Gram Positive Bacilli PDF

Significant Bacterial Isolates: Gram-Positive Bacilli  2 Significant Bacterial Isolates: Gram-Positive Bacilli Gram-positive bacilli are a taxonomically diverse group of organisms, most of which are foun...

Significant Bacterial Isolates: Gram-Positive Bacilli  2 Significant Bacterial Isolates: Gram-Positive Bacilli Gram-positive bacilli are a taxonomically diverse group of organisms, most of which are found in the environment and rarely cause human disease. Many of them are used in the industry for the production of enzymes, antibiotics, and some are important food spoilage organisms.   The following organisms are non- acid fast. Most of these organisms NON-SPORE FORMING, are part of the normal human flora NON-BRANCHING, and they colonize various parts of the human body. They are also found CATALASE POSITIVE in the environment and are BACILLI associated with various animals.  Corynebacteria  Rothia  Listeria Corynebacteria General Characteristics Usually found in fresh & salt-water, soil & air. This genus includes many species of aerobic and facultative gram-positive rods. Lipophilic Corynebacteria are considered fastidious organisms that This genus is closely related to Mycobacteria may demonstrate slow growth on and Nocardia with regards to the composition standard culture media. of their cell wall. o These 3 organisms typically exhibit a meso- diaminopimelic acid (m-DAP) and mycolic acid cell wall.  5 3 Groups: Human Pathogen Animal and plant pathogen Nonpathogen Gram staining morphology: From the Greek word “koryne” meaning club, the genus Corynebacterium has a typical club-shaped morphology. They are small and pleomorphic. Pleomorphic because aside from the cells having club ends, they also form Chinese letter or palisade arrangements. Medically important Corynebacterium are catalase positive, non-motile, and spores are not formed. Growth is generally best under aerobic conditions on media enriched with blood or other animal products, but many strains also grow anaerobically. There are also strains that form glucose and maltose through carbohydrate fermentation (Glucose and maltose fermenters), except for C. urealyticum and C. pseudodiphtheriticum. Corynebacterium diphtheriae General Characteristics Most notable pathogen of Corynebacteria and is Fragment B – Binding fragment. Binds to the causative agent of diphtheria. receptors of the cells and mediates the entry of Fragment A. Most common site of infection: Tonsils and The toxin is not toxic until it is exposed to Pharynx trypsin through a process called Virulence factor: Diphtheria toxin (DT) trypsinization, wherein the toxin inhibits protein synthesis of cells. Produced by bacteria infected with a lysogenic B-phage, which carries the tox Generally, Corynebacterium diphtheriae is gene. enhanced considerably when the bacteria are grown in low iron conditions. The diphtheria toxin is a heat stable polypeptide composed of 2 fragments: Fragment A and Fragment B. Fragment A (cytotoxicity) – Active fragment.  Clinical Infections 7 In order for Corynebacterium diphtheriae to cause Respiratory Diphtheria disease, the organism must ▪ Is a pharyngitis characterized by the Invade and development of an exudative membrane Proliferate in local tissues which is known as pseudomembrane. Infection Associated: Diphtheria Pseudomembrane – a gray white pseudomembrane that adheres to Transmitted by droplet spread, direct contact with the mucous membrane cutaneous infections and to a lesser extent, by fomites In situations that the pseudomembrane spreads to the Characterized by a characteristic lesion which ranges nasal mucosa, the pharyngeal wall, from a simple pustule to a chronic non-healing ulcer an edema involving cervical lymph and membrane formation nodes in the neck producing a “bull neck” appearance occurs  8 TREATMENT (directed at neutralization of the toxin): ❑Anti-toxin (commercially produced from horses) Accomplished by promptly administering a diphtheria anti-toxin which is an anti-serum produced in horses Must be administered early because it only neutralizes circulating toxin Has no effect on toxin that is already fixed or that is already within the cells ❑DOC: Penicillin/Erythromycin Corynebacterium diphtheriae is susceptible to a variety of antimicrobials including Penicillin and Erythromycin Erythromycin: most effective antimicrobial  9 Loeffler’s methylene blue staining: Babes-Ernst granules/Volutin granules These granules may be demonstrated clearly Presence of metachromatic granules with the use of Special stains such as Albert’s, when stained with Loeffler’s methylene Neisser’s and Ponder’s stain blue These granules take up a bluish-purple color from the stain and they are often situated at the poles of the bacilli and are called polar bodies Left: Babes-Ernst granules These granules are composed of Polymetaphosphates and they are more Right: Gram Stain strongly gram-positive than the rest of the bacterial cells  10 Biochemical Tests: Catalase positive (+) Nitrate reduction (+) Urease test (–) Beta hemolysis (V) - variable for Beta hemolysis Hydrolysis o Gelatin (–) o Esculin (–) CHO fermentation o Glucose (+) o Maltose (+) o Sucrose (–)  11 Cultural Optimal growth at 37ºC (multiplication at 15-40ºC) The optimum temperature for growth is 37ºC; it ranges from 15-40ºC and this organism is an aerobe and a facultative anaerobe. Corynebacterium species usually grow on 5% sheep’s blood and chocolate agar. Culture media with enrichments such as blood, serum, or egg, is necessary for good growth. However, if diphtheria is already suspected, selective and differential media should be used. The usual media employed for the cultivation of the diphtheria bacillus are Loeffler serum agar, CTBA, and Modified Tindsdale Agar. Loeffler’s serum agar or Pai agars – enriched medium Diphtheria bacilli grow on Loeffler serum very rapidly. They can usually be seen after 6-8 hours of incubation. The colonies may appear small at first, but after continued incubation, they may appear large and they may acquire a distinct yellow tint. Loeffler’s medium contains serum and egg, which stimulates the growth of Corynebacterium diphtheriae and the production of metachromatic granules in the cells as well.  13 Tindsdale Agar (cystine-tellurite blood agar) CTBA (cystine-tellurite blood agar) Modified Tindsdale Agar (sheep’s blood, bovine serum, cystine potassium tellurite) The two (2) selective differential media that are commonly used for the purpose of identifying Corynebacterium diphtheriae are cystine-tellurite blood agar (CTBA) and Modified Tindsdale Agar. When grown on CTBA, Corynebacteria form black to brownish colonies from the reduction of tellurite to tellurium.  Differentiating characteristics: ❖ Corynebacterium diphtheriae also produces a halo on both media. On “brown halo/Tindsdale halo” – CTBA, the halo produced appears produced by the Corynebacteria brown as a result of the organism breaking down the cystine. due to (cystinase activity – CTBA; ❖ On Modified Tindsdale Agar, the hydrogen sulfide – Modified halo, also brown-colored, is Tindsdale Agar) produced when the organism uses tellurite to produce hydrogen sulfide.  16 (+) brown halo on CTBA – C. diphtheriae, C. ulcerans, C. pseudotuberculosis Aside from the diphtheria bacilli, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis also produce a brown halo on CTBA.  17 Treatment and Prevention Administration of antitoxin (horse) Immunization (DPT- 6, 10, 14 weeks/16-24 months, 5-6 years) This is done with diphtheria toxoid as it induces anti-toxin production in the body. So, here we make use of the DPT or the diphtheria-pertussis-tetanus toxoid vaccine- which is given at 6,10 and 14 weeks of birth followed by two booster doses at 16 to 24 months and 5 years. Drug of Choice: Penicillin or erythromycin Surgical with tracheostomy tube if needed Listeria  19 Pathogenesis of Listeria monocytogenes Listeria monocytogenes has the ability to survive within phagocytes by production of different virulence factors: Clinical Infections  21 Laboratory Diagnosis Specimen: Blood, CSF, Swabs from lesions Listeriosis is usually diagnosed when a bacterial culture grows (L. monocytogenes from a body tissue or fluid such as Blood, CSF, and Swabs from lesions) 22  23 Cultural  Optimal growth temp: 30-35 degree C  CAMP Test (Christie-Atkins-Munch-Peterson Test) – in order to differentiate L. monocytogenes from other  SBA, Chocolate agar, NAP, and Brain Heart beta-hemolytic listeria species. Infusion Agar (BHIB)  New Oxoid Chromogenic Listeria Agar – allows for the  Beta-hemolytic, small, color white, smooth, differentiation of the pathogenic Listeria species from translucent colonies (after 1-2 days incubation) the nonpathogenic ones. In this medium, identification  Cold enrichment (4 C for several weeks) is carried out based on the activity of the enzymes  CAMP test – positive (“block” pattern type of which are responsible for the pathogenicity of Listeria hemolysis) species. Rhodococcus equi Here, Listeria spp. grows in the form of turquoise colonies, developed as a result of degradation of  New Oxoid Chromogenic Listeria Agar glucosidase (+) Turquoise colonies with halo Both of the pathogenic Listeria species can grow in this medium  24  Pathogenic Listeria species: Listeria monocytogenes Listeria Ivanovii  In order to differentiate the two pathogenic species, we can also take a look at the colonies. They can be further differentiated based on their capability for the synthesis of the enzyme PHOSPHOLIPASES This enzyme, phospholipase, hydrolyzes Lecithin from the culture medium and develops an opaque zone or halo around colonies ❑(+) Turquoise colonies with halos = Listeria monocytogenes  25 Different Diagnosis Differentiation of L. monocytogenes and Gram (+) bacteria Treatment and Prevention  28 Erysipelothrix rhusiopathiae General Characteristics 3 species:  E. rhusiopathiae  E. tonsillarum  E. inopinata o Out of the three species in this genus, the only considered human pathogen is Erysipelothrix rhusiopathiae  Usual route: skin cuts or scratches Infections are typically a result of a puncture wound or skin abrasion  Erysipelothrix means “red skin, thread”  Rhusiopathiae – red disease This organism causes erysipelas or erysipeloid, from the word “erysipelothrix” meaning ‘red skin thread’ and “rhusiopathiae” meaning ‘red disease’ This organism is the cause of erysipeloid which is a veterinary infection, considered as occupational hazard for those handling animals Clinical Infections Human infections caused by Erysipelothrix ❑ Cutaneous infection (generalized, rhusiopathiae may result in three distinct entities diffuse) – rare o Septicemia – Erysipeloid (most common), septicemia and associated with endocarditis, 38% generalized cutaneous infection) mortality rate 3 types of diseases: ❑ Infection can also disseminate in Erysipeloid – localized skin infection usually immunocompromised patients in the hands or fingers; resembles resulting in bacteremia or septicemia streptococcal erysipelas; self-limiting (usually between 3-4 weeks); low incidence, most common infection ❑ This lesion is acquired as a result of skin abrasion, injury or bites from infected animals particularly domestic swine Clinical Infections 31 Specimen: Tissue biopsy and aspirates from skin lesions o Skin lesions for erysipelothrix should be collected by biopsy or by aspirates Microscopic: Gram positive, thin, pleomorphic rods (chains, singly or in a V shape) Organism stains as both thin, short rods, pleomorphic rods like the coryneform bacilli Tendency to form long filaments, which are easily decolorized, may appear gram variable Biochemical Tests:  33 Cultural Aerobic or facultative anaerobic Clinical specimens can be plated on Sheep Blood Agar or Chocolate Blood Agar, but patients with sepsis, their blood specimen can be inoculated into Nutrient Broth with 1% glucose incubated in 5% CO2 at 35oC Nutrient broth with 1% glucose incubated in 5% CO2 at 35oC SBA and Chocolate agar – produce nonhemolytic or alpha hemolytic, pinpoint colonies from systemic infections  34 Treatment and Prevention Therapeutic options for this organism, it is susceptible to: Penicillin Cephalosporins Erythromycin Clindamycin  35 Lactobacillus General Characteristics ❑L. acidophilus surgery ❑L. fermentum, L. vaginalis ❑Have roles as probiotic bacteria, promoting beneficial health effects ❑L. salivarius, L. plantarum ❑Genus Lactococcus ❑Gram-positive pleomorphic rod (coccoid or spiral-shaped) ❑Normally isolated from dairy products ❑Aerotolerant anaerobe; non-motile ❑Have also been isolated from endocarditis ❑Important for maintaining proper pH balance cases in vaginal secretions ❑Reported cases of infections (e.g., endocarditis, bacteremia) are associated with immunocompromised individuals (e.g., HIV disease) and those who had gastrointestinal Lactobacillus acidophilus General Characteristics Biochemical Catalase (-) Oxidase, indole, nitrate (-) Hydrogen sulfide (H2S) (-) Ferments glucose, maltose, sucrose (+) Since Lactobacillus helps maintain normal acidic pH of vaginal secretions, this acidic environment inhibits the growth of ❖ Mobiluncus ❖ Prevotella ❖ Gardnerella vaginalis ❖ Candida albicans  38 Gardnerella vaginalis General Characteristics First described in 1953 Normal biota in human urogenital tract; natural inhabitant of the human vagina Anaerobic, Gram (+) rod May also colonize distal urethra of males Cell Wall Gram positive cell wall Thinner peptidoglycan Significantly thinner Contains less peptidoglycan than the typical Gram (+) bacteria Clinical Infection ❖ Bacterial vaginosis (BV) These anaerobes increase the vaginal pH greater than 4.5 BV is recognized as one of the most common causes of vaginal discharge Also associated with maternal and fetal morbidity during pregnancy Characterized as foul smelling, excessive purulent vaginal discharge (malodorous discharge)  40 Clinical Infection Specimen: vaginal discharge, can be isolated from urine Urine specimen can be used since G. vaginalis is an occasional cause of Urinary Tract Infection Microscopic: BV can be differentiated from other vaginal infection by gram stain Appear short, pleomorphic gram positive rod or coccobacillus that often stains gram-variable Characteristic Finding: Presence of “clue cells”  41 Amine test or Whiff test = (+) result is fishy amine-like Cultural: odor GROW on 5% sheep blood and chocolate agars but they do NOT GROW on MacConkey agar ❑ Sample of discharge is placed on a glass slide Columbia CNA(colistin-nalidixic acid) agar - contains biotin, ❑ 10% potassium hydroxide (KOH) solution is added folic acid, niacin, thiamine Addition of 10% KOH will cause the release of Antibiotics: colistin and nalidixic acid prevent the overgrowth of gram-negative organisms volatile amines HBT (Human Blood Bilayer Tween) - beta-hemolytic Will enhance and it will confirm if there is the Selective medium that can be used to isolate Gardnerella presence of the typical fish like odor vaginalis from female genital tract specimens Biochemical: Is a CNA medium with the addition of Amphotericin B Catalase negative, Oxidase negative Amphotericin B: prevent the growth of yeasts and filamentous fungi Hippurate hydrolysis - positive All of these culture media should be incubated at 35 degrees celsius in 5 – 10% carbon dioxide within 48 hours of inoculation Drug of Choice: Metronidazole Ampicillin - it is also susceptible to this antibiotic  42 Differential Diagnosis Laboratory Diagnosis: Vaginal Infections  43 NON-SPORE FORMING, BRANCHING AEROBIC ACTINOMYCETES Nocardia Other Actinomycetes NOCARDIA GENERAL CHARACTERISTICS Commonly found in soil and several have been implicated in human infections usually occurring in immunocompromised patients Gram-positive often with a beaded appearance, they are partially acid fast or modified acid fast positive, catalase positive & strictly aerobic organisms On gram stain, they produce branching filaments and the cell walls of these organisms contain Mycolic acids that are shorter chained than those of Mycobacteria spp They are considered to be partially acid fast since they stain with the routine acid fast reagent Carbolfuchsin  44 Clinical Infections In most cases, Nocardiosis is an opportunistic infection Pulmonary:  Pulmonary Nocardiosis is the main clinical presentation caused by N. asteroides, since inhalation is the primary route of bacterial exposure On immunocompromised patients, its common manifestation is a confluent bronchopneumonia that is usually chronic but may also be acute or relapsing Laboratory Diagnosis 45 Treatment and Prevention Drainage and surgery are possible for treating Nocardia infection along with antibiotics such as sulfonamides, trimethoprim- sulfamethoxazole except with penicillin Susceptible to sulfonamides, trimethoprim-sulfamethoxazole; resistant to penicillin SPORE FORMING, GRAM POSITIVE BACILLI Bacillus Obligate aerobes that have non-bulging spores Clostridia Obligate anaerobes with bulging spores Bacillus General Characteristics There are more than 100 species within the genus Bacillus It remains the largest genus within this group of gram-positive bacilli It contains the most important medically relevant organisms Widely distributed in the soil and environment Large gram-positive or gram-variable rods in singles or pairs The spores do not stain and may appear as intracellular or extracellular clear oval structures like “empty spaces” Bacillus spp. are aerobic or facultative anaerobic bacilli Bacillus subtilis, stained Has peritrichous flagella causing these organisms to be motile Except: Bacillus anthracis and Bacillus mycoides (both are non-motile) The growth temperature range of Bacillus spp. is -5 to 75 deg C Some spp. are thermophiles having a growth temperature range at 55 deg C or higher  49 Bacillus anthracis Clinical Infections The primary virulence factors of Bacillus anthracis Furthermore, the capsule of Bacillus anthracis is are TWO exotoxins. very unusual, it is not a polysaccharide but rather is composed of amino acid residues. It is a glutamic ❑ EF (Edema factor/toxin)- causes local edema or acid capsule, which for some reason does not swelling, and interferes with phagocytosis by stimulate a protective response by the immune macrophages. system. This capsule is anti-phagocytic, it protects ❑ LF (Lethal factor/toxin)- specifically targets and the organism from phagocytosis kills macrophages which disables the immune defense of the human host.  50 Anthrax is primarily a disease of herbivores. It is endemic among developing countries in Africa, central and south America. Enzootic: Africa, Central America, South Africa. The largest/longest outbreak of anthrax occurred in Zimbabwe In animals the portal of entry is the mouth and the gastrointestinal tract usually the spores come from contaminated soil Spread: animals feeding on plants contaminated with spores. Human: occupational exposure to animals/products In addition to that, anthrax has also been used as a weapon, a bioterrorism agent or a bioterrorism weapon around the world for nearly a century. In 2001, powdered anthrax spores were deliberately put into letters that were mailed through the U.S. postal system. There were few people who have died during this event, and the attack prompted institutions to implement or modify bioterrorism readiness plans Postal workers-mail= spore-tainted material (powder in envelopes)  51 Cutaneous Anthrax Spore penetration- black eschars The infection results from close contact and inoculation of endospores through a break on the skin, following incubation and inoculation of approximately 2 to 3 days in most cases, a small papule appears that progresses to a ring of vesicles, after that the vesicles develop into an ulceration and the typical presentation is a black necrotic lesion known as an eschar. Referred to as “malignant pustule”- a small papule appears at the site of incubation 2 to3 days after exposure A ring of vesicles coalesces to form an erythematous ring.  52 Pulmonary Anthrax  Also known as respiratory anthrax or inhalation anthrax is due to inhalation of the spores  Wool Sorter’s Disease - is used to describe respiratory infections that result from the exposure to endospores during the handling of animal products  The disease develops from flu-like symptoms to (severe phase-2 to 3 days post- infection) respiratory distress, dyspnea, cyanosis, and having pleural effusion  Patients may also be disoriented and may lead to coma and death after 24 hours of infection.  53 Gastrointestinal anthrax  Ingestion of spores, toxin contaminated meat  Initial Symptoms (non-specific): Progresses to abdominal pain, nausea, vomiting, bloody diarrhea  The mortality rate of this type of anthrax is higher than that of cutaneous anthrax Because it is usually attributed to toxemia and sepsis  54 Laboratory Diagnosis Specimen:  Blood, Lung tissue  Specimens that was collected from a patient suspected of having anthrax should be placed in: leak-proof containers and placed in a secondary container  Cutaneous Anthrax Specimens should be collected from underneath the eschar It is also possible to have a vesicular fluid that should also be collected from underneath the lesion  Pulmonary Anthrax o Blood cultures, pleural fluid and other serum specimens for serology  55  Microscopic:  Young cultures o Large o spore-forming central rod o square-ended o gram positive  Old cultures o Rods found singly or in chain o gram variable rods  Resembles “String of pearls” o Large spherical bacilli in chains  Unstained spores with ends fit snugly together may appear like bamboo rods  Gram-stain preparations of clinical samples: 56  57 Treatment Anthrax can be successfully treated if the disease is promptly recognized and appropriate therapy is initiated early. So first we have the antibiotic regimen for treatment it consists of: Penicillin, Gentamicin, Erythromycin, Tetracycline, Chloramphenicol Ciprofloxacin (2000) – post exposure prophylaxis It is an antibiotic for post-exposure prophylaxis as well as doxycycline Doxycycline - post exposure prophylaxis and is preferred if the strain is penicillin sensitive For prevention, the general control measures include: the disposal of animal carcasses by burning or by deep burial and peats the contamination of animal products usually by autoclaving the use of protective clothing and gloves for handling potentially infectious materials Bacillus cereus: “Fried Rice Bacilli” General Characteristics Other bacillus species other than what we've just tackled the anthrax bacillus most of them are non-pathogenic except for bacillus cereus. Bacillus cereus is a normal habitant of: Soils, common air or dust-born contaminant Virulence: enterotoxin spores Clinical infections: common cause of food poisoning (reheated rice, potato) Widely associated with food items & is an important agent of food poisoning in man. TWO TYPES OF TOXIN: Diarrheal toxin (Diarrheal type of food Emetic toxin (Emetic type of Food poisoning) poisoning) Clinical features: abdominal cramps Ingestion of meat or poultry and vomiting Ingestion of fried rice/Chinese fried Clinical features: Abdominal rice pain/cramps, fever and diarrhea It is a heat-stable pre-formed toxin Incubation period: usually between 8 resembling staphylococcus aureus enterotoxin and unlike diarrheal toxin to 16 hours this toxin acts immediately on It is heat labile and food items intestine contaminated are usually meat or The incubation period of food poisoning is shorter usually one to six poultry, vegetables, dried beans and hours cereals  60 Eye infections (endophthalmitis, panophthalmitis, keratitis with abscess formation, poor visual outcomes) most common type of non-gastrointestinal infections, resistant to penicillin Other conditions such as ocular diseases which are the most common type of non-gastrointestinal infections and causes severe keratitis following trauma to the eye that may lead to loss of vision other conditions which rarely occur such as systemic infections such as endocarditis, meningitis, osteomyelitis and pneumonia Laboratory Diagnosis  62 Other Bacillus Species General Bacillus stearothermophilus - autoclave indicator Bacillus subtilis o Rare cause of GI illness o (biological type of indicator) Common laboratory contaminant Bacillus thuringiensis Interpretation: Presence of yellow color change = it reported to cause food Bacillus licheniformis poisoning bacteremia indicates a failed test and a positive bacterial growth Bacillus pumilus meningitis pneumonia and other infections No color change = indicates a passing test and proper Bacillus sphaericus sterilization of the sample However, they are more commonly seen as contaminants  63 Laboratory Diagnosis Differentiation of B. anthracis and B. cereus  64 Clostridia General Characteristics Clostridium botulinum 65 General Characteristics  66 Clinical Infections  Clostridium tetani 67 General Characteristics  68 Produces 2 exotoxins Tetanolysin Tetanospasmin aka Tetanus toxin ❑neurotoxin responsible for disease manifestations ❑binds to the receptors present on our motor nerve terminals ❑Following toxin internalization, it prevents the release of inhibitory neurotransmitters Which leads to spastic muscle contractions or Spastic paralysis  Clostridium perfringens 69 General Characteristics  A commensal in the large intestine of human beings and animals.  Also found as saprophyte in soil, dust and air.  It is a capsulated, non-motile, gram-positive bacillus and it bears subterminal bulging spores.  Organisms multiply in tissue and produce gas and multiple toxins.  It has four major toxins: Alpha Beta Epsilon Iota And other toxins such as the enterotoxin.  70  Among the four major toxins, alpha toxin is the one that destroys cell membranes.  Enterotoxin is cytotoxic which is also identical to the protein found in the spore coat and is a cause of food poisoning.  Clinical infections: Gas-gangrene ❑Particularly associated with Clostridium perfringens which is described as a rapidly spreading edematous myonecrosis. ❑Alpha toxin which is known as the principal virulence factor is the one that mediates gas-gangrene. Food poisoning ❑Caused by clostridium perfringens type A enterotoxin. 71  72 Clostridium difficile General Characteristics Gram-positive bacilli are a taxonomically diverse group of organisms, most of which are found in the environment and rarely cause human disease. Many of them are used in the industry for the production of enzymes, antibiotics, and some are important food spoilage organisms.  73 Usually pathogenesis of clostridium difficile is toxin mediated. Clostridium difficile may be harbored as a commensal in the intestine. However, only the toxigenic strains can cause pseudomembranous colitis. It produces two powerful exotoxins: ❑Toxin A or enterotoxin ❑Toxin B or cytotoxin. Both toxins A and B are secreted in the intestine. ❑Toxin A results in watery diarrhea. ❑Exotoxin B damages colonic mucosa. Microscopically: they are gram-positive bacilli, pink staining bacilli.  74 Laboratory Diagnosis Cultures for clostridium difficile are plated on a ❖CCFA or Cycloserine Cefoxitin Fructose special selective medium such as: Agar ❖Egg yolk agar which contains: A selective and differential medium. Lecithinase which is responsible for white zone of opacity. Basically, it is just an egg yolk base Lipase the colony is covered with an with cycloserine, cefoxitin and iridescent multicolored sheen fructose Proteolytic E which is responsible for the A positive growth shows a yellow colonies, complete narrow clearing. the pink agar turns yellow in the vicinity of colonies