09-Aerobic_and_Facultative_Gram-Positi____ve_Bacilli_v1-_3

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Aerobic and Facultative Gram-Positive Bacilli:

Aerobic and Facultative Gram-Positive Bacilli Dr. John R. Warren Department of Pathology Northwestern University Feinberg School of Medicine June 2007

An Algorithm for Aerobic Gram-Positive Bacilli:

An Algorithm for Aerobic Gram-Positive Bacilli Gram’s stain morphology Catalase reaction (positive or negative) -hemolysis with sheep blood agar (present or absent) Aerobic sporulation (present or absent)

Gram’s Stain Morphology:

Gram’s Stain Morphology Irregular rods: Bacilli whose longitudinal edges are curved and not parallel Coryneform rods: Club-shaped bacilli Diphtheroid arrangement: Bacilli arranged in palisades of parallel cells, V or L shaped pairs of bacilli, and clusters of these morphologies forming “Chinese letters” Regular rods: Bacilli whose longitudinal edges are straight and parallel

Irregular Gram-Positive Rods:

Irregular Gram-Positive Rods Corynebacterium species 1 Catalase positive Non- -hemolytic Arcanobacterium haemolyticum 2 Catalase negative -hemolytic 1 Coryneform rods in diphtheroid arrangement 2 Previously Corynebacterium haemolyticum

Regular Gram-Positive Rods:

Regular Gram-Positive Rods Listeria monocytogenes ( -hemolytic, catalase positive) Bacillus species 1 (aerobic sporulation, catalase positive 2 ) Erysipelothrix rhusiopathiae (Non- hemolytic, catalase negative) 1 Bacillus anthracis non--hemolytic, B . non- anthracis species -hemolytic 2 Most strains

Taxonomy of Corynebacterium:

Taxonomy of Corynebacterium Belong to the class Actinobacteria and the family Corynebacteriaceae Corynebacterium species contain cell wall mycolic acids with 22 to 36 carbon atoms 16S rRNA gene sequences indicate a close relationship of Corynebacterium to the family Mycobacteriaceae ( Mycobacterium , Nocardia , and Rhodococcus )

Taxonomy of Corynebacterium:

Taxonomy of Corynebacterium Corynebacterium species not acid-fast positive Corynebacterium species have high DNA G+C content in a wide range (46-74 mol%) indicating genetic diversity Corynebacterium composed of 59 species of which 36 are medically relevant

Pathogenic Corynebacterium Species:

Pathogenic Corynebacterium Species Corynebacterium diphtheriae 1 C . ulcerans 1 C . pseudotuberculosis 1 C . jeikeium 2 C . amycolatum 2 C . urealyticum 2 1 Toxin-producing strains cause diphtheria 2 Variably associated with invasive infection

Corynebacterium: Natural Habitats:

Corynebacterium : Natural Habitats Corynebacterium diphtheriae establishes a carrier state by infection of the human nasopharnyx or skin (not considered a constituent of normal microbial flora, no environmental or animal source) C . ulcerans associated with bovine mastitis, C . pseudotuberculosis associated with suppurative lymphadenitis, abscess formation, and pneumonia in cattle

Corynebacterium: Natural Habitats:

Corynebacterium : Natural Habitats Many species normal commensals of the human skin (including C . jeikeium , C . amycolatum , and C . urealyticum ) C . jeikeium and C . amycolatum also present in the inanimate hospital environment

Corynebacterium: Modes of Infection:

Corynebacterium : Modes of Infection Corynebacterium diphtheriae spread person-to-person by exposure to respiratory droplets from convalescent or healthy carriers Corynebacterium ulcerans infection associated with cattle or raw milk Corynebacterium pseudotuberculosis associated with close animal contact or drinking raw milk, human infection rare

Corynebacterium: Modes of Infection:

Corynebacterium : Modes of Infection Corynebacterium jeikeium , C. amyco- latum , and C . urealyticum as skin flora can be introduced systemically from infected catheter wounds in debilitated patients (prolonged hospitalization, multiple antibiotics, neutropenia) Corynebacterium urealyticum as an anterior urethral commensal can invade the urinary tract in debilitated patients

Corynebacterium: Types of Infectious Disease:

Corynebacterium : Types of Infectious Disease Diphtheria is an acute pharyngitis with formation of an inflammatory pseudomembrane. Systemic absorption of diphtheria toxin causes sterile myocarditis with a mortality of 10-30%. Diphtheria toxin catalyzes the transfer of ADP-ribose from NAD to elongation factor-2 (EF-2) with inactivation of EF-2 in ribosomal protein synthesis.

Corynebacterium: Types of Infectious Disease:

Corynebacterium : Types of Infectious Disease Toxigenic strains of C . diphtheriae lyso- genized by tox + -prophage most often cause diphtheria. Occasional strains of C . ulcerans and C . pseudotuberculosis also produce toxin, but only C . ulcerans is associated with diphtheria-like illness. Tox – strains of C . diphtheriae cause pharyngitis and endocarditis.

Corynebacterium: Types of Infectious Disease:

Corynebacterium : Types of Infectious Disease C . jeikeium , C . amycolatum , and C . urealyticum produce wound infection, bacteremia, and endocarditis in hospital patients. C . urealyticum is a urease producer that causes alkaline-encrusted cystitis due to deposition of ammonium magnesium phosphate crystals in damaged bladder mucosa with ulceration and infection.

Corynebacterium: Resistance to -Lactam Drugs:

Corynebacterium : Resistance to -Lactam Drugs Corynebacterium amycolatum , C . jeikeium , and C . urealyticum are characteristically resistant to penicillin and other -lactam drugs, and uniformly vancomycin susceptible

Characteristics of Corynebacterium:

Characteristics of Corynebacterium Gram-positive bacilli with non-parallel sides and wider ends resulting in club shaped forms (coryneform) Arranged as single cells, pairs, V, L, and Y forms, palisades, and “Chinese letters” (diphtheroid) Non- -hemolytic on sheep blood agar Uniformly catalase positive

Characteristics of Corynebacterium:

Characteristics of Corynebacterium Grow well on sheep blood but not enteric agar (MacConkey) Lipophilic species ( C . jeikeium , C . urealyticum ) demonstrate optimal growth in presence of the non-ionic detergent Tween 80 (polysorbate 80)

Laboratory Detection of Corynebacterium diphtheriae:

Laboratory Detection of Corynebacterium diphtheriae Translucent, gray, or white non-hemolytic colonies up to 2 mm in size after 18-24 hours in 5% CO 2 at 37 o C Deep blue or red metachromatic granules (accumulated inorganic polyphosphates) by methylene blue stain after incubation on a Loeffler’s serum slant (heat inspissated serum and whole egg medium) (not specific for C . diphtheriae )

Laboratory Detection of Corynebacterium diphtheriae:

Laboratory Detection of Corynebacterium diphtheriae Black colonies (tellurite reductase) are surrounded by a brown halo (cystinase) on Tinsdale agar specific for C . diphtheriae. Tinsdale cystine-tellurite blood agar is selective and differential by containing potassium tellurite. Staphylococcus and Proteus can produce black colonies, but the colonies lack a brown halo, and the colonies demonstrate gram-positive cocci ( Staphylococcus ) or gram-negative rods ( Proteus ) by Gram’s stain.

Corynebacterium diphtheriae: Identification:

Corynebacterium diphtheriae : Identification Forms black colonies surrounded by a brown halo on Tinsdale medium Produces acid from glucose and maltose Biotypes variably positive for nitrate reduction (biotypes gravis , intermedius , and mitis positive, biotype belfanti negative) and lipophilic growth (biotype intermedius positive)

Corynebacterium diphtheriae:Biotypes:

Corynebacterium diphtheriae :Biotypes Four biotypes: intermedius , gravis , mitis , belfanti Biotype intermedius shows small, gray, or translucent, lipophilic colonies, other biotypes larger (up to 2 mm at 24 hours), white or opaque, non-lipophilic colonies Biotype intermedius rarely occurs in clinical infection and belfanti rarely contains the tox+ gene

Elek Assay for Diphtheria Toxin:

Elek Assay for Diphtheria Toxin Paper strip or disk saturated with diphtheria antitoxin is placed in molten agar at 55 o C, and allowed to sink to the bottom of the plate. The agar is allowed to solidify by cooling to room temperature. Streaks of unknown test organisms are placed at a right angle to the strip, or around the periphery of the disk.

Elek Assay for Diphtheria Toxin:

Elek Assay for Diphtheria Toxin Development of an agar precipitin line within 1-2 days of incubation at 35 o C is a positive result for diphtheria toxin. In an Elek test using a strip, the precipitin line forms at a 45 o angle to the strip. In an Elek test using a disk, the precipitin line forms between the disk and test organism.

Corynebacterium amycolatum: Identification:

Corynebacterium amycolatum : Identification Fermentative, growth not lipophilic Growth resistant to the vibriocidal compound O/129 (150- g disks) Acid production from glucose, variable acid from maltose and sucrose Variably positive for urease

Corynebacterium jeikeium: Identification:

Corynebacterium jeikeium : Identification Oxidative, lipophilic growth Growth resistant to a 10 unit penicillin disk Acid production from glucose (broth supplemented with rabbit plasma or Tween 80), variable acid from maltose

Corynebacterium urealyticum: Identification:

Corynebacterium urealyticum : Identification Oxidative, lipophilic growth Positive for urease Asaccharolytic (glucose, maltose, sucrose, mannitol, xylose)

Species Identification of Corynebacterium1:

Species Identification of Corynebacterium 1 F/O LIP URE GLU MAL SUC C . jeikeium 2 O + – + 5 +/– 5 – 5 C . amycolatum 3 F – +/– + +/– +/– C . urealyticum 4 O + + – – – 1 F/O=fermentative/oxidative; LIP=lipophilic; URE=urease; GLU, MAL, SUC=production of acid from glucose, maltose, sucrose (Andrade’s) 2 Isolated from blood and vascular catheter wounds, growth uniformly resistant to 10-u penicillin disk 3 Isolated from blood and vascular catheter wounds, most strains growth resistant to 150- µg O/129 disk 4 Isolated from urine 5 Andrade’s supplemented with 2-3 drops coagulase rabbit plasma

Species Identification of Corynebacterium: Criteria1:

Species Identification of Corynebacterium : Criteria 1 Isolate from normally sterile specimen Isolate from more than one specimen of the same type especially blood Isolate from urine as only (>10 4 /mL) or predominant (>10 5 /mL) organism Presence of coryneform bacteria by Gram’s stain with many leukocytes 1 Including C . diphtheriae in invasive infection (rare)

Arcanobacterium:

Arcanobacterium Arcanobacterium closely related but genomically distinct from Corynebacterium A . haemolyticum type species and medically relevant, A . pyogenes and A . bernardiae also clinically significant

Arcanobacterium:

Arcanobacterium Arcanobacterium haemolyticum associated with streptococcal-like pharyngitis, mixed wound and tissue infections, A . pyogenes with abscess formation, wound and soft tissue infection, and A . bernardiae with polymicrobial abscess formation

Arcanobacterium:

Arcanobacterium Irregular, sometimes branching gram-positive rods -hemolysis on sheep blood agar Catalase negative Fermentative

Arcanobacterium:

Arcanobacterium A . haemolyticum demonstrates reverse CAMP test A. haemolyticum, A. pyogenes, A. bernardiae produce acid from glucose, only A . pyogenes produces acid from xylose, A . bernardiae produces acid more quickly from maltose than glucose

Characteristics of Listeria:

Characteristics of Listeria Genetic taxonomy places Listeria closest to Bacillus and Staphylococcus ( Listeria - Brochothrix family). Seven species of Listeria , of which only Listeria monocytogenes is pathogenic for humans.

Characteristics of Listeria:

Characteristics of Listeria Regular short gram-positive rods, often coccobacillary, occur singly and in short chains Facultatively anaerobic, fermentative for glucose, Voges-Proskauer and methyl red positive, catalase positive Motile at 20 o -25 o C

Listeria monocytogenes: Natural Habitats:

Listeria monocytogenes : Natural Habitats Primary habitat is soil and decaying vegetable matter Widely present in foods including fresh and processed poultry and meat, raw milk, cheese, ice cream, raw fruits and vegetables Transiently colonize the gastrointestinal tract in 2-20% of humans without symptoms

Pathogenesis of Listeriosis:

Pathogenesis of Listeriosis Two modes of infection: Ingestion of food contaminated by L . monocytogenes (fecal organism); Transmission to fetus in mother bacteremic with L . monocytogenes Internalin of L . moncytogenes binds to intestinal epithelial cells followed by transmucosal invasion and lympho-hematogenous dissemination

Pathogenesis of Listeriosis:

Pathogenesis of Listeriosis Infection of mononuclear phagocyte system (macrophages of spleen, lymph nodes, liver, and bone marrow) Listeriosis observed clinically as a bacteremia in pregnancy, AIDS, and renal transplantation with high-dose steroids Meningitis secondary to bacteremia in neonates and immunosuppresed adults

Pathogenesis of Listeriosis:

Pathogenesis of Listeriosis L. monocytogenes is a facultative intracellular parasite that infects macrophages The exotoxin listeriolysin O binds to membrane cholesterol and disrupts phagolysosomes of infected macrophages

Pathogenesis of Listeriosis:

Pathogenesis of Listeriosis Listeria bacilli escape phagolysosomes into the iron-rich cytoplasm and rapidly divide with a doubling time of ~1 hour Listeria bacilli induce polymerization of actin in the infected cell that propels bacilli through the plasma membrane with penetration and infection of adjacent cells

Listeria monocytogenes: Types of Infectious Disease:

Listeria monocytogenes : Types of Infectious Disease Maternal bacteremia during pregnancy with transplacental fetal infection and abortion, stillbirth, or premature birth Bacteremia and (less frequently) meningitis in immune compromised individuals (AIDS, lymphoma, corticosteroid suppression therapy in organ transplantation)

Clinical Presentation of Listerosis:

Clinical Presentation of Listerosis Most frequently transient bacteremia with no clinical signs or symptoms, or mild “flu-like” symptoms Bacteremia in immunocompromised host (especially AIDS) can manifest as sepsis with fever, tachycardia, and neutrophilic leukocytosis, often accompanied by headache, myalgia, and arthralgia

Clinical Presentation of Listerosis:

Clinical Presentation of Listerosis Meningitis acute but can be subacute as a mimic of tuberculous meningitis Nuchal rigidity absent in 15-20% of adult patients Ataxia, tremors, myoclonus, and seizures occur in 15-25% CSF: Gram’s stain negative in ~60%, glucose not depressed in >60%, and mononuclear cell pleocytosis in ~1/3rd

Listeria monocytogenes:

Listeria monocytogenes Gram-positive bacillus with narrow-zone β - hemolysis on sheep blood agar Catalase positive Ferments glucose by butylene glycol pathway (Voges Proskauer positive) Demonstrates esculin hydrolysis Enhanced motility at cooler temperatures (20 o -25 o C) (end-over-end in hanging drop, umbrella pattern in soft agar)

Hemolytic Patterns and CAMP Test for Listeria monocytogenes:

Hemolytic Patterns and CAMP Test for Listeria monocytogenes L . monocytogenes produces a narrow zone of -hemolysis on sheep blood agar barely extending beyond the edge of colonies 1 L . monocytogenes shows a rectangular zone of syngergistic -hemolysis in the CAMP test with Staphylococcus aureus 1 1 Identical pattern seen for non-pathogenic β -hemolytic L . seeligeri

Fermentation Patterns of Listeria1:

Fermentation Patterns of Listeria 1 Listeria monocytogenes 2 Positive for rhamnose fermentation, negative for xylose fermentation Listeria seeligeri 2 and Listeria ivanovii 3 Negative for rhamnose fermentation, positive for xylose fermentation 1 β -hemolytic species of Listeria 2 Narrow zone hemolysis 3 Wide zone hemolysis, negative with CAMP test

Erysipelothrix rhusiopathiae:

Erysipelothrix rhusiopathiae The genus Erysipelothrix classified as a regular non-spore forming gram-positive rod with Listeria and Lactobacillus Erysipelothrix rhusiopathiae carried by animals including mammals, birds, and fish, and most commonly associated with pigs

Erysipelothrix rhusiopathiae:

Erysipelothrix rhusiopathiae E . rhusiopathiae causes erysipeloid, a localized cellulitis acquired by infection of skin abrasions, injuries, or bites in individuals who handle animals or animal products E . rhusiopathiae forms pinpoint (<0.5 mm) non-hemolytic, catalase-negative colonies on sheep blood agar, and produces H 2 S in TSI agar

Lactobacillus:

Lactobacillus Lactobacillus is a microaerophiic or occasionally anaerobic lactic acid-producing organism with a close phylogenetic relationship to Streptococcus , Leuconostoc , and Weissella . Lactobacilli are catalase-negative and form pinpoint -hemolytic colonies on sheep blood agar with a strong resemblance to viridans streptococci.

Lactobacillus:

Lactobacillus Gram’s stain varies with long slender rods and parallel sides to slight curved or coryneform coccobacilli Identified by sheep blood agar colony morphology, Gram’s stain characteristics, and negative catalase reaction

Lactobacillus:

Lactobacillus Lactobacilli widely distributed in nature and present as mucosa-associated human saprophytes of the oral cavity, gastro- intestinal tract, and female genital tract Lactobacillus causes endocarditis, and is associated with polymicrobial abscesses of the mouth, abdomen, and pelvis

Taxonomy of Bacillus:

Taxonomy of Bacillus Family Bacillaceae tentatively consists of Bacillus , Listeria , and Staphylococcus Bacillus consists of 70 species of which B . anthracis and B . cereus are medically important

Bacillus: Natural Habitats:

Bacillus : Natural Habitats Bacillus spores ubiquitously distributed in soil Vegetative forms of Bacillus species other than B . anthracis widely present in soil and water Bacillus spores and vegetative forms of species other than B . anthracis are frequent colonizers of the skin and gastrointestinal tract

Bacillus: Modes of Infection:

Bacillus : Modes of Infection Natural infection by Bacillus anthracis by introduction of spores into skin abrasions or cutaneous wounds by contact with infected herbivorous animals or animal products (especially wool), inhalation of spores, or ingestion of spores Spread of anthrax spores through the US postal system (2001) with contact and inhalational infection (bioterrorism)

Bacillus: Types of Infectious Disease:

Bacillus : Types of Infectious Disease Anthrax caused by Bacillus anthracis and occurs in three forms: cutaneous, pulmonary, and gastrointestinal. Cutaneous anthrax most common natural form (~95% of human cases). Papules develop at skin inoculation sites, progress to hemorrhagic vesicles that rupture and form a black eschar surrounded by edema (malignant pustule).

Bacillus: Types of Infectious Disease:

Bacillus : Types of Infectious Disease Mortality of untreated cutaneous anthrax is ~20%. Pulmonary anthrax has a mortality of 100% due to a severe hemorrhagic mediastinitis and bacteremia. Pulmonary and cutaneous anthrax occurred during the 2001 bioterrorist attack on the US postal system.

Bacillus: Types of Infectious Disease:

Bacillus : Types of Infectious Disease Gastrointestinal anthrax results in a severe hemorrhagic ascites. Bacillus cereus causes a rapid, virulent, and destructive endophthalmitis following penetrating trauma of the eye or hematogenous spread. B. cereus bacteremia can develop secondary to intravenous drug abuse.

Bacillus: Types of Infectious Disease:

Bacillus : Types of Infectious Disease Most frequently (>99%) a blood culture positive for the growth of Bacillus is due to a skin contaminant. A food-poisoning syndrome with acute emesis is caused by heat-stable exotoxin of B . cereus . Clinically mimics staphylococcal food-poisoning. Secretory diarrhea is produced by heat stable exotoxin of B . cereus .

Characteristics of Bacillus:

Characteristics of Bacillus Large (0.5 x 1.2-2.5 x 10 m) gram-positive bacilli with square or concave ends containing endospores that by Gram’s stain appear as intracellular non-staining (clear) ovoid areas (spores do not stain by crystal violet, safranin, or carbolfuchsin) Bacillus becomes gram-variable or gram-negative with aging in culture, but unlike true gram-negative bacteria, Bacillus susceptible to growth inhibition by a 30 µg-vancomycin disk

Characteristics of Bacillus:

Characteristics of Bacillus Bacillus grows well on 5% sheep blood agar and chocolate agar but not MacConkey agar Catalase positive Aerobic growth of large spore-forming gram-positive rods positive for catalase strongly presumptive for Bacillus

Characteristics of Bacillus:

Characteristics of Bacillus Colonies of B . anthracis on sheep blood agar large (4-5 mm), flat, and white to gray with irregular edges and no -hemolysis Colonies of B . cereus large (3-8 mm) and raised with a gray to green frosted-glass appearance and undulate margins with distinct -hemolysis

Characteristics of Bacillus:

Characteristics of Bacillus Bacillus anthracis non-motile and sensitive to penicillin (growth inhibited by a 10-unit penicillin disk) Bacillus cereus motile and resistant to penicillin (growth inhibited by a 10-unit penicillin disk)

Red Flags for Bacillus anthracis:

Red Flags for Bacillus anthracis Flat or slightly convex 2-5 mm non-hemolytic tenacious colonies on sheep blood agar after overnight incubation at 35 o C Catalase positive Gram-positive bacilli in long chains with oval spores that do not swell the cells Non-motile

Recommended Reading:

Recommended Reading Winn, W., Jr., Allen, S., Janda, W., Koneman, E., Procop, G., Schreckenberger, P. Woods, G. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Sixth Edition, Lippincott Williams & Wilkins, 2006: Chapter 14. Aerobic and Facultative Gram-Positive Bacilli.

Recommended Reading:

Recommended Reading Murray, P., Baron, E., Jorgensen, J., Landry, M., Pfaller, M. Manual of Clinical Microbiology, 9 th Edition, ASM Press, 2007: Funke, G., and Bernard, K.A. Chapter 34. Coryneform Gram-Positive Rods. Bille, J. Chapter 33. Listeria and Erysipelothrix . Logan, N.A., Popovic, T., and Hoffmaster, A. Chapter 32. Bacillus and Other Aerobic Endospore-Forming Bacteria.

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