Aerobic Non-Spore Forming Gram Positive Bacilli

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Non-spore forming gram positive rods

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• Corynebacterium
• Listeria
• Erysipelothrix
• Lactobacillus
• Arcanobacterium
• Gardnerella
• Nocardia spp, Rhodococcus, Mycobacterium

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Spore Forming gram positive rods

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• Bacillus (aerobic)
• Clostridium (anaerobic)

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General characteristics of Corynebacterium Species

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• Found as free-living saprophytes: water, soil, air, resistant to drying
• Catalase positive
• Non-motile
• Opportunistic pathogens
• Facultative anaerobes

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What's the most significant Corynebacterium species

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Corynebacterium diphtheriae: difficult to speciate 16s rDNA gene sequencing

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Microscopic colony morphology of Corynebacterium Species

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• Gram-positive, non-spore-forming rods: arrange in palisades, "club-shaped", cell walls contain mesodiaminopimelic acid and short chain mycolic acids

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Babes-Ernst granules

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• Methylene blue stain
• Polymerized phosphates
• aka: metachromatic granules

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Geographical distribution of C. diphtheriae

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Toxigenic Corynebacterium diphtheriae; worldwide distribution

rare in places where vaccination programs exist

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What is the major virulence factor of C. diphtheriae

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Exotoxin:

diphtheria toxin, antigenic (uses IgG to block toxin effect)

Only toxin producing bacteria can cause diphtheria

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Toxin production in C. diphtheriae

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Toxin production is inversely proportional to iron availability

Alkaline pH (7.8-8.0)

Oxygen concentration

Iron concentration less amt is needed

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Two fragments of toxin

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A-B toxin linked by disulfide bond

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A: Active fragment

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• Inhibits protein synthesis
• NAD+ +EF-2--> adenosine diphosphoribose (ADPR)-EF-2 + Nicotinamide + H+

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EF2

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required for protein synthesis

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B: Binds to specific cell membrane receptors

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• Binds to specific cell membrane receptors
• Mediates entry of Fragment A

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EF2 ADPR.EF

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inactivated: catalyzes transfer of ADPR to link with them

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Other toxigenic strains of Corynebacteria

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C. ulcerans

C. pseudotuberculosis

Can produce toxin when lysogenized with Beta-phage containing tox gene

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Clinical Infections of Diphtheria: Respiratory

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• Acquired by droplet spray
• Aerosol or hand to mouth contact
• Unimmunized individuals are susceptible

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Symptoms of Clinical Infections of Diphtheria

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• Incubation lasts 2-4 days
• Low grade fever, malaise, mild sore throat: local toxin production causes tissue necrosis, forms a tough white to gray psuedomembrane: starts at tonsils and spreads downward into the larynx and trachea, may obstruct breathing

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Systemic effects of Clinical infections of Diphtheria

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• Toxin is absorbed in the bloodstream and carried systemically
• Affects the kidneys, heart, and nervous system: sometimes causing demyelinating peripheral neuritis resulting in paralysis
• Death occurs due to cardiac failure

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Treatment of Diphtheria

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Administer antitoxin: relieves toxin symptoms, but must use antibiotics to clear the infection

Produced in horses: occasionally a hypersensitivity reaction (10%)

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Most cases of Diphtheria in the US

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unimmunized

inadequately immunized persons

Higher in older populations

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Nonrespiratory disease of C. diphtheria

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Cutaneous form: more prevalent in the tropics,

Infections occur at the site of minor abrasions,

Less severe than respiratory disease: non-healing ulcers, mostly prevalent in native American populations and in the homeless

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Loeffler's serum agar

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Used to demonstrate pleomorphism and metachromatic granules: Babes-Ernst granules

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Cystine-tellurite blood agar (CTBA)

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• Modified Tinsdale medium
• Selective and differential: Potassium tellurite inhibits many non-coryneform bacteria, brown or grayish to black halos around the colonies

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Laboratory Diagnosis: ID of Corynebacteria

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Confirm id by fermentation reactions

Urease

Nitrate

Toxigenicity testing: must have toxin for disease

Elek test: immunodiffusion test, iron poor media

PCR for Tox gene

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Elek test

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Immunodiffusion test

Iron poor media

(Positive control, toxigenic unknown, negative control, unknown nontoxigenic)

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C. jeikeium

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Infections from catheters or prosthetic devices

Sepsis, prosthetic valve endocarditis, meningitis, and peritonitis predominately in immunosuppressed in patients

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Identification of Corynebacterium jeikeium

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• Pleomorphic
• Catalase-positive
• gram-positive bacilli
• growth on lipid containing media
• resistant to multiple antibiotics
• API 20S
• Minitube RSU broth-neg for urease and sucrose

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RSU broth

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• Urease production and sucrose utilization
• minimal amount of inoculum
• results in 3 hours

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Identification of other Corynebacteria, Speciated if:

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• Isolated from normally sterile site
• Predominant organism from properly collected specimen
• Urine samples if pure culture of >10^4 organisms or predominant organism at >10^5

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Rothia dentocariosa

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Normal oral flora: Endocarditis

Nitrate positive, non-motile, esculin hydrolysis positive, and urease negative

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R. mucilaginosa

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Improtant cause of endocarditis

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Undesignated CDC coryneform groups

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• Bacteremia
• Endocarditis
• Wound Infections
• Eye Infections

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Two pathogenic species of Listeria

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L. monocytogenes- Human pathogen

L. ivanovii- Animal pathogen

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Listeria monocytogenes

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Gram-positive, nonsporulating rods

Coccobacillus that often appears like cocci-short rods

Widespread in nature, infect animals

Human exposure is limited to: dairy products because it grows at low temp and meats

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Listeriosis infection in adults

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• Likely develop disease from penetration of the intestine and systemic spread
• Septicemia/meningitis in Neonates and the compromised/elderly-endocarditis
• Mild flulike syndrome in pregnant women could be fatal to fetus

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Listeriosis in pregnant women

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Neonatal- early onset from intrauterine transmission results in sepsis; high mortality rate

Late onset manifests as meningitis; low mortality rate

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Listeriosis in Immunocompromised

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• CNS infection

• Meningitis

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Virulence Factors: L. monocytogenes

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• Hemolysin (listeriolysin O)
• Catalase
• Superoxide dismutase
• Phospholipase C
• Surface Protein 60- Induces phagocytosis through adhesion and penetration in mammalian cells

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Laboratory Diagnosis: L. monocytogenes

Microscopic morphology

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Gram-positive

Non-spore-forming

Coccobacillary

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Culture Characteristics L. monocytogenes

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• Grows well on enriched media such as blood agar
• Colonies produce a narrow zone of beta-hemolysis- Similar to group B Streptococcus: Differentiated from GBS by catalase reaction

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Identifying L. monocytogenes

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• Cold enrichment-Grows at 4 deg
• Catalase positive- Rules out Streptococcus
• Motility- motile at 25 deg "umbrella" type motility, tumbling motility in hanging drop preparations

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Other Identifications of L. monocytogenes

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• CAMP test- produces a "block" type of hemolysis in contrast to "arrow" by group B streptococcus
• Bile esculin
• Hippurate hydrolysis positive

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Erysipelothrix rhusiopathiae

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• Gram positive
• Non-spore-forming
• Pleomorphic rods
• Distributed in nature-can cause disease in animals
• Infection through occupational exposure

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Erysipeloid

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• Self-limiting localized infection at the site of inoculation
• Produces painful swelling, usually on the hands or fingers
• Heals within 3 to 4 weeks

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Endocarditis

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May occur in those who have had valve replacement

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Erysipelothrix rhusiopathiae Identification

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Catalase negative

Production of H2S on TSI

Culture: CO2 is required

Grows on CA

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Erysipelothrix rhusiopathiae colony morphology

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Colonies may appear gray or translucent with alpha hemolysis

Pleomorphic, gram-positive thin rods that may form very long filaments-singly, in short chains, or in a V shape

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A. haemolyticum infections

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Throat cultures: often indistinguishable from Group A streptococci infection- rash resembling Scarlet fever

Also, soft tissue, endocarditis, sepsis

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Arcanobacterium

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All are catalase negative

Narrow zone beta-hemolysis- black dot on agar surface under the colony

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Lactobacillus

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• Gram positive rod
• Aerotolerant anaerobe
• Important normal flora in the vagina
• Contaminants in urine- cannot survive in urine
• Antibiotics
• Can rarely be a pathogen in immunocompromised patients

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Gardnerella vaginalis

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• Short, pleomorphic gram-positive rod or coccobacillus
• Infection- Bacterial vaginosis (BV)

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Bacterial vaginosis

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• Malodorous discharge
• Vaginal pH of >4.5
• Associated with bacterial vaginosis but most now feel this organism is not responsible

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Treatment of Gardnerella vaginalis

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• Metronidazole
• Clindamycin

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Laboratory diagnosis of Gardnerella vaginalis

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• Culture is not the "gold standard" for the diagnosis of BV, can be recovered from healthy women
• Direct examination of vaginal secretions: presence of clue cells, an abundance of mixed flora consisting of small gram variable rods and an absence of lactobacilli

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Gram Stain Characteristics Gardnerella vagianlis

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• Gold standard includes gram stain and wet prep, not culture
• Gram Stain: gram variable pliomorphic bacilli
• Biochemical: not applicable, indentification done by direct visualization

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Clue Cells

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Gram stain showing epithelial cell coated with gram variable bacilli

Gram negative curved bacilli and regular GNR

Straight GPR lactobacilli are absent

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Nugent Score

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• Gram stain scoring system for pap test to diagnose bacterial vaginosis
• Calculated by assessing for the presence of: Gram-positive rods, small Gram-variable rods, curved Gram-variable rods, range from 0 to10
• A score of 7 to 10 is consistent with bacterial vaginosis

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Gardnerella vaginalis Culture Characteristics

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SBA or chocolate agar:

5%-7% CO2 at 35-37deg

Slow growth, pinpoint colonies at 48 hours

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Length of mycolic acid in gram positive rods

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• Corynebacteria: Short (22-36 C atoms)
• Nocardia: Medium (50-62)
• Mycobacteria: Long (70-90)

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Acid fast bacteria

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• Retained by mycolic acid and resist decolorization with dilute acid or acid alcohol
• "acid-fast" resist decolorization

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Modified Acid Fast Stain

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• Allows the presumptive diagnosis of Nocardia
• Distinguishes Nocardia from Mycobacteria
• Other actinomycetes are generally modified acid fast negative

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Modified Acid Fast

Primary Stain:

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Stain 3 minutes with carbol fuchsin

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Modified Acid Fast

Decolorize

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10 seconds with 1% HCl or Sulfuric acid

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Modified Acid Fast:

Counter Stain

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Stain 30 seconds with methylene blue

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Acid Fast Stain:

Primary Stain

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Stain 5 min with carbol fuchsin

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Acid Fast Stain:

Decolorize

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• 3 min with 3% HCl
• Rinse

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Acid Fast Stain:

Counter Stain

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Methylene blue 3-4 min

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MAF Positive

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• Mycobacteria
• Nocardia
• Rhodococcus
• Gordonia
• Tsukamurella

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MAF Negative

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• Actinomyces
• Streptomyces
• Actinomadura
• Nocardiopsis

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General Characteristics: Nocardia

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• Medium mycolic acid stain
• Beaded branching on Gram Stain
• Have a peptidogylcan cell wall contains: meso-diaminopimelic acid

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Taxonomy: Nocardia

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• Historically, based on hydrolysis of xanthine tyrosine, casein, and hypoxanthine on quad plates
• Antibiotic susceptibility patterns
• PCR restriction enzyme analysis of 441-bp region of the hsp65 gene
• 16s rRNA sequencing

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Nocardia: Transmission

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• Cutaneous infection caused by traumatic inoculation
• Severe pulmonary or CNS disease in immunocompromised host
• Health care transmission is rare
• Not communicable

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Older laboratory testing for Nocardia

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• Relied on hydrolysis of casein, xanthine, starch, and tyrosine
• Replaced by sequencing or MALDI-ToF

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Tropheryma

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Whipple's disease

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What is Actinomadura

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is an aerobic actinomycetes

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What was Actinomadura once confused with

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fungus

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Common cause of Actinomadura

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actinomycotic mycetoma

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Clinically relevant species of Actinomadura

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A. madurae and A. pelletieri

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What is a characteristic formation of Actinomadura

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granules containing branched filaments

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Growth of Actinomadura

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Slow growth on routine mycologic or mycobacteriologic media under aerobic conditions

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Chronic infection of Actinomadura

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Chronic infection

• subcutaneous tissue
• muscles and bones particularly of lower extremities

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Lowenstein-Jensen medium

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Actinomadura

• 2 weeks incubation,
• aerial hyphae may develop on the surface

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Actinomadura madurae v. A. pelletieri

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A. madurae: produce acid from cellobiose

• Gram positive
• Nonacid-fast
• Filamentous
• Aerial "hyphae"

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Streptomyces

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• Aerobic
• Peptidoglycan
• Gram positive
• Catalase positive
• Chalky aerial mycelium with branching
• Produce one or more antibiotics
• Colonies: white to gray to yellow-pigmented
• Can cause mycetoma (fungus tumor)
• Subcutaneous, can penetrate bone

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Gordonia bronchialis

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• Aerobic, catalase positive, Gram positive/variable, partially acid fast
• Non-motile
• nocardioform- mycelial form fragments into rods or coccoid elements
• Variable colony morphology
• Nitrate reduction
• Associated with IV catheters

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Gordonia bronchialis antibiotics

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susceptible to a broad range of antibiotics

• B-lactams
• quinolones
• aminoglycosides
• macroslides

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Tsukamurella

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• Gram Postive, aerobic, catalase positive, partially acid fast
• requires extensive biochems and 16s rRNA
• Chronic lung infections, subcutaneous abcesses, cutaneous lesions, catheter bacteremia, peritonitis, prosthesis infections
• Susceptible to B-lactams and aminoglycosides

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Rhodococcus

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• Facultative, intracellular, non-motile, non-spore-forming, gram positive rod
• red pigment (salmon-colored)
• weakly acid-fast similar to diphtheroids
• Grouped with aerobic actinomycetes
• Necrotizing pneumonia
• Immunocompromised host

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Tropheryma whipplei

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• Facultative intracellular pathogen
• Gram positive actinomycete
• Environmental, human feces, saliva, gastric secretions
• ID by PCR

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Whipple Disease

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• Diarrhea, weight loss, arthralgia, abdominal pain
• Fatal if untreated
• Rx: Trimethoprim-sulfamethoxazole: 1yr regimen of antibiotics tatracycline associated with relapses

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Bacillus Species

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• Found in nature
• Most are saprophytic
• Isolated as contaminants
• Gram-positive large rods with "empty" spaces
• Endospores-resistant to heat, drying, chemicals
• Catalase positive

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Group 1 Bacillus species

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• Oval or cylindrical spores
• Central or terminal location
• does not distend the vegetative cell

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Group 2 Bacillus species

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• Oval spores
• Central or terminal location
• Causes the vegetative cell to swell

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Group 3 Bacillus species

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• Round spores
• Terminal location
• Swollen spore

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Bacillus anthracis

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• Anthrax
• Humans aquire via infection by:
• contamination of wound
• ingestion
• inhalation spores

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Morphology Bacillus anthracis

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• Large, spore-forming gram positive bacilli
• Spores viable for >50 years
• Nonhemolytic SBA

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Virulence factor Bacillus anthracis

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Plasmids: Capsule & Exotoxin

• Polypeptide capsule (pX02)
• Potent exotoxin (pX01):

1. Edema factor
2. Protective antigen
3. Lethal factor

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Is there a vaccine for Bacillus anthracis

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Yes, for those with a high risk

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Incidence of infection in Bacillus Anthracis

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• Fewer than five cases a year
• Woolsorter's disease/Ragpicker's disease:

1. Handling fibers
2. hides
3. animal products

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Most common infection of anthrax

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Cutaneous:

• Enter through cuts causing a localized infection
• Malignant pustules or black eschar (scab):

1. painless
2. nonpus producing
3. permanent scar

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Gastrointestinal Anthrax

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• Ingestion of contaminated meat
• Inoculates intestinal mucosa lesion
• Abdominal pain, nausea, anorexia, vomiting, and sometimes bloody diarrhea
• More fatal than cutaneous but less likely to occur

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Pulmonary anthrax (Woolsorter's Disease)

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• Inhalation of spores
• Mild fever, fatigue, and malaise for 2-5 days after exposure
• Sudden respiratory distress, disorientation, coma, and death: may last less than 24 hours from onset to death

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Microscopic morphology B. anthracis

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• Large, square-ended gram-positive rods
• bamboo appearance
• in blood specimens long filamentous rods

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Colonial morphology B. anthracis

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• Non-hemolytic SBA
• Raised, large, grayish white, irregular
• Fingerlike edges
• "Medusa head"
• "beaten egg whites"

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Suspecting anthrax

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• Work in a BSC
• Non-hemolytic BAP
• Non motile
• Produces lecithinase
• Exposure to penicillin causes large spherical bacilli in chains: string of pearls morphology

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Treatment of anthrax

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• Most susceptible to penicillin
• Resistance due to beta-lactamase production
• Ciprofloxacin for treatment: until susceptibility results are known

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Bacillus cereus: Food poisoning

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• Enterotoxins
• Diarrheal syndrome: ass. with meat, poultry, and soups
• Incubation period of 8-16 hours, fever uncommon, resolves within 24 hours
• Emetic form: ass. with fried rice, abdominal cramps and vomiting
• Incubation 1-5 hours, resolves in 9 hours

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Infections of B. cereus

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Immunosuppressed hosts:

• Opportunistic infections of the eye
• Meningitis, septicemia, and osteomyelitis
• Found as a contaminant in drug paraphernalia

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Other Bacillus species associated with disease

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Food Poisoning, bacteremia, pneumonia

• B. subtilis
• B. licheniformis
• B. circulans
• B. pumilus
• B. sphaericus

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What does L. monocytogenes look like?

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Group B strept

L. monocytogenes is catalase positive

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High risk group for Erysipelothrix rhusiopathiae

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Fish handlers

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What is A. hemolyticum indistinguishable from?

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Group A strept; but does not lancefield group type

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Main contaminant in urine

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Lactobacillus

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Clue cell

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coated in bacteria

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In clue cell what can be absent, what does this lead to?

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Lactobacillus; the amount that is present is scored via nugent score 0-4

Gram-variable rods 0-4

Curved gram-variable road 0-2

7-10 consistent with bacterial vaginosis

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Nocardia distribution

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ubiquitous in the environment and can be found world wide as saprophytic components

Cutaneous infection

Severe pulmonary or CNS disease in immunocompromised

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One way to identify Nocardia

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Paraffin bait test, only Nocardia can break it down as a substance

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Gordonia closely realated to

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Rhodococcus

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Anthrax

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disease in livestock, Select Agent A

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What is needed for B. anthracis virulence

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Both plasmids: capsule and exotoxin