Clinical Bacteriology - 03 Gram Positive Bacilli

• CLINICAL BACTERIOLOGY MODULE 3 - GRAM POSITIVE SPORE FORMING AND NON-SPOREFORMING BACILLI OUTLINE • • • • Introduction • Non-Banching, Catalase (+), Gram (+) Bacilli, Genus Bacillus o Microscopic Characteristics o Tests for Identification o Clinical Significance Listeria, Eysipelothrix, Lactobacillus o Listeria monocytogenes o Ersipelothrix rhusiopathiae o Lactobacillus Corynebacterium o Microscopic Characteristics o Tests for Identification o Clinical Significance • • • TESTS FOR IDENTIFICATION • INTRODUCTION • • • Bacillus spp. are aerobic, gram positive, spore forming rods. Bacillus anthracis, the most notorious pathogen of this genus, inhabits the soil. Humans acquire infections when they are inoculated with the spores, either by inhalation during exposure to contaminated animal products, such as hides, or by traumatic introduction. All other Bacillus spp. are generally considered to be opportunistic pathogens of low virulence and are usually only associated with compromised patients exposed to contaminated materials. The two most potent pathogens classified as catalase positive gram positive rods but not spore forming are Listeria monocytogenes and Corynebacterium diphtheriae. These two species differ markedly in epidemiology. L. monocytogenes is widely distributed in nature and occasionally colonizes the human gastrointestinal tract. C. diphtheriae is only carried by humans but rarely , is isolated from healthy individuals. L. monocytogenes, by virtue of its ability to survive within phagocytes, and C. diphtheriae, by production of an extremely potent cytotoxic exotoxin, are the most virulent species. Other gram positive bacilli are Lactobacillus and E. rhusiopathiae. Lactobacillus is highly pleomorphic, occuring in long chain rods, as well as in coccobacilli and spiral forms. It is found in the environment in water and sewage and as normal flora of many animals. E. rhusiopathiae is a an occupational hazard for those handling meat, poultry , fish and rabbits. Human infections , or erysipeloid, can develop from direct contact or ingestion of contaminated water or animal product. NON-BRANCHING, CATALASE (+) GRAM (+) BACILLI, GENUS BACILLUS • • • • Large gram-positive rods in singles, pairs, or serpentine changes Capable of producing endospores in the presence of oxygen Species differentiation within the genera is based on the size of the vegetative cell, sporulation resulting in swelling of the vegetative cell, and biochemical analysis CEU Malolos BSMT 2023 Gram Staining o The only specific procedure for the direct detection of Bacillus spp. in clinical specimens o Capable of producing endospores in the presence of oxygen Triple Sugar Iron (TSI), Urea or Nutrient Agar with 5 mg/L Manganese Sulfate o Induce production of spores Special Stains o Used to visualize endospores 1. Smear is covered with malachite green 2. Filter paper is placed over the stain 3. Heat slide for several minutes to force dye into cell walls of spore 4. Counterstain with Safranin. 5. Endospores stain green and the vegetative cells will appear pink from the secondary stain, safranin. CULTIVATION • Blood Agar Media o Colonies are non-haemolytic o Narrow zone of hemolysis • Chocolate Agar, routine blood culture media and commonly used nutrient broths can also be used Will Not grow on MacConkey agar. Incubation Conditions and Duration o Will produce detectable growth within 24 hrs. incubation o 35˚C in ambient air or in 5% C0₂ o *Bicarbonate agar must be incubated in C0₂ • • MICROSCOPIC CHARACTERISTICS • B. anthracis, B. cereus, B. mycoides, and B. megaterium o Width of vegetative cell is more than 1 µm o Spores do not cause swelling B. pumilus, and B. licheniformis o Vegetative cell width is less than 1 µm o Spores do not cause the cell to swell B. circulans, B. coagulans, B. sphaericus, B. brevis, P. macerans, P. alvei, and P. polymyxa o Cell width is less than 1 µm o Spores cause the cell to swell Bacillus thuringiensis o Harbors cereulide enterotoxin BACILLUS ANTHRACIS • • Irregular, round raised dull opaque grayish white colonies with a frosted glass appearance Edge of the colony is composed of long, interlacing chains of bacilli, resembling locks of matted hair- “Medussa Head Appearance” BACT - M3 • 1 • • Gelatin stab culture - “ inverted fir tree” appearance with slow liquefaction starting from top. Medussa Head Appearance - wavy colonies with small projections CLINICAL SIGNIFICANCE Bacillus thuringiensis • • • • Occupational exposure to insecticides and pesticides containing the organism Presence of organism in feces w/o gastrointestinal symptoms Toxins are commercialized for the control of insects that cause agricultural damage, e.g. moths, beetles, flies, and parasitic worms (nematodes) Rare cases: wound, burn, pulmonary, and ocular infections Bacillus anthracis • • • INVERTED FIR TREE 2 Special Media used for isolation and identification of B. anthracis PLET(polymyxin-lysozyme-EDTA-thallous acetate) o selective media used for the isolation of Bacillus anthracis from contaminated specimens. Bicarbonate agar- to induce capsule formation • • • SERODIAGNOSIS • • Indirect hemagglutination and enzyme-linked immunosorbent assays - detect antibodies to B. anthracis Not used to diagnose infections caused by other Bacillus spp. • RED LINE ALERT TEST • • • • • • Immunochromatographic test For the rapid, in vitro qualitative presumptive identification of Bacillus anthracis from non-hemolytic Bacillus colonies Cultured on sheep blood agar plates. The test is intended for use in o clinical, o public health, and o Hospital laboratories in conjunction with other markers and testing for the presumptive identification of Bacillus anthracis Gives sentinel labs the ability to significantly shorten the time from when the plate is streaked until PCR confirmation is completed at the LRN reference lab DIFFERENTIATION OF CLINICALLY SIGNIFICANT BACILLUS • Virulence factor: Capsule exotoxins 3 Forms of Anthrax o Cutaneous anthrax o Pulmonary anthrax o Gastrointestinal anthrax Cutaneous anthrax o From an erythematous papule to ulceration and formation of a black scar (i.e., eschar) o May progress to toxemia and death o Occurs at site of spore penetration 2-5 days after exposure Pulmonary anthrax (’Woolsorters’ Disease’’) o Malaise with mild fever and non-productive cough to respiratory distress, massive chest edema, cyanosis, and death o Acquired through inhalation of spores o Woolsorters’ disease and Ragpickers’ disease are used to describe respiratory infections Gastrointestinal anthrax o Causes toxemia and overwhelming sepsis o Acquired through ingestion of spores o Affects either the oropharyngeal or the abdominal area Bacillus cereus • • • • • • Fried rice bacillus Most commonly encountered species of Bacillus in opportunistic infections including posttraumatic eye infections, endocarditis, and bacteremia Infections of other sites are rare and usually involve intravenous drug abusers or immunocompromised patients Produces several toxins implicated in the diarrheal symptoms, including hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe), and cytotoxin K (CytK). Virulence factors: Enterotoxins and pyogenic toxin Food poisoning of two types: o Diarrheal type - characterized by abdominal pain and watery diarrhea o Emetic type - manifested by profuse vomiting Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, other Bacillus spp. • • • • • CEU Malolos BSMT 2023 Unknown virulence factors Food poisoning has been associated with some species but is uncommon These organisms may also be involved in opportunistic infections similar to those described for B. cereus B. subtilis - hay bacilli, halophilic, tolerate up to 7% NaCl o Source of bacitracin antibiotic B. pumilus - biological indicator for sterilization BACT - M3 • 2 Erysipelothrix rhusiopathiae Listeria, Erysipelothrix, Lactobacillus Listeria monocytogens • • Characteristics o Normal human flora of vagina and intestines in human o Found in the environment o Causes spontaneous abortion and meningitis in animals (e.g. sheep) o A potent pathogen o Has the ability to survive within phagocytes o Media of choice: 5% Sheep Blood Agar and Chocolate Agar o Microscopically: short gram-positive rod that may occur singly or in short chains resembling Streptococci • • Gram Stain Clinical manifestation and pathogenesis o MOT: By ingestion of contaminated food (meat and dairy products) o Listeriosis o Bacteremia o Meningitis – usual outcome of infection o Infections – usually occurs in neonates and immunocompromised o Granulomatosis infantiseptica • • Listeriosis Laboratory diagnosis o Catalase (+) o Motility (+) o Esculin hydrolysis (+) o Hippurate (+) o CAMP test (+) o VP test (+) o H2S on TSI (-) o 5% Sheep Blood Agar: small, white, translucent, smooth, moist o SBA: Narrow band of beta hemolysis o On McBride’s media: blue green o Semisolid media: umbrella like growth 5% SBA CEU Malolos BSMT 2023 Umbrella like growth Characteristics o Non-motile o Catalse negative o Facultatively anaerobic gram positive bacilli o Microscopically: short rods with rounded ends; singly, in short chains or in non-braching filaments Gram stain Clinical manifestation and pathogenesis o MOT: abrasion or inoculation thru skin wounds with exposure to infected animals o Human infections ▪ Erysipeloid – localized skin infection; occupational hazard for those handling meat, poultry and fish ▪ May also present as bacteremia or endocarditis Erysipeloid Laboratory diagnosis o Best specimen for culture: Biopsy and tissue aspirates from erysipeloid lesions o Oxidase (-) o Catalase (-) o Urease (-) o Esculin hydrolysis (-) o Gelatin (-) o o o BAP On TSI: H2s (+) On BAP: Alpha hemolytic On Gelatin Stab: produces “pipe cleaner”/”test tube brush” pattern growth (very short projections, uniform length) Gelatin Stab / TSI BACT - M3 • 3 • Antimicrobial Susceptability o Susceptible to: ▪ Penicillin – treatment of choice for localized and systemic infection ▪ Cephalosporins ▪ Erythromycin ▪ Clindamycin ▪ Chloramphenicol o Resistant to: ▪ Sulfonamides ▪ Aminoglycosides ▪ Vancomycin Lactobacillus • Characteristics o Normal human flora o Widely distributed in foods o Gram positive non-spore-forming rods o Non-motile, anaerobic bacteria o Microscopic appearance: pleomorphic rods or cocco bacilli • • • • • • • • • • • Clinical Significance o Infections are rare, since it is frequently encountered as contaminant o Some Lactobacillus spp may cause bacteremia in immunocompromised patients Laboratory diagnosis o Medium: 5% Sheep Blood Agar and Chocolate Agar; CNA; Anaerobic BAP o Usual identification based on: o Colony o Gram stain morphology o Catalase test (-) o Urease, Nitrate reduction and beta hemolysis (-) o Glucose fermentation (+) o Maltose fermentation (+) o Mannitol fermentation (v) o Sucrose fermentation (+) Antimicrobial Susceptibility o Resistant to: ▪ Cephalosporin ▪ Penicillin ▪ Vancomycin • • Corynebacterium freneyi o On gram stain: Typical coryneform gram (+) rods o On 5% sheep blood agar: Whitish, dry, rough Corynebacterium jeikeium o On gram stain: Pleomorphic; Club-shaped arranged in Palisades o On 5% sheep blood agar: Small, white-yellow, convex, non-hemolytic Corynebacterium imitans o On gram stain: Typical coryneform gram (+) rods o On 5% sheep blood agar: White-gray, glistening, convex, creamy Corynebacterium minutissimum o On gram stain: With single cells, palisading and Chinese letters o On 5% sheep blood agar: Small, circular, shiny and moist Corynebacterium pseudodiphtheriticum o On gram stain: Typical Coryneform gram (+) rods o On 5% sheep blood agar: Small-medium, slightly dry Corynebacterium pseudotuberculosis o On gram stain: Typical Coryneform gram (+) rods o On 5% sheep blood agar: Yellowish white, opaque, convex, matted surface Corynebacterium striatum o On gram stain: Shows banding o On 5% sheep blood agar: White, moist and smooth Corynebacterium ulcerans o On gram stain: Typical Coryneform gram (+) rods o On 5% sheep blood agar: Gray-white, dry, waxy Corynebacterium urealyticum o On gram stain: Gram (+) coccobacilli o On 5% sheep blood agar: Pinpoint after 24 hours, white, smooth Corynebacterium xerosis o On gram stain: Can show banding o On 5% sheep blood agar: Granular, dry, yellowish TESTS FOR IDENTIFICATION GRAM STAINING • Corynebacterium spp. o gram positive rods, pleomorphic, slightly curved with tapered or clubbed ends. o May occur singly or in pairs, often in a “V” formation forming “Chinese letters”. o Usually stain weakly and unevenly giving a beaded appearance Corynebacterium MICROSCOPIC CHARACTERISTICS • • Corynebacterium amycolatum o On gram stain: V forms or Chinese letters o On 5% sheep blood agar: Small, white to gray, dry Corynebacterium diphtheriae group o On gram stain: Irregular stain, pleomorphic o On 5% sheep blood agar: o (there are 3 biotypes of C. diphtheria) ▪ 1. intermedius - small, gray, translucent. ▪ 2. mitis and 3. gravis - medium, white, opaque CEU Malolos BSMT 2023 A - Microscopic Gram stain of diphtheroid; B - Microscopic Loefler methylene blue stain of Corynebacterium spp. BACT - M3 • 4 REVERSE CAMP TEST • • • C. diphtheriae with metachromatic granules Incubation period: at 37ºC for 24-48 hours in aerobic conditions Result: "arrowhead" hemolysis is seen between the growth of C. pseudotuberculosis due to alpha toxin produced interacts with CAMP factor and produce synergistic hemolysis. It can be used for determination of C. ulcerans & C. pseudotuberculosis. CULTURING TWEEN 80 TEST • • • • 5% Sheep’s Blood Agar - most common media of choice for Corynebacterium spp., Incubated at 35°C with 5-10% CO2 for 48 hours the organism may have a very small zone of β-hemolysis. A lipophilic Coryneform bacterium grows more slowly requiring 3 or more days to show growth. • TEST FOR TOXIGENICITY • • • • • o Corynebacterium diphtheriae on SBA Selective and differential media for C. diphtheriae (incubated at 35-37°C for 16-48 hours.) Cystine-Tellurite Blood Agar: colonies appear black or gray. Useful preliminary identification of a certain coryneform bacteria, such as C. jeikeium and C. urealyticum are by the enhancement of growth by lipids. Elek Test – routinely used for the detection of exotoxin from toxigenic strains of Corynebacterium diphtheriae. Principle: 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 incubated at 35°C and examined after 18, 24, and 48 hours. NOTE: 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. SCHICK TEST • • • • Used to determine susceptibility or immunity to diphtheria in humans. Involves injecting a very small dose of the toxin under the skin of the forearm and evaluating the injection site after 48 hours. A positive test/inflammatory reaction: indicates susceptibility (nonimmunity) A negative test/no reaction: indicates immunity (antibody neutralizes toxin). SUMMARY OF SOME Corynebacterium SPP. WITH THEIR RESPECTIVE RESPONSE TO DIFFERENT BIOCHEMICAL TEST • Modified Tinsdale Agar: colonies are black with brown halos. • (+) positive; (-) negative; (rev) reverse CAMP test; (v) variable BIOCHEMICAL TEST • • Loeffler’s medium – may stimulate the growth of C. diphtheria and the production of metachromatic granules. Other routine blood culture and nutrient broth used for Corynebacterium spp.: o Thioglycollate broth and Brain-Heart Infusion. o NOTE: If broths are infused with rabbit serum, lipophilic coryneform bacteria will enhance growth. CEU Malolos BSMT 2023 BACT - M3 • 5 CLINICAL SIGNIFICANCE • • • • • • • Corynebacterium diphtheriae o Inhabits human nasopharyn and not considered as normal flora o MOT: Exposure to contaminated respiratory droplets or direct contact with infected cutaneous lesions o Two forms of Diphtheria ▪ Respiratory diphtheria – a pharyngitis characterized by the development of an exudative membrane that covers the tonsils, uvula, palate and pharyngeal wall ▪ Cutaneous diphtheria – characterized by nonhealing ulcers and membrane formation Corynebacterium jeikeium o Commonly in the inguinal, axillary and rectal sites o MOT: Introduced during placement or improper care of intravenous catheters o Caused Septicemia and wound infections Corynebacterium ulcerans o Normal flora of humans and cattles o MOT: Contact with animals especially during summer o Causes diphtheria like sore throat Corynebacterium pseudotuberculosis o Associated with infections in sheep , goats and horses o MOT: Close animal contact o Causes Suppurative granulomatous lymphadenitis Corynebacterium pseudodipthericum o Normal humal pharyngeal and skin flora o MOT: Access of patient’s endogenous strain to normally sterile site o Causes Endocarditis, pneumonia and lung absceses Corynebacterium minutissimum o Normal skin flora o MOT: Access of patient’s endogenous strain to normally sterile site o Causes Erythrasma – a puritic skin infections Corynebacterium urealyticum o Normal skin flora o MOT: Access of patient’s endogenous strain to normally sterile site o Causes Urinary tract infections and wound infections of compromised patients • CEU Malolos BSMT 2023 BACT - M3 • 6