Lecture 13 Microbiology 8-12-23 PDF

Lecture 13 Microbiology: Actinomycetes, Mycoplasma and cell wall-defective bacteria  Content 1. Actinomycetes, norcardiosis and actinomycetoma 2. Mycoplasma and cell wall-defective bacteria Mycoplasma pneumoniae & atypical pneumonias Ureaplasma urealyticum...

Lecture 13 Microbiology: Actinomycetes, Mycoplasma and cell wall-defective bacteria  Content 1. Actinomycetes, norcardiosis and actinomycetoma 2. Mycoplasma and cell wall-defective bacteria Mycoplasma pneumoniae & atypical pneumonias Ureaplasma urealyticum Cell wall-defective bacteria  Actinomycetes Elongated G+ rods Branching Slow growing (4-10 days) Microaerophilic or strictly anaerobic Source: oral intestinal endogenous flora Disease: chronic celullitis, draining sinuses The lesion is composed of inflammatory sinuses, which ultimately discharge in the surface. Most infections due to A. israelii  Actinomycosis Part of normal intestinal microflora Endogenous infection - conditions for growth require displacement into tissue (surgery, trauma, etc.) Sinus tracts contain pus and sulfur granules Little evidence of immunity Treatment: Penicillin G  Aerobic actinomycetes Genus : aerobic actinomycetes G+ branching filamentous bacteria Poorly stained Slow growing (2-3 Days) Subgroups: Nocardia, Actinomadura, Streptomyces, Rhodococcus, Gordonia, Tsukamurella and Tropheryma whipplei  Nocardia :History Edmond Nocard, 1888 Aerobic actinomycetes from cattle with bovine farcy Growth on blood agar  Nocardia At least 13 species : cause human infection 7most important 1. Nocardia asteroides complex :80% of noncutaneous dz. :most systemic & CNS nocardiosis *** 2. Nocardia farcinica :less common,more virulent :more antibiotic-resistant member 3.Nocardia nova 4.Nocardia brasiliensis: skin,cutaneous,lymphocutaneous 5.Nocardia pseudobrasiliensis:systemic infections, CNS 6.Nocardia otitidiscaviarum 7.Nocardia transvalensis.  Nocardia :ECOLOGY& EPIDEMIOLOGY Ubiquitous environmental saphrophyte Soil, organic matter, water Tropical and subtropical regions :Mexico, Central and South America, Africa and India  Nocardia :ECOLOGY& EPIDEMIOLOGY Nearly all cases :sporadic Human-to-human Animal-to-human not documented Outbreaks : Contamination of the hospital environment, solutions, drug injection equipment. Occurance in immunocompromised is increased Source: soil  Nocardia :ECOLOGY& EPIDEMIOLOGY The risk of pulmonary or Transmission disseminated disease *deficient cell-mediated * -Alcoholism Inhalation -Diabetes Skin -Lymphoma -Transplantation -Glucocorticoid therapy -AIDS  Nocardia : PATHOLOGY Acute pyogenic inflammatory reaction. Branching, beaded, filamentous bacteria G/S from a nocardial lung abscess G/S from nocardial pneumonia  Nocardia :PATHOGENESIS Neutralization of oxidants Prevention of phagosome-lysosome fusion Prevention of phagosome acidification. Mycolic acid polymers: associated with virulence  CLINICAL MANIFESTATIONS : 4 main forms Lymphocutaneous syndrome - follows minor trauma Pulmonary :Pneumonia CNS : Brain abscess Disseminated disease CNS, Eyes (particularly the retina🡪Keratitis), Skin& subcutaneous, Kidneys, Joints, bone Heart  Nocardia keratitis Nocardia infections of eye. (a) Shows nocardia keratitis with characteristic wreath-like lesion. (b) Shows a case of advanced keratitis with dry-looking infiltrate mimicking fungal infection. (c) Shows nocardia keratitis with suppurative lesion. (d) Shows suppurative nodular scleritis  Lymphocutaneous syndrome -Cellulitis -Lymphocutaneous syndrome -Actinomycetoma Ubiquitous in soil🡪 inoculation injuries, Insect and animal bites 🡪contaminated abrasions N. brasiliensis : most common N. asteroides : self-limited Days to months ,typical: distal limb Cutanious nocardiosis Nocardial actinomycetoma swelling, multiple sinus tracts,  Pulmonary disease Pneumonia Subacute(more acute in immunosuppressed) Cough** Small amounts of thick, purulent sputum Fever, anorexia, weight loss, malaise Endobronchial inflammatory mass Lung abscess Cavitary disease Inadequate therapy 🡪Progressive fibrotic diseases Cerebral imaging, should be performed in all cases of pulmonary and disseminated nocardiosis  Pulmonary nocardiosis Nocardial pneumonia. Discrete nodular in midlung on both sides CT scan (A),CXR (B) from : multiple abscesses : Nocardia farcinica  CNS : Brain abscess Insidious presentations : mistaken for neoplasia !!! Granulomatous , abscesses Cerebral cortex, basal ganglia and midbrain*** Less commonly: spinal cord or meninges. Brain tissue diagnosis in pulmonary nocardiosis : not necessary However,cerebral biopsy: considered early in immunocompromised brain abscess ; Nocardia farcinica Nocardial abscess :rt. occipital lobe  LABORATORY DIAGNOSIS Gram-positive, beaded, branching filaments usually weak acid fast +ve. Standard blood culture :48 hrs to several wks, but typical = 3 to 5 days Colonization of sputum :underlying pulmonary dz + not receiving steroid therapy🡪 no specific therapy Susceptibility testing -Deep-seated /disseminated dz. fail initial therapy -Relapse after therapy -Alternatives to sulfonamides are being considered  MANAGEMENT :Medication Sulfonamides : the mainstay of therapy treatment of choice :N. brasiliensis N. asteroides complex N. transvalensis. severely ill patients, CNS /disseminated/ immunosuppressed patients🡪 =/> 2 drugs Amikacin and Carbapenem or 3rd generation cephalosporin.  Genus: Actinomycetes Gram positive bacteria Anaerobic Branching Part of normal flora of GI tract Most infections are due to A. israelii Conditions for growth require displacement into tissue The lesion is composed of inflammatory sinuses, which ultimately discharge in the surface. Sinus tracts contain pus and sulfar granules  Actinomycosis Sulfur granules G/S :sulfur granule  Genus : Actinomyces Slowly progressive infection Colonize : mouth, colon, vagina Infection : mucosal disruption In vivo : Grains / Sulfur granules The most misdiagnosed disease 3 clinical presentations 1.chronicity, progress across tissue boundaries, mass-like 2. develop sinus tract, resolve and recur 3. refractory/relapsing after a short course therapy  Etiologic Agents A. israelii*** A. naeslundii/viscosus A. odontolyticus A. viscosus A. meyeri A. gerencseriae pelvic disease ass. IUCDs & “lumpy jaw” 16S rRNA gene sequencing led to identification of an ever-expanding list of Actinomyces spp  Epidemiology Members of oral, GI, and genital flora Never been cultured from nature No document of person-to-person transmission The peak incidence : mid-decades Male > Female (poorer dental hygiene & oral trauma )  Risk Factors Foreign bodies : IUCDs Abnormal host defense : HIV Post transplantation Radio-Chemotherapy Ulcerative mucosal infection: HSV/CMV  Clinical Manifestations Oral-Cervicofacial Disease Thoracic Disease Abdominal Disease Pelvic Disease Central Nervous System Disease Musculoskeletal & Soft tissue infection Disseminated Disease  Oral-Cervicofacial Disease Cervicofascial forms usually linked to poor dental hygiene Most frequently site Soft tissue swelling / mass/ abscess : mistaken for a neoplasm Most common site : Angle of jaws Dx: mass lesion/relapsing infection in head &neck Complication :-Otitis, sinusitis, and canaliculitis Most common site : Angle of jaws  Thoracic Disease Thoracic and abdominal actinomycoses are rare and follow aspiration or traumatic (including surgical) introduction Diagnosis is usually delayed Chest pain, fever, and weight loss *** The firm fibrous masses are often initially mistaken for a malignancy  A:Chest wall mass D:Purulent pleural fluid (aspiration) B and C: Chest x-ray + CTscan :pulmonary infiltrate, pleural effusion, pleural and chest wall extension (arrow).  Abdominal Disease(1) Usually pass from inciting event Appendicitis Diverticulitis PUD Foreign bodies Bowel surgery ascension from IUCD-associated pelvic disease Abscess, mass, mixed lesion : mistaken—tumor??? 🡪CT: heterogeneous enhance+ thick adjacent bowel. Sinus tracts 🡪 abd. wall / perianal/ between bowel (Mimic inflammatory bowel disease) Clue : Recurrent dz /wound or fistula : fails to heal Imaging and percutaneous techniques :Therapeutic diagnosis  Abdominal Disease(2) Kidney, Ureter, Bladder (KUB) Disease All levels: can be infected - pyelonephritis - renal and perinephric abscess ❑ Bladder involvement:usually due to pelvic disease ❑ urine : stains and cultures  Pelvic Disease Risk: IUCD in place >1year-months after removed S&S: Typically indolent fever, weight loss, abdominal pain, abnormal vaginal bleeding or discharge Endometritis Removed as early as possible :but not removal of the IUCD (Intrauterine Contraceptive Device) unless a suitable contraceptive An IUCD encased by endometrial fibrosis (solid arrowhead) paraendometrial fibrosis (open arrow)  Diagnosis Avoid unnecessary surgery Aspirations & Biopsy Material for C/S + microscopic identification Sulfur granules Biopsy shows characteristic clubbed lesions Penicillin may have to be used empirically  Special Group # 1. Mollicutes (Mycoplasmas): The mollicutes represent a group of eubacteria. The common name for this group has traditionally been mycoplasmas. However, this usage invites confusion, since it is often not clear whether “mycoplasma” is being used to represent a genus or to refer to a member of the genus Mycoplasma. To avoid this confusion, the more recently coined term “mollicutes” to designate the members of this eubacterial group is being adopted here. Genera: Mollicutes  Mollicutes The distinguishing property of the mollicutes is the lack of a definite cell wall in them. They all are sensitive to osmotic lysis, resistant to penicillin and other wall-synthesis attacking antibiotics, pleomorphic in shape to at least some extent and parasites to eukaryotic organisms. Their colonies that develop on solid culture media are often small, typically having a nipped or “fried-egg” appearance. Fried egg shape colonies  Mycoplasma At present, more than 36 mycoplasmal representatives have been isolated; the most typical representatives of the pathogenic species are the causative agents of: pleuropneumonia in cattle (Mycoplasma mycoides), acute respiratory infections (Mycoplasma hominis), and a typical pneumonia in humans (Mycoplasma pneumoniae).  Pleuropneumonia organism The mycoplasmas are essentially bacteria lacking a rigid cell wall during their entire life cycle, although they are also much smaller than bacteria. The plasma membrane, which binds mycoplasmal cell in absence of cell wall, is tri-layered and contains cholesterol The studies suggest that they are derived from Gram-positive bacteria by reductive evolution. The first organism of this type was associated with pleuropneumonia of cattle, and was originally called the pleuropneumonia organism (PPO).  General Characteristics smallest known free-living organisms. Because of the absence of cell walls, they do not stain with the Gram stain, and they are more pleomorphic and plastic than eubacteria. Giemsa stain (contains separate fixative (methanol) and stain solutions) – they appear as tiny pleomorphic cocci, short rods, short spirals, and sometimes as hollow ring forms. Their diameter ranges from 0.15 u to 0.30 u. Mycoplasma pneumoniae Filamentous and Bottle shaped form of Mycoplasma  Mycoplasma, Ureaplasma very small (0.2 x 0.8 um) – pass through a 0.45 um filter No DNA homology with the known prokaryotes No Cell wall: plasma membrane only – resistant to antibiotics that interfere with the integrity of cell wall; penicillins, cephalosporins, vancomycin, bacitracin susceptible to tetracycline, erythromycin  Structure The cell is enclosed by a limiting membrane which is more similar to that of animal cells than that of bacterial cells because of sterols present in the membrane. The cytoplasm contains ribosomes, but lacks mesosomes. There is no nuclear membrane. In some strains, amorphous material on the outer surface of the membrane suggests the existence of a capsule.  Structure Mycoplasma pneumoniae has a terminal organelle which mediates attachments and gliding motility. This structure contains a number of proteins P1, P30 responsible for attachment.  Mycoplasma requires sterols for growth, can be grown on laboratory media most are facultatively anaerobic – Exception M. pneumoniae replication controversial – replication time 1-6 hours  Toxin production Mycoplasma pneumoniae produces an ADP-ribosylating toxin called the Commonly Associated Respiratory Disease Syndrome (CARDS) toxin. In the lab colonies of Mycoplasma bind red blood cells onto the surface of agar plate cultures (hemadsorption)  Mycoplasma Also known as Eaton’s agent – aerobic but very slow growing extracellular pathogen: attaches to respiratory epithelium by an attachment factor called P1 interacts with a glycoprotein receptor on the epithelial cell surface ciliostasis is followed by epithelial cell destruction  Clinical Syndrome Pneumonia Low infectious dose (< 100) – walking pneumonia frequently confused with virus infection – primary atypical – clinical Tracheobronchitis Pharyngitis – differential diagnosis from Strep throat  Spread of Mycoplasma Infections The disease is world wide, and found in all age groups. Transmission by droplet infection of nasopharyngeal secretions. Spread is associated with close contact of infected person Important infection in Military personal. Epidemics occur with intervals of 4-6 years Even the persons recovered from infection will harbor the pathogens for 2 moths or more The most common age range for symptomatic M.pneumoniae infection is between 5 and 15 years, and the disease accounts for more than 1/3 of all classes of pneumonia in teenagers (but is also seen in older persons). Infections in children younger than 6 months are uncommon. Children most susceptible No seasonal incidence  Clinical Manifestations Generalized aches and pains Fever (usually 102°F) Cough - Usually non-productive Sore throat (nonexudative Pharyngitis) Headache/ myalgias Chills but not rigors Nasal congestion with coryza Earache General malaise  Pathogenesis M.pneumonia infection involves the trachea, bronchi, bronchioles and peribronchiol tissues Initially M. pneumoniae attaches to the cialia and microvilli of the cells lining to bronchial epithelium. The attachments is associated by protrusion associated proteins (P1, P30). Other proteins bind to elements of extracellular matrix like fibronectin. The CARDS toxin interferes with ciliary action and causes nuclear vacuolization and fragmentation of tracheal epithelial cells. This leads to inflammation and desquamation of the involved mucosa  ADP-ribosylating and vacuolating by CARDS toxin Effect of rCARDS TX on CHO cell morphology. Cells were grown to 60% monolayer confluence before addition of 10 μg of rCARDS TX for 16–40 h. Control CHO cells were treated with 10 μg of heat-inactivated rCARDS TX for 40 h. (Magnification: ×200).  Radiological presentation Mycoplasma has a variable presentation on CXR, most commonly presenting as bilateral lower lobe consolidation with small pleural effusions. The opacities usually start as partly mottled, partly node-like peribronchial opacities, which may gradually develop to involve whole segments or lobes  Immunity Immunity is incomplete Re-infection may occur Clinical disease appears to be more sever in older than in younger children, which has led to the suggestion that many of clinical manifestations of disease are the result of immune responses rather than invasion by organism.  Urethritis 1/2 of urethral infections not caused by Chlamydia or N. gonorrhoeae. Caused by – Mycoplasma hominus – Ureaplasma  Laboratory diagnosis Culture: – fried egg colonies on medium containing sterols – Most mycoplasmas require a rich medium containing a sterol and serum proteins for growth. Serology: – Complement Fixation test, Hemagglutination  Laboratory Diagnosis Culture Mycoplasma from sputum, mucous membrane swabbings or other specimens direct inoculation into liquid or solid media containing serum, yeast extract and penicillin to inhibit contaminating bacteria.  Cultural Characteristics Despite the lack of a cell wall, they do not require a medium of very high osmotic pressure. On solid media, they form minute, transparent colonies. – looks like a fried egg. The different strains vary in their growth rate may take from two days to several weeks to form a colony.  Fried Egg Colonies Stain intensely with neutral red or tetrazolium or methylene blue.  serology: complement fixation on acute and convalescent serum. patient’s serum heated to 56OC to eliminate complement combine patient’s serum and known Mycoplasma antigen in presence of added complement. Mix. Incubate - add indicator system – Red cells and anti-red cell antibody – hemolysis occurs if complement is unused  Hemagglutination Cold agglutinin test - Positive in Mycoplasma (Primary Atypical ) Pneumonia The patients sera agglutinated human O group erythrocytes at 4 O C the agglutination being reversible at 37 0 C hemabsorption & B-hemolysis of guinea pig red blood cells.  Identification conclusively identified by staining its colonies with fluoresceint-labelled antibody.  M. pneumoniae Nucleic Acid Probes specific recombinants to oligonucleotide sequences that are only found in Mycoplasma pneumoniae.  L - forms Some bacteria readily give rise spontaneously to variants that can replicate in the form of small filterable protoplasmic elements with defective or absent cell walls. These organisms, called L-forms, can also be formed by many species when cell wall synthesis is impaired by antibiotic treatment or high salt concentration.  L forms vs Mycoplasma contain a rigid cell wall, at least at one stage of their life cycle no sterols in their cytoplasmic membrane.  Pleuropneumonia-like organisms Several organisms with similar morphological characteristics and cultural properties have been isolated. These are commonly referred to as pleuropneumonia-like organisms or PPLO. A certain group of mycoplasmas produce extremely tiny colonies on agar plates, and are called the T-strains.  Multiplication In the absence of a rigid cell wall, the pattern of replication is quite different from that of typical bacteria, whose division starts with the formation of a well-defined septum. Thank you for attention