Clinical Bacteriology - 04 Gram Positive Acid Fast Bacilli

CLINICAL BACTERIOLOGY MYCOBACTERIA ACID FAST AND PARTIALLY ACID FAST, GRAM POSITIVE BACILLI OUTLINE • Introduction • Nocardia, Streptomyces, Rhodococcus and Similar • Mycobacteria Organisms o General Characteristics o General Characteristics o Mycobacterium Tuberculosis o Partially Acid-Fast Aerobic Complex Actinomycetes o Non-tuberculosis o Non–Acid-Fast Aerobic Mycobacteria Actinomycetes o Specimen Collection o Direct Detection Methods o Direct Detection Methods o Cultivating Methods o Cultivation o Biochemical Testing o Conventional Test INTRODUCTION • • • • • • • • • • Classified as gram positive-acid fast bacilli is the genus Mycobacterium . This genera is composed of approximately 100 recognized & proposed species. The most familiar are M. tuberculosis and M. leprae which are agents of tuberculosis and Hansen's disease (leprosy) respectively.. Both diseases have long been associated with chronic illness and social stigma. Tuberculosis remains a major cause of morbidity and mortality in the world today. additionally, the growing number of immunocompromised patients worldwide has led to resurgence of tuberculosis as well as diseases caused by non-tuberculous mycobacteria (NTM). Classified as an aerobic actinomycetes are members of the Genus Nocardia. Nocardia species are unique in their ability to retain carbol fuchsin after decolorization with mild acid. They are thus termed " partially acid fast". The species most frequently found in human infections is Nocardia asteroides. Other species include N. brasiliensis and N. caviae. Infections are usually chronic in nature and most frequently onvolve the respiratory tract and central nervous system. The organism typically infects immunocompromised hosts, and infection is characterized by necrosis and abscesses, Nocardia can also cause skin infections. Because the organism can resist pahgocytosis, it can remain in the host indefinitely. No Motile, Non-Encapsulated, Non-spore forming Strictly aerobic; catalase (+); produce Musch granules (inclusion bodies) Cell wall contain N-glycomuramic acid and hs very high lipid content Resist decolorization with acid-ethanol (acid-fastness); resistant to heat, cold, drying Most species associated with diseases grow slowly (2-6 weeks incubation) but the rapidly growing mycobacteria grow in 2-3 days; some species require 5-10% Culture: smooth smooth and soft or a rough and friable appearance - egg based media pH requirement - 6.5-6.8 (culture media generation time: >12 hours - grow slowly due to hydrophobic cell surface GENERAL CHARACTERISTICS • • • • • • • • • Belongs to the family of Mycobacteriaceae. Very thin, Rod shaped and non – motile. Has an unusual cell wall structure and are difficult to stain with basic aniline dyes. Referred to as Acid Fast Bacilli Grows slowly compared to other human pathogenic bacteria. Colonies become visible in 2-60 days at optimum temp. Many species are found in water and soil. Divided in to two Major groups: (based on fundamental differences in epidemiology and association with diseases) o Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. africanum) o Non – tuberculous Mycobacteria (NTMs) Culture media - egg based agar o Colonies - smooth MYCOBACTERIUM TUBERCULOSIS COMPLEX • • General characteristics: o M. tuberculosis, M. bovis, M. africanum, M. canettil, M. microti are all capable of causing tuberculosis. o Slow growers and colonies are not pigmented. Pathogenesis: o Inhalation of a single viable organism has been shown to lead to infection. o Diseases usually develop years after the initial infection o M. bovis may penetrate the gastrointestinal mucosa or invade the lymphatic tissue of the oropharynx. o A strain of M. bovis, Bacille Calmette – Guèrin is used to immunize susceptible individuals against tuberculosis. o Pott’s Disease ▪ Known as the tuberculosis spondylistis or skeletal TB of the spine ▪ Grave form of tuberculosis caused by the invasion of M. tuberculosis into the spinal vertebrae o Millary tuberculosis ▪ Systemic; extrapulmonary TB which refers too the seeding of many organs outside the pulmonary tree with AFB through hematogenous spread ▪ Occurs shortly after primary pulmonary disease but can take place anywhere in the course of acute or chronic TB ▪ Common sites of spread: spleen, liver, lungs, bone marrow, kidney, adrenal gland, and eyes NON – TUBERCULOUS MYCOBACTERIA • • • • MOTT - Mycobacterium other than tuberculosis This includes all other types if mycobacterial species that do not belong to M. tuberculosis complex. Little is known about how infection is acquired. Slow growing NTM o Photochromogens = colonies are pigmented when exposed to light. o Scotochromogens = colonies are pigmented when grown in the light or dark. o Non – Photochromogens = Colonies produce no pigment whether they are grown in the light or dark. o M. avium, M. kasasli, M. marinum, M. ulcerans, M. gordonae, M. xenopi, M. terrae • Rapidly Growing NTM o Colonies appear on solid media within 7 days or less. o May appear as weakly – gram positive rods resembling diptheriods. MYCOBACTERIUM AVIUM COMPLEX • • • • • General characteristics: o M. avium complex comprises of M. avium, M. intracellulare, M. paratuberculosis, M. lepraemurium, and the “wood pidgeon” bacillus. o The term M. avium complex is most commonly used to report the isolation of M. avium – intacellelare. Epidemiology: o Potentially pathogenic (Pulmonary) in immunocompromised patients. o Isolated from natural soil, water, soil, dairy products, pigs, chicken, cats and dogs. o Water reservoir is the major reservoir for human infection. o Infection is acquired by inhalation or ingestion. o Can tolerate extreme temperatures. M. avium can replicate in protozoa with amoeba grown M. avium being more invasive toward human epithelial and macrophage cells. Cultures of M. avium complex exhibit opaque and translucent or transparent colony morphology. M. paratuberculosis (M. avium subs. paratuberculosis) is known to cause an inflammatory diseases referred to as Johne’s disease in cattle, sheep, and goats. NON – CULTIVABLE NTM – M. leprae • • • • Causes Leprosy (A.K.A. Hansen’s Disease) General characteristics: o It has not been cultivated in vitro. o It can only be cultivated in the armadillo and foot pads of mice. Pathogenesis: o Infection is both bacterial and immunological. o M. leprae infection passes through many stages. (Characterised by clinical and histopathologic features) o Primary stages include a silent phase during which leprosy bacilli multiply in the skin within the macrophages. o An intermediate phase in which the bacilli multiply in peripheral nerves and begin to cause sensory impairment. More severe states may follow. Spectrum of disease: o Spectrum of diseases may range from subclinical infection to intermediate stages of disease to a full – blown disease. o Two major forms: ▪ Tuberculoid leprosy = do not have immune defect. Localised to the skin and nerves. ▪ Lepromatous leprosy = defect in cell immunity. Extensive skin lesions. • • • • DIRECT DETECTION METHODS ACID – FAST STAINS • • • • • • • • • Pulmonary (Sputum, gastric lavage. Tracheal aspiration, bronchoscopy, laryngeal swab) Gastric lavage (Bronchial lavages, washings, and brushings) Urine specimen (Early morning specimens) Mycolic acid (present in cell wall) contributes to the bacteria’s acid – fastness. When gram – stained, they appear as poorly stained, beaded gram positive bacilli. Acid – fastness is also affected by colony age, medium on which growth occurs, and ultraviolet light. Rapid growing species are acid – fast variable. 3 types of staining procedure is used: Fluorochrome, Ziehl – Neelsen and Kinyoun. Fluorochrome = more sensitive than carbolfuchsin stains. Fuchsin Acid – Fast stains = Ziehl – Neelsen (A.K.A. Hot Stain) and Kinyoun (A.K.A. Cold Stain). Positive results appear Purple to red. ANTIGEN – PROTEIN DETERMINATION • • Detection of microbial products or components. (Ex: M. tuberculosis = tuberculostearic acid) Detected by gas chromatography / mass spectrometry NUCLEIC ACID AMPLIFICATION • • Molecular techniques such as conventional and real – time PCR. 2 test kits approved by FDA: o The Amplicor Mycobacterium tuberculosis test = Uses PCR to detect M. tuberculosis directly in the respiratory system. o Amplified Mycobacterium tuberculosis direct test = based on ribosomal RNA amplification. CULTIVATION • • • • SPECIMEN COLLECTION • Fecal specimen Tissue and Body Fluid Specimen (CSF, Peritoneal and Pericardial fluids) Blood specimen Wounds, Skin lesions and Aspirates (Dry swabs are unacceptable and Aspirates are the best specimen) • Use at least 1 solid media in addition to a liquid medium to optimize recovery of Mycobacteria. Solid Media (Agar Based) o Middlebrook 7H10 and Middlebrook 7H10 Selective o Middlebrook 7H11 and Middlebrook 7H11 Selective o Middlebrook biplate (7H10/7H11S agar) Solid Media (Egg Based) o Löwenstein – Jensen (L – J) o L – J Gruft o L – J with pyruvic acid o L – G with Iron Liquid Media: o BATEC 12B Medium o Middlebrook 7H9 Broth o Septi – Check AFB o Mycobacteria growth indicator tube o Commercially prepared broths Solid Media: o Recommended because of characteristic, reproducible colonial morphology, good growth from a small inocula and low contamination rate. • Liquid Media: o Reduces turn around time for isolation of acid – fast bacilli to approx. 10 days. CONVENTIONAL TEST • • • • • • • • • • Growth characteristics o The preliminary identification of mycobacterial isolates depends on their rate of growth, colonial morphology, colonial texture, pigmentation, and in some instances, permissive incubation temperatures of mycobacteria. Growth rate o Rapid growers usually produces colonies within 3 to 4 days after subculture, however, it may take longer than 7 days to initial produce colonies due to harsh decontaminating procedure. Pigment production o To achieve optimum photochromogenecity, colonies should be young, actively metabolizing, isolated and well – aerated. o M. Kansasii turns yellow after a few hours of light exposure Biochemical testing o It is used to definitively identify the species. Nitrate Reduction o Valuable test for the identification of M. tuberculosis, M. kansasii, M. szulgai and M. fortuitum. Catalase test o Most species of Mycobacteria, except for certain strains of M. tuberculosis and M.gastri produces the enzyme catalase. Tween 80 hydrolysis o Slow growing scotochromogen and non photoschromogens prodeces a lipase to hydrolyse Tween 80. Tellurite Reduction Arylsulfatase Growth inhibition by Thiophene – 2 – Carboxylic Acid Hydrazide (TCH) o This test is used to distinguish M. bovis from M. tuberculosis because M. bovis does not grow in the presence of 10mg per ml TCH. PARTIALLY ACID-FAST AEROBIC ACTINOMYCETES Nocardia • • • • • • NOCARDIA, STREPTOMYCES, RHODOCOCCUS AND SIMILAR ORGANISMS • GENERAL CHARACTERISTICS • • The genera Nocardia, Rhodococcus, Gordonia, and Tsukamurella are partially acid-fast aerobic actinomycetes. Nocardia and Rhodococcus belong to the family Nocardiacea while Gordonia and Tsukamurella are in the Gordoniaceae and Tsukamurellaceae families CLINICALLY RELEVANT AEROBIC ACTINOMYCETES Organisms belonging to the genus Nocardia are grampositive Often with a beaded appearance, variably acid-fast, Catalase-positive, and strictly aerobic. Nocardiae also are characterized by the presence of mesodiaminopimelic acid (DAP) and the sugars arabinose and galactose in their cell wall peptidoglycan. Epidemiology and Pathogenesis o Nocardia infections can be acquired by either traumatic inoculation or inhalation. o Nocardia, particularly N. asteroides, are facultative intracellular pathogens that can grow in various human cells. Nocardia spp. In immunocompetent individuals o Nocardia spp. cause three types of skin infections: o Mycetoma, a chronic, localized, painless, subcutaneous infection o Lymphocutaneous infections o Skin abscesses or cellulites o Of note, N. brasiliensis is the predominant cause of these skin infections. o Mycetoma caused by N. brasiliensis Nocardia spp. In immunocompromised individuals o Nocardia spp. can cause invasive pulmonary infections and disseminated infections. o Pulmonary infection can lead to complications such as pleural effusions, empyema, mediastinitis, and soft tissue infection. o An acute inflammatory response follows infection, resulting in necrosis and abscess formation Rhodococcus, Gordonia, Tsukamurella • • gram-positive, aerobic, catalase-positive, partially acidfast branching, filamentous bacteria that can fragment into rods and cocci. • BIOCHEMICAL TESTING Epidemiology and Pathogenesis o Rhodococcus equi has been the organism most commonly associated with human disease, particularly in immunocompromised patients, such as those infected with human immunodeficiency virus (HIV). o Gordonia spp. and Tsukamurella have little clinical pathogenic mechanisms but they are able to cause opportunistic infections in humans NON–ACID-FAST AEROBIC ACTINOMYCETES: STREPTOMYCES, ACTINOMADURA, DERMATOPHILUS, NOCARDIOPSIS, AND THE THERMOPHILIC ACTINOMYCETES • • • gram-positive, branching filaments that do not have mycolic acids present in their cell envelopes and are therefore non–acid-fast. Epidemiology and Pathogenesis o Mycetoma is an infection of subcutaneous tissues resulting in tissue o swelling and draining sinus tracts. o Acquired by traumatic inoculation of organisms (usually in the lower limbs) and are usually caused by fungi. o If a mycetoma is caused by an actinomycete, the infection is then called actinomycetoma. DIRECT DETECTION METHODS • Gram Stain o if gram-positive, branching or partially branching organisms are observed, a modified acid-fast stain should be performed (i.e., o 1% sulfuric acid rather than 3% hydrochloric acid as the decolorizing agent) o For histopathologic specimen, Gomori’s methenamine silver (GMS) stain can be used for detection of actinomycetes. CULTIVATING METHODS • • • sheep blood chocolate, Sabouraud dextrose, and brainheart infusion agar Although Nocardia spp. Has a slower growth (minimum 48-72hrs), they also grow well on Sabouraud dextrose agar as well as on fungal media containing cycloheximide such as Mycosel. Typical Gram-Stain Morphology and Colonial Appearance\ •