MEDT 14 | CLIN. BACTERIOLOGY (LEC) LESSON 16.

RICKETTSIACEAE,
Jonnel P. Andaya || 3rd Year CHLAMYDIACEAE, AND CELL WALL
Transcribed by: De Los Reyes, J. D. M. | Ybanez, T. M. C.
DEFICIENT BACTERIA
 Causes most serious rickettsial infection
OUTLINE:  Etiologic agents: R. conorii, R. rickettsia
I. Family Rickettsiaceae  Rocky Mountain Spotted Fever
o Most serious rickettsial infection.
A. Rickettsia
o Humans are accidental hosts, ticks are the main
B. Orientia Tsutsugamushi vector an reservoir
C. Anaplasma phagocytophila o Rashes develop on the palms of the hands and soles
D. Bartonella of the feet
E. Coxiella burnetti  Bountonneuse Fever
F. Ehrlicia o Presence of tache noires (black spots) in patients
o Rashes are observed on the face and extremities
G. Laboratory Diagnosis o Rickettsialpox
H. Differential Characteristics of  Pathogenesis:
Rickettsiaceae and Related Organisms o When infected vectors such as ticks, brown ticks, or
II. Family Chlamydiaceae dog ticks bite humans, it will release bacteria and
A. Chlamydia these bacteria can get access to our bloodstream. In
the bloodstream, they have the ability to bind with the
B. Chlamydia trachomatis lining of our blood vessels.
i. Laboratory Diagnosis o They can infect the endothelial cells found in the blood
C. Chlamydia Psitacci vessels for certain and unknown signal.
D. Chlamydia Pneumoniae o They induces phagocytosis.
E. Differential Characteristics of Chlamydia o Inside the endothelial cells, there is a formation of
phagosome, and inside these are the rickettsial
spp. bacteria.
III. Cell Wall Deficient Bacteria o To inhibit phagolysosome formation, they’re going to
A. MYcoplama and Ureaplasma (Mollicutes) secrete phospholipase A (a virulence factor) to
B. Mycoplasma Pneumoniae degrade the membrane of the phagosome.
C. Laboratory Diagnosis o It will then be released in the cytoplasm of the
endothelial cell. And in the cytoplasm of the cell, they
D. Comparison between the Common and will utilize the ATP for replication until it overloads the
Atypical Bacteria endothelial cells. Once overloaded, they can leave the
cell to infect other endothelial cells leaving a damage
and there will be a necrosis.
FAMILY RICKETTSIACEAE o As a result, the patient will manifest rashes.
Sometimes, rashes are in form of black spots (tache
RICKETSSIA noires)
● Extremely small organisms o If RMSF and Bountonneuse Fever are left untreated, it
● Transitional organisms between bacteria and viruses can lead to fatal conditions. Due to increased
● Obligate intracellular parasites. They urvive briefly outside permeability in blood vessels, it can cause edema or
their host. hypobulimia. And if there are vascular disturbances in
o They have functional genes for protein synthesis the kidneys, it may result to kidney failure.
and ATP production.
o They need host to survive because of their limited Typhus Group
amount of ATP for survival.  Causative agent of Endemic typhus
o After the consumption of their ATP, they need to  Mode of transmission: Inhalation of aerosol from dried
utilize their host’s ATP so they can survive and infected flea feces with Rickettsia typhi and Rickettsia
reproduce in the cytoplasm of their host. prowazekii.
● Gram-negative bacteria, motile, and will not grow in cell-  Endemic typhus:characterized by rashes on the face,
free media. palms, and soles of the feet of the sick.
● Requires living cell for growth
● Reproduces through binary fission ORIENTIA TSUTSUGAMUSHI
● Mode of Acquisition: bite of an arthropod vector  It was categorized as a separate genus due to absence of
● Mode of Prevention: avoiding contact with the respective lipopolysaccharides and peptidoglycan, and the presence
vectors. of 54- to 58 kDa major surface proteins.
● Accidental host: humans  Reproduce inside the cell
● Agents of bioterrorism: R. prowazekii, R. rickettsii  It replicates in the cytoplasm of its host cell and is released
through a process that involved “pinching off” the host cell
Three Groups  Mode of acquisition: Bite of an infected arthropod vector
● Spotted Fever  Vector: Leptotrombidium delicense (chigger), specifically,
● Transitional the larval stage of thrombiculid mites
● Typhus  Usually infects ground mammals and ground feeding birds
 Accidental host: Humans and rats
Transmission and Spread  Etiologic agent of scrub typhus. Scrub is a type of
vegetation like grassfields.
● The organisms gain access into humans through skin
 It is characterized by nonspecific signs and symptoms:
abrasions or arthropod bites.
fever, chills, malaise, headache, maculopapular rash, and
● The organisms are disseminated homogenously, thus
one of the clinical manifestations of scrub typhus is black
causing vasculitis in the blood vessels eschar found in auxillary, perianal, and thigh region.
● The members of the typhus group reproduce in the
 If left untreated, it can lead to complications such as
cytoplasm cellular injury and death interstitial pneumonitis, meningitis, encephalitis,
disseminated intravascular coagulopathy, cardiac
Spotted Fever involvement.
 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | RICKETTSIACEAE, CHLAMYDIACEAE, AND CELL WALL DEFICIENT
BACTERIA
 Antibiotics: Tetracycline, but in severe cases, doxycycline
and chloramphenicol
ANAPLASMA PHAGOCYTOPHILA
 It was categorized as a separate genus due to absence of
lipopolysaccharides and peptidoglycan, and the presence
of 54- to 58 kDa major surface proteins.
 Reproduce inside the cell
 It replicates in the cytoplasm of its host cell and is released
through a process that involved “pinching off” the host cell
 Mode of acquisition: Bite of an infected arthropod vector
 Vector: Leptotrombidium delicense (chigger), specifically,
the larval stage of thrombiculid mites
 Usually infects ground mammals and ground feeding birds
 Accidental host: Humans and rats
 Etiologic agent of scrub typhus. Scrub is a type of
vegetation like grassfields.
 It is characterized by nonspecific signs and symptoms:
fever, chills, malaise, headache, maculopapular rash, and
one of the clinical manifestations of scrub typhus is black
eschar found in auxillary, perianal, and thigh region.
 If left untreated, it can lead to complications such as
interstitial pneumonitis, meningitis, encephalitis,
disseminated intravascular coagulopathy, cardiac
involvement.
 Antibiotics: Tetracycline, but in severe cases, doxycycline
and chloramphenicol
BARTONELLA
 The species of this genus are facultative, intracellular, and
Gram-negative bacilli.
 They live within the RBC in their natural mammalian hosts
 The species can be cultivated in CAP with 5% CO2 or in
charcoal yeast extract agar
 Some species exhibit a “twitching motility” in wet mounts
(B.bacilliformis and B. hensalae).
COXIELLA BURNETTI
 It is the only species in the genus Coxiella.
 It is causative agent of Q (Query) fever which is a systemic
infection of the lungs causing pneumonia. It can also
cause hepatitis, liver granuloma, hepatomegaly without
jaundice, encephalitis, meningitis, and endocarditis.
Hepatitis is common in Europe while pneumonia is
common in North America.
 It is extremely contagious and can be considered as a
potential bioterrorism agent
 It can survive extracellularly because of its endospore-like
body.
 It can infect birds and rodents, which in turn excrete the
organisms via their urine, feces, and birth products
 It is not transmitted by arthropod vectors.
 It does not multiply in routine bacteriologic culture media.
 Hard to diagnose
 Modes of acquisition: Inhalation of contaminated aerosol
from dried animal feces and ingestion of contaminated
unpasteurized milk
 Animal reservoir: Cattle, goat, and sheep
EHRLICIA
 It belongs to the family Anaplasmataceae
 The species of the genus are Gram-negative coccobacilli
that undergo an intracellular development cycle following
the infection of circulating WBC’s (replicate ion occurs in
the leukocytes)
 Microscopy: Presence of intravacuolar microcolony that
resembles “mulberries” or a morula (identified through
Wrights-Giemsa Staining)
 It has three developmental stages:
a. Elementary body (infective form)
b. Initial bodies
c. Morulae
 Natural host: humans and animals (deer, dogs)
 Primary vector: Amblyomma americanum (lone star tick)
 Species: E. chaffeensis, and E. ewingii
 Related infection: Human monocytic ehrlichiosis (HME)
LABORATORY DIAGNOSIS
 Specimens: Biopsy of skin tissue, peripheral blood, and
CSF
 Processing of specimens should be done in a biosafety
level 3 laboratory
Direct method
 Immunohistology (Immunofluorescence or
immmunoenzyme stains) of skin biopsy material is a
sensitive and specific test that is used to visualize
Rickettsia in tissue sections
 Giemsa stain is used for the detection of morulae during
the febrile stage of ehrlichiosis.
Culture
 Culture media: Yolk sacs or embryonated eggs and tissue
cultures
 Rickettsia, Ehrlichia, and Anaplasma can be isolated from
human in an antibiotic free, centrifugation-enhanced shell
vial cell culture.
 Columbia blood agar with 5% defibrinated blood is used for
the cultivation of B. bacilliformis
 Lung tissue cell are the preferred medium for C. burnetti.
Serologic Test
 It is the only test that is performed for the diagnosis of
rickettsial diseases ∙ It is used to confirm ricketssioses
during convalescent stage
 Antibodies to Rickettsia (except for R. ricketsii) cannot be
detected until at least two weeks after infection.
 Rickettsioses are seldom diagnosed serologically during
the acute stage of the illness due to the absence of an
early antibody response
 . Indirect immunofluorescent antibody (IFA) assay
i) . Indirect immunofluorescent antibody (IFA) assay
o It is the gold standard serologic test or
reference method for rickettsioses and Q
fever
ii) Weil-Felix reaction
o It is presumptive test for rickettsioses
o The agglutination of certain strains of Proteus
vulgaris by serum from patients with
rickettsial diseases in diagnostic
o Individuals with Q-fever, ehrlichiosis, and
rickettsialpox do not produce Weil-Felix
antibody
iii) Microimmunofluorescent dot test
o It has an excellent sensitivity for detecting
antibodies to Rickettsia
o It is used for the early diagnosis of RMSF
after the onset of symptoms.
iv) Other serologic tests:
o Latex agglutination
o Enzyme immunoassay
o Line blot
 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | RICKETTSIACEAE, CHLAMYDIACEAE, AND CELL WALL DEFICIENT
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Nucleic acid amplification (Polymerase chain reaction or o Reticulate body (RB)

PCR)  It is replicative and non-infectious form
 Cat scratch disease can be confirmed through PCR testing  It is intracellular and metabolically active
of lymph node aspirates and biopsies. form of Chlamydia.
 It directs the reproduction of host cells to
 PCR is a diagnostic tool for ehrlichioses
their own metabolic needs so they
multiply by binary fission
DIFFERENTIAL CHARACTERISTICS OF RICKETTSIACEAE o Elementary body (EB)
AND RELATED ORGANISMS  Infectious form
 It is extracellular form of Chlamydia and
ORGANISMS INFECTION/DISEASE VECTOR/MODE is spherical in shape
OF  It resembles Gram-negative bacilli with a
TRANSMISSION rigid cell wall.
SPOTTED FEVER  It infects the host cell by inducing active
Rickettsia Boutonneuse fever or Ticks phagocytosis
conorii Meditterranean (Rhipicephalus  Its two components are the major outer
spotted fever sanguineus) membrane protein (MOMP) and the
Rickettsia Rocky mountain  Wood ticks lipopolysaccharide antigen
rickettsii spotted fever (RMSF) (Dermacentor  Pathogenesis
andersoni) o Elementary body infects epithelial cell.
 Dog ticks o Epithelial cells are non-professional phagocytic cell.
(Dermacentor They won’t be able to engulf cell. But chlamydia can
variabilis) induce phagocytosis.
 Brown dog o There will be a endocytosis and the EB form of
ticks chlamydia inside epithelial cells.
(Rhipicephalus o In the cytoplasm, these EB forms will be converted
sanguineus into reticulated bodies. These will utilize the ATP
and found in the host cell and they will be going to
Amblyomma replicate until it overloads the epithelial cells.
cajennense) o Once overloaded, they are ready to leave the
TRANSITIONAL epithelial cells to infect other cells but they will convert
Rickettsia Rickettsialpox Mouse mite themselves back to EB form so that after the release
akari (Liponyssoides extracellularly, they will be able to withstand extreme
sanguineus) environmental conditions.
Flea-borne spotted
Rickettsia felis Flea bite or feces o There will be a reverse endocytosis that will release
fever them extracellularly. Leaving cells damage and having
necrosis.
SCRUB TYPHUS GROUP
Orientia Scrub typhus Chigger
CHLAMYDIA TRACHOMATIS
tsutsugamuchi (Leptotrombidium
delicense) bite  It is one of the major sexually transmited pathogens
ANAPLASMATACEAE  It is one of the principal causes of pelvic inflammatory
disease (PID) and ocular trachoma
Ehrlichia Human monocytic Lone star tick
chaffeensis (Amblyomma  Pelvic Inflammatory Disease
ehrlichiosis
americanum) o Includes endometritis and salpingitis
 Can infect epithelial cells found in cervix of women and
Anaplasma Human Deer tick (Ixodes
can ascend in endometrium and fallopian tube.
phagocytophila granulocytotropic scapularis and
anaplasmosis Ixodes pacificus)  Can cause strong inflammation
Coxiella Q fever Inhalation of  Chronic inflammation can cause scarring and occlusions
burnetti aerosol from that’s why it can be associated with infertility and ectopic
infected animals pregnancy
 It can travel through the birth canal where infants can be
Bartonella Trench fever Feces of body
quintana louse (Pediculus infected during birth.
humanus  It is associated with infertility and ectopic pregnancy
corporis)  Ocular trachoma
o One of the leading cause of blindness
Bartonella Cat scratch disease Kitten scratch or
hensalae bite o It can affect the conjunctiva (conjunctivitis), and
cornea
Bartonella Oraya fever and Sandly o Can be vertical transmission
bacilliformis verruga peruana (Lutzomyia) bite
 Natural host: Humans
 Unique features: 10 stable plasmids
FAMILY CHLAMYDIACEAE  Biovars: Lymphogranuloma venereum (LGV), mouse and
CHLAMYDIA pneumonitis trachoma ∙ Serovars of C. trachomatis based
 The species of this genus are non-motile, small (0.2 to 1.5 on the MOMP antigenic differences:
um) and have a Gram negative cell wall o Serotypes A, B, Ba, C- endemic trachoma
 They are obligate, intracellular organisms that require o Serotypes L1, L2, L2a, L2b, L3- Lymphogranuloma
living cells for growth. Venereum (LGV)
 They do not possess cytochrome and cannot synthesize o Serotypes D to K, Da, Ia, Ja- Pelvic Inflammatory
their own ATP Disease, Urethritis, cervicitis, epididymitis,
 They are called “energy parasites” because they depend inclusion conjunctivitis
on the eukaryotic cells of their hosts for metabolism,
growth, and reproduction. Related Infections and Diseases
 They replicate by binary fission in the cytoplasm of infected  Chlamydia infection is transmitted sexually, as well as
cells during birth, from the mother to the infant (vertica)
 Species: Chlamydia trachomatis, Chlamydophila psittaci,  The intraperitoneal spread may cause peritonitis or
and Chlamydophila pneumoniae perihepatitis (Fitz-Hugh-Curtis Syndrome).
 ∙ Chlamydia has two morphologic forms:
 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | RICKETTSIACEAE, CHLAMYDIACEAE, AND CELL WALL DEFICIENT
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o Re-complication of PID trachomatis

o Extrapelvic manifestation  It is the preferred method for identification of
antibodies to C.trachomatis
1. Trachoma  It can be used for the diagnosis of LGV,
o It is chronic inflammation of the conjunctiva that leads trachoma, inclusion conjunctivitis, and
to blindness. chlamydia neonatal infection.
o It can cause distortion of the eyelids (eyelashes  (+) Result: IgM titer of 1:32
become misdirected and turned-in).
o Modes of transmission: Contact with contaminated Immunoassay
objects, hand-to-hand contact with carriers, and  Enzyme immunoassay (EIA) is the most commonly used
through vectors (flies) rapid antigen assay.
2. Lymphogranuloma venereum (LGV)  EIA detect either the outer membrane LPS chlamydia
o It is sexually transmitted disease which has a multi- antigen or the MOMP antigen.
system involvement.
o A small, painless ulcer or papule appears initially and CHLAMYDIA PSITACCI
then nodules (buboes) develop after several weeks  Chlamydola psitacci
3. Inclusion conjunctivitis  It is causative agent of psittacosis or ornothosis
o It is characterized by an abundant eye discharge and  It is endemic pathogen of birds species such as parrots,
a swollen conjunctiva. parakeets, chickens and ducks
o It affects infants who acquire it through aspiration or  Modes of acquisition: Inhalation of infected aerosol from
ocular exposure during birth dried bird excreta or handling of infected birds
 Commonly used test: Complement fixation with a titer
of >1:32
LABORATORY DIAGNOSIS
 Sensitive method: Direct microimmunofluorescence
 Specimen: Urethra and cervical secretion, conjunctiva  Precautionary measures: Only laboratories with biosafety
discharge, nasopharynx and rectal swabs; and materials level 3 facilities can perform the isolation and cultivation of
aspirated from fallopian tubes and epididymis the specimens.
 The use of Dacron or rayon-tipped swabs is preferred.
CHLAMYDIA PNEUMONIAE
Culture  It is formerly known as the Chlamydia pneumoniae strain
 Cannot be cultivated easily using routine culture medium TWAR
because they are obligate intracellular organisms. Living  It is a human pathogen that is transmitted through aerosol
tissue cells for culture is preferred droplets
 Culture media: Buffalo green monkey kidney cells, HeLa  It is one of the major causes of infectious respiratory
229 cells, Hep-2 cells, McCoy cells, and cycloheximide- diseases.
treated McCoy cells  It is associated with pneumonia, bronchitis, pharyngitis,
 Procedure prior to culture: Centrifugation of the and sinusitis.
specimen onto the cell monolayer  Specimen for isolation: Nasopharyngeal aspirates,
 The shell via technique facilitates the adherence of sputum and throat swabs
elementary bodies.  Culture media: HeLa cells or HEp-2 cell lines
 After 48 to 72 hours of incubation, the monolayers are  Preferred method: Microimmunofluorescent assay
stained with iodine or immunofluorescent stain to identify
the presence of inclusion bodies. DIFFERENTIAL CHARACTERISTICS OF CHLAMYDIA
SPECIES:
Cytologic examination
 Cell scrapings from the conjunctiva of newborns or person Properties C. trachomatis C. psittaci C.
with ocular trachoma are used as specimens. pneumonia
 A direct fluorescent antibody (DFA) staining method that e
utilizes the fluorescein isothiocyanate-conjugated Host range Human Birds Humans
monoclonal antibodies is used to identifying the organisms Elementary Round Round Pear-
in infected cells. body shaped
Inclusion Round, Variable, Round,
Antigen detection and nucleic acid amplification morphology vacuolar, dense dense
 Nucleic acid amplification is the most sensitive method for and Halbertstaede Levinthal-
detecting C. trachomatis inclusion r Cole-Lillie
 It is more reliable in symptomatic patients bodies Prowazek bodies
 Lipopolysaccharide (LPS) antigen is the major antigen that bodies
is detected in genus specific serologic tests for chlamydial Stains used Lugol’s iodine Macchiavell Giemsa
infection. stain o stain and stain
 Specimens: Endocervical and urethral swabs Giemsa
stain
Serodiagnosis Glycogen- Present Absent Absent
 The extractable LPS or elementary body with keto- containing
deoxyoctonate is the primary antigen that can be identified inclusions
in genus-specific tests. Susceptibilit Susceptible Resistant Resistant
 A negative test result can exclude chlamydial infection. y to
o Complement fixation sulfonamides
 It identifies family- reactive antibodies Diseases Trachoma, Psittacosis Pneumonia,
 It is used to diagnose LGV LGV, and Pharyngitis
 Genus- specific antigen can be detected by inclusion
using this method Conjunctivitis
 (+) Result: > 1:64 titer Number of 20 10 1
o Microimmunofluorescence (Micro-IF) assay serovars
 It is used for the type-specific antibodies of C.
 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | RICKETTSIACEAE, CHLAMYDIACEAE, AND CELL WALL DEFICIENT
BACTERIA

CELL WALL DEFICIENT BACTERIA o M. hominis is the only species that is capable of
MYCOPLASMA AND UREAPLASMA (MOLLICUTES) growing on BAP and CAP.
 The species of these genera are considered as the o M. pneumoniae requires a bisphasic culture system
and inocubation of up to three weeks in a chamber
smallest free-living organisms that are capable of growing
with 5% to 10% CO2
in artificial media.
 Urea and/or arginine can be incorporated into the media to
 They are pleomorphic organisms that lack a cell wall. detect the presence of U. urealyticum and M. hominis and
 They are found mainly in the oropharyngeal, upper produce an alkaline reaction.
respiratory, and genitourinary tracts  Growth of mycoplasma is Shepard’s 10B arginine medium
 They are slow-growing, fastidious, and facultative is exhibit as red colorations
anaerobes that replicate by binary fission  Blood culture is not recommended for mycoplasmas
 They require sterols (cholesterol) for membrane function  Overlaying suspicious colonies with 0.5% guinea pig RBC
and growth. in PO4-buffered saline is the definitive identification
 They are common parasite of the genital tract and their method of M. pneumoniae
transmission is related to sexual activity.
 Species: M. pneumoniae, M. hominis, M. fermentans, M. Serodiagnosis
pirum, M. penetrans and U. urealyticum  ELISA Is the most commonly used serological method for
diagnosis of Mycoplasma and Ureaplasma.
MYCOPLASMA PNEUMONIAE  The detection of specific IgM in a single serum sample is
 It is the etiologic agent of primary atypical pneumonia or diagnostic of the acute infection that is caused by M.
walking pneumonia pneumoniae.
 It is not included in the normal commensals and it is
extremely fastidious. Manganese chloride urea test
 Mode of acquisition: Inhalation of contaminated aerosol  It is rapid identification test for U. urealyticum
droplets  The reaction for the test is observed under a dissecting
 Initial of disease: Attachment to respiratory mucosal cells, microscope.
evasion form phagocytosis, and modulation of the immune  (+) Result: Dark brown precipitate of manganese oxide
system around the colonies
 U. urealyticum utilizes manganese chloride in the presence
 Culture: SP4 broth- Colonies exhibit a yellow color
of urea
Primary atypical pneumonia: COMPARISON BETWEEN THE COMMON AND ATYPICAL
 It can occur as separate incidents or as outbreaks in BACTERIA
closed population such as in school, military camps, and
within family members Characteristi Bacteria Rickettsi Chlamy Mycopla
cs eae diae smas
A. MYCOPLASMA HOMINIS AND UREAPLASMA Obligate - + + -
UREALYTICUM Intracellular
 The species can be recovered from the genital tract of
DNA and + + + +
healthy adults.
RNA
 They can cause prostatitis and pelvic inflammatory disease.
Ribosomes + + + +
 Colonization among infants occurs during passage through
an infected birth canal and results in the isolation of these Peptidoglyca + - + -
organisms from their nose and throat n
 M. hominis causes postabortal fever and postpartum fever Replication + + + +
in women. by binary
fission
 Culture:
o M. hominis- Colonies exhibit ”fried-egg” appearance Growth on + - - +
on a plate medium non-living
o U. urealyticum- Colonies exhibit dark-brownish lumps media
on an A7 or A8 agar medium. Sensitivity to + + + +
antibiotics
LABORATORY DIAGNOSIS
 Specimens: Throat swab, serum, bronchoalveolar lavage,
putum, and lung tissue for <.
o Pneumoniae; urethral, vaginal or endocervical swab,
blood, urine, prostatic secretions and semen for
genital mycoplasmas.

Direct method
 No direct method or Gram staining can be used for
identification

Culture
 Culture media: Beef or soybean protein with serum, fresh
yeast extract, bisphasic SP4 medium, pleuropneumonia-
like organisms (PPLO) broth or agar with yeast extract
and horse serum, A8 agar, Shepard’s 10B arginine broth
and modified New York City Medium
 Swabs for sample collection: Calcium alginate and
Dacron swabs
 Transport medium: SP4 (sucrose phosphate buffer,
horse serum, and neutral red)
o Mycoplasma and Ureaplasma grow slowly than most
of the other bacteria