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Rickettsiae ,Chlamydiae & Mycoplasmas B Y A LT A Y I B Z A K A R I A MSC IN MDICAL MICROBOBOGY  Rickettsiae GENERAL PROPERTIES Rickettsiae comprise of group of small non- motile gram negative coccobacilli that possess the following common characteristi...

Rickettsiae ,Chlamydiae & Mycoplasmas B Y A LT A Y I B Z A K A R I A MSC IN MDICAL MICROBOBOGY  Rickettsiae GENERAL PROPERTIES Rickettsiae comprise of group of small non- motile gram negative coccobacilli that possess the following common characteristics: I. They are obligate intracellular organisms. II. They are non cultivable in artificial media, although they can grow in cell lines, or by animal and egg inoculation. III. They are transmitted by a11 arthropod vector, such as tick. mite, flea or louse. GENUS RICKETTSIA Species of Rickettsia can be categorized into two groups based on the clinical manifestations : 1. Typhus group 2. Spotted fever group Antigenic Structure : The cell wall of rickettsiae is similar to any gram-negative bacteria, composed of peptidoglycan, lipopolysacchaaride, and an outer membrane containing few outer membrane proteins. I. Species specific outer membrane proteins (OMP) II. Group specific alkali stable lipopolysaccharide (LPS) antigen Pathogenesis 1. Transmission: All rickettsiae are transmilled to humans by arthropod vectors. Tick and mite Louse and flea borne 2. Spread: Rickensiae spread through the lymphatics from the portal of entry, multiply in the regional lymph nodes and then spread via bloodstream. 3. Target sites: For all rickensiae, the final target site is the endothelial cells. 4. Phagocytosis: Adhesion to the endothelial cells is mediated by outer membrane proteins; OmpA and OmpB present on rickettsial surface. Following adhesion, the organisms are phagocytosed. 5. Intracellular locations: Following phagocytosis, rickettsiae remain inside a vacuole. 6. Multiplication: Inside the host cells, they multiply slowly by binary fission. 7. Cell-to-cell spread: Spotted fever rickettsiae can spread from cell-to-cell by actin polymerization. ln contrast other rickettsiae accumulate in the cell until the lysis of the cell takes place. 8. Reason for obligate intracellular survival: it is not understood. 9. Endothelial cell Injury: this occurs via lipid peroxidation of host-cell membranes. 10. Release: once these bacteria are released from the host cells, they are unstable and die quickly.  LABORATORY DIAGNOSIS  Serology (antibody detection)  Non-specific test (Weil Felix test)-Rickettsial antibodies detected  Specific antibody detection - by Indirect IF, CFT ELISA and latex agglutination test  Histopathological examination  Isolation-by inoculating into cell lines (Vero, Wl-38, Hela), egg (yolk sac), or animal (guinea pig) Neil Mooser reaction-intraperitoneal inoculation into Guinea pig.  PCR-detecting 16S rRNA or Omp genes TREATMENT Doxycycline is the drug of choice for treatment of most rickettsial illnesses. It is given as 100 mg twice a day orally for 1- 5 days. Chloramphenicol is used as alternative.  Chlamydiae GENERAL DESCRIPTION Chlamydiae are obligate intracellular bacteria that cause a spectrum of diseases in man such as trachoma, lymphgrmnuloma venereum (LGV), conjutivitus, pneumonia and psittacosis and can also cause widespread diseases in birds and mammals. Classilfication Based on genetic characteristics, family Chlamydiaceae has undergone recent taxonomic changes , Species of medical importance are : 1. C.trachomatis. 2.C. psittaci 3. C. pneumoniae.  Chlamydiae are Bacteria, Not Viruses  Chlamydiae were once thought to be viruses because of possessing a few viral properties, such as: 1. They are obligately intracellular. 2. They cannot be grown in artificial media (however they can grow in cell lines, embryonated egg or animals). 3. Filterable- small enough to pass through bacterial filters 4. Produce intracytoplasmic inclusions.  However, chlamydiae are now confirmed to be bacteria, because they have many other properties similar to that of bacteria, as follows: 1. Possess both DNA and RNA. 2. Their cell wall is similar to that of gram-negative bacteria. 3. Multiply by binary fission. 4. Contain prokaryotic 70S ribosomes. 5. Capable of synthesizing their own nucleic acid, lipids and proteins. 6. Susceptible to a wide range of antibacterial antibiotics.  Life Cycle  Chlamydiae exist in two distinct morphological forms elementary hody (EB) and reticulate body (RB)  Chlamydiae have specific tropism for squamous epithelial cells and macrophages of the respiratory tract.  Life cycle steps. 1. Attachment: Elementary bodies attach to the specific receptors on the surface of host cells following which they are endocytosed 2. Intracellular survival: Elementary body resides inside the vacuole (phagosome), where the entire growth cycle is completed 3. Transform to reticulate bodies: By 12 hours of infection the elementary bodies enlarge containing diffuse nucleoid. 4. Replication: Reticulate bodies start to divide by binary fission with in 18 hours. 5. Transform back to elementary bodies: By 18- 24 hours of infection, the reticulate bodies gradually reorganize to form elementary bodies 6. Inclusion body: the vacuoles gradually increase in size to form inclusion body which can be readily detected by histologic stains. 7. Release: Mature inclusion body contains 100-500 elementary bodies which are ultimately released from the host cell by 48 hours. 8. Persistent infection: Sometimes, the development is arrested at the reticulate body stage, leading to persi51en1 ( or latent) infection. Antigenic Structure Chlamydiae possess the following antigen s: 1. Genus/group specific antigen 2. Species specific protein antigens 3. Serovar-specific antigens: they are the major outer membrane proteins. 4. Other antigens: Such as outer membrane complex proteins, type Ill secretory system and heat shock proteins play important role in pathogenesis. 1. CHLAMYDIA TRACHOMATIS  Chlamydia trachomatis is primarily a human pathogen, causing ocular, urogenital and neonatal infections.  C. trachomatis, 18 serovars have been identified affecting humans.  Serovars A, B, Ba and C are associated primarily with ocular disease called trachoma.  Serovars D-K are associated with oculogenital disease, which may be transmitted to neonates.  Serovars Ll-L3 causes a sexually cransmined disease, lymphogranuloma venereum (LGV). A. C. trachomatls Serovars A, 8, B and C (Trachoma)  Trachoma is a chronic keraroconjunctivitis, caused by C. trachomatis serovars A, B, and C.  Mode of transmission: Trachoma is transmitted through direct contact.  Age: Infection is acquired by 2-3 years of age and active disease is most common among young children.  Acute infection presents as: Follicular conjunctivitis (inflammation of conjunctival lymphoid follicles) and papillary hyperplasia. Follicles rupture to leave shallow pits termed Herbert's pits.  Cornea gets infected (keratitis). B. C. trachomatis Serovars D-K  The infections produced by C. trachomatis serovars D-K are as follows. 1. Genital Infections A. Nongonococcal urethritis (NGU): Chlamydia trachomatis is the most common cause of nongonococcal urethritis (NGU), responsible for 30- 50% of cases of NGU. B. Postgonococcal urethritis (PGU)  C. tradiomatis is the most common cause of PGU. Urethritis develops in men 2-3 weeks after recovery from GU. C. Epididmitis and proctitis: C. trachomatis is the most common cause of epididymitis in males. D. Reactive arthritis 2. lndusion Conjunctivitis C. trachomatis serovars D-K cause die following ocular infections: A. Ophthalmla neonatorum (or inclusion blennorrhea) occurs in the new borne. B. Adult inclusion conjunctvitis: It is an acute follicular conjunctvitis, that may occur in adults following swimming (swimming pool conjunctivitis) 3. Infant Pneumonia C. trachornatis Serovarl 1, 1.2, and L3 C. trachomatis serovar Ll, U, and U are the agents of lymphogranuloma venereum (LGV).  CHLAMYDOPHILA PSITTACI  C. psittaci is a pathogen of parrots and other psittacine birds causing psittacosis.  Reservoirs: Pet birds (parrots, parakeets, macaws, and cockatiels) and poultry (turkeys and ducks) act as natural reservoir of infection  Mode of transmission: C. psitlaci can be rransmined to humans by Inhalation of aerosols from avian nasal discharges.  Direct contact with infected birds.  there is no person 10 person transmission. Clinical manifestations: Incubation period is usually 5-19 days. It can present as: Respiratory manifestation is the most common form. Septicemia gasrroinrestinal symptoms. Typhoid-like syndrome CHLAMYDOPHILA PNEUMONIAE C. pneumoniae is an exclusively human pathogen. Clinical findings : 1. Atypical pneumonia: C. pn.et,moniae is a common cause of atypical (interstitial) pneumonia 2. Atherosclerosis: there is strongevidence of association between C. pneumonia.e and atherosclerosis of coronary and other arteries.. 3. Asthma and COPD: C. pn.eumon.iae may cause exacerbations of bronchial asthma and COPD (chronic obstructive pulmonary disease).  LABORATORY DIAGNOSIS 1. Specimen: Depends on the type of lesions. 2. Microscopy: Detectschlamydial inclusion bodies by : A. Gram staining B. Direct IF 3. Antigen detection (LPSantigens): By enzyme immuno assays. 4. Culture: The gold standard method in the past. A. Egg (yolk sac). B. Cell lines of choice 5. Nuclek acid amplification tests (NAAT) 6. Serology (antibody detection):  Mycoplasma Mycoplasmas are the smallest microbes capable of free living in the environment and self-replicating on artificial culture media. They have the following characteristics : 1. Size: They are very small, 150-350 mu in size 2. They are filterable bv bacterial filters They differ from viruses as: A. They are free living in the environment B. They can grow on artificial cell-free culture media 4. They lack a rigid cell wall 5. They are completely resistant to antibiotics acting on cell wall 6. Pleomorphic: They are highly pleomorphic, exist ill coccoid, bacillary or filamentous or even in helical. 7. They are poorly gram-negative, better stained by Giem sa stain. 8. They reproduce by binary fission and budding 9. They are non-sporing and non-fagellated. 10. Contaminants of cell cultures  Classifications :  Family Mycoplasmataceae comprises of two genera Mycoplasma and Ureaplasma; both infect humans and animals.  Out of 16 species of Mycoplasma and Ureaplasma infecting humans  The human pathogenic species are: 1. Mycoplasma pneumoniae causing pneumonia 2. Others cause genital tract infections, such as: a. Mycoplasma hominis b. Mycoplasma genitalium MYCOPLASMA PNEUMONIAE M. pnettmoniae is the causative agent of primary atypical pneumonia. Antigens : Mycoplasma possesses several cell membrane antigens which probaly play an essential role in the host response to infection. 1. Glycolipid antigen 2. Membrane bound proteins Pathogenesis Pathogenesis of M. pneumoniae involves the following events: 1. Adhesion: Attachment to respiratory mucosa is the most important step in pathogenesis 2. Induces injury to host respiratory tissue: this is mediated by producing: 1. Hydrogen peroxide 2. Cytotoxin. 3. Lipoproteins. Epidemiology Mycoplasma pneumoniae infection occurs worldwide.  Transmission: M. pneumoniae is transmitled by respiratory droplets expectorated during coughing.  Facilitating factors: the transmission is favored by close contacts as in families, military bases, boaschools, and summer camps.  lnfeclions tend to be endemic, with periodic epidemics every 4-7 years.  Incubation period is about 2-4 weeks.  Clinical Manifestations  M. pneumoniae produces various infections; which are as  follows: 1. Upper Respiratory Tract Infections (URTI)  URTI manifests as pharyngitis, tracheobronchitis or rarely otilis media. A. Pneumonia  M. pneumoniae causes "atypical" community acquired interstitial pneumonia similar to pneumonia call~ed by other agents, such as C. pneumoniae, Legion.el/a pneumophila and viraI pneumonia.  It is characterized by wheeze or rales, dry cough and peribronchial pneumonia with thickened bronchiaI markings and streaks of inerstitial infiltration on chest X-ray. 2. Extrapulmonary Manifestations  they are rare, occur either as a result of active Mycoplasma infection (e.g. septic arthritis) or due to post infectious autoimmune phenomena (e.g. Guillain-Barre syndrome). 3. Various manifestations include: a. Neurologic: Meningoencephalitis, encephalitis, Guillain-Barre syndrome and aseptic meningitis. b. Dermatologic: Skin rashes including erythema. C. Cardiac: Myocarditis, pericarditis D. Rheumatologic: Reactive arthritis E. Hematologic: Anemia and hypercoagulopatliy.  LABORATORY DIAGNOSIS 1. Specimen:Throat swabs and nasopharyngeal aspirates 2. Culture: A. Solid medium containing PPLO agar: Produces fried egg appearance colonies B. Liquid medium containing PPLO broth: Produces turbidity and a color change. 3. Antigen detection: By Direct IF.antigen capture ELISA 4. Antibody detection in serum:  Specific CFT, indirect-IF, LAT and ELISA using protein Pl antigens  Non-Specific: Cold agglutination test and Streptococcus MG test 5. Molecular methods: Detects 16SrRNA and Pladhesin gene TREATMENT Macrolides are drug of choice (oral azithromycin, 500 mg on day 1.  Alternative drugs are : Doxycycline Respiratory fluoroquinolones such as levofloxacin, moxifloxacin and gemifloxacin. Thank you