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Term Three Revision Medical Microbiology term Three 1) Rickettsia, Chlamydia, Mycoplasma, Rickettsia Classification – Family Rickettsiaceae with 3 medically important genera Rickettsia Coxiella Rochlimaea Morphology and cultural characteristics All are obligate intracellular parasites (except Rochlimaea) that can grow in both phagocytic and nonphagocytic cells. Rochlimaea can be cultivated on artificial media containing blood Rickettsia Others are grown in embryonated eggs or tissue culture Cultivation is costly and hazardous because aerosol transmission can easily occur Small, pleomorphic coccobacilli Gram stain poorly, but appear to be GStain readily with Giemsa All, except Coxiella, are transmitted by arthropod vectors. Rickettsia Diagnosis/serological tests Direct detection of Rickettsia in tissues (Giemsa stain or direct fluorescent antibody test) Weil-Felix reaction – in certain rickettsial infections (typhus group) antibodies are formed that will agglutinate OX strains of Proteus vulgaris. This is used as a presumptive evidence of typhus group infection, however, the test is not very sensitive or specific and many false positives occur. Rickettsia Agglutination or complement fixation tests using specific Rickettsial antigens are better serological diagnostic tests. Virulence factors Rickettsial sp. Induced phagocytosis Recruitment of actin for intracellular spread Coxiella burnetti Is resistant to lysosomal enzymes Clinical significance – the diseases caused by Rickettsia are all characterized by fever, headache, myalgias, and usually a rash. Typhus fevers – incubation is 5-18 days. Symptoms include a severe headache, chills, fever, and after a fourth day, a maculopapular rash caused by subcutaneous hemorrhaging as Rickettsia invade the blood vessels. The rash begins on the upper trunk and spread to involve the whole body except the face, palms of the hands, and the soles of the feet. The disease lasts about 2 weeks and the patient may have a prolonged convalescence. Two types of typhus may occur: Epidemic typhus – caused by R. prowazekii and transmitted by human lice as it bites and defecates in the wound. This occurs in crowded areas causing epidemics. Mortality rates are high in untreated cases. Following an initial attack, some individuals may harbor the organism of a latent infection with occasional relapses = Brill-Zinsser disease Endemic typhus – caused by R. typhi and transmitted to man by rat fleas. The disease occurs sporadically, but is clinically the same, but less severe than epidemic typhus. Rickettsia responsible for spotted fevers Rocky mountain spotted fever – caused by R. rickettsii and transmitted by ticks that must remain attached for hours in order to transmit the disease. An incubation of 2-6 days is followed by a severe headache, chills, fever, aching, and nausea. After 2-6 days a maculopapular rash develops, first on the extremities, including palms and soles, and spreading to the chest and abdomen. If left untreated, the rash will become petechial with hemorrhages in the skin and mucous membranes due to vascular damage as the organism invades the blood vessels. Death may occur during the end of the second week due to kidney or heart failure. Rocky mountain spotted fever Rickettsia Rickettsial pox – caused by R. akari and transmitted by a mouse mite. After a 1-2 day incubation a papule develops at the entry site and within 1-2 weeks a fever, malaise, and headache develop followed by a rash. The disease is mild and usually not fatal. Q fever – caused by Coxiella burnetii. The infection is acquired by inhalation of infectious material. After an incubation of 14-26 days there is a sudden onset of fever, chills, and headache, but no rash. The disease is characteristically an atypical pneumonia lasting 5-14 days with a low mortality rate. Trench fever – caused by Rochalimaea quintana and transmitted by body lice. Was a major problem during WW I and WW II. After an incubation of 6-22 days, the patient experiences a headache, exhaustion, leg pains, and a high, relapsing fever. A roseolar rash occurs and the patient usually recovers. Treatment/antimicrobic therapy Chloramphenicol or tetracycline Wear protective clothing and use insect repellents. Chlamydia Classification – order Chlamydiales – contains one medically important genus – Chlamydia Are obligate intracellular parasites Cell walls are similar to the cell walls of G-B, but lack muramic acid Have a complex developmental cycle The infectious form is called an elementary body (EB) which is circular in form and is taken into the cell by induced phagocytosis. Inside the phagocytic vesicle replication takes place Chlamydia Over the next 6-8 hours, the EB reorganizes into the noninfectious, but metabolically active reticulate body (RB) which is larger and less dense than the EB. For 18-24 hours the RB synthesized new materials and divides by binary division to form inclusion bodies that reorganize and condense into EBs. Between 48-72 hours, the cell lyses and releases the EB which begin the cycle again. Chlamydia Are energy parasites that use ATP produced by the host cell A Giemsa stain can be used to visualize chlamydial inclusions in tissues. Identification Direct methods – stain tissues with Giemsa or use a direct fluorescent antibody technique. The most sensitive method is to culture the organisms in tissue cultures and then stain the infected tissue culture cells Chlamydia A complement fixation serological test is available as are DNA based tests. Virulence factors Toxicity from attachment and penetration Clinical significance Chlamydia trachomatis – serotypes A-K and L1,2,3; the serotype determines the clinical manifestation. Genital tract infection (serotypes D-K) – is the major cause of nongonococcal urethritis; is sexually transmitted and frequently found concomitantly with N. gonorrhoeae In males symptoms include urethritis, dysuria and it sometimes progresses to epididymitis Chlamydia In females symptoms include mucopurulent cervical inflammation which can progress to salpingitis and PID. Inclusion conjunctivitis – this occurs in both newborns and adults and a genital tract infection is the source of the infection (serotypes D-K); is a benign, self-limited conjunctivitis which heals with no scarring Newborns – are infected during the birth process and the infection manifests 1-2 weeks after birth as a mucopurulent discharge that lasts2 weeks and then subsides. Some may develop an afebrile, chronic pneumonia Chlamydia In adults – causes an acute follicular conjunctivitis with little discharge. Trachoma (serotypes A-C) – is the single, greatest cause of blindness in underdeveloped countries. Transmission is by direct contact and in poor, less developed countries children may be infected in the first three months of life. Chronic infection and reinfection are common and result in conjunctival scarring and corneal vascularization. The scars contract causing the upper lid to turn in so that the eyelashes cause corneal abrasions. This leads to secondary bacterial infections and results in blindness. Chlamydia Lymphogranuloma venereum (serotypes L1, 2, 3) is a venereal disease that occurs in poor, tropical areas. Upon infection, widespread dissemination takes place and a primary, painless lesion (either a vesicle or an ulcer) occurs at the site of entry within a few days. This heals with no scarring. A secondary stage occurs 2-6 weeks later with symptoms of regional suppurative lymphadenopathy (buboes) that may drain for a long time and be accompanied by fever and chills. Arthritis, conjunctival, and CNS symptoms may also occur. A tertiary stage may occur and is called the urethrogenital perineal syndrome. This is characterized by structural changes such as non-destructive elephantiasis of the genitals and rectal stenosis. Chlamydia psittaci – naturally infects avian species and non-primate animals causing mild to severe illness. In man causes psittacosis (ornithosis) and is acquired by contact with an infected animal. Infection can range from subclinical to fatal pneumonia. Most commonly causes an atypical pneumonia with fever, chills, dry cough, headache, sore throat, nausea, and vomiting. Chlamydia Treatment/antimicrobic susceptibility C. trachomatis – Trachoma – systemic tetracycline, erythromycin; long term therapy is necessary Genital tract infections and conjunctivitis – tetracyclines and erythromycin C. psittaci – same as above Mycoplasma Classification – order Mycoplasmatales; family Mycoplasmataceae; 2 medically important genera Mycoplasma Ureoplasma Three common clinical isolates – M. pneumoniae, M. hominis, and U. urealyticum Morphology and cultural characteristics Do not possess the distinctive cell wall of bacteria Plasma membrane is the outermost part of the organism and is unique in bacteria in that it has a high content of sterols that act to prevent osmotic lysis Very small in size (too small to be seen with an ordinary light microscope) and highly pleomorphic Don’t stain with a Gram’s stain Non-motile May possess a capsule Although some are free living, most are closely adapted parasites Grow on media enriched with serum (need cholesterol) Grow beat at 35-370 C either aerobically or anaerobically M. pneumoniae grows in 5-14 days, M. hominis in 2-4 days, and U. urealyticum in 24-28 hours. M. pneumoniae colonies resemble fried eggs and can be stained with Dienes stain (they stain blue) Identification M. pneumoniae Isolation in culture Ability of colonies to hemolyze guinea pig RBCs Rise in specific antibody titer Cold agglutinin test – a nonspecific test in which the patient produces cold reacting antibodies that agglutinate type O human RBCs at 40 C, but not at 370 C A single titer of 1:128 is significant and occurs in 7 days and disappears in 6 weeks. M. hominis Isolation in culture No hemolysis of guinea pig RBCs U. urealyticum Urease production Virulence factors Not invasive and simply colonize cell surfaces through specific binding Damage to host tissues may be due to toxic metabolic products Clinical significance M. pneumonia – the major cause of primary, atypical pneumonia (walking pneumonia) Transmitted by droplet infection After a 2-3 week incubation, the disease begins as a mild, upper respiratory tract infection and progresses to fever, headache, malaise, and a dry cough which is usually mild and self-limited. 3-10% develop clinically apparent pneumonia with occasional complications of arthritis,rashes, cardiovascular problems, or neurological problems. Genital tract infections - caused by M. hominis and U. ureolyticum which may also be found as part of the NF in the genital tract May cause nongonococcal urethritis, PID, post-partum fever, infertility, stillbirth, spontaneous abortion, and acute urethral syndrome Treatment M. pneumonia – tetracycline or erythromycin Genital infections – tetracycline Parasites Medical parasitology: “the study and medical implications of parasites that infect humans” A parasite: “a living organism that acquires some of its basic nutritional requirements through its intimate contact with another living organism”. Parasites may be simple unicellular protozoa or complex multicellular metazoa Eukaryote: a cell with a well-defined chromosome in a membrane-bound nucleus. All parasitic organisms are eukaryotes Protozoa: unicellular organisms, e.g. Plasmodium (malaria) Metazoa: multicellular organisms, e.g. helminths (worms) and arthropods (ticks, lice) An endoparasite: “a parasite that lives within another living organism” – e.g. malaria, Giardia An ectoparasite: “a parasite that lives on the external surface of another living organism” – e.g. lice, ticks Key definitions: What is ….? Host: “the organism in, or on, which the parasite lives and causes harm” Definitive host: “the organism in which the adult or sexually mature stage of the parasite lives” Intermediate host: “the organism in which the parasite lives during a period of its development only” Zoonosis: “a parasitic disease in which an animal is normally the host - but which also infects man” Vector: “a living carrier (e.g.an arthropod) that transports a pathogenic organism from an infected to a non-infected host”. A typical example is the female Anopheles mosquito that transmits malaria The burden of some major parasitic infections Examples of important intestinal protozoa Parasites Transmitted by the faecal-oral route and cause diarrhoea. Protozoa Giardia lamblia: world-wide distribution, lives in the small intestine and results in malabsorption Entamoeba histolytica: may invade the colon and cause bloody diarrhoea – amoebic dysentery. Also causes ameobic liver abscess. Cryptosporidium parvum: more prevalent in the immunocompromised Examples of important systemic protozoa Parasites Detected in the blood Plasmodium: the cause of malaria. There are 4 species that infect man: P. falciparum, P. vivax, P. ovale and P. malariae Toxoplasma gondi: transmitted by the ingestion of oocysts from cat faeces. Infection can lead to ocular problems and is also a cause of neonatal toxoplasmosis Leishmania: transmitted by sand flies, can lead to visceral, cutaneous and mucocutaneous leishmaniasis Trypanosoma: haemoflagellates which cause In Africa - sleeping sickness (transmitted by the Tsetse fly) In South America - Chagas disease (transmitted by the Reduviid bug) Helminths 1. Nematodes : Examples of important metazoa – intestinal nematodes Trichuris (whipworm) A soil transmitted helminth prevalent in warm, humid conditions Can cause diarrhoea, rectal prolapse and anaemia in heavily-infected people Ancylostoma and Necator (hookworms) A major cause of anaemia in the tropics Strongyloides inhabits the small bowel infection more severe in immunospressed people (e.g. HIV/AIDS, malnutrition, intercurrent disease) Enterobius (pinworm or threadworm) prevalent in cold and temperate climates but rare in the tropics found mainly in children Ascaris (roundworm) Found world-wide in conditions of poor hygiene, transmitted by the faecal- oral route Adult worms lives in the small intestine Causes eosinophilia 2. Platyhelminths Intestinal flatworms – 2a) Cestodes (“tapeworms”) Intestinal flatworms Taenia saginata Human/ Beef tapeworm: worldwide acquired by ingestion of contaminated, uncooked beef a common infection but causes minimal symptoms Taenia solium Human / pork tapeworm worldwide acquired by ingestion of contaminated, uncooked pork that contains cystercerci Less common, but causes human cystercicosis – a systemic disease where cysticerci encyst in muscles and in the brain – may lead to epilepsy Systemic flatworm Echinococcus granulosus (dog tapeworm) and Echinicoccus multilocularis (rodent tapeworm) Hydatid disease occurs when the larval stages of these organisms are ingested The larvae may develop in the human host and cause space-occupying lesions in several organs, e.g. liver, brain Ruptured cysts can cause death via anaphylaxis Metazoa –trematodes (flukes) Intestinal Fasciola hepatica (liver fluke)- Primarily, a parasite of sheep, humans become infected when they ingest metacercariae that have encysted on watercress. The adult trematode lives in the intra-hepatic bile ducts of the liver. “Fascioliasis” can lead to severe anaemia in humans Clonorchis sinensis (liver fluke)- Widespread in China, Japan, Korea and Taiwan, this parasite is acquired by ingestion of infective metacercariae in raw or pickled fish Paragonimus westermani ( lung fluke)- Widespread in the Far East and South east Asia, the parasite is acquired by ingestion of infective metacercariae in raw or pickled crustaceans Schistosoma haematobium, S. mansoni and S. japonicum – see below Genetic change of Bacteria The short generation span of bacteria facilitates their evolutionary adaptation to changing environment Bacteria have Circular DNA, plus Plasmids, binary fission (produce clones), 1) Mutations The major component of the bacterial genome is one double-stranded, circular DNA molecule. – For E. coli, 100 times more DNA than in a typical virus and 1,000 times less than in a typical eukaryote cell. In addition, many bacteria have plasmids, much smaller circles of DNA. – Each plasmid has only a small number of genes, from just a few to several dozen. 2) Genetic recombination Three natural processes of genetic recombination in bacteria a. Transformation b. Transduction c. Conjugation and plasmids Replication of the bacterial chromosome Oswald T. Avery and colleagues spent several years identifying the transforming principle. They finally published the work in 1944. They treated the extract from the S-strain bacteria in various ways to destroy different types of substances (e.g., proteins, nucleic acids, carbohydrates, or lipids) but retain others. When DNA was destroyed, the transforming activity was lost, but when DNA was left intact, the transforming activity survived. Transformation Transformation = Process of genetic transfer during which a bacterial cell assimilates foreign DNA from the surroundings Take up naked DNA from the surroundings Assimilate foreign DNA Progeny will carry a new combination of genes E. coli can be artificially induced to take up foreign DNA by incubating This technique is used by Biotechnology industry to introduce foreign genes into bacterial genomes BACTERIAL TRANSFORMATION Not all bacteria take up free-floating DNA in the environment. The genera that generally exhibit transformation include: Bacillus, Streptococcus, Azotobacter, Haemophilus, Neisseria, and Thermus. The recipient cells must be competent (able to transform). Competence is a phenotype conferred by one or more proteins. It has been shown that competence occurs late in the exponential phase of bacterial growth. The duration of competence varies from a few minutes in Streptococcus to hours in Bacillus TRANSFORMATION ds DNA binds to the surface of a competent cell and is cleaved into fragments of about 15 kb. The double stranded DNA, which is still external to the cell, is then separated into ss DNA. One fragment of the ss DNA is degraded, while the other is transported across the membrane and into the cell. Upon entry into the cell, a single strand of the foreign DNA is incorporated into the recipient cell via recombination Transduction Transduction = Gene transfer from one bacterium to another by a bacteriophage Generalized transduction = occurs when random pieces of host cell DNA are packaged within a phage capsid during the lytic cycle of a phage This process can transfer almost any host gene When the phage infects a new cell, the donor cell DNA can recombine with the recipient cell DNA Specialized transduction (restricted transduction) Transduced bacterial genes are restricted to specific genes adjacent to the prophage insertion site Transductions Conjugation and plasmids Conjugation= The direct transfer of genes between two cells that are temporarily jointed Plasmid = A small, circular, double-stranded, self-replicating molecule ring of DNA in some bacteria Transposon (Transposable Genetic Element)= DNA sequence that can move from one chromosomal site to another Conjugation and recombination in E. coli Conjugation and recombination in E. coli Insertion of a transposon and creation of direct repeats Chemical Control The Ideal Drug* 1. Selective toxicity: against target pathogen but not against host LD50 (high) vs. MIC and/or MBC (low) 2. Bactericidal vs. bacteriostatic 3. Favorable pharmacokinetics: reach target site in body with effective concentration 4. Spectrum of activity: broad vs. narrow 5. Lack of “side effects” Therapeutic index: effective to toxic dose ratio 6. Little resistance development Chemical Control Disinfectants Very toxic damaging to skin Used on surfaces and inert material Gasses : Formaldahyde, Ethylene oxide, Ozone Liqids: Phenol, Zepherin, Hydroperoxide Antiseptics Can be used on skin Betadine, Chlorhexidine, physohex (hexachlorophene) cf. controversy In intestine if not absorbed eg nystatin Antibiotics: Safe for internal use Susceptibility Tests Minimum Inhibitory Concentration (MIC) Minimum Biocidal Concentration (MBC) Agar diffusion Kirby-Bauer Disk Diffusion Test Diffusion depends upon: 1. Concentration 2. Molecular weight 3. Water solubility 4. pH and ionization 5. Binding to agar Susceptibility Tests “Kirby-Bauer Disk-plate test” Zones of Inhibition (~ antimicrobial activity) depend upon: 1. pH of environment 2. Media components Agar depth, nutrients 3. Stability of drug 4. Size of inoculum 5. Length of incubation 6. Metabolic activity of organisms Standardization of Kirby-Bauer Disk-plate test” The amount of antibiotic in each paper disc is adjusted so that each disc produces a 1 cm zone 0of inhibition with a standard test organism. Ie a particular strain of Staph.aurius or E. coli Results of test are recorded as (S) more sensitive or ( R ) more resistant than the standard organism Antibiotic Mechanisms of Action Mechanism of Action ANTIMETABOLITE ACTION Mechanism of Action 2. ALTERATION OF CELL MEMBRANES Polymyxins and colistin destroys membranes active against gram negative bacilli serious side effects used mostly for skin & eye infections Mechanism of Action 3. INHIBITION OF PROTEIN SYNTHESIS: Steps in synthesis: 1. Initiation 2. Elongation 3. Translocation 4. Termination Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS Tetracyclines bind to 30S subunit and interferes with the attachment of the tRNA carrying amino acids to the ribosome effective against: Chlamydia Rickettsia Mycoplasma Brucella Mechanism of Action 4. INHIBITION OF DNA/RNA SYNTHESIS Rifampin binds to RNA polymerase active against gram positive cocci bactericidal for Mycobacterium used for treatment and prevention of meningococcus Mechanism of Action INHIBITION OF DNA/RNA SYNTHESIS Metronidazole breaks down into intermediate that causes breakage of DNA active against: protozoan infections anaerobic gram negative infections Mechanism of Action 5. CELL WALL SYNTHESIS INHIBITORS Steps in synthesis: 1. NAM-peptide made in cytoplasm 2. attached to bactoprenol in cell membrane 3. NAG is added 4. whole piece is added to growing cell wall 5. crosslinks added 6. the β-Lactams the non β-Lactams Mechanism of Action 5. CELL WALL SYNTHESIS INHIBITORS β-Lactam Antibiotics Penicillins Cephalosporins Carbapenems Monobactams Resistance Plasmids independent replicons o circular DNA dispensable several genes drug resistance metabolic enzymes virulence factors host range o restricted or broad Viral Structure Nucleic acid, Capsids, Envelopes Envelopes Composition Lipids from host cell membrane Proteins Glycoproteins Function Camouflage? Recognition/attachment to host cell Viral Shape Helical , polyhedra and complex Viral Shape determined by protein capsid Classification of Viruses Host range Very specific Enveloped or nonenveloped Type of nucleic acid Shape Bacteriophage Viruses that infect bacteria. Propagation of Bacteriophage Lytic or Lysogenic Phaes Animal Viruses Multiplication Cycle of An Animal Virus 1. Attachment Glycoprotein spikes bind to receptors on host cell surface Multiplication Cycle: Entry II 2. Entry (Fusion of cell membrane with viral envelope) Multiplication Cycle 3. Maturation/Assembly New nucleocapsids self-assemble Mode of infection Persistent Infections Persistent Infections Transformation of Host Cells Cultivation Tissue culture Embryonated Chicken Eggs Signs of viral propagation Death of embryo Pocks on membranes 4. Viroids Infectious RNA that attacks plants. Catalyze hydrolysis of RNA.