Download Rickettsia, Chlamydia, Mycoplasma

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
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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.
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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.
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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
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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
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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.
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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
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 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
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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
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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
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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.