Lec 10 Bacteria with unusual growth requirement.pdf

MODULE 10. BACTERIA WITH UNUSUAL GROWTH REQUIREMENT Human infections caused by chlamydias, rickettsiae, and mycoplasmas are discussed separately because the responsible pathogens differ from most other bacteria in several ways: 1. The organisms are smaller 2. Th...

MODULE 10. BACTERIA WITH UNUSUAL GROWTH REQUIREMENT Human infections caused by chlamydias, rickettsiae, and mycoplasmas are discussed separately because the responsible pathogens differ from most other bacteria in several ways: 1. The organisms are smaller 2. The structure of their cell walls is different 3. Chlamydias and many rickettsiae are obligately intracellular parasites. Cell Wall-Deficient Bacteria: Mycoplasma and Ureaplasma “soft skin” Mycoplasmataceae smallest known free-living forms (approximately 0.3-1.0 μm in diameter). DO not have a cell wall highly fastidious slow growing, facultative anaerobes require nucleic acid precursor molecules, fatty acids, and sterols such as cholesterol for growth believed to be highly reduced genetically - fewer opportunities to adapt to environmental changes (Have lower guanine/cytosine (G/C) ratio than most bacteria (higher G/C ratio more bonded and stable) human microbiota and are found mainly in the oropharynx, upper respiratory tract, and genitourinary tract. MOT: a. direct sexual contact, b. transplanted tissue from donor to recipient, c. from mother to fetus during childbirth or in utero M. genitalium accounts for approximately 15% to 20% of nongonococcal urethritis (NGU); cervicitis and pelvic inflammatorydisease (PID) M. fermentans has been isolated from specimens such as bronchoalveolar lavage, bone marrow, peripheral blood, and the throats of children with pneumonia. M. amphoriforme has been detected in the lower respiratory tract in patients with chronic respiratory disease and antibody deficiencies Have well-established roles in human infections: M. pneumoniae – isolated from respiratory tract isolated from the internal organs of of Ureaplasma urealyticum, - genitourinary tract stillborn, premature, and spontaneously Mycoplasma hominis - genitourinary tract aborted fetuses Unique among bacteria because they have no cell wall! 1. Pleomorphic 2. Spherical or pear shaped to filamentous with branching 3. Resistant to beta-lactams (Penicillin-resistant) (cannot synthesize cell wall components) 4. Lack of a reaction to Gram stain (Gram-negative) 5. Replicate by binary fission. 6. They require sterols (cholesterol) for membrane function and growth 7. Contains both DNA and RNA 8. can replicate on their own and grow on artificial media  9. ability to ferment glucose, utilize arginine and hydrolyze urea. Mycoplasma pneumoniae ▪ aka Eaton agent ▪ Colonizes the mucosa of the respiratory tract ▪ Causes: 1. Primary Atypical Pneumonia (PAP) / Walking Pneumonia ▪ CAP & tracheobronchitis in children & young adults ▪ Most frequently in persons aged 5–20 years ▪ Notable non-purulent sputum and dry cough Walking pneumonia is an informal term for pneumonia that isn't severe enough to require bed rest or hospitalization. You may feel like you have a cold. The symptoms are generally so mild that you don't feel you need to stay home from work or school, so you are out walking around. M. pneumoniae strongly attaches to the mucosal cells and may reside intracellularly (specialized attachment organelle a P1 adhesin protein that primarily interacts with host cells) within host cells, resulting in a chronic persistent infection that may last for months to years ➔ long-term inflammatory syndromes 2. Extra-respiratory Manifestations Hemolytic anemia (very high titers of cold agglutinins➔ premature destruction of red blood cells) Arthritis, acute glomerulonephritis, etc Determinants of Pathogenicity 1. Gliding motility: penetrate through respiratory secretions 2. P1 protein For attachment of M. pneumoniae to host cells Attaches to glass, RBC & respiratory EC 3. Hydrogen peroxide and Superoxide Injure mucosal cells, causing ciliostasis (loss of cilia/movement) and sloughing of superficial cells 4. Filamentous, flexible form facilitate localization in crypts and folds and between microvilli and cilia, where it is protected from phagocytosis Initiation of disease: a. attachment to respiratory mucosal cells, b. evasion from phagocytosis c. modulation of the immune system Acceptable specimens: ▪ “Body fluids, tissues, wound aspirates and swabs of wounds, the throat, nasopharynx, urethra, cervix, or vagina.” ▪ Throat swab and blood for M. pneumoniae ▪ Other respiratory specimens such as BAL, sputum and lung tissue is acceptable Serologic tests ▪ More rapid tests and are most useful ▪ Detection of specific IgM in a single serum sample is DIAGNOSTIC of acute infection (M. pneumoniae) ▪ Cold Agglutination Reaction, CF, ELISA, etc * No direct method or Gram staining can be used for identification Culture clinically non-contributory  Isolation of M. pneumoniae may require several weeks Culture media ▪ SP-4 Mycoplasma medium – for 1° isolation ▪ Shepard’s ▪ A7B ▪ Edward-Hayflick agar (E-agar) – typical” fried egg” colonies with a dense and translucent periphery * Culture is difficult and lengthy process that’s why serological identification methods have been primary method for id. Swabs for sample collection ▪ Calcium alginate and Dacron swabs - without the application of any disinfectants, analgesics, or lubricant ▪ Transport medium - they are highly susceptible to drying (no cell wall) - Liquid specimens such as body fluids do not require transport media if inoculated to appropriate media within 1 hour of collection a. SP4 (10% heat-inactivated calf/horse serum sucrose + phosphate buffer, pH 7.2 + neutral red) b. Shepard’s 10B broth, c. 2 SP ▪ Cultivation media: a. Stuart’s medium, b. trypticase soy broth supplemented with 0.5% bovine serum albumin, c. A3B broth ▪ DEFINITIVE IDENTIFICATION: Overlaying suspicious colonies with 0.5% guinea pig RBC on PO4- buffered saline (Adherence of RBCs to colonies) Except blood Can be stored indefinitely at -80°C if diluted in transport media after centrifugation. Genital Mycoplasma A. Ureaplasma spp. 1. Ureaplasma urealyticum 2. Ureaplasma parvum B. Mycoplasma spp. 1. Mycoplasma hominis 2. Mycoplasma genitalia Ureaplasma spp. ▪ U. urealyticum is known as T (Tiny) strain Mycoplasma ▪ Found colonizing the vagina and cervix in 40%–80% of adult women ▪ Cause urinary calculi and non-gonococcal urethritis (NGU) ▪ Intrarenal abscess formations in the transplanted kidney ▪ Vaginal colonization with the genital mycoplasmas in pregnant women is not associated with disease ▪ Presence of mycoplasma in the placental membranes or amniotic fluid is associated with chorioamnionitis, preterm birth, spontaneously aborted fetuses, stillborns and several neonatal disorders (perinatal pneumonia and sepsis in preterm infants) Mycoplasma spp. 1. Mycoplasma hominis √ Associated with postpartum fever & post-abortal fever ▪ Related to both pelvic inflammatory disease (PID) and Pelvic inflammatory disease (PID) - pyelonephritis infection of one or more of the 2. Mycoplasma genitalia upper reproductive organs, including the uterus, fallopian tubes ▪ Linked to NGU in males only and ovaries. Manganese Chloride Urea Test: U. urealyticum ▪ A rapid identification test for U. urealyticum ▪ The reaction for the test is observed under a dissecting 0.5nM MnSo4 microscope ▪ (+) Result: Dark brown precipitate of manganese oxide around colonies GM (Genital Mycoplasma) agar 1nM MnSo4 Urease Spot Test ▪ No commercial serologic assays are available for the genital mycoplasmas ▪ Use of serology is confined to research. *Pleuro Pneumonia Like Organisms BAP, strains of M. hominis, but not of U. urealyticum, produce nonhemolytic, pinpoint colonies that do not Gram stain. These colonies can be stained with the Dienes or acridine orange stains  Mycoplasma ferments glucose to lactic acid; ➔ pH change is detected by a color change in a dye indicator * Colonies of Mycoplasma Colonial growth characteristics of pneumoniae visualized under Mycoplasma in agar medium 100x magnification. Mycoplasma hominis is an opportunistic urogenital pathogen in vertebrates. -non-glycolytic species that produces energy via Treatment: arginine degradation M. pneumoniae and M. genitalium: macrolides, tetracycline, ketolides, and fluoroquinolones. M. hominis and U. urealyticum: tetracycline Prevention: no vaccine!! THUS barrier protection for STDS  Obligate Intracellular and Nonculturable Bacterial Agents 1. Chlamydia, Three other organisms are difficult to cultivate 2. Rickettsia, or are noncultivable: 3. Orientia, 1. Coxiella, 4. Anaplasma, and 2. Calymmatobacterium granulomatis 5. Ehrlichia 3. Tropheryma whipplei * are prokaryotes that differ from most other bacteria with respect to their very small size and obligate intracellular parasitism. CHLAMYDIA family Chlamydiaceae obligate intracellular (once regarded as viruses,➔ require biochemical resources of eukaryotic host cells to fuel their metabolism for growth and replication by providing high- energy compounds such as adenosine triphosphate. similar to gram-negative bacilli in that they have lipopolysaccharide (LPS) as a component of the cell wall (has little endotoxic activity) have a Major Outer Membrane Protein (MOMP) that is very diverse. unique developmental life cycle reminiscent of parasites, with an: a. intracellular, replicative form, the reticulate body (RB), b. extracellular, metabolically inert, infective form, the elementary body (EB). 1. After infection of a host cell, the EB differentiates into an RB. 2.The RB divides by binary fission within vacuoles. 3. As the numbers of RB increase, the vacuole expands, forming an intracytoplasmic inclusion. 4. The RB then revert to EB 5. 48 to 72 hours postinfection, the EB are released from the host cell Chlamydia trachomatis biovar LGV Causes Lymphogranuloma Venereum (LGV) aka Lymphogranuloma Inguinale, Climactic Bubo (the swelling of the lymph nodes), tropical bubo, Esthiomene (elephantiasis of the female genitals) STD Presents as vesicular lesion with painful & enlarged femoral lymph nodes Contains four serovars: L1, L2, L2a, and L3 Frei test: intradermal skin test for LGV A heat inactivated LGV 0.1ml grown in yolk sac of embryonated egg is injected intra dermally on the forearm control material: noninfected yolk sac on the other forearm i.e. control. Result: After 48–72 hours, an inflammatory nodule more than 6mm in diameter develops at the test site. ➔ indicates delayed hypersensitivity to an intradermal standardized antigen prepared from chlamydia grown in the yolk sac of a chick embryo. Chlamydia: The Leading Cause of STD More cases of STD are caused by Chlamydia trachomatis than by any other bacterial pathogen, making C. trachomatis infections an enormous public health problem throughout the world (WHO). Chlamydia is a common STD caused by a bacterium (CDC). Transmitted through anal, vaginal, or oral sex The figures 2.86 million infections occur annually in the United States 1 in 15 sexually active females aged 14-19 years has chlamydia. What COMPLICATIONS can result from Chlamydial infection? Women: Pelvis sepsis leading to abscess formation Chronic and recurrent pelvic inflammatory disease Ectopic pregnancy Infertility Chronic pelvic pain Men: Chronic genital tract infection, possibly resulting in infertility. In children: Pneumonia Eye infection Chlamydia trachomatis biovar trachoma ▪ Serovar A, B, Ba, and C ▪ Endemic trachoma ▪ primary infectious cause of blindness in trachoma-endemic regions of the Middle East, sub-Saharan Africa, and Asia ▪ Serovar D–K, Da, Ia, and Ja ▪ Sexually transmitted Disease ▪ C. trachomatis is the most common cause of STD in the US ▪ Causes TRIC (Trachoma Inclusion Conjunctivitis) Specimens: ▪ Urethral and cervical secretions, conjunctiva discharge, nasopharyx and rectal swabs; and materials aspirated from fallopian tubes and epididymis ▪ Dacron-Rayon-tipped swabs are preferred *Wood in cotton swab may be toxic to C. trachomatis Chlamydia psittaci ▪ Formerly Chlamydophila psittaci ▪ Causes of Psittacosis / Ornithosis ▪ Parrot fever ▪ Disease of birds ✓ Esp. psittacine birds: parrots, parakeets, cockatoos, etc. ▪ Occasionally transmissible to humans ▪ Transmission to humans occurs via inhalation of infectious aerosols derived from feces, fecal dust, and secretions of C. psittaci–infected birds ▪ Cells infected with C. psittaci usually are severely damaged, and the organisms are released by cell lysis within 48 hours. ▪ Lungs are the primary target of infection ▪ Diagnosis: ▪ Grows best in L 929 cells ▪ Usually diagnosed serologically ▪ Inclusion bodies: ✓ Levinthal-Cole-Lillie bodies Chlamydia pneumoniae Illness is mild and self-limited – Causes at least 10% of CAPs – Associated with mild respiratory tract infections Also TWAR (from first two isolates) – TW-183, isolated in 1965 from the eye of a control child in a trachoma vaccine trial in Taiwan – AR-39, recovered the same year from the throat of a student with pharyngitis at the University of Washington Diagnosis: – Difficult to grow in cell culture lines – Diagnosis is based predominantly on serologic tests RICKETTSIAE ▪ Gram negative coccobacilli ▪ Obligate intracellular parasites ▪ All can’t survive outside animal host or insect vector except Coxiella ▪ All require living cells for growth except Rochalimea quintana which can be cultivated in cell free media ▪ All are destroyed by heat/drying/chemicals Signs of infection (basic symptoms) 1. Fever 2. Chills 3. Headache 4. Malaise 5. Characteristic rash Manifestations 6. Conjunctivitis 7. Pharyngitis 8. Mild Respiratory Distress Infections caused by organisms of the genus RICKETTSIA Rocky Mountain Spotted Fever caused by Ricketsia rickettsii Geographic distribution: North, Central, and South America Vector: Hard tick: Dermacentor variabilis (dog tick) and Dermacentor andersoni (wood tick) Rickettsiae are injected via the infected tick’s salivary gland secretions into the patient’s dermis Most severe of all the rickettsioses!!! ▪ Fatality rate of 5% ▪ Vascular endothelium is the target of intracellular infection ▪ Renal failure is a feature of severe illness. ▪ Seizures and coma occur in 8%–10% of cases overall ▪ Spread of rash is from extremities to the trunk (centripetally) and commonly involves the palms & soles ▪ In addition to the basic symptoms GIT complaints, arthralgias, conjunctivitis, stiff neck, DIC, etc...  ▪ Rickettsial pox is characterized by a local eschar Transitional Group Rickettsial Fevers Newly recognized transitional group Considered to be relatively distant members of the spotted fever group, with which they share LPS ags. Typhus fever ▪ Rash appear as first on the trunk and later to the extremities (proximodistal) ▪ Epidemic typhus ▪ Systemic infection and prostration are severe ▪ Disease often fatal ▪ Endemic typhus ▪ Milder and rarely fatal LABORATORY TESTS FOR RICKETTSIAL DISEASE ▪ Rickettsial diseases are usually diagnosed acutely purely on the basis of clinicoepidemiologic suspicion ▪ Specimens ▪ Blood – primarily ▪ Sputum & urine – for Q fever ▪ Vesicular fluid – for rickettsial pox Serology  ▪ Serology is seldom useful in assisting therapeutic decisions because antibodies appear later in the course ▪ often mistakenly sought early in the course of illness, provides the majority of laboratory-confirmed diagnoses ▪ Serologic diagnosis is the usual approach to the diagnosis of human ehrlichiosis ▪ The “gold standard” serologic test for rickettsioses is the Indirect Immunofluorescent Antibody (IFA) Assay Other Laboratory Methods 1. Stain for smears Giemsa, Machiavello and Gimenez (the best!) 2. PCR – a diagnostic tool for ehrlichioses 3. Biopsies Immunohistology—immunofluorescence or immunoenzyme staining 4. Isolation: tissue culture in antibiotic-free, centrifugation- enhanced shell vial cell culture in reference and research laboratories with biosafety level 3 Gimenez stain of tick hemolymph cells infected with R. rickettsii. containment and specialized expertise. Agents of Bioterrorism! ▪ R. prowazekii and R. rickettsii are select agents, possession of which is restricted by law to registered scientists in approved institutions Case fatality rates of 15%–25 Weil-Felix test ▪ Measure agglutination of Proteus vulgaris strains OX-19 and OX-2 ▪ Rickettsiae & Proteus share certain antigens ▪ Insensitive and nonspecific! ▪ Should not be used except in developing countries in which no other method can be performed Principle: based on cross-reactivity of the patient’s antibodies with polysaccharide antigens present on the (share antigens) Proteus vulgaris OX-19 and OX-2 strains Proteus mirabilis OX-K - detects anti-rickettsial antibodies in patient’s serum Infections caused by ORIENTIA TSUTSUGAMUSHI and COXIELLA BURNETII Orientia tsutsugamushi Scrub typhus Resembles epidemic typhus clinically Skin rash heralded by an ESCHAR – From a small papule/vesicle into a punched-out ulcer covered with a blackened scab Ehrlichia and Anaplasma Characteristics: 1. Small (0.5 μm) 2. Tick-borne 3. Obligately intracellular 4. Gram-negative coccobacilli 5. Reside in a cytoplasmic vacuole of white blood cells 6. This intravacuolar microcolony of bacteria stained by the Wright-Giemsa method resembles a mulberry and thus is called a morula (Latin for “mulberry”) Ehrlichia ▪ It has three development stages: 1. Elementary body (infective form) 2. Initial bodies 3. Morulae ▪ Natural hosts: Humans and animals (dogs and deer) ▪ Primary vector: Amblyomma americanum (lone star tick) ▪ Related infection: Human monocytic ehrlichiosis (HME) Ehrlichioses 1. Neorickettsia sennetsu – causes Sennetsu rickettsiosis – aka Infectious mononucleosis, glandular fever, hyuganetsu and kagaminetsu – Clinical findings: fever, lymphadenopathy, atypical lymphocytosis 2. Ehrlichia canis – causes Canine ehrlichiosis or Tropical canine pancytopenia – MOT: tick bites – Clinical findings 1. resembles RMSF but only few have rashes 2. leukopenia & thrombocytopenia NONCULTIVABLE ORGANISM Coxiella burnetii ▪ distant phylogenetically from other pathogenic rickettsiae ▪ Resembles influenza, nonbacterial pneumonia, hepatitis, or encephalopathy rather than typhus ▪ No rash or lesions involved! Infections caused by organisms of the genus BARTONELLA, EHRLICHIA and ANAPLASMA Bartonella ▪ Rochalimaea quintana is now Bartonella quintana ▪ Gram-negative ▪ Genus Bartonella has been removed from the order Rickettsiales ▪ despite their historical association with rickettsiology and arthropod transmission, Bartonella organisms are cultivable in cell-free medium and do not belong in the order Rickettsiales Klebsiella granulomatis ▪ Previously known as Calymmabacterium granulomatis ▪ Gram-negative bacillus ▪ Encapsulated ▪ Pleomorphic ▪ Usually observed in vacuoles in the cells of large mononuclear cells ▪ MOT: Sexual Intercourse ▪ Causes granuloma inguinale or donovanosis Granuloma inguinale ▪ Characterized by the presence of the following: – Enlarged subcutaneous nodules on the genitalia that evolve to form beefy, erythematous, granulomatous, painless lesions that bleed easily. – Inguinal lymphadenopathy – have been mistaken for neoplasms Laboratory Diagnosis: ▪ Lesions are scraped and stained with Wright’s or Giemsa stain and observed for Donovan bodies. ▪ Donovan bodies: √ Group of organisms that are seen within mononuclear endothelial cells √ Stains as a blue rod with prominent polar granules, giving rise to a “safety pin” appearance, surrounded by a large, pink capsule. Antibiotic Susceptibility Testing and Therapy ▪ No antibiotic susceptibility testing is performed ▪ Trimethoprim/sulfamethoxazole and doxycycline are the most effective drugs for the therapy of granuloma inguinale. ▪ Ciprofloxacin, azithromycin, or erythromycin (in pregnancy) also provides effective treatment for granuloma inguinale Tropheryma T. whipplei General Characteristics ▪ Gram-positive actinomycete ▪ Not closely related to any other genus known to cause infection  ▪ Nonculturable ▪ making the development of “traditional” diagnostic assays difficult (e.g., serology or antigen detection) Whipple’s Disease ▪ Tropheryma whipplei was identified as the causative agent ▪ affect the mucosal lining of your small intestine first, forming small sores (lesions) within the wall of the intestine ➔ IMPAIR breakdown of food ▪ Characterized by the presence of periodic acid-Schiff (PAS) staining macrophages (indicating mucopolysaccharide or glycoprotein) in almost every organ system ▪ Found primarily in middle-aged men Signs and Symptoms Diarrhea Weight loss Arthralgia Lymphadenopathy Hyperpigmentation Long history of joint pain, Distended and tender abdomen Neurologic and sensory changes Endocarditis Laboratory Diagnosis ▪ Detection is limited to only a few laboratories using conventional and real-time PCR Antimicrobial Susceptibility Testing: ▪ The organism is non-culturable resulting in the inability to perform susceptibility testing Treatment: Patients usually respond well to long-term therapy with antibacterial agents: Trimethoprim/sulfamethoxazole Macrolides, Aminoglycosides Tetracycline Penicillin Colchicine (to control symptoms) Differential Characteristics of Rickettsiaceae and Related Organisms