow on CHOC, BAP, not MAC
• Presumptive Identification
• Small pleomorphic gram variable coccobacilli
• Catalase and oxidase negative
• Hippurate hydrolysis positive

25. Corynebacterium species
Corynebacterium species – “diphtheroids”
• General characteristics and morphology
i. Widely distributed in nature
ii. Many species normal flora of skin, mucous membranes
iii. Most species are non-pathogenic (referred to collectively as
“diphtheroids”)
• Gram stain morphology
i. Club-shaped and beaded with irregularly staining
granules, pleomorphic (many sizes and shapes),
palisading (Chinese letters) gram-positive rods
ii. Non-spore forming

26. Corynebacterium diphtheriae
• Aerobic
• Extracellular
• Rods; club-shaped, has granules
“V”, “L”, or “Chinese Letter”
• Colonies = Dark gray or black
on potassium tellurite medium

27. C. diphtheriae
• Not motile
• No glycocalyx
• Bacterial toxin damages major organs, resulting in a high death rate.
a) Toxigenic C. diphtheriae strains are infected with a
bacteriophage that contains the gene for the diphtheria
toxin.
b) Nontoxigenic C. diphtheriae strains lack the bacteriophage
gene and do not produce the diphtheria toxin.
• Exotoxins = diphtheria toxin
– An ADP-Ribosyltransferase
• Peptide B binds to host cells to transport peptide A inside
• Peptide A has the enzymatic activity

28. • Attaches ADP ribose and prevents ribosome movement
along mRNA
• Blocks host EF2 (a protein synthesis elongation factor
tRNA translocase)
• Blocks protein synthesis
• Can kill host’s Natural Killer (NK) cells
• Coded on viral DNA which gets integrated into the
bacteria by lysogeny

29. Symptoms:
• Upper respiratory tract infections and skin lesions
• Pseudomembrane formation in the throat; exudate forms a
tough gray membrane which can lead to stridor (respiratory
sound), respiratory distress, cyanosis, lymphadenopathy
• Can be fatal
• Humans are the only reservoir for C. diphteria.
• Horizontal transmission occurs via respiratory droplets

30. Pseudomembrane

31. • Intoxication consequences of diphtheria toxin
– cardiac toxicity - occurs weeks after initial infection
– myocarditis, arrhythmias - Can be fatal
– neurologic toxicity - occurs only following severe
infection - Early (first few days) – paralysis of soft
palate and pharynx

32. Virulance factors
• Diphtheria exotoxin
• Storage granules (metachoromatic bodies)
– Contain phosphate polymers for high-energy
reserve
– Stain with metachromatic dye : methylene blue
Albert Staining

33. Media
I. Cystine-tellurite: Corynebacterium spp. form black colonies
from hydrolysis of tellurite.
II. Tinsdale's agar: Corynebacterium spp. form brown to black
colonies with halos from hydrolysis of tellurite.
III. Loeffler agar is a nonselective medium that supports growth
and enhances pleomorphism and the formation of metachromatic
granules. Most Corynebacterium spp. produce small, white to
gray colonies
IV. C. diphtheriae will grow on SBA as small, white, dry colonies.
Most strains are nonhemolytic.

34. Growth on tellurite medium-
black colonies
Loeffler agar

35. In vitro toxigenicity test (Elek Test)
• In a strip of sterile filter paper
antitoxin is impregnated
• A heavy inoculum is streaked on
a agar surface
• Antitoxinated paper is placed on
the surface of the agar medium at
right angles to the inocula.
• Allowed to incubate for 24
hours.
• If the organisim is toxigenic a
visible line of Ag-Ab precipitate
will form

36. • Part of the indigenous human flora (Mucous membranes of
the skin, respiratory tract, urinary tract, and conjunctiva)
• Opportunistic pathogen
I. C. jeikeium
II. C. urealyticum
III.C. amycolatum
IV.C. ulcerans (Closely related to C. diphtheriae )
V.C. pseudotuberculosis
• C. jeikeium, C. urealyticum, and C. amycolatum:
• Resistant to most antibiotics
• Vancomycin must be given
Other Corynebacterium species

37. • Strong urease producer
• C. urealyticum is the most common urease-
producing Corynebacterium species
• Makes the urine alkaline, leading to the formation
of renal stones
• Urinary tract infections, septicemia, endocarditis,
wound infections
• Is rapid urease positive and grows very slowly
Corynebacterium urealyticum

38. Treatment
• Penicillin G to kill the organism
• Erythromycin in penicillin allergy
• Diphtheria antitoxin (horse or human-derived antibodies to the
toxin) must be administered immediately
Vaccine & toxoid
Toxoid - inactive toxin is given as part of the DPT vaccine

39. Corynebacterium jeikeium
• C. jeikeium is an important cause of nosocomial
infections and produces infections after prosthetic
device implants and infections in immunocompromised
patients.
• Pyrazidamidase positive
• Resistant to most antimicrobial agent

40. Listeria
• Gram-positive rod which is not capable of forming endospores.
• Small and smooth colony on blood with a narrow zone of beta-
hemolysis
• Aerobic, Catalase +ve Oxidase +ve
• Several species exist, but two species are important for human
pathogenic significance:
– L. monocytogenes
– L. ivanovii.

41. Listeria monocytogenes
• Normal inhabitant of the gastrointestinal tract and of
animal feces
• Found responsible in several food poisoning epidemics.
– infected patients suffer from vomiting, nausea, and
diarrhea.
• High risk group:
– newborns,
– pregnant women and their fetuses,
– the elderly,
– immune compromized

42. Clinical findings:
– septicemia
– meningitis
– listeriosis: is an inflammation of the brain.
Laboratory diagnosis:
 Catalase +
 Bile esculin hydrolysis
 Beta-hemolysis
 Ability to grow;
• At broad temperature range (1 oC to 45 oC)
• In a wide pH range
• In a high concentration of salt
Treatment:
• Antibiotics are recommended for treatment of infection
• Most strains of Listeria are sensitive to ampicillin and
gentamicin

43. • Detection of listeriae in specimens contaminated with rapidly
growing bacteria;
- Selective media
- Cold enrichment (storage of the specimen in the refrigerator for a
prolonged period)
• CAMP test positive
Hippurate hydrolysis
Esculin positive
Treatment:
Antibiotics are recommended for treatment of infection
most strains of Listeria are sensitive to ampicillin and gentamicin

44. Erysipelothrix
• E. rhusiopathiae, the only species of this genus,
• Found in many farm animals such as pigs, horses,
and turkeys
• Is better known as a veterinary pathogen than as a
human pathogen.
• When cultured on blood agar or some other
nutrient medium, Erysipelothrix forms notably
large colonies.
• Occasionaly, it can infect a human host and cause
an inflammatory skin disease, Erysipeloid.
• Treatment usually consists of penicillin G,
ampicillin, or cephalothin.
• Laboratory diagnosis:
– Can grow on BA and CA
– Catalase -ve
– Non-motile
– TSI : H2S + /(the only GPR that is H2S
positive)

45. Arcanobacterium
• Six species of Arcanobacterium have been named; three are clinically
significant:
– A. haemolyticum,
– A. pyogenes, and
– A. bernardiae.
• The natural habitat of these organisms has not been confirmed,
although.
• The clinically significant Arcanobacterium spp. form small beta
hemolytic colonies on SBA and are catalase negative and non-motile.
• A. haemolyticum can be identified by the CAMP inhibition test.
• This bacterium produces phospholipase D, which inhibits the activity
of the Staphylococcus aureus beta-lysin.
• Corynebacterium pseudotuberculosis also exhibits this phenomenon

46. Arcanobacterium haemolyticum
• Formerly Corynebacterium
• Causes pharyngitis, and wound and tissue infections and is
found on mucous membranes of cattle, sheep, and swine and
is linked to abscess formation, wound, and soft tissue
infections in cattle and humans
• Facultative anaerobe
• Beta hemolysis
• Catalase negative
• Reverse CAMP positive
• inhibits S. aureus hemolysis

47. Reverse CAMP

48. Nocardia spp.
• Found in soil
• Usually infect immunocompromised
• Pulmonary
• Cutaneous
• Pulmonary
N. asteroides complex
• Pneumonia ► abscess ► necrosis- May
disseminate
• Cutaneous (mycetomas)
N. brasiliensis
• Abscess ► invasive
• Draining pus with sulfur granules
• Aerobic GPR morphologically similar to fungus
• Form beaded, branching filaments on gram stain
• Weakly acid fast

49. Nocardia spp. Microscopic
• Grow slowly (1 week or more) on nonselective media
• Waxy, chalky, crumbly colonies

50. Nocardia spp. Colony Morphology on SBA

51. Nocardia ID
• Filamentous, branching organism
• Weakly acid fast
• Acid-fast stains (Ziehl Neelsen uses heat or Kinyoun
Stain uses detergent)
• Primary stain carbolfuchsin (minutes)
• Rinsed and decolorized with acid
• Rinsed and counterstained with methylene blue
• Reddish purple filaments (partially acid fast), blue:
negative
• Nocardia is positive with Modified acid fast stain

52. Nocardia asteroides
• Nocardia asteroides
– Generally found in immunocompromised patients with chronic
pulmonary disorders
– N. asteroides is the most clinically relevant species; other species
include N. brasiliensis and N. otitidiscaviarum.
• Identifying characteristics
 Pleomorphic, branching gram-positive bacilli in chains that
produce a beading arrangement, appear fungal-like
 Partially acid-fast, catalase positive, nonmotile
 Requires up to 6 weeks for growth
 Exudate contains masses of filamentous organisms with pus that
resemble sulfur granules.

53. Other Actinomycetes
• Tropheryma whipplei
• Whipple Disease
• Intracellular pathogen
• Uniformly fatal if untreated
• Diarrhea, weight loss, malabsorption
• Identify by PCR or 16S Rrna
• Streptomyces
• Actinomadura
• Gordonia
• Tsukamurella
• Rhodococcus equi