NON-SPORE-FORMING GRAM-POSITIVE BACILLI

I. Genus Corynebacterium
- coryneforms or diphtheroids; found worldwide in fresh and salt water, soil and air
- closely related to mycobacteria and nocardiae; these 3 groups collectively may be referred to as the CMN group (they
have common cell wall structure)
- Discovered by Pierre Bretonneau first recognized diptheria

Corynebacterium diphtheria (KLEBS-LOEFFLER BACILLUS)

- diphtheria bacillus; premier pathogen of the group
- humans are the only natural hosts; found in the upper resp tract, spread by droplet infection or hand-to-mouth contact
- G (+), aerobic bacilli, non motile, non sporeforming, pleomorphic (vary in shaped)
- cardinal feature\; club forms due to immular swelling of one end of organism, when present in both ends, they are called polar bodies
- beaded appearance is due to BABES- ERNST GRANULES
- Gram stain cells arranged as “Chinese letters”

- virulence factor: diphtheria toxin

- exceedingly potent; *toxicity is due to its ability to block protein synthesis
- has two fragments (A* and B)
- toxin production depends on a lysogenic state in which the bacterium is infected with a bacteriophage that carries
the tox+ gene, which codes for the production of diphtheria toxin
- only toxin-producing C. diphtheriae causes the infection diphtheria
- nontoxigenic strains can be converted to tox+ by infection with the appropriate bacteriophage (-phage)
- C. ulcerans and C. pseudotuberculosis which belong to "C. diphtheriae group" may also produce the toxin when
they become infected with the tox-carrying bacteriophage
- production of the toxin in vitro depends on a number of environmental conditions:
a. an alkaline pH 7.8 - 8.0
b. oxygen
c. most importantly, iron concentration in the medium
- the amount of iron needed for optimal toxin production is less than that needed for optimal growth
- toxin is released in significant amounts only when the available iron in the culture medium is exhausted
- causes diphtheria
- the identification of an isolate as C. diphtheriae does not mean that the patient has diphtheria; diagnosis of
diphtheria depends on showing that the isolate produces diphtheria toxin
- occurs in two forms: respiratory and cutaneous
- incubation period averages 2-5 days; characterized by low-grade fever, malaise, and a mild sore throat
- most common site of infection: tonsils or pharynx
- the organisms rapidly multiply on the epithelial cells and trigger an inflammatory reaction
- the infecting toxigenic strain produces toxin locally, causing tissue necrosis and exudate formation
- this combination of cell necrosis and exudate forms a very tough gray to white pseudomembrane that attaches to
the tissues; it may appear on the tonsils and then spread downward into the larynx and trachea

- there is the potential for suffocation if the membrane spreads and blocks the air passage or if
it is dislodged
- the toxin is also absorbed and produces systemic effects - involve the kidneys, heart and
nervous system
- death is often a result of cardiac failure
- treated by prompt administration of antitoxin; commercial diphtheria antitoxin is produced
in horses
- antibiotics have no effect on toxin that is already circulating, but they do serve to eliminate
the focus of infection
as well as to prevent the spread of the organism; drug of choice: penicillin
** DPT vaccine

Lab Diagnosis:
- g+rods; non-spore-former or nonsporulating; nonmotile
- facultative anaerobe; ferments glucose and maltose, reduces nitrate to nitrite
- highly pleomorphic and appears in palisades or as individual cells lying at sharp angles to one another
in V and L formations; this particular arrangement has been described as "Chinese characters"
- often stain irregularly, esp when stained with methylene blue, giving them a beaded appearance; the metachromatic areas
of the cell, which stain more intensely than other parts, are called Babes-Ernst granules; their presence indicates
accumulation of food reserves

Culture Media: (growth requirements are complex, with 8 amino acids being essential)
1. Tinsdale agar
- contains sheep's blood, bovine serum, cystine, and potassium tellurite
- used as both a selective and differential medium
- they form black or brownish colonies; Staphylococcus and Streptococcus also produce black colonies

- a brown halo surrounding the colony is a useful differentiating feature because only C. diphtheriae, C. ulcerans,
and C. pseudotuberculosis produce a brown halo on Tinsdale agar
- C. diphtheriae can be differentiated from the other 2 species by its lack of urease production
- three biotypes of C. diphtheriae that can be distinguished by their growth characteristics on Tinsdale agar ---
mitis, intermedius, and gravis

2. Cystine-tellurite agar - tellurite salts inhibit the growth of most normal respiratory flora
- garlic-like odor colonies
3. Loeffler serum agar - characteristic microscopic morphology is demonstrated well when they are grown on this medium
- colonies are minute. Glistening, grayish-white
4. Pai slant – to enhance morphology
Tests for Toxigenicity:
1. in vivo test (rarely done) animal inoculation
- carried out in guinea pigs
- approx 24 hours prior to the test, one guinea pig is injected with diphtheria antitoxin
- the next day, the protected guinea pig and an untreated guinea pig are injected with a suspension of the
suspected organism prepared from Loeffler slants
- if the isolate produces diphtheria toxin, the untreated animal will die within 3-5 days, and the antitoxin-
treated animal will survive

2. Elek test (in vitro - Gel immunodiffusion test)

- organisms (controls and unknowns) are streaked on media of low iron content to optimize toxin production
- organisms are each streaked in a single straight line parallel to each other and 10 mm apart on an Elek’s plate
- **KL virulence agar
- a filter paper strip impregnated with diphtheria antitoxin is laid along the center of the plate on a line at right
angles to the lines of control and unknown organisms
- plate is incubated at 35 C and examined after18, 24, and 48 hours
- the white precipitin lines start about 4-5 mm from the filter paper strip and are at an angle of about 45 degrees to
the line of growth
- if an isolate is positive for toxin production, and it is placed next to the positive control, the toxin line of the
positive control should join the toxin line of the positive unknown to form an "arch of identity"

3. Shick’s Test – a skin test which determines whether the patient is immune or not

Other Corynebacterium species:

C. ulcerans C. pseudodiphtheriticum
C. pseudotuberculosis C. urealyticum
C. xerosis C. striatum
C. jeikeium C. kutscheri
C. auris
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C. diphtheriae C. ulcerans C. pseudotuberculosis Other corynebacteria

Tinsdale (brown halo) + + + -

Catalase + + + +

Urease - + + +/-

II. Genus Listeria

Listeria monocytogenes (serotypes Ia, Ib, IVb)
- only human and animal pathogen among the 7 species
- has been recovered from soil, water, vegetation, and animal products
- the portal of entry has not been determined, studies seem to indicate that the ingestion of contaminated food with
subsequent systemic spread via the intestine is likely
- causative agent of listeriosis
1. in pregnant women
- most commonly seen during the 3rd trimester; responsible for spontaneous abortion and stillborn neonates
- flu like
2. in newborn (granulomatosis infantisepticum)
- extremely serious; fatality rates are high
- two forms of neonatal listeriosis: early-onset --- sepsis; late-onset --- meningitis
3. in the immunosuppressed host
- meningitis, meningoencephalitis, or sepsis
- infection of apparently healthy individuals may occur via the intestinal tract when they eat contaminated food
- outbreaks have occurred as a result of eating contaminated cheese, coleslaw, and chicken
- manifestation in these cases is nearly always meningitis, and the fatality rate is high
- virulence factors: Anton’s test : (+) development of purulent conjunctivitis in rabbits eye listeriolysin O, catalase, superoxide
dismutase, phospholipase C, surface protein p60

Lab Diagnosis:
- blood, CSF, or swabs of lesions
- g+ coccobacillus that often appears coccoid in nature
- non spore-former; aerobic
- B-hemolytic on sheep's blood agar
- colonies are small, smooth, and translucent, and they show a very narrow zone of beta hemolysis
- may be confused with Group B streptococci (Streptococcus agalactiae) because the resemblance is striking
- CAMP test (+), similar to that of group B streptococci when S. aureus is utilized to augment hemolysis; a more
pronounced CAMP reaction is seen with L. monocytogenes when Rhodococcus equi is used in place of S. aureus
- catalase (+), which differentiates it from Streptococcus
- motile at room temperature
- in wet mount preparations, it exhibits "tumbling motility" when viewed microscopically
- the use of motility medium demonstrates the characteristic "umbrella" pattern when the organism is incubated at
room temp (25 C) but not at 35 C

- Hippurate hydrolysis (-)

- Cold Enrichment
- because L. monocytogenes grows at 4 C, an unusual characteristic, this technique may be used to isolate the
organism from clinical specimens
- this technique calls for inoculation of the specimen into broth and incubation at 4 C for several weeks

III. Genus Erysipelothrix

Erysipelothrix rhusiopathiae
- only species in the genus
- commensal or a pathogen in a very wide variety of vertebrates and invertebrates; domestic swine are the major reservoir
- human cases are rare, with infections resulting from occupational exposure
- those whose work involves handling fish and animal products are most at risk
- the usual route of infection is through cuts or scratches on the skin
- the organism is resistant to salting, pickling, and smoking and survives well in environmental sources such as water, soil,
and plant material

Clinical infections: three types of disease in humans

- septicemia
- endocarditis,
- erysipeloid
- localized skin infection that resembles streptococcal erysipelas
- lesions are usually seen on hands or fingers because the organisms usually are inoculated through work activities
- infected area is painful and swollen and gives rise to a characteristic lesion - a sharply defined, slightly elevated,
purplish red zone that spreads peripherally as discoloration of the central area fades

Lab diagnosis:
- the specimen is from a tissue biopsy or aspirate from skin lesions
- g+ pleomorphic rod that has a tendency to form long filaments
- H2S (+) in TSI / KIA
- nonmotile; nonspore-former;
- non-hemolytic or alpha-hemolytic
- catalase (-); indole(-); oxidase(-)
- Neomycin resistant; vancomycin resistant
IV. Genus Lactobacillus
- non pathogenic
- gram (+) bacilli, normally present in the gastrointestinal tract and urogenital tract
- non-sporeforming, microaerophilic rods, nonpathogenic
- produce lactic acid from CHO, prefer a more highly acidic environment (pH 5)
- associated in dental carries

Lactobacillus acidophilus (Boas-Oppler Bacillus)

- normal inhabitants of the intestines and are increased by a diet rich in milk or carbohydrate
- Doderlein’s bacilli: lactobacilli normal flora of the vagina

Lactobacillus bulgaris
- Isolated from Bulgarian milk
- Prevent intestinal putrefaction thereby prolonging life

Lactobacillus casei
- Used commercially in bioassay of the B complex vitamins and certain amino acids

Lactobacillus bifidus
- Seen in intestines of breast fed infants

V. Genus Propionibacterium

Propionebacterium acnes
- Pleomorphic, non-sporeforming, gram + bacilli
- Catalase and indole (+), non motile and ferment carbohydrates producing propionic acid
- Found in plants and animals
- Normally colonized the skin, respiratory tract, urogenital tract of humans
- Seen on skin, RT, GIT, UGT
- Diseases
- Acne (teenagers and young adults)
- Opportunistic infections (patient with prosthetic devices or intravascular lines
 AEROBIC GRAM-POSITIVE BACILLI
AEROBIC ACTINOMYCETES
- morphology similar to fungi; demonstrate filamentous hyphae in culture
- true bacteria and can be differentiated from fungi

I. Genus Nocardia
- found in soil and plant material
- g+rods, but often stain gram-variable; may be weakly acid fast / partially acid-fast
- often form branched hyphae; aerobic
- species of medical importance: N. asteroides, N. brasiliensis, N. caviae
- clinical infections: infection occurs by two routes: pulmonary and cutaneous
- not transmitted from person to person
1. pulmonary (“farmer’s lung”)
- due to inhalation of the organism, which is present in dust or soil
- majority are due to N. asteroides
- manifestation: bronchopneumonia
- no sulfur granules or sinus tract formation (unlike w/ infection by anaerobic actinomycetes, w/ sulfur granules)
2. cutaneous
- inoculation of the organism into the skin or subcutaneous tissues
- most frequently caused by N. brasiliensis
- usually seen in the hands and feet as a result of outdoor activity
- infection begins as a localized subcutaneous abscess that is invasive destructive of the tissues and underlying bone;
these lesions are termed mycetomas
- mycetomas are characterized by swelling, draining sinuses and granules
- about half of the mycetomas are caused by the actinomycetes; the other half, caused by fungi
- as infection progresses, burrowing sinuses open to the skin surface and drain pus; the pus may be pigmented and
contain sulfur granules (sulfur granules are masses of filamentous organisms bound together by calcium phosphate;
they often appear yellow or orange and have a distinct granular appearance)
Treatment:
- involves drainage and surgery as well as antimicrobials
- resistant to penicillin; susceptible to sulfonamides; antifungal agents have no activity against Nocardia

Lab Diagnosis:
1. Microscopy
a. G/S - sputum and exudates, or aspirates from skin or abscesses
b. Acid-fast staining - partially acid-fast (1 – 4% sulfuric acid may be a better decolorizer)
c. Wet mounts
- granules may be seen in spns from cutaneous infection; tissue and pus from draining sinuses
- granules are visualized by separating them from the pus w/ an inoculating needle and then washing in sterile saline
- granules of N. asteroides, N. brasiliensis, and N. caviae are soft, white to cream-colored and 0.5-1 mm in size
- may be crushed between 2 glass slides to see the branching and cellular morphology, comprised of g+ interwoven,
thin filaments
- the granules of a fungal mycetoma (eumycotic mycetoma) are composed of broad, interwoven, septate hyphae that
are wider (2-5 mm) than those of the actinomycetes
2. Colonial characteristics
- chalky or velvety; dry, crumbly appearance that is likened to that of bread crumbs
- growth may take 3-6 days
- use non-selective media or Saboraud dextrose agar(SDA)
- "paraffin bait technique" increases the possibility of isolating Nocardia
- an isolate showing branching filaments that are g+ and partially acid-fast should be suspected of belonging to Nocardia

II. Genus Actinomadura

- formerly classified as members of Nocardia; A. madurae, A. pelletieri, A. dassonvillei
- etiologic agents of mycetoma, which is identical to that caused by Nocardia
- microscopic and colonial morphology is similar to that of Nocardia
- A. madurae is cellobiose and xylose (+); Nocardia do not produce acid from these two CHO
- treatment same with Nocardia

III. Genus Streptomyces

- Streptomyces griseus is the 3rd most common aerobic actinomycete, after N. asteroides and N. brasiliensis
- ID is done by reference lab
IV. Genus Actinomyces
- species of medical importance: A. israelii and A. naeslundii
- facultative anaerobes
- grow best in increased CO2
- produce “spider” colonies during the first week then becoming “MOLAR TOOTH” colonies after the first week

- infections produce sulfur granules

- granules contain macrophages, tissue cells, fibrin, bacteria (**club-shaped enlargements of the bacterial cell)

clinical findings:
1. cervicofacial disease – swollen, erythematous process in the jaw area (Lumpy Jaw)
2. thoracic – pulmonary disease
3. abdominal
4. genital actinomycosis – due to contaminated IUD