MEDT 14 | CLIN. BACTERIOLOGY (LEC)
Jonnel P. Andaya || 3rd Year
Transcribed by: Last Name, FN | Last Name, FN
OUTLINE:
I. INTRODUCTION
II. GENERAL CHARACTERISTICS
III. TREPONEMA
A. T. PALLIDUM SUBSP. PALLIDUM
B. LABORATORY DIAGNOSIS
C. T. PALLIDUM SUBSP. PERTENUE
D. T. PALLIDUM SUBSP. ENDEMICUM
E. T. CARATEUM
F. T. DENTICOLA & T. SOCRANSKI
IV. BORRELIA (BLOOD SPIROCHETES)
A. B. RECURRENTIS
B. B. HERMSII, B. TURICATE, B. DUTONI & B.
PARKERI
C. B. BURGDORFERI SENSU STRICTO, B. GARINII
& B. AFZELII
D. RELATED INFECTION AND DISEASES
E. LABORATORY DIAGNOSIS
V. LEPTOSPIRA
A. LEPTOSPIROSIS
B. LABORATORY DIAGNOSIS
INTRODUCTION
● Spirochetes are gram-negative, motile, spiral bacteria,
from 3 to 500 m (1 m = 0.001 mm) long. Spirochetes are
unique in that they have endocellular flagella (axial fibrils,
or axial filaments), which number between 2 and more than
100 per organism, depending upon the species. Each axial
fibril attaches at an opposite end and winds around the cell
body, which is enclosed by an envelope. Spirochetes are
characteristically found in a liquid environment and some of
which are seriou spathogens for humans, causing diseases
such as syphilis, yaws, Lyme disease, and relapsing fever.
Examples of genera of spirochete include Spirochaeta,
Treponema, Borrelia, and Leptospira.
GENERAL CHARACTERISTICS
● Group of bacteria that belongs to the order Spirochetes
● Organisms have unusual morphologic features
● Facultatively anaerobic or aerobic
● Exhibit various types of motility patterns
● Have fibrils of axial filaments which are flagella-like
organelles that wrap around the bacterial cell wall and
facilities motility (exhibiting a “corkscrew-like” winding) of
the organisms.
● General microscopy: slender, helical, and unicellular
bacteria
● Genera: Treponema, Borrelia, and Leptospira
Varying Characteristics of the Genera within the order
Spirochetes
1. Number of axial fibrils or periplasmic flagella
2. Number of insertion disks
a) An insertion disk is a plate-like structure where fibrils
are attached. It is found near the terminal end of the
cell.
3. Biochemical and metabolic features
TREPONEMA
● The name of the genus came from the Greek words
Trepein, which means “to turn,” and nema, which means
“thread” that when combined means” turning thread”
● The species are difficult to visualize with a bright field
microscope but cab be observed with a dark field
microscope
LESSON 17.
SPIROCHETES
● They only affects human and have not been observed to
be cultivated after multiply passage in vitro
● Most species stain poor with Gram or Giemsa stains
● Reproduction: Transverse fission
● Microscopy:
o Thin, spiral organisms (4 to 14 spirals/ organisms)
with three axial filaments and one insertion disk
o Cell ends are pointed and covered with sheath
o (+) Corkscrew motility
● Species: T. pallidum and T. carateum
TREPONEMA PALLIDUM SUBSP. PALLIDUM
● Causative agent of syphilis
● Microaerophilic which survives longer in the presence of
3% to 5% oxygen
● Killed rapidly at 42 degrees Celsius, so it is used as a basis
for syphilis therapy.
● Remains visible in whole blood or plasma for at least 24
hours, which is of potential importance in blood transfusion
● Can cross intact mucous membranes and the placenta,
and spread throughout the body.
● Dark field microscopy: Appears white against a dark
background and long with fine spirals that have 10 – 13
coils and three fibrils/periplasmic flagella.
● Generation time: 30 hours
● The two types of antibodies are in used in the diagnostic
test for the species are as follow:
o Treponema reagin
o Non-treponema reagin
SYPHILIS (French disease/Italian disease/ The Great Pox)
● Disease of the blood vessels and the perivascular areas.
● Also known as the “great imitator” because it can copy
and assume many clinical manifestations.
● Some treated syphilitic patients exhibit a biologic cures and
loses serologic activity.
● Transmission: Sexual contact, congenital (vertical)
transmission; skin contact with an active lesion (primary or
secondary syphilitic lesion); transfusion of fresh blood;
injuries form contaminated needle sticks; and handling of
specimens
● Symptoms: Chancre, fever, sore throath, headache,
rashes (palms and soles), and gummas on skin
● The stages of syphilis are as follows:
o Primary syphilis
▪ Characterized by the appearance of
hunterian or hard chancre, which is an
infectious primary lesion that is painless
and is usually seen at the site of
inoculation (most commonly on the
genitalia).
▪ Develops 10 – 90 days after infection.
▪ No systemic sign and symptoms are
evident in this stage.
o Secondary syphilis
▪ Develops 2 to 12 weeks after the
appearance of chancre.
▪ All lesions that are observe seen in this
phase are highly infectious.
▪ In this stage, the chancre heals but the
organisms are still disseminated in
various tissues via the bloodstream
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▪ Symptoms: Fever, sore throat, weight
loss, headaches, and rashes (palms and
soles)
o Latent stage
▪ Is the period in which the disease
becomes subclinical but not necessarily
dormant
▪ Occurs within more than a year of
infection.
▪ In this stage, diagnosis can be made
only by serologic tests.
o Tertiary stage/ Late syphilis
▪ tissue-destructive phase
▪ appears 10 to 25 years after the initial
infection
▪ In this stage, individuals are not usually
infectious.
▪ Complications: Central Nervous Disease
(Neurosyphilis), Cardiovascular
abdnormalities, eye disease, and
granuloma-like lesions (gummas)
LABORATORY DIAGNOSIS
● Specimen: Skin lesions (cleaned with saline)
● Oral lesion should not be examined because non-
pathogenic spirochetes will lead to false-positive results.
1. Microscopic examination
● Direct microscopic examination of exudates is
recommended
● The demonstration of motile treponemes from the chancre
specimens is diagnostic for primary syphilis
● The fluorescent antibody staining of the specimen can also
be performed
● Definitive test: Dark field microscopy for the observation
of motility
● Stain used: Levaditi’s stain and Fontana-Tribondeau
stains
● Direct detection in lesion: Fluorescein isothiocyanate-
labeled (FITC) antibodies
2. Serodiagnosis
It is basis of diagnosis especially in the secondary and tertiary
stages of the syphilis.
● Non-treponemal test/ non-specific test- screening test
o It detects the presence of non-specific antibodies
or antibody-like proteins (regain or Wassermann
antibodies) to lipoprotein materials from damaged
cells and cardiolipin from treponemes.
o It is used to monitor the treatment of the syphilis
o Principle: Reagin binds to the test antigen
cardiolipin-lecithin disease research laboratory
(VDRL) test, unheated serum regain (USR),
toluidine red unheated serum test (TRUST),
ELISA
o Sample: Serum CSF
o Reagent: Combination of cardiolipin
(phospholipid), lecithin, cholesterol, choline
chloride, and charcoal
o (+) Result: Flocculation
o Causative of false-positive RPR and VDRL test
result: Old age, pregnancy, hepatitis, rheumatic
fever, systematic lupus erythematosus (SLE) and
infectious mononucleosis
● Treponemal test/Specific test- confirmatory test
o It detects the presence of antibodies to
treponemal antigens
o There are two (2) types of treponemal/specific test:
⮚ Indirect fluorescent antibody test
(FTA-ABS)
● The procedure involves the
overlaying of whole treponemes
(T. pallidum Nichols strain) that
are fixed to a slide with a
heated serum from possible
syphilis patients.
● It is used to confirm positive
VDRL or RPR
● (+) Result: Fluorescent of the
treponemes
⮚ Treponema pallidum particles
agglutination
● It uses gelatin particles that are
sensitized with T. pallidum
antigens
● (+) Result: Agglutination in the
presence of anti-treponemal
antibodies
3. Molecular Test
● PCR is used for neurosyphilis detection (AIDS patients)
● Western blot is used for the detection of congenital syphilis
NOTES TO REMEMBER:
 RPR does not require the heating of the serum and is not
recommended for
 CSF VDRL is recommended for the diagnosis of
neurosyphilis using CSF specimens
 Reagents for the VDRL must be freshly prepared and the
patient’s serum must be heated at 56 dgeree Celsius for
30 minutes (complement inactivation).
 Charcoal particles are used for the visualization of
flocculation.
TREPONEMA PALLIDUM SUBSP. PERTENUE
● Causative agent of yaws or frambesia tropica.
● Acquired by direct contact through skin breaks with an
infected lesion.
● Yaws is a non-venereal infection that is characterized by
random bodily chronic ulcerative sores which eventually
lead to tissue and bone destruction and then to crippling if
left untreated.
TREPONEMA PALLIDUM SUBSP. ENDEMICUM
● Causative agent of endemic non venereal syphilis or
bejel.
● Transmitted by direct contact with active lesions and
contaminated fingers and utensils.
● Bejel is a non-venereal syphilis that is characterized by the
appearance of a primary lesion usually on or near the
mouth and followed by the development of pimple-like
sores on the trunk, arms, and legs.
TREPONEMA CARATEUM
● Causative agent of pinta or carate.
● Acquired by contact with infected skin.
● Pinta is an infection of the skin that is characterized by the
appearance of a primary lesion or a gradually enlarging
papule with enlargement of the regional lymph nodes. This
is followed by a generalized red to slate-blue macular rash
in 1 to 12 months.
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TREPONEMA DENTICOLA & TREPONEMA SOCRANSKI o There is a presence of recurring chronic arthritis
● They cause ulcerative gingivitis and chronic (acrodermatitis chronica atrophicans) that may
periodontitis continue for years.
o Infected individuals may also develop
● Related to T. pallidum. demyelination of neurons with symptoms of
neurons with symptoms of Alzheimer’s disease
BORRELIA (BLOOD SPIROCHETES) and multiple sclerosis.
● Slow-growing spirochetes that multiply by binary fission.
● Composed of 3 to 10 loose coils and is actively motile LABORATORY DIAGNOSIS
1. Microscopic Examination
● Species have 15 ot 20 axial filaments and two insertion
disks. ● Leptospira species can be stained with Giemsa or Wright’s
● Stain well with Giemsa stain and can be visualized by stain.
using brightfield microscopy. ● Dark field microscopy can be used for the detection of the
● Species that have been grown in vitro are microaerophilic. organism in blood cultures after two to three weeks of
incubation at 35 degrees Celsius
● Species: B. recurrentis, B. dutoni, B. hermsii, B. turicatae,
B. parkeri, B. afzelii, B. burgdorferi, and B. garinii A. Relapsing fever
BORRELIA RECURRENTIS ⮚ Specimen of choice: Peripheral blood
● Agent of louse-borne/epidemic/European relapsing fever. ⮚ Spirochetes in peripheral blood are
stained with Giemsa or Wright’s stains
● Vector: Louse (Pediculus humanus) where the organisms appear blue-
● Reservoir: Humans colored.
B. Lyme Disease
B. HERMSII, B. TURICATE, B. DUTONI, & B. PARKERI ⮚ Specimens: Blood, CSF and biopsy
● Agents of tick-borne relapsing fever/endemic/American specimens
relapsing fever. ⮚ For tissue sections, Warthin-Starry stain
● Vector: Soft ticks (Ornithodoros) is used
⮚ For blood and CSF, acridine orange or
B. BURGDORFERI SENSU STRICTO, B. GARINII, & B. Giemsa stain is used
AFZELII
● Agents of Lyme disease 2. Culture
● Vector: Hard ticks (Ixodes)- Ixodes pacificus, Ixodes ● Culture media: Barbour-Stoenner-Kelly medium or chick
scapularis (deer ticks), Ixodes persulcatus, Ixodes dammini) embryo
● Transmission: Bite of the Ixodes ticks ● The organism is slow –grower and requires 7 to 14 days at
● Natural hosts of ticks: Deer and rodents (Peromyscus 35 degrees Celsius.
leucopus or white-footed mouse)
● All stages of tick (larval, nymph, and adult) can harbor the 3. Serodiagnosis
organisms and transmit disease. ● Relapsing fever
● The organisms are maintained in the environment by o Serological test reveals increased titers to Proteus
horizontal transmission from infected nymphal ticks. OXK antigens (up to 1:80).
● Lyme Disease
RELATED INFECTION AND DISEASE o Serology is the standard method for the
● Relapsing fever diagnostic testing of this disease.
o Acute infectious disease with recurring febrile o IgM and IgG antibodies are detected in the
episodes (2 to 10 relapses) serum
o Symptoms: Fever, headache, myalgia ( 2 to 15 o Serology test: ELISA and IFA
days after infection) o ELISA and IFA are used as the first-line tests
for the Lyme disease antibody.
● Lyme disease
o Acute, recurring inflammatory infection involving
4. Molecular Test
the large joints like those of the knees.
o The hallmarks of the infection are erythema ● PCR is important in diagnosis of B. burgdorferi DNA in
migrans (bull’s eye lesion on the skin) and urine
swelling.
LEPTOSPIRA
Three (3) stages of Lyme Disease ● Genus belongs to the family Leptospiraceae under the
● First stage order Spirochaetales.
o The presence of erythema migrans or a red, ring- ● Obligate aerobes which can be grown in artificial media.
shaped lesion with a central clearing or a “bulls- ● Tightly coiled and are highly motile with hooked ends.
eye-like” lesion at the site of the tick bite is
diagnostic of Lyme disease. ● Live in the lumen of the renal tubules and shed into the
o Signs and symptoms: Headache, fever, muscle, urine
joint paint, and malaise ● Recommended animals for cultivation: Hamster and
● Second stage guinea pigs
o It may start weeks to months after infection. ● Microscopy: Tightly coiled, thin flexible organisms with
o There is dissemination of the organisms to the two long axial filaments that exhibit a spinning motility
other parts of the body. ● Species: L. interrogans (pathogenic species) L. biflexa
o Symptoms: Neurologic disorders (Meningitis) and (non-pathogenic species)
nerve palsy
● Virulence factor: Hemolyis (L. interrogans)
● Third stage
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● Growth factor: Hemoglobin and thiamine ● The diagnosis of leptospirosis is made by demonstrating
● Generation time: 6 to 16 hours seroconversion.
● Animal reservoir: Rats and dogs 4. Molecular Test
● Related disease: Leptospirosis
● It detects leptospiral DNA in infected patients.
LEPTOSPIROSIS (INFECTIOUS JAUNDICE) ● Methods: Polymerase chain reaction (PCR) and
● Zoonotic disease in humans that is caused by L. hybridization techniques
interrogans.
● Can be acquired in home and recreational settings
(swimming pools)
● Symptoms: Fever, headache, myalgia, anorexia, and
vomiting
● Mode of transmission:
o Entry through breaks in skin, mucous membrane,
or conjunctiva
o Direct contact with urine of carriers like rats
o Contact with bodies of water that are
contaminated with the urine of the carriers
o Upon entry, leptospires rapidly invade the
bloodstream and spread throughout the CNS and
kidneys

Types of Leptospirosis
1. Icteric leptospirosis or Weils syndrome
a) It is severe form of illness that affect the liver and
kidney, and cause vascular dysfunction.
b) Death can occur in up to 10% of the cases.
2. Anicteric leptospirosis
a) Symptoms: Septicemic stage of infection, high fever,
and severe headache (3 to 7 days) followed by the
immune stage.
b) Hall mark of immune stage: Aseptic meningitis

LABORATORY DIAGNOSIS
● Specimens: Blood, CSF, and tissue for the bacterimic
phase (first week); urine for the immune phase (second
week)

1. Microscopic Examination
● Darkfield microscopy can be used for the detection of
motile leptospires in the specimens.

2. Culture
● Culture media: Fletcher’s medium, Ellinghausen-
McCullough-Johnson-Harris (EMJH) medium, Bovine
serum albumin, Stuarts broth, and Noguchi’s medium
● Fletchers and EMJH media are semi-solid media
● The culture media are used in the direct inoculation of
leptospirosis form the blood, CSF, and urine specimens.
● Fletcher’s and EMJH are semi-solid media
● Urine should be inoculated immediately because the
acidity may harm the spirochetes within the specimen.
● The organisms grow below the surface, and the plates
should be examined weekly for the presence of growth

3. Serodiagnosis
● Commonly used methods for antigen detection: ELISA,
radioimmunoassay (RIA) and immunomagnetic capture
● Methods for antigen detection in tissues:
Immunofluorescens and immunohistochemistry (IHC)
● Reference method: microscopic agglutination (MA) using
living cells
● Immunoglobulin antibodies (IgM) are identified within the
first week of the disease or until several months later
● The microscopic agglutination test identifies leptospiral
antibodies.