Corynebacterium spp. (Coryneform Bacteria) By Dr. Rand Riadh Hafidh Department of Microbiology/College of Medicine/ Baghdad University 2018-2019Learning Objectives: In the end of this lecture the student should be able to: * Identify coryneform bacteria species. * Describe diphtheria and its causative agent(s). * Evaluate the differences between toxigenic and nontoxigenic strains. * Identify diphtheria toxin; the virulence factor, and the reason for diphtheria pathology. * List laboratory diagnosis steps. * Outline the steps for toxigenicity detection. * Determine the prevention and treatment means for diphtheria infection. * Evaluate the new medial importance of other coryneform species.Clinical Scenari Severe pharyngitis, particularly with difficulty swallowing, or signs of systemic disease including myocarditis or generalized weakness. The presence of a pharyngeal pseudomembrane or an extensive exudate should prompt consideration of diphtheria.Clinical Scenario: Nonhealing or enlarging skin ulcer characterized by punched-out ulceration with necrotic sloughing or pseudomembrane formation, which most commonly occur on the extremitiesGeneral Characteristics: Corynebacteria spp. -Non-spore-forming gram-positive bacilli. -Unencapsulated and nonmotile bacilli. -Some Corynebacteria are part of the normal flora of humans.General Characteristics: Corynebacteria spp. - Coryneform bacteria are group of bacteria identified by the rod beaded and club-shaped appearance due to the presence of the metachromatic granules. od ee g 2... < =F V7 \ = s 4 S —- 2 I we S_-.:General Characteristics: Corynebacteria spp. Coryneform bacteria in stained smears tend to lie parallel or at acute angles to one another referred as Chinese Characters.General Characteristics: Corynebacteria spp. The principal human pathogen of the Coryneform group is “OV < Corynebacterium diphtheriae Is the causative agent of diphtheria (a nasopharyngeal and skin infection)General Characteristics: Corynebacteria spp. * C. diphtheriae previously is considered a strictly human pathogen. © C. ulcerans infections in humans can also be respiratory or cutaneous.Diphtheria (Infectious Diseases Related to Travel WORLD POLITICAL ar e@ mote oc feo an B ler Projection "arencano @|~ * ‘ 3 ae ATLANTIC. monte PACIFIC oo =o . i OCEAN EAN | x mo le ; wa Ko INDIAN ism ten sua rata ‘NOIAN i a ut, Oa cn roesin “un ed sca “ - yy... “ wi - souTH | SOPH ATLANTIC wtyaotth PACIFIC + OCEAN ~ OCEAN SOUTHERN OCEAN « ‘Scale - 1:170,052,868 free ee) ~— ye 7 agement FE pam sr Ge eae oo ew eweCorynebacterium diphtheriae * Four biotypes of C. diphtheriae have been widely recognized: gravis, mitis, intermedius, and belfanti. These variants have been classified on the basis of growth characteristics such as colony morphology, biochemical reactions, and, severity of disease produced by infection. In general, var gravis tends to produce more severe disease than var mitis, but similar illness can be produced by all types.Corynebacterium diphtheriae (ulcerance) Two phenotypes: TRATES -Nontoxigenic _(tox-) || No-Diphtheria-Town and toxigenic (tox*). 2 -Toxigenic strains produce an_ exotoxin that causes local and systemic effect. DO NOT LOSE THIS TICKETCorynebacterium diphtheriae (ulcerance) * The toxin is associated with the formation of pseudomembranes in the pharynx during respiratory diphtheria or in skin lesions during cutaneous diphtheria. * Toxigenic strains most frequently cause pharyngeal diphtheria. * Nontoxigenic strains commonly cause cutaneous diphtheria. + Recently, studies proved that toxigenic or nontoxigenic strains can cause both nasopharyngeal and skin infections.Corynebacterium diphtheriae (ulcerance) Corynebacteriophage beta carries the structural gene (tox*) encoding diphtheria toxin, and this corynebacteriophage is responsible for toxigenic conversion of (tox) C. diphtheriae to the (tox*) phenotype. : Step 1. Attachment A Phage attaches to O) thecoll surface of ‘bacterium. f = ‘Step 2. Penetration “Y= Phage DNA enters the bacterial cell, ) ‘Step 3. Integration Phage DNA Integrates into bacterial DNA. Step 4. Replication f Integrated prophage ()) tates nen ‘ bactrlONA —" replicates. These cells may exhibit new properties ‘Saunders College PublishingCorynebacterium diphtheriae (ulcerance) Route of Infection: «In nature, C diphtheriae occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers. * There are No significant reservoirs other than humans.Corynebacterium diphtheriae (ulcerance) Route of Infection: * Corynebacterium ulcerans usually involve close animal contact. * Reservoirs include animals from farms and domestic and natural settings. * Corynebacterium ulcerans may carry the diphtheria tox gene. The toxigenic C. ulcerans can cause disease similar to clinical diphtheriaCorynebacterium diphtheriae (ulcerance) Route of Infection: * It is spread by droplets or by contact to susceptible individuals; the bacilli then grow on mucous membranes or in skin abrasions, and those that are toxigenic start producing toxin.Corynebacterium diphtheriae (ulcerance) * Carriers are defined as individuals who have positive cultures for C. diphtheriae (ulcerance) and either are asymptomatic or have symptoms but lack pseudomembranes. In other word, they carry nontoxigenic strains.Corynebacterium diphtheriae (ulcerance) Pathogenesis: - Short incubation period (2-5 days). -C infects mucous membranes, most commonly in the respiratory tract, and also invades open skin lesions resulting from insect bites or trauma. -All toxigenic C. are capable of elaborating the same disease-producing exotoxin.Corynebacterium diphtheriae: Exotoxin inhibition of protein synthesis diphtheria toxin oe - cell membrane SSE Unc cae ior Sener S Soe TE reaeal cell death prevents inactivates protein elongation — x > synthesis factor-2 by ribosomeCorynebacterium diphtheriae (ulcerance): Exotoxin Diphtheria toxin is a heat-labile polypeptide that can be lethal in a dose of 0.1 pg/kg. The molecule can be split into two fragments A and B. Fragment B has no independent activity but is required for the transport of fragment A into the cell. Fragment A_ inhibits polypeptide chain elongation by inactivating the elongation factor EF-2. It is assumed that the abrupt arrest of protein synthesis is responsible for the necrotizing and neurotoxic effects of diphtheria toxin.Corynebacterium diphtheriae (ulcerance) Pathogenesi * Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial inflammatory response. * The necrotic epithelium becomes embedded in exuding fibrin and red and white cells, so that a grayish “pseudomembrane" is formed— commonly over the tonsils, pharynx, or larynx. Any attempt to remove the pseudomembrane exposes and tears the capillaries and thus results in bleeding. * Obstruction of the airway may follow.Corynebacterium diphtheriae (ulcerance) Pathogenesis: * The —pseudomembrane contains large numbers of C. organisms, but the bacteria are rarely isolated from the blood or internal organs. * The regional lymph nodes in the neck enlarge, and there may be marked edema of the entire neck. 9/25/20 Dr, Racid RiadCorynebacterium diphtheriae (ulcerance) Pathogenesis: DIPHTHERIA * The diphtheria bacilli within the membrane continue . to produce toxin actively. This is absorbed and results I$ deadly- in distant toxic damage, particularly parenchymatous NW degeneration, fatty infiltration, and necrosis in heart muscle, liver, kidneys, and adrenals, sometimes accompanied by gross hemorrhage. * The toxin also produces nerve damage, resulting often in paralysis of the soft palate, eye muscles, or extremities. * Wound or skin diphtheria occurs chiefly in the tropics. IMMUN ATION A membrane may form on an infected wound that fails ~~ isthe to heal. However, absorption of toxin is usually slight on \ and the systemic effects negligible. The small amount We safeguard of toxin that is absorbed during skin infection | ra promotes development of antitoxin antibodies. \ we sehen Dr, Racid Riad 4Corynebacterium diphtheriae (ulcerance) Virulence factor: -The "virulence" of diphtheria bacilli is due to their capacity for establishing infection, growing rapidly, and then quickly elaborating toxin that is effectively absorbed. * C. does not need to be toxigenic to establish localized infection—in the nasopharynx or skin, but nontoxigenic strains do not yield the localized (formation of membrane) or systemic toxic effects. * C. does not actively invade deep tissues and practically never enters the bloodstream.Corynebacterium diphtheriae (ulcerance) Lab. Diagnosis: * Swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered. Swabs should be collected from beneath any visible membrane. The swab should then be placed in semisolid transport media such as Amies. 952038 Dr, Racid RiadCorynebacterium diphtheriae (ulcerance) Lab. Diagnosis: 1. Screening Tests: a) Direct Gram stain from swab b) Culture on: — Loeffler’s slant medium — Blood agar medium — Tellurite agar medium 2. Confirmatory Tests: a) Tinsdale agar medium (highly selective medium) b) Toxigenicity detection Tests: - EleK test (Immunodiffusion or immunoprecipitation assay) - Polymerase chain reaction-based method (PCR) - Enzyme-linked immunosorbent assay (ELISA) ~ Animmunochromographic strip assay (ICA)Corynebacterium diphtheriae (ulcerance) Lab. Diagnosis: . Smears stained with alkaline methylene blue or Gram stain show beaded rods in typical arrangement.Corynebacterium diphtheriae (ulcerance) Lab. Diagnosis: -A presumptive C diphtheriae isolate should be subjected to testing for toxigenicity. Such tests are performed only in reference public health laboratories. There are several methods, as follows: - Sn The isolated organism is Corynebacterium diphtheriae Is it Toxigenic or Not?Corynebacterium diphtheriae Lab. Diagnosis: (4) Elek test: A filter paper disk containing antitoxin is placed on an agar plate. After 48 hours of incubation, the antitoxin diffusing from the paper disk has precipitated the toxin diffusing from toxigenic cultures and has resulted in precipitate bands between the disk and the bacterial growth. This is the modified Elek method described by the World Health Organization (WHO) Diphtheria Reference Unit. @ Aan Antitssin-soaked filer paper wa aS Texigenic Nonioxgenic Conners CdphanerizeCorynebacterium diphtheriae (ulcerance) Lab. Diagnosis: (2) PCR: have been described for detection of the diphtheria toxin gene (tox). PCR assays for tox can also be used directly on patient specimens before culture results are available. (3) ELISA: can be used to detect diphtheria toxin from clinical C. diphtheriae isolates. (4) ICA: allows detection of diphtheria toxin in a matter of hours. This assay is highly sensitive.Prevention: * Itis preventable by vaccination. * Routine booster doses with Td (tetanus-diphtheria) should be given to all adults every 10 years. * Booster is particularly important for travelers who will live or work with local populations in countries where diphtheria is endemic. « Antimicrobial prophylaxis (erythromycin or penicillin) is recommended for close contacts of patients.Treatment: Equine diphtheria antitoxin (DAT) is the mainstay of treatment and is administered after specimen testing, without waiting for laboratory confirmation. Specific antitoxin is life-saving if administered in first 24 hours of symptom onset. An antibiotic (erythromycin or penicillin) should be used to eliminate the causative organisms, stop exotoxin production, and prevent transmission. Administration of diphtheria toxoid vaccine is indicated at recovery since sustained immunity does not develop in all patients after infection In non-vaccinated individuals, and especially if proper treatment is delayed, death can occur in up to 10% of clinical cases despite antibiotics and the use of anti-sera.Other Coryneform Bacteria * Corynebacterium jeikeium can cause’ disease in immunocompromised patients and is important because it produces infections, including bacteremia, that have a high mortality rate and because it is resistant to many commonly used antimicrobial drugs. * Corynebacterium urealyticum is a multiple resistant to antibiotics. It is urease-positive. It has been associated with acute or chronic encrusted urinary tract infections manifested by alkaline urine pH and crystal formation. * C. striatum is commonly found on human skin and mucous membranes and shows emerging evidence as a pathogen in immunocompromised patients or patients with prosthetic devices. Concerns exist as person-to-person transmission has been reported. Pneumonia, endocarditis and septicemia are s-the. most common complications,Conclusion: * The term Coryneform bacteria referred to a group of bacteria with specific shape; i.e. club-shaped. * C.diphtheriae (newly, C.ulcerance) are the most important human pathogen. * Diphtheria is a nasopharyngeal and skin infection. * Both toxigenic (tox*) strains and nontoxigenic (tox) strains can cause nasopharyngeal or skin infections. * Tox+ strains responsible for the formation of pseudomembrane and distant toxic damage while tox strains associated with local lesion only. * Lab. Diagnosis is depended mainly on the isolation of the bacteria and the detection of tox* gene in the isolated one. * The disease is preventable by vaccination. Treatment is mainly by DAT + antibiotics.References for further readings: Brooks, G. F. (Ed.) 2007. Jawetz, Melnick, & Adelberg's Medical Microbiology, New York, McGraw-Hill Companies, Inc. Kasper, D. L. & Harrison, T. R. 2005. Harrison's principles of internal medicine, New York, McGraw-Hill, Medical Pub. Division. Gillespie, S. H. & Hawkey, P. M. 2006. Principles and practice of clinical bacteriology, Chichester, West Sussex, England; Hoboken, John Wiley & Sons. Centers for disease control and prevention: http://www.cde.gov/diphtheria/index.html European Center for disease prevention and control :http://ecdc.europa.eu/en/healthtopics/diphtheria/pages/index.aspx#st hash.Xh51Hp2v.dpuf