Chair of Microbiology, Virology, and Immunology
Spiral-shaped
pathogenic bacteria
Lecturer Prof. S.I. Klymnyuk
Groups of spiral shaped bacteria,
which are pathogenic for human
Vibrio
 Campylobacter
 Helicobacter
 Spirilla
 Spirochetes

– Treponema
– Borrelia
– Leptospira
Evolution of spiral-shaped bacteria
Vibrio, Campylobacter, Helicobacter and
Spirilla develop together with Eubacteria.
Spirochetes are differed according to their
morphological and physiological signs.
There is hypothesis about their
intermediate evolution position between
Protozoa and Bacteria
Morphlogy of Campylobacter
Morphlogy of Campylobacter
S-shaped
form
S-form
Comma-shaped form
Spiral-shaped
form
Campylobacter jejuni
42oC 48h microaerophilic conditions
Main biochemical differences between
Campylobacter and Helicobacter
Species
Cata- Н2S Hippurate Oxilase
test
dase
Urease
Growth
at 42 0С
C. jejuni
+
+
+
+
-
+
C.coli
+
+
-
+
-
+
C. fetus
+
+
-
+
-
-
H.pylori
+
+
-
+
+
-
Campylobacteriosis in human





Diarrhea (enterococlites), sometimes bloodly
Endoracditis, pericarditis, meningitis,
encephalitis, septicemia
Disease of genitourinary tract
Pathology of pregnancy (abortion, premature
delivery)
Autoimmune pathology with alteration of
central nervous system
Helicobacter pylori



History
In 1982 following the Easter holidays in
the Microbiology Dept of the Royal
Perth Hospital a culture of a spiralshaped bacterium from gastric biopsies
of patients with gastritis was isolated
The discovery was by Dr.Robin Warren
and Dr.Barry Marshall
Noble
Prize inNobel
2005 prize in 2005
•2005

This discovery have revolutionised the diagnosis, treatment
and prognosis of upper gastrointestinal disease

H. pylori causes gastritis in over half of the world`s population
and is the aetiological agent of 95% of duodental ulcers, 70-80%
of gastric ulcers and has casual rate in probably up to 60-70% of
Gastric Cancer
World-wide prevalence of H. pylori
H. pylori diseases in human

Chronic gastritis

Gastric and duodenal ulcer

Adenocarcinoma and BLymphoma of stomach

Alterations of
cardiovascular system?
Ulcerated gastric adenocarcinoma
Evidances of etiologic role of
Helicobacter pylori in pathogenesis of
ulcer disease
> 95% patients with duodenal ulcer and
> 80 % patients with gastric ulcer are infected
with H. pylori: this level is more then in healthy
persons;
 In persons, which are infected with H. pylori,
duodenal ulcer develops more frequently then in
non-infected ones;
 Antibacterial drugs have medicinal effect and
safely prevent ulcer disease relapses

Morphology of Helicobacter
Helicobacter pylori in mucous
membrane of a stomach
Helicobacter pylori
Peculiarities of Helicobacter pylori






Microaerophilic bacterium (5% О2, 5-10 % СО2);
They require special nutrient media, a surface of
medium must be moist);
Selective medium required for isolation-10%
sheep blood agar + selective antibiotic
supplement
They give growth in 48-72 hours, when
antibiotics are used – in 13 days;
They have high urease, catalase and oxidase
activity;
They are motile;
They are polymorphic bacteria (comma-shaped,
spiral-shaped, coccal-shaped forms)
Cultivation of Helicobacter pylori
Growth of H. pylory
Pathogenesis of ulcer disease
1.
Infection develops
in antral part of
stomach
2.
Inflammation
causes gastritis,
duodenitis, but
sometimes it is
without any
symptoms
3.
Ulcer is result of
inflammation, its
complication perforation and
hemorrhage
Pathogenicity factors of H.pylori
Enzymes of virulence:
– Urease
– Phospholipase А
– Proteases
 Аdhesins
 Toxins:
– Endotoxin
– Exotoxin-cytotoxin (factor which is responsible
for formation of vacuoles)

Bacteriology
Catalase: antioxidant and protects from
toxic oxygen metabolites from activated
neutrophils
 Protease: further degrade mucus
 Phospholipase: alter phospholipid content
of gastric barrier to change surface
tension, hydrophobicity, and permeability
 Receptor-mediated adhesion: Cag genes
encode bacterial membrane proteins

Detection of vacuolizing factor in cell
culture
Positive results
Negative results
фактору
Survival factors of
Helicobacter pylori in a stomach





Neutralization of acidic
contents of the stomach near
bacteria (urease, “altruistic
autolysis”)
Active invasion in mucous
layer which covers stomach
epithelium (lophotrichates)
Ability to adhere to
epitheliocytes of stomach
(fimbria with hemagglutinin
activity)
Micro-aerophilism – for
survival within the mucous gel
Evasion of Immune response
Immune Response
Recall H Pylori is non-invasive but stimulates
hearty inflammatory/immune response
 Marked increase in platlet activation and
aggregation: contribute to microvascular
dysfunction and inflammatory cell recruitment
 Antigenic substances: heat shock protein,
urease, lipopolysaccharide (all activate T cells)

– Cellular disruption at epithelial tight junctions
enhances antigenic presentation

Increased IL-1, IL-6, TNF-alpha, and most
notably IL-8
– IL-8: chemotactic, activates, recruits neutrophils
Immune Response
Also stimulates B cell response (IgG and IgA)
locally and systemically (role of local antibodies
unclear)
 IgM antibodies: insensitive indicator of acute
infection (and not clinically useful)
 IgA and IgG remain present while infection
active and decrease after infection cured
 Antibodies to CagA protein detectable in gastric
tissue and serum (more virulent organism)

Reservoir
Humans: major reservoir but seen in
primates and domestic cats (may transfer
to humans!)
 Sheep: natural host?

– H pylori seen in milk and gastric tissue
– Higher infection rate in shepherds!
Transmission
Person to person: isolations of genetically
identical strains from multiple family members
 Fecal/oral: contaminated water in developing
countries (h pylori can remain viable in water
for several days)
 Oral/oral: seen in dental plaque but prevalence
is low

– dentists don’t have higher prevalence

Iatrogenic infection: from endoscopes
– GI docs/nurses increased risk for H pylori infection!
Diagnosis of Helicobacter pylori
Bacteriologic
Isolation of causative agent
Hystologic
Examination of causative
agent in histological sections
and biopsy specimen
Urease
Examination of biochemical
activity in biopsy specimen
Respiratory
test
Examination of of urease
activity of causative agent in
the stomach with help of C
isotopes (13С, 14С)
Immunologic
ELYSA, examination of
bacterial antigens in feces
Chain
Biopsy specimen
polymerase reaction
Diagnosis
MODALITY
ADVANTAGES
DISADVANTAGES
Endoscopy with
Biopsy ( must be
performed
symtoms of
Gastriccarcinoma
or
> 50Years)
Culture
Permits inspection of
pathology, allows detention of
ulcers, neoplasms.
Invasive, expensive, time
consuming.
Permits determination of
antimicrobial susceptibilities
and pathogenic features of
isolates.
Not optimally sensitive in
most laboratories.
Requires several days for
results.
Generally more sensitive than
culture. Allows direct
visualisation of organism and
extent and nature of tissue
involvement.
Gastritis may be patchy
and biopsy may be
performed on wrong
area. Insensitivity to
detect small numbers of
organisms. Requires
several days for results.
Performed in those
Who have failed 2nd
Line therapy
Histology
Diagnosis
Urease Detection
Rapid, most positives seen
within 2 hours
Increased sensitivity
requires longer incubation.
May be false positives with
bacterial overgrowth.
Serology
Not reliable in young
children
Noninvasive, rapid,
quantitative, inexpensive.
No determination of
lesions or pathology, no
antimicrobial
susceptibility. Not rapidly
responsive to therapy.
Urea Breath Tests
90% sensitivity and
specificity
Relatively non-invasive,
relatively rapid, quantitative,
rapidly responsive to therapy.
Most valuable for assessing
response to eradication therapy
after 4-8 weeks.
Involves expensive
instrumentation or
administration of
radioisotopes. More
invasive and less
convenient than serology.
No determination of
lesions or pathology, no
antimicrobial
susceptibility.
Urease test for diagnosis of
helicobacteriosis
Test-system for diagnosis of
helicobacteriosis
Standard triple therapy-Eradication therapy, which
is probably the most widely used treatment for eradication
of H. pylori.-7days mimimal
Proton pump inhibitor
B.D. (e/g Lansoprazole 30 mg BD)
+
Clarithromycin 500mg
B.D.
1/52 90% plus effective
+
Amoxycillin 1g
B.D.
Or If penicillin allergic
Proton pump inhibitor B.D. (e/g Lansoprazole 30 mg)
Clarithromycin 500mg B.D.
Metronidazole 400 mg B.D
If treatment failure refer to Gasterenterologist
Sodoku (Spirillum minor)
Rat-bite fever

A febrile, systemic illness usually acquired by
direct contact with rats or small rodents

Streptobacillus moniliformis
 Spirillum minor
 Bites of lab rats are an increasing source of
infection
 Two distinct forms
 “sodoku” – S. minus
 Semticemia – S. moniliformis, epidemic
arthritic erythema or Haverhill fever
Rat-bite fever






Clinical manifestations are similar
Both produce a systemic illness characterized
by fever, rash, and constitutional symptoms
Clinical differentiation is possible
Semticemia – S. moniliformis, generalized
morbilliform eruption to include palms and
soles, may become petechial
“sodoku” – S. minus, bite site is often
inflamed and may become ulcerated
Eruption begins with erythematous macules
on the abdomen, which enlarge, become
purplish red and forming extensive indurated
plaques
Taxonomy
Order: Spirochaetales
Family: Spirochaetaceae
Genus: Treponema
Borrelia
Family: Leptospiraceae
Genus: Leptospira
Main pathogenic bacteria of
Spirochetaceae
Genus
Species
Subspecies
Treponema
T.pallidum
T.pallidum
T. pallidum
T.сarateum
T.vincentii
Borellia
B.recurrentis
B.caucasica,
B.duttoni,
B.persica та
ін
Epidemic relapcing fever
Endemic relapsing fever
B.burgdorferi
Lyme disease
Icterohaemorrhagiae
Leptospirosis
Leptospira
pallidum
endemicum
Pertenue
Disease
Syphilis
Bejel
Yaws
Pinta
Vincent’s angina
General Overview of Spirochaetales
 Gram-negative spirochetes
• Spirochete from Greek for “coiled hair”
Extremely thin and can be very long
 Tightly coiled helical cells with tapered ends
 Motile by periplasmic flagella (a.k.a., axial fibrils
or endoflagella)
 Outer sheath encloses axial fibrils wrapped around
protoplasmic cylinder
• Axial fibrils originate from insertion pores at both poles of cell
• May overlap at center of cell in Treponema and Borrelia, but
not in Leptospira
• Differering numbers of endoflagella according to genus &
species
Spirochetes structure
CrossSection of
Spirochete
with
Periplasmic
Flagella
NOTE:
endoflagella,
axial fibrils or
axial
filaments.
Spirochetes morphology
Treponema
Leptospira
Borrelia
Spirochetes ultrastructure
Borrelia
Leptospira
Classification of Human
Treponema

Non-pathogenic
– T.denticola
– T.macrodenticum
– T.orale

Conditionally
pathogenic
– T.vincentii

Pathogenic
– T.pallidum pallidum
(syphilis)
– T.pallidum endemicum
(bejel)
– T.pallidum pertenue
(yaws)
– T.carateum (pinta)
The treponemes responsible for these diseases cannot be distinguished
serologically, morphologically, or by genome analysis, and they have not been
successfully cultivated on artificial media.
Vincent’s angina
Noma (gangrenous stomatitis)
Historical Background


Most accepted theory is
that Christopher
Columbus and his Crew
brought back the
bacteria which caused
the disease Syphilis
from the journey to the
New World.
No distinctive signs of
syphilis were found in
Europe before he came
back.
http://www.solarnavigator.net/history/christopher_columbus.htm
Background
Between 1905 -1910, Schaudinn &
Hoffman identified T pallidum as the
cause of syphilis
 The name "syphilis" was coined by
Italian physician and poet Girolamo
Fracastoro in his Latin written poem
“Syphilis sive morbus gallicus”
("Syphilis or The French Disease") in
1530

General Characteristics of
Treponema pallidum
 Too thin to be seen with light microscopy in
specimens stained with Gram stain or Giemsa stain
• Motile spirochetes can be seen with darkfield
micoscopy
• Staining with anti-treponemal antibodies labeled with
fluorescent dyes
 Intracellular pathogen
 Cannot be grown in cell-free cultures in vitro
• Koch’s Postulates have not been met
 Do not survive well outside of host
• Care must be taken with clinical specimens for
laboratory culture or testing
Treponema in tested material
Cell attacked by treponema
Virulence Factors of T. pallidum
 Outer membrane proteins promote adherence
 Hyaluronidase may facilitate perivascular
infiltration
 Antiphagocytic coating of fibronectin
 Tissue destruction and lesions are primarily
result of host’s immune response
(immunopathology)
Epidemiology of T. pallidum
 Transmitted from direct sexual contact or from
mother to fetus
 Not highly contagious (~30% chance of acquiring
disease after single exposure to infected partner) but
transmission rate dependent upon stage of disease
 Long incubation period during which time host is
non-infectious
• Useful epidemiologically for contact tracing and
administration of preventative therapy
 Prostitution for drugs or for money to purchase drugs
remains central epidemiologic aspect of transmission
Pathogenesis of T. pallidum
 Tissue destruction and lesions are primarily a
consequence of patient’s immune response
 Syphilis is a disease of blood vessels and of the
perivascular areas
 In spite of a vigorous host immune response the
organisms are capable of persisting for decades
• Infection is neither fully controlled nor eradicated
• In early stages, there is an inhibition of cell-mediated
immunity
• Inhibition of CMI abates in late stages of disease, hence
late lesions tend to be localized
Pathogenesis of T. pallidum (cont.)
Primary Syphilis
Primary disease process involves invasion of mucus
membranes, rapid multiplication & wide
dissemination through perivascular lymphatics and
systemic circulation
Occurs prior to development of the primary lesion
10-90 days (usually 3-4 weeks) after initial contact the
host mounts an inflammatory response at the site of
inoculation resulting in the hallmark syphilitic lesion,
called the chancre (usually painless)
• Chancre changes from hard to ulcerative with profuse
shedding of spirochetes
• Swelling of capillary walls & regional lymph nodes w/ draining
• Primary lesion heals spontaneously by fibrotic walling-off
within two months, leading to false sense of relief
Hard chancre
Primary syphilis lesion, chancre
57
Pathogenesis of T. pallidum (cont.)
Secondary Syphilis
 Secondary disease 2-10 weeks after primary
lesion
 Widely disseminated mucocutaneous rash
 Secondary lesions of the skin and mucus
membranes are highly contagious
 Generalized immunological response
Clinical findings of Syphilis
Secondary syphilis
Generalized
Mucocutaneous
Rash
Hetchinzone
teeth
Manifestations of syphilis
60
Pathogenesis of T. pallidum (cont.)
Latent Stage Syphilis
Following secondary disease, host enters latent
period
•First 4 years = early latent
•Subsequent period = late latent
About 40% of late latent patients progress to
late tertiary syphilitic disease
Pathogenesis of T. pallidum (cont.)
Tertiary Syphilis
 Tertiary syphilis characterized by localized
granulomatous dermal lesions (gummas) in which
few organisms are present
• Granulomas reflect containment by the immunologic
reaction of the host to chronic infection
 Late neurosyphilis develops in about 1/6 untreated
cases, usually more than 5 years after initial infection
• Central nervous system and spinal cord involvement
• Dementia, seizures, wasting, etc.
 Cardiovascular involvement appears 10-40 years
after initial infection with resulting myocardial
insufficiency and death
Tertiary syphilis
Progression of Untreated Syphilis
Late benign Gummas in skin and soft tissues
Tertiary Stage
Pathogenesis of T. pallidum (cont.)
Congenital Syphilis
 Congenital syphilis results from transplacental
infection
 T. pallidum septicemia in the developing fetus and
widespread dissemination
 Abortion, neonatal mortality, and late mental or
physical problems resulting from scars from the
active disease and progression of the active disease
state
Congenital syphilis - early evidence of
infection - bullae and vesicular rash
Multiple, punched out, pale, blistered lesions, with
associated desquamation of palms & plantars
Intraoral mucous
Multiple, punched out, pale, blistered
patches & facial skin lesions, with associated desquamation
lesions
of palms & plantars
Late-onset CS

Manifestations include neurosyphilis and
involvement of teeth, bones, eyes, and 8th
cranial nerve

E.g.: Frontal bossing, short maxilla, high
palatal arch, Hutchinson triad, saddle
nose, and perioral fissure (Rhagades =
bacterial infection of skin lesions )
1.
2.
3.


Hutchinson triad
Deafness (10 – 40 years)
Hutchinson’s teeth = centrally notched, widely-spaced
peg-shaped upper central incisors
Interstitial Keratitis → blindness (5-20 years)
Manifestations include neurosyphilis and involvement
of teeth, bones, eyes, and 8th cranial nerve
E.g.: Frontal bossing, short maxilla, high palatal arch,
Hutchinson triad, saddle nose, and perioral fissure
(Rhagades = bacterial infection of skin lesions )
Notched incisors known as
Hutchinson’s teeth
Metaphyseal osteomyelitis
Radiolucent distal radius & ulna with cupping distal ulna
“Sabre shins” = Osteoperiostitis Tibia
Interstitial keratitis
Possible Complications

Blindness

Deafness

Facial deformity

Neurological problems
Microbiologic diagnosis of Syphilis

Microscopy (native an
fixed material)
– Romanovsky-Giemsa stain
– Phase contrast
– Dark field
Chain polymerase
reaction

Serologic diagnosis
– Wassermann’s test (CFT)
– Sedimentation reartions
(Каhn’s and SachsWitebsky’s tests)
– Microreaction of
Treponema pallidum
immobilization
– IFT, ELYSA, IHAT
Diagnostic Tests for Syphilis
(Original Wasserman Test)
NOTE: Treponemal antigen tests indicate experience with a treponemal
infection, but cross-react with antigens other than T. pallidum ssp.
pallidum. Since pinta and yaws are rare in USA, positive treponemal
antigen tests are usually indicative of syphilitic infection.
Conditions Associated with False
Positive Serological Tests for Syphillis
Prevention & Treatment of Syphilis
 Penicillin remains drug of choice
•
•
•
WHO monitors treatment recommendations
7-10 days continuously for early stage
At least 21 days continuously beyond the early stage
 Prevention with barrier methods (e.g., condoms)
 Refraining from sexual contact with a individual infected
with Syphilis will prevent spreading
 Prophylactic treatment of contacts identified through
epidemiological tracing
Facts
The group with the highest infection rate in
1998 was between the ages of 20 to 39.
 If you were African-American you were three
times greater to be infected with syphilis than
the white population.
 Factors that may increase your risk of being
infected are poverty levels, availability of
medical treatment, and access to educational
opportunities.

Tropic trepanematoses:
pinta (T.carateum), bejel (T.endemicum), yaws (T.pertenue)
Yaws (tropic
granuloma)
Bejel (endemic
syphilis)
Treponema pallidum ssp. endemicum
Bejel (a.k.a. endemic syphilis)
• Initial lesions: nondescript oral lesions
• Secondary lesions: oral papules and mucosal patches
• Late: gummas (granulomas) of skin, bones &
nasopharynx
 Transmitted person-to-person by contaminated
eating utensils
 Primitive tropical/subtropical areas (Africa, Asia &
Australia)
Treponema pallidum ssp. pertenue
(May also see T. pertenue)
Yaws: granulomatous disease
• Early: skin lesions (see below)
• Late: destructive lesions of skin, lymph nodes & bones
 Transmitted by direct contact with lesions
containing abundant spirochetes
 Primitive tropical areas (S. America, Central Africa, SE Asia)
Papillomatous Lesions of
Yaws: painless nodules widely
distributed over body with
abundant contagious
spirochetes.
Treponema carateum
Pinta: primarily restricted to skin
•
•
•
1-3 week incubation period
Initial lesions: small pruritic papules
Secondary: enlarged plaques persist for
months to years
• Late: disseminated, recurrent
hypopigmentation or depigmentation of
skin lesions; scarring & disfigurement
 Transmitted by direct contact with skin
lesions
 Primitive tropical areas
(Mexico, Central & South America)
Hypopigmented Skin Lesions
of Pinta: depigmentation is
commonly seen as a late sequel with
all treponemal diseases
Pinta
Borrelia morphology
Borrelia in blood smear
Epidemiology of Borrelia Infections
Borrelia
recurrentis
Pediculus humanus
Ornithodoros spp.
Borrelia spp.
Borrelia
burgdorferi
Ixodes spp.
Epidemiology of Relapsing Fever
 Associated with poverty, crowding, and warfare
 Arthropod vectors
• Louse-borne borreliosis = Epidemic Relapsing Fever
 Transmitted person-to-person by human body lice
(vectors) from infected human reservoir
 Infect host only when louse is injured, e.g., during
scratching
 Therefore, a single louse can only infect a single person
 Lice leave host that develops a fever and seek normal
temperature host
• Tick-borne borreliosis = Endemic Relapsing Fever
 Sporadic cases
 Transmitted by soft body ticks (vectors) from small
mammal reservoir
 Ticks can multiply and infect new human hosts
Pathogenesis of Relapsing Fever
 Relapsing fever (a.k.a., tick fever, borreliosis, famine
fever)
• Acute infection with 2-14 day (~ 6 day) incubation period
• Followed by recurring febrile episodes
• Constant spirochaetemia that worsens during febrile
stages
 Epidemic Relapsing Fever = Louse-borne borreliosis
• Borrelia recurrentis
 Endemic Relapsing Fever = Tick-borne borreliosis
• Borrelia spp.
Clinical Progression of
Relapsing Fever
Lyme disease
Causative agents:
Borrelia burgdorferi
Borrelia garinii
Borrelia afzelii
B. burgdorferi
Epidemiology of Lyme Borreliosis
 Lyme disease was recognized as a syndrome in
1975 with outbreak in Lyme, Connecticut
 Transmitted by hard body tick (Ixodes spp.)
vectors
• Nymph stage are usually more aggressive feeders
• Nymph stage generally too small to discern with
unaided eye
• For these reasons, nymph stage transmits more
pathogens
 White-footed deer mice and other rodents, deer,
domesticated pets and hard-shelled ticks are most
common reservoirs
Pathogenesis of Lyme Borreliosis
 Lyme disease characterized by three stages:
i.
Initially a unique skin lesion (erythema chronicum
migrans (ECM)) with general malaise
 ECM not seen in all infected hosts
 ECM often described as bullseye rash
 Lesions periodically reoccur
ii. Subsequent stage seen in 5-15% of patients with
neurological or cardiac involvement
iii. Third stage involves migrating episodes of nondestructive, but painful arthritis
 Acute illness treated with phenoxymethylpenicillin
or tetracycline
Erythema chronicum migrans of
Lyme Borreliosis
Bullseye rash
Diagnosis of Lyme Borreliosis
Leptospira morphology
Obligate aerobes
Characteristic
hooked ends (like
a question mark, thus
the
species epithet –
interrogans
Leptospirosis Clinical Syndromes
 Mild virus-like syndrome
 (Anicteric leptospirosis) Systemic with aseptic
meningitis
 (Icteric leptospirosis) Overwhelming disease
(Weil’s disease)
Vascular collapse
Thrombocytopenia
Hemorrhage
Hepatic and renal dysfunction
NOTE: Icteric refers to jaundice (yellowing of skin and mucus
membranes by deposition of bile) and liver involvement
Clinical findings of leptospirosis
Clinical Progression of Icteric (Weil’s
Disease) and Anicteric Leptospirosis
(pigmented
part of eye)
Epidemiology of Leptospirosis
 Mainly a zoonotic disease
• Transmitted to humans from a variety of wild and
domesticated animal hosts
• In USA most common reservoirs rodents (rats), dogs,
farm animals and wild animals
 Transmitted through breaks in the skin or intact
mucus membranes
 Indirect contact (soil, water, feed) with infected
urine from an animal with leptospiruria
 Occupational disease of animal handling
Pathogenesis of Icteric Leptospirosis
 Leptospirosis, also called Weil’s disease in humans
 Direct invasion and replication in tissues
 Characterized by an acute febrile jaundice &
immune complex glomerulonephritis
 Incubation period usually 10-12 days with flu-like
illness usually progressing through two clinical
stages:
i. Leptospiremia develops rapidly after infection (usually
lasts about 7 days) without local lesion
ii. Infects the kidneys and organisms are shed in the urine
(leptospiruria) with renal failure and death not
uncommon
 Hepatic injury & meningeal irritation is common