Lab2

Sterilization and Disinfection especially for dental

instruments ( Assistant Professor Ali Fakhriddeen)
Sterilization is defined as the process where all the living
microorganisms, including bacterial spores are killed.
Sterilization can be achieved by physical, chemical and
physiochemical means.

Disinfection is the process of elimination of most pathogenic

microorganisms (excluding bacterial spores) on inanimate
objects. Disinfection can be achieved by physical or chemical
methods.

For hygiene and sanitary safety purposes, all instruments not

marked “sterile” must be cleaned, disinfected and sterilized
before each usage to prevent any contamination.

Single use marked products are not approved for re-use .

For your own safety, please wear personal protective

equipment (gloves, safety glasses, etc.) .

Limitations and restrictions on reprocessing

The individual DFU indicates if the useful life of a device might

be reduced by the number of reprocessing cycles. Furthermore,
the appearance of defects such as cracks, deformations (bent,
twisted), corrosion, loss of color coding or marking, are
indications that the devices are not able to fulfill the intended
use with the required safety level .

The water quality has to be convenient to the local regulations

especially for the last rinsing step or with a washer-disinfector .

1
Lab2

Hand instruments and NiTi instruments are degraded by

Hydrogen Peroxide (H2O2) solution .

NiTi Instruments are degraded if immerged more than 5

minutes in a solution of NaOCl at more than 5% .

1- Pre-disinfection or Decontamination

2- Automated Cleaning Disinfection.

3- Manual Cleaning Disinfection

4-Inspection

5- Packaging

6- Sterilization

7-Storage

2
Lab2

Sterilization and Disinfection of Dental Instruments

According to the Centers for Disease Control, dental

instruments are classified into three categories depending on
the risk of transmitting infection.

1- Critical instruments : are those used to penetrate soft tissue

or bone, or enter into or contact the bloodstream or other
normally sterile tissue. They should be sterilized after each use.
Sterilization is achieved by steam under pressure (autoclaving),
dry heat, or heat/chemical vapor. Critical instruments include
forceps, scalpels, bone chisels, scalers and surgical burs .

2- Semi-critical instruments : are those that do not penetrate

soft tissues or bone but contact mucous membranes or non-
intact skin, such as mirrors, reusable impression trays and
amalgam condensers. These devices also should be sterilized
after each use. In some cases, however, sterilization is not
feasible and, therefore, high-level disinfection is appropriate.

3- Non-critical instruments : are those that come into contact

only with intact skin such as external components of x-ray
heads, blood pressure cuffs and pulse oximeters. Such devices
have a relatively low risk of transmitting infection; and,
therefore, may be reprocessed between patients by
intermediate-level or low-level disinfection. An intermediate-
level disinfectant is EPA-registered as a "hospital disinfectant"
and will be labeled for "tuberculocidal" activity (e.g., phenolics,
iodophors, and chlorine-containing compounds). A low-level
disinfectant is EPA- registered as a "hospital disinfectant" but is
not labeled for "tuberculocidal" activity (e.g., quaternary
ammonium compounds). The tuberculocidal claim is used as a
benchmark to measure germicidal potency. Germicides labeled

1
Lab2

as "hospital disinfectant" without a tuberculocidal claim pass

potency tests for activity against three representative
microorganisms: Pseudomonas aeruginosa, Staphylococcus
aureus, and Salmonella choleraesuis .

Processing Instruments

All critical and semi-critical dental instruments that are heat

stable should be sterilized after each use by steam under
pressure (autoclaving), dry heat, or chemical vapor. Before
sterilization or high-level disinfection, instruments should be
cleaned so that any debris is removed. Enzymatic and non-
enzymatic solutions facilitate instrument cleaning. Heavy- duty
gloves should be worn when handling contaminated
instruments. Instruments should soak in water or
disinfectant/detergent as soon as possible after use to prevent
drying of debris. Instrument cassettes and mechanical cleaning
(e.g., ultrasonic cleaners) may be used to reduce direct
handling of contaminated instruments. Applying rust inhibitors
will protect instruments from corrosion that may result from
autoclaving. Packaging rinsed and dried instruments before
sterilization protects them from contamination after they are
removed from the sterilizer and during transport chair side or
to storage.

2
NCBI ( This research is summarized by Assistant
Professor Ali Fakhriddeen to students of Faculty
of Dentistry )
Introduction

Cross infection remains one of the major challenges in the

dental profession, especially in field settings. Transmission of
hepatitis B, hepatitis C, and human immunodeficiency virus
.have raised a major concern for patients and dental staff.

A major issue in these settings is infection control. This is an

area where blood or saliva contamination can easily occu r. As
there is use of small, sharp instruments contaminated with
blood or other fluids, there is ample opportunity for
transmission of hepatitis B, hepatitis C and human
immunodeficiency virus (HIV). Dentistry potentially exposes
much of the population to blood contact with infected patients.
Thus, a wide range of dental equipment may pose an
unacceptable risk of cross infection.

If the protocol is not followed strictly, contamination of

instruments may result. Interruption of the cycle results in
inadequately sterilized instruments that cannot be considered
safe. After the sterilization cycle, the sterilizer must
depressurize, and the packs remain in the sterilizer for drying.
The drying phase may take an additional 20-45 min. The unit
must only be opened after completion of the drying cycle
making it more time consuming in field settings. Improperly
sterilized instruments utilized in patient care can result into

1
surgical site infection and pose a serious threat to the patient's
safety leading to life-threatening infection or even death.

In general, heat sterilization method is preferred to chemical

disinfection. However, with certain instruments that are
repeatedly used, frequent chemical disinfection may be
necessary since heat sterilization can lead to corrosion. Further,
it is not possible to carry out heat sterilization in field surveys
.due to time constraints

In these situations, the disinfection using chemical disinfectants

may be considered an alternate for heat sterilization to reduce
the risk of cross contaminations. Glutaraldehyde is a dialdehyde
that displays potent bactericidal, fungicidal, mycobactericidal,
sporicidal and virucidal activities. Mechanism of its action is
based on its interaction with amino groups in proteins and
enzymes. Glutaraldehyde is normally used as a 2% solution,
which is sufficient to achieve a sporicidal effect. It is used as an
as immersion solution for metallic instruments, face masks,
heat sensitive plastic rubbers, and fiber optics.

Hydrogen peroxide (H2O2) is commonly employed for

disinfection, sterilization, and antisepsis and is effective against
bacteria, viruses, yeast and spores. It is commercially available
in concentrations ranging from 3% to 90%. H2O2 is
environmental friendly, because it can rapidly degrade in to
harmless products that is, water and oxygen. H2O2 acts as an
oxidant by producing hydroxyl free radicals (•OH), which attack
cell components, including lipids, proteins, and DNA. Proposed
mechanism of action is based on its ability to target exposed
sulfhydryl groups and double bonds

2
Alcohol is an effective skin antiseptic and disinfectant for
medical instruments. A number of alcohols have shown
effective antimicrobial activity but, ethyl alcohol, isopropyl
alcohol and n-propanol are the most widely used. Alcohols
exhibit rapid broad-spectrum antimicrobial activity against
vegetative bacteria (including mycobacteria), fungi, and viruses
but they lack sporicidal activity hence are not recommended
for sterilization. In general, the antimicrobial activity of alcohols
is optimum in the range of 60-90%, but it becomes significantly
lower at concentrations below 50%. The exact mode of action
of alcohols is unclear, but it is generally believed that they
cause membrane damage leading to cell lysis and result into a
rapid denaturation of proteins.

The lack of published literature comparing the effectiveness of

various chemical disinfectants has prompted us to undertake
this study to evaluate the disinfecting efficacy of three chemical
solutions in reducing the contamination of diagnostic
.instruments that are routinely used in dental screening camps

3
 Clostridia
Assistant Professor Ali Fakhriddeen

There are at least 118 species, the clinically important species:

Clostridia tetani

Clostridia perfringens

Clostridia botulinum

Clostridia difficile

Clostridium tetani
Gram-positive rods that form terminal spores

❖ Culture: clustering growth with hemolysis occurs on blood agar.

❖ Clostridia tetani is found in soil. It is occasionally found in intestinal flora of

humans and animals.

❖ Clostridia tetani is the cause of tetanus when the spores enter wounds.
Clostridia tetani produces two exotoxins called tetanus toxins:

Tetanolysin: its virulent role remains unknown.

Tetanospasmin: it is a neurotoxin with strong toxicity and the most

important virulent factor of the bacterium.

Pathogenesis of tetanospasmin
one heavy chain (H chain)

C end：binds to the ganglioside receptors of inhibitory neurones

N end：helps in entrance to the cells

one light chain (L chain)

It contains a zinc endopeptidase

Blocks the release of inhibitory neuronal mediators g-GABA

and glycin
Stops inhibitory nerve impulse to skeletal muscles, resulting in
persistent muscle contraction.

Typical symptoms include sardonic smile, lockjaw, neck rigidity,

opisthotonos and dyspnea

Among all animal species, horses and humans, are most susceptible to
tetanospasmin

If not treated in time, about 20% of the patients are died of suffocation
and respiratory failure

Among all animal species, horses and humans, are most susceptible to
tetanospasmin

If not treated in time, about 20% of the patients are died of suffocation
and respiratory failure
 Treatment
Although antibiotics (streptomycin and erythromycin) are used as part of
the treatment, tetanus patients must be promptly treated with tetanus
antitoxin (TAT) to neutralize free tetanospasmin.
1500 ~ 3000 U for prevention

100.000 ~ 200.000 U for therapy

Tetanus toxoid is a component of DPT vaccine (diphtheria toxoid, killed

whole cell pertussis, tetanus toxoid).

Clostridia perfringens
Gram-positive rods, have capsule, can form terminal spores

Form double hemolysis circles on blood agar plates.

The α-hemolysis is caused byα-toxin while the β-hemolysis byθ-toxin.

Diseases

Wound contaminated by soil (main source) and mammalian feces

Gas gangrene refers to serious tissue swelling due to release of gas

(fermentation product of the bacterium) and tissue necrosis
The death can occur within 2 days if untreated. Treatment includes
debridement, antitoxin and antibiotic therapy.

Diseases
Common diseases:

• Gas gangrene

• Food poisoning

Other diseases:
 • Necrotizing Enteritis

• Cellulitis

• Septicemia

Food poisoning
Marked hypersecretion in jejunum and ileum with loss of fluids and electrolytes
in diarrhea.

Necrotizing enteritis
an acute necrotizing process in the jejunum with symptoms of abdominal
pain, bloody diarrhea and peritonitis.

The death rate of this disease is as high as approximately 50%.

Virulent factors
Clostridium perfringens produces over 10 types of toxins. Some toxins are
hemolytic, proteolytic, saccharolytic enzymes. Some are lethal and necrotic.

alpha-toxin is the most important, it lyses erythrocytes, platelets,

leukocytes and endothelial cells.

According to antigenic differences of 4 major toxins, the bacterial strains

can be divided into A~E toxic types.

Type A is clinically the most important. Type A can also produce

enterotoxin to cause food poisoning.

Treatment
Debridement (Gas gangrene)

A large dose of antibiotics (penicillin)

Antitoxin against alpha-toxin and hyperbaric oxygenation .

No vaccine is available
 Clostridium botulinum
Clostridium botulinum is a Gram-positive, anaerobic, spore-forming
bacillus.

It produces an enterotoxin (botulinum toxin) that causes food poisoning

(botulism).

According to the antigenicity of botulinum toxin, The microbe can be

divided into A~G types.

Among the 7 types, type A and then type B strains cause most disease.

The foods that are easily contaminated by the toxins or spores are
sausage and canned foods abroad, and fermented-bean preparations in
China.

In adult, the toxin or spore causes botulism (death rate 50-70%).

In infant (especially younger than 6 months), the toxin or spore causes

infant botulism (death rate 1-2%) .

Pathogenesis of botulinum toxin

Botulinum toxin is a nuerotoxin. It binds to other non-toxic proteins to
form a complex. It is released, when the bacteria are dead and broken.

When the complex enters intestines, the alkaline condition makes

botulinum toxin released.

Botulinum toxin binds the cellular receptor of neuromuscular junction and

then enters the cells to block the release of acetylcholine.

Acetylcholine is a neurotransmitter, which mediates nerve impulses.

Therefore, flaccid paralysis occurs.
The typical symptoms are double vision and squint (eye), dyscatabrosis
(throat), and dyspnea. The patients are usually died of respiratory failure.

10 ng of Botulinum toxin can kill an adult.

The patients must be promptly treated with A, B, E multi-valent antitoxin

to neutralize the free toxins.

Sometimes antibiotic therapy is needed.

Vaccination does not protect hosts from botulism.

General Characteristics of Bacillus
➢ ~ 60 species; Gram-positive or Gram-variable bacilli

• Large (0.5 x 1.2 to 2.5 x 10 um)

• Most are saprophytic contaminants or normal flora

• Bacillus anthracis is most important member

➢ Produce endospores

➢ Aerobic or facultatively anaerobic

➢ Catalase positive (most)

• Rapidly differentiates from Clostridium

• Bacillus spp. are ubiquitous

• Soil, water, and airborne dust

• Thermophilic (< 75°C) and psychrophilic (>5-8°C)

• Can flourish at extremes of acidity & alkalinity (pH 2 to 10)

Diseases Associated with Bacillus

Laboratory Characteristics of Bacillus
➢ On blood agar

• Large, spreading, gray-white colonies, with irregular margins

• Many are beta-hemolytic (helpful in differentiating various Bacillus species from B. anthracis)

➢ Spores seen after several days of incubation, but not

typically in fresh clinical specimens

Summary of B. anthracis Infections

Summary of B. anthracis Infections (cont.)
Epidemiology of Bacillus anthracis
➢ Rare in the US (1974-1990, 17 cases reported by CDC)

➢ Enzootic in certain foreign countries (e.g., Turkey, Iran, Pakistan,and Sudan)

➢ Anthrax spores infectious for decades

• Biologic warfare experiments (annual tests for 20 years)

✓ Gruinard, off western coast of Scotland

✓ 4 x 10e14 fully virulent spores exploded

✓ Eliminated in 1987 (formaldehyde & seawater)

➢ Three well-defined cycles

• Survival of spores in the soil

• Animal infection

• Infection in humans

Epidemiology of Bacillus anthracis (cont.)

➢ Primarily a disease of herbivorous animals

➢ Most commonly transmitted to humans by direct contact with animal products (e.g., wool and hair)

➢ Also acquired via inhalation & ingestion

• Increased mortality with these portals of entry

➢ Still poses a threat

• Importing materials contaminated with spores from these countries (e.g., bones, hides, and other
materials)

• Usually encountered as an occupational disease

• Veterinarians, agricultural workers

Clinical Presentation of Anthrax Cutaneous Anthrax

➢ 95% human cases are cutaneous infections

➢ 1 to 5 days after contact

➢ Small, pruritic, non-painful papule at inoculation site

➢ Papule develops into hemorrhagic vesicle & ruptures

➢ Slow-healing painless ulcer covered with black eschar surrounded by edema

➢ Infection may spread to lymphatics w/ local adenopathy

➢ Septicemia may develop

 ➢ 20% mortality in untreated cutaneous anthrax

Clinical Presentation of Anthrax Inhalation Anthrax

➢ Virtually 100% fatal (pneumonic)

➢ Meningitis may complicate cutaneous and inhalation forms of disease

➢ Pharyngeal anthrax

• Fever

• Pharyngitis

• Nneck swelling

Clinical Presentation of Anthrax Gastrointestinal (Ingestion) Anthrax

➢ Virtually 100% fatal

➢ Abdominal pain

➢ Hemorrhagic ascites

➢ Paracentesis fluid may reveal gram-positive rods

Treatment & Prophylaxis

➢Treatment

• Penicillin is drug of choice

• Erythromycin, chloramphenicol acceptable alternatives

• Doxycycline now commonly recognized as prophylactic

➢Vaccine (controversial)

✓ Laboratory workers

✓ Employees of mills handling goat hair

✓ Active duty military members

✓ Potentially entire populace of U.S. for herd immunity

Summary of B. cereus Infections

Summary of B. cereus Infections (cont.)

Foodborne Diseases of B. cereus
(Intoxication) (Foodborne Infection)
Enterobacteriaceae

Lesson 1 : Assistant Professor

Ali Fakhriddeen
Enterobacteriaceae

 Morphology and General Characteristics

 Gram-negative, non-sporing, rod shaped bacteria
 Oxidase –
 Ferment glucose and may or may not produce gas
in the process (aerogenic vs anaerogenic)
 Reduce nitrate to nitrite (there are a few
exceptions)
Enterobacteriaceae
 Are facultative anaerobes
 If motile, motility is by peritrichous flagella
 Many are normal inhabitants of the intestinal tract
of man and other animals
 Some are enteric pathogens and others are urinary
or respiratory tract pathogens
 Differentiation is based on biochemical reactions
and differences in antigenic structure
Enterobacteriaceae

 Most grow well on a variety of lab media

including a lot of selective and differential
media originally developed for the the
selective isolation of enteric pathogens.
 Most of this media is selective by incorporation
of dyes and bile salts that inhibit G+ organisms
and may suppress the growth of nonpathogenic
species of Enterobacteriaceae.
 Many are differential on the basis of whether or
not the organisms ferment lactose and/or
produce H2S.
Enterobacteriaceae

 On CBA they all produce similar colonies

that are relatively large and dull gray. They
may or may not be hemolytic.
 The three most useful media for screening
stool cultures for potential pathogens are
TSI, LIA, and urea or phenylalanine agar.
 The antigenic structure is used to
differentiate organisms within a genus or
species.
 Three major classes of antigens are found:
Enterobacteriaceae
 Somatic O antigens – these are the heat stable
polysaccharide part of the LPS.
 Variation from smooth to rough colonial forms is
accompanied by progressive loss of smooth O Antigen.
 Flagellar H antigens – are heat labile
 Envelope or capsule K antigens – overlay the surface O
antigen and may block agglutination by O specific
antisera.
 Boiling for 15 minutes will destroy the K antigen and unmask
O antigens.
 The K antigen is called the Vi (virulence) antigen in
Salmonella typhi.
Antigenic Structure of
Enterobacteriaceae
Enterobacteriaceae

 Escherichia coli
 Normal inhabitant of the G.I. tract.
 Some strains cause various forms of
gastroenteritis.
 Is a major cause of urinary tract infection and
neonatal meningitis and septicemia.
 May have a capsule.
 Biochemistry
 Most are motile.
E. coli
 May be hemolytic on CBA – more common in pathogenic
strains
 KEY tests for the normal strain:
 TSI is A/A + gas
 LIA K/K
 Urea –
 Indole +
 Citrate –
 Motility +
 There is an inactive biotype that is anaerogenic, lactose –,
and nonmotile.
E. coli
 Antigenic structure - has O, H, and K antigens. K1
has a strong association with virulence, particularly
meningitis in neonates.
 Virulence factors
 Toxins
 Enterotoxins – produced by enterotoxigenic strains of E. coli
(ETEC). Causes a movement of water and ions from the
tissues to the bowel resulting in watery diarrhea. There are
two types of enterotoxin:
 LT – is heat labile and binds to specific Gm1
gangliosides on the epithelial cells of the small intestine
where it ADP-ribosylates Gs which stimulates adenylate
cyclase to increase production of cAMP.
 Increased cAMP alters the activity of sodium and
chloride transporters producing an ion imbalance that
results in fluid transport into the bowel.
E. coli toxins
 ST – is heat stable and binds to specific receptors
to stimulate the production of cGMP with the same
results as with LT.
E. coli toxins
 Both enterotoxins are composed of five beta
subunits (for binding) and 1 alpha subunit (has the
toxic enzymatic activity).
E. coli toxins
 Shiga-type toxin – also called the verotoxin -produced
by enterohemorrhagic strains of E. coli (EHEC) – is
cytotoxic, enterotoxic, neurotoxic, and may cause
diarrhea and ulceration of the G.I. tract.
 There are two types shiga-like toxin 1 and shiga-
like toxin 2.
 Inhibit protein synthesis by cleaving a 28S rRNA
that’s part of the 60S subunit
E. coli toxins
 Enteroaggregative ST-like toxin – produced by
enteroaggregative strains of E. coli (EAEC) – causes watery
diarrhea.
 Hemolysins – two different types may be found: cell bound
and secreted.
 They lyse RBCs and leukocytes and may help to inhibit
phagocytosis when cell bound.
 Endotoxin
 Type III secretion system to deliver effector molecules
directly into the host cells.
 Involved in inducing uptake of EIEC into intestinal cells.
 Involved in development of an attachment and effacing
lesion in EPEC characterized by microvilli destruction and
pedestal formation.
E. coli
 Adhesions – are also called colonization factors and
include both pili or fimbriae and non-fimbrial factors
involved in attachment (e.g. intimin).
 There are at least 21 different types of adhesions.
 Antibodies to these may protect one from colonization.
 Virulence factors that protect the bacteria from host
defenses
 Capsule
 Iron capturing ability (enterochelin)
 Outer membrane proteins - are involved in helping the
organism to invade by helping in attachment (acting as
adhesion) and in initiating endocytosis.
E. coli

 Clinical significance
 Is the leading cause of urinary tract infections
which can lead to acute cystitis (bladder
infection) and pyelonephritis (kidney infection).
Ascending urinary tract infection
Urinary tract infections (UTI)

 New evidence in women who suffer from

recurrent UTIs suggests that this is due to
the formation of pod-like E. coli biofilms
inside bladder epithelial cells.
 Bacteria living on the edges of the biofilms nay
break off leading to a round of infection.
E. coli infections
 Neonatal meningitis – is the leading cause of neonatal
meningitis and septicemia with a high mortality rate.
 Usually caused by strains with the K1 capsular antigen.
 Gastroenteritis – there are several distinct types of E. coli
that are involved in different types of gastroenteritis:
 enterotoxigenic E. coli (ETEC),
 enteroinvasive E. coli (EIEC),
 enteropathogenic E. coli (EPEC) ,
 enteroaggregative E. coli (EAEC), and
 enterohemorrhagic E. coli (EHEC).
E. coli gastroenteritis
 ETEC – is a common cause of traveler’s diarrhea and diarrhea in
children in developing countries.
 The organism attaches to the intestinal mucosa via
colonization factors and then liberates enterotoxin.
 The disease is characterized by a watery diarrhea, nausea,
abdominal cramps and low-grade fever for 1-5 days.
 Transmission is via contaminated food or water.
 EPEC – Bundle forming pili are involved in attachment to the
intestinal mucosa.
 The type III secretion system inserts the tir (translocated
intimin receptor) into target cells, and intimate attachment of
the non-fimbrial adhesion called intimin to tir occurs.
 Host cell kinases activated to phosphorylate tir which then
causes a reorganization of host cytoskeletal elements resulting
in pedestal formation and development of an attaching and
effacing lesion
 The exact mode of pathogenesis is unclear, but it is probably
due to the attachment and effacement.
 Diarrhea with large amounts of mucous without blood or pus
occurs along with vomiting, malaise and low grade fever.
 This is a problem mainly in hospitalized infants and in day care
centers.
E. coli gastroenteritis
 EIEC – The organism attaches to the intestinal mucosa via
pili and outer membrane proteins are involved in direct
penetration, invasion of the intestinal cells, and destruction
of the intestinal mucosa.
 There is lateral movement of the organism from one cell
to adjacent cells.
 Symptoms include fever,severe abdominal cramps,
malaise, and watery diarrhea followed by scanty stools
containing blood, mucous, and pus.
 EAEC – Mucous associated autoagglutinins cause
aggregation of the bacteria at the cell surface and result in
the formation of a mucous biofilm.
 The organisms attach via pili and liberate a cytotoxin
distinct from, but similar to the ST and LT enterotoxins
liberated by ETEC.
 Symptoms include watery diarrhea, vomiting,
dehydration and occasional abdominal pain.
E. coli gastroenteritis
 EHEC – The organism attaches via pili to the intestinal mucosa and
liberates the shiga-like toxin.
 The symptoms start with a watery diarrhea that progresses to
bloody diarrhea without pus and crampy abdominal pain with
no fever or a low-grade fever.
 This may progress to hemolytic-uremic syndrome that is
characterized by low platlet count, hemolytic anemia, and
kidney failure.
 This is most often caused by serotypes O157:H7.
 This strain of E. coli can be differentiated from other strains of
E. coli by the fact that it does not ferment sorbitol in 48 hours
(other strains do).
 A sorbitol-Mac (SMAC) plate (contains sorbitol instead of
lactose) is used to selectively isolate this organism.
 One must confirm that the isolate is E. coli O1547:H7 using
serological testing and confirm production of the shiga-like
toxin before reporting out results.
 Serotypes of E. coli other than O157H7 have now been found
to cause this disease
E.coli
 Antimicrobic therapy- E. coli is usually susceptible
to a variety of chemotherapeutic agents, though
drug resistant strains are increasingly prevalent.
 It is essential to do susceptibility testing.
 Treatment of patients with EHEC infections is not
recommended because it can increase the release
of shiga-like toxins and actually trigger HUS
Shigella species
 Shigella
 Contains four species that differ antigenically and, to a lesser
extent, biochemically.
 S. dysenteriae (Group A)
 S. flexneri (Group B)
 S. boydii (Group C)
 S. sonnei (Group D)
 Biochemistry
 TSI K/A with NO gas
 LIA K/A
 Urea –
 Motility -
 All ferment mannitol except S. dysenteriae
 S. sonnei may show delayed lactose fermentation
Shigella species
 Antigenic structure
 Differentiation into groups (A, B, C, and D) is based on O
antigen serotyping; K antigens may interfere with
serotyping, but are heat labile.
 O antigen is similar to E. coli, so it is important to ID as
Shigella before doing serotyping.
 Virulence factors
 Shiga toxin – is produced by S. dysenteriae and in smaller
amounts by S. flexneri and S. sonnei.
 Acts to inhibit protein synthesis by inactivating the 60S
ribosomal subunit by cleaving a glycosidic bond in the 28S
rRNA constituents.
 This plays a role in the ulceration of the intestinal mucosa.
Shigella species
 Outer membrane and secreted proteins
 These proteins are expressed at body temperature
and upon contact with M cells in the intestinal mucosa
they induce phagocytosis of the bacteria into
vacuoles.
 Shigella destroy the vacuoles to escape into the
cytoplasm.
 From there they spread laterally (Polymerization of
actin filaments propels them through the cytoplasm.)
to epithelial cells where they multiply but do not
usually disseminate beyond the epithelium.
Shigella
 Clinical significance
 Causes shigellosis or bacillary dysentery.
 Transmission is via the fecal-oral route.
 The infective dose required to cause infection is very low (10-200
organisms).
 There is an incubation of 1-7 days followed by fever, cramping,
abdominal pain, and watery diarrhea (due to the toxin)for 1-3
days.
 This may be followed by frequent, scant stools with blood,
mucous, and pus (due to invasion of intestinal mucosa).
 It is rare for the organism to disseminate.
 The severity of the disease depends upon the species one is
infected with.
 S. dysenteria is the most pathogenic followed by S. flexneri, S.
sonnei and S. boydii.
Shigella

 Antimicrobial therapy
 Sulfonamides are commonly used as are
streptomycin, tetracycline, ampicillin, and
chloramphenicol.
 Resistant strains are becoming increasingly
common, so sensitivity testing is required.
Salmonella
 Salmonella
 Classification has been changing in the last few years.
 There is now 1 species: S. enteritica, and 7 subspecies: 1,
2 ,3a ,3b ,4 ,5, and 6.
 Subgroup 1 causes most human infections
 Clinically Salmonella isolates are often still reported out as
serogroups or serotypes based on the Kauffman-White scheme
of classification.
 Based on O and H (flagella) antigens
 The H antigens occur in two phases; 1 and 2 and only 1 phase is
expressed at a given time.
 Polyvalent antisera is used followed by group specific antisera (A,
B, C1, C2, D, and E)
 Salmonella typhi also has a Vi antigen which is a capsular antigen.
Salmonella
 Biochemistry
 TSI K/A + gas and H2S: S. typhi produces only a small
amount of H2S and no gas , and S. paratyphi A produces
no H2S
 LIA K/K with H2S with S. paratyphi A giving K/A results
 Urea –
 Motility +
 Citrate +/-
 Indole -
 Virulence factors
 Endotoxin – may play a role in intracellular survival
 Capsule (for S. typhi and some strains of S. paratyphi)
 Adhesions – both fimbrial and non-fimbrial
Salmonella virulence factors
 Type III secretion systems and effector molecules – 2
different systems may be found:
 One type is involved in promoting entry into intestinal
epithelial cells
 The other type is involved in the ability of Salmonella to
survive inside macrophages
 Outer membrane proteins - involved in the ability of
Salmonella to survive inside macrophages
 Flagella – help bacteria to move through intestinal mucous
 Enterotoxin - may be involved in gastroenteritis
 Iron capturing ability
Salmonella

 Clinical Significance – causes two different

kinds of disease: enteric fevers and
gastroenteritis.
 Both types of disease begin in the same way,
but with the gastroenteritis the bacteria remains
restricted to the intestine and with the enteric
fevers, the organism spreads
 Transmission is via a fecal-oral route, i.e., via
ingestion of contaminated food or water.
Salmonella
 The organism moves through the intestinal
mucosa and adheres to intestinal epithelium.
 Effector proteins of the type III secretion system
mediate invasion of enterocytes and M cells via
an induced endocytic mechanism.
 Salmonella multiplies within the endosome.
Salmonella
 The endosome moves to the basal side of the cell and
Salmonella are released and may be phagocytosed by
macrophages.
 For gastroenteritis the Salmonella multiply and their
presence induces a strong inflammatory response which
causes most of the symptoms seen in gastroenteritis (mild to
moderate fever with diarrhea and abdominal cramps).
 The inflammatory response prevents the spread
beyond the GI tract and eventually kills the bacteria.
 In enteric fevers (typhoid and paratyphoid) the Salmonella
disseminate before they multiply to high enough levels to
stimulate a strong inflammatory response so the initial
symptoms are only a low-grade fever and constipation.
Salmonella

The bacteria move via the lymphatics and bloodstream to

the liver and spleen where phagocytosis and
multiplication occurs.
 The bacteria re-enter the bloodstream to disseminate
throughout the body to all organs causing fever,
headaches, myalgia, and GI problems.
 Rose spots (erythematous, muculopapular lesions) are
seen on the abdomen. Osteomyelitis, cystitis, and gall
bladder infections may occur.
 Symptoms of paratyphoid fevers (due to S. paratyphi A,
B, or C) are similar to but less severe than those that
occur with typhoid fever (due to S. typhi)
Salmonella
 Diagnosis of typhoid fever
 Blood cultures are positive during the first week and
after the second week
 Stool cultures and sometimes urine cultures are positive
after the second week
 The Widal test is a serological test for antibodies
against Salmonella typhi. One looks for a 4-fold rise in
titer between acute and convalescent stages.
 10% of those infected become short term carriers and a
smaller % become long-term carriers due to persistence
of the bacteria in the gallbladder or urinary bladder.
Salmonella

 Antimicrobial therapy
 Enteric fevers – use chloramphenicol usually.
Resistant strains have emerged making
antimicrobial susceptibility testing essential.
 Gastroenteritis – usually doesn’t require
antimicrobic therapy.
 Replace lost fluids and electrolytes.
Enterobacteriaceae

 Klebsiella
 NF of GI tract, but potential pathogen in other areas
 TSI A/A + gas
 LIA K/K
 Urea +
 Citrate +
 MR-, VP+
 Motility -
 Has both O and K antigens
Klebsiella
 Virulence factors
 Capsule
 Adhesions
 Iron capturing ability
 Clinical significance
 Causes pneumonia, mostly in immunocompromised
hosts.
 Permanent lung damage is a frequent occurrence (rare in
other types of bacterial pneumonia)
 A major cause of nosocomial infections such as
septicemia and meningitis
Enterobacteriaceae

 Proteus, Providencia, and Morganella

 Are all part of the NF of the GI tract (except
Providencia).
 All motile, with Proteus swarming
 PA +
 Lysine deamination + (LIA R/A)
 Urea + for most, strongly + for Proteus
 TSI variable (know the reactions for each in the
lab!)
 Indole – only P. mirabilis is -
Proteus, Providencia, and
Morganella
 Virulence factors
 Urease – the ammonia produced may damage the
epithelial cells of the UT
 Clinical Significance
 UT infections, as well as pneumonia, septicemia, and
wound infections
 Yersinia
 Three species are important pathogens in man
 Yersinia pestis – causes plague
 Yersinis enterocolitica – enteropathogenic
 Yersinia pseudotuberculosis – enteropathogenic
Yersinia species
 Identification
 Y. pestis can be separated from Y. enterocolitica and Y.
pseudotuberculosis by the fact that it is non-motile. Y.
enterocolitica and Y. pseudotuberculosis are both non-
motile at 370 C, and motile at 220 C.
 Y. pestis is identified based on the following:
 Non-motile
 Bipolar staining
 Slow growth of small colonies on ordinary culture media – it
grows better at lower temperature (25-300 C)
Yersinia species
 TSI K/A no gas
 LIA K/A
 Urea –
 Guinea pig or mouse pathogenicity studies: LD50<10
 Direct fluorescent antibody test
 New DNA probe test
 Yersinia pestis – virulence characteristics
 Endotoxin – is responsible for many of the symptoms
 Murine toxin – causes edema and necrosis in mice and rats,
but has not been shown to play a role in human disease
Y. pestis
 Fraction 1 – a protein component of the
antiphagocytic protein capsule. Also blocks flea
digestion.
 V antigen – a secreted protein that controls
expression of many of the virulence genes plus it
appears to have another unknown function that is
essential for virulence
 Pla – a protease that activates plasminogen activator
(acts as a fibrinolysin) and degrades C3b (prevents
formation of complement membrane attack complex)
and C5a (prevents attraction of phagocytes)
 Psa – a pilus adhesion for attachment
 Iron acquisition and sequestering system
 Type III secretion system
 YopB and YopD – disrupt actin cytoskeleton in
phagocytic cells to evade phagocytosis
Y. pestis
 Y. pestis – clinical significance
 In man plague occurs in two forms; bubonic and pneumonic
 Bubonic plague – transmitted by fleas from an infected rodent
(is endemic in our local mountains).
 The bacteria travel in the blood to the nearest lymph node
where they are engulfed by fixed macrophages.
 A high fever develops and the lymph nodes in the groin and
armpit become enlarged (buboes) as the bacteria proliferate
and stimulate an inflammatory response.
 The bacteria growing in the lymph node leak into the
bloodstream.
 Lysis of the bacteria releases LPS, causing septic shock.
 Subcutaneous hemorrhages, probably due to LPS causing DIC
gave the disease the name, the black death, in the middle
ages.
 The untreated mortality rate is quite high.
Buboes and pneumonia
Y. pestis
 Eventually bacteria reach the lungs where they are
ingested by lung macrophages to cause pneumonic
plague.
 Pneumonic plague – this can be transmitted
directly to others via aerosol. Direct inhalation of
aerosols containing the organism produces a form
of the disease that progresses much more rapidly
and the mortality rate is close to 100%.
 Treatment for plague
 Streptomycin or tetracycline are effective
Yersinia species
 Yersinia enterocolitica and Yersinia
pseudotuberculosis identification –
 Both are motile at 22-250 C, but non-motile at 370 C
 Both exhibit bipolar staining
 Both grow better at lower temperatures and produce
small colonies at 370 C
 TSI A/A (sucrose, not lactose fermentation) for Y.
enterocolitica; K/A for Y. pseudotuberculosis
 LIA K/A for both
 Urea + for both
 ODC + for Y. enterocolitica only
Yersinia species
 Cefsulodin-irgasan-novobiocin (CIN) agar is a selective
media developed specifically for the isolation of Y.
enterocolitica from gastrointestinal specimens.
 The media also contains mannitol and phenol red to
differentiate mannitol from non-mannitol fermenting
organisms.
 The media is incubated at room temperature and
Yersinia are the only Enterobacteriaceae that will grow
on the media.
 Aeromonas and Pleisiomonas, both members of the
Vibrionaceae will also grow.
 After 48 hours at RT, Y. enterocolitica and Y.
pseudotuberculosis both produce typical pink (from
mannitol fermentation) colonies with a bulls-eye
appearance.
Y. enterocolitica growth on CIN
Yersinia species
 Y. enterocolotica – virulence factors
 Enterotoxin similar to E. coli ST (increases cGMP leading to
watery diarrhea)
 Adhesions – include both fimbrial and non-fimbrial
adhesions.
 At least four different adhesions have been identified
thus far.
 Antiphagocytic proteins – include both outer membrane and
secreted proteins.
 Some are actually injected directly into the host via a
type III secretion mechanism.
 Some interfere with signal transduction in host cells,
thus interfering with the ability of PMNs to respond to
signals leading them to the invading bacteria.
 Others disrupt the actin cytoskeleton and lead to death
of the PMNs.
Yersinia species
 V antigen - a secreted protein that controls expression of
many of the virulence genes plus it appears to have another
unknown function that is essential for virulence
 Iron capturing ability
 Yad A – an outer membrane protein that interferes with C3b
binding to bacteria thus preventing the formation of a
membrane attack complex.
 Endotoxin
 Y. pseudotuberculosis – virulence factors
 Has all of the same virulence factors as Y. enterocolitica
except the enterotoxin.
Yersinia species
 Yersinia enterocolitica and Y. pseudotuberculosis –
clinical significance
 Both are acquired by ingestion of contaminated food or
water.
 Y. enterocolitica is a common cause of human disease,
whereas, Y. pseudotuberculosis is mainly a disease of other
animals.
 Both cause a disease involving fever and abdominal pain.
Y. enterocolitica also causes a watery diarrhea.
 After ingestion, the bacteria invade the intestinal epithelium
by invasion of M cells.
 They are transcytosed through the M cells and released
at the basal surface.
 Once through the intestional epithelium, the bacteria
penetrate into the underlying lymphoid tissue, where
they multiply both inside and outside host cells.
Yersinia species
 Multiplication of the bacteria produces an
inflammatory response that is responsible for the
extreme pain associated with the infections
(resembles acute appendicitis)
 Fever is due to the activity of the LPS endotoxin.
 Sometimes they drain into adjacent mesenteric
lymph nodes, causing mesenteric lymphadenitis.
 Reactive arthritis may occur in some people following
Y. enterocolitica infection.
 It is thought to be due to cross reacting T cells or
antibodies that attack the joints.
 Enterobacteriaceae
Characteristics
GNR Gamma Proteobacteria
Motile = flagella
Capsule/slime layer
Nitrate reduction
FA
Oxidase (-)
Epidemiology
Source
GI
Water
Soil
Decaying vegetation
Enterobacteriaceae
Lesson 2 :
Assistant Professor
Ali Fakhriddeen
Enterobacteriaceae
Groups
Gammaproteobacteria:
Enterics
Coliforms
Noncoliforms
Pathogens
FA
Oxidase (-)
Reduce nitrate
(cocco)bacilli
Coliforms
Normal GI microbiotica
Groups
Escherichia
Klebsiella
Serratia
Enterobacter
Hafnia
Citrobacter
Biochemical tests
Ferment lactose
Noncoliform
Opportunistic
Nosocomial
Diseases
UTI
Kidney stones
Groups
Proteus
Morganella
Providencia
Edwardsiella
Biochemical
Non lactose fomenters
 Pathogenic Enteric Bacteria
Characteristics
NLF
Virulence
Type III secretion
Toxins
Groups
Salmonella
Shigella
Yersinia
 Brucella
Characteristics
coccobacillus
Location
Intracellular parasite
Animal hosts
Pathogenicity
Prevent phagolysosome
Examples
B. melitensis
B. abortus
B. suis
B. canis
Brucella
Epidemiology
Unpasteurized dairy
Animal blood / urine
Reproductive organs
Disease
Undulant fever (Bangs)
Tx: AB
Prevention
Animal vaccination
Haemophilus
Characteristics
GNR
Pleomorphic
Location
MM parasite
Examples
H. influenza
H. ducreyi
H. aphrophilus
H. parainfluenza
H. aegyptius
Haemophilus influenzae
Pathogenicity
Capsule
K antigen
Various strains
Disease
Meningitis
Infantile arthritis
Cellulitis
Epiglottitis
Ocular and Aural (OM)
Sinusitis
URTI (bronchitis. Pneumonia)
Tx: AB
Prevention
vaccination
Haemophilus parainfluenza
Characteristics
Epidemiology
Component of dental
plaque
Pathogenesis
Disease
POD
Valvular endocarditis
Dx
Haemophilus aegyptius
Purpuric Fever
South America
Children
Disease
Conjunctivitis 
N/V/D 
Shock 
Death
Haemophilus ducreyi

STD
Pathogenicity
toxin
Disease
Genital ulcer (chancroid)
 Vibrio
Characteristics
GNR
Agent: V. cholera
Epidemiology
carriers
Fecal contamination
Water
Food
Pathogenesis
Serotypes: O1 and O139
Adhere to intestinal mucosa
Toxin: Choleragen (from bacteriophage)
Subunit A
Activates adenylate cyclase
Hypersecretion of Cl- and H20
Subunit B
Binds to intestinal receptors
Disease
Muscle cramps
Profuse diarrhea
Circulatory shock and collapse
Dx: culture of feces, Agglutination Rxn
Tx: supportive; AB
Prevention: water sanitation
Lactobacillus
Assistant Prof. Ali Fakhriddeen

Lactobacillus is a genus of Gram-positive, facultative

anaerobic or microaerophilic, rod-shaped, non-spore-
forming bacteria. They are a major part of the lactic
acid bacteria group (i.e. they convert sugars to lactic
acid). In humans, they constitute a significant
component of the microbiota at a number of body
sites, such as the digestive system, urinary system,
and genital system. In women of European
ancestry, Lactobacillus species are normally a major
partofthe vaginal
microbiota. Lactobacillus forms biofilms in the vaginal
and gut microbiota, allowing them to persist during
harsh environmental conditions and maintain ample
populations. Lactobacillus exhibits
a mutualistic relationship with the human body as it
protects the host against potential invasions
by pathogens, and in turn, the host provides a source
of nutrients. Lactobacillus is the most common
probiotic found in food such as yogurt, and it is
diverse in its application to maintain human well-
being as it can help treat diarrhea, vaginal infections
and skin disorders such as eczema.

Many lactobacilli operate using homofermentative

metabolism (they produce only lactic acid from
sugars), and some species use heterofermentative
metabolism (they can produce either alcohol or lactic
acid from sugars). They are aerotolerant despite the
complete absence of a respiratory chain. This
aerotolerance is manganese-dependent and has been
exploredin Lactobacillus plantarum. Many species of

1
this genus do not require iron for growth and have an
extremely high hydrogen peroxidetolerance.

–Antioxidant
–Inhibits β-amyloid fibril formation
Maintains mucosal reactivity:
↑IL-22 production
Associated with vascular disease:
↑Oxidative stress
↑Smooth muscle cell proliferation
↑Aortic wall thickness and calcification
Associated with chronic kidney disease:
↑Renaldysfunction
–Uremic toxin
Kidneys
According to metabolism, Lactobacillus species can
be divided into three groups:

• Obligately homofermentative (group I) including:

• L. acidophilus, L. delbrueckii, L. helveticus, L.
salivarius
• Facultatively heterofermentative (group II)
including:
• L. casei, L. curvatus, L. plantarum, L. sakei
• Obligately heterofermentative (group III) including:
• L. brevis, L. buchneri, L. fermentum, L. reuteri

Human health
Vaginal tract
The female genital tract is one of the principal
colonisation sites for human microbiota, and there is
interest in the relationship between the composition
of these bacteria and human health, with a domination
by a single species being correlated with general
welfare and good outcomes in pregnancy. In around
70% of women, a Lactobacillus species is dominant,

2
Interactions with other pathogens
Lactobacillus species produce hydrogen
peroxide which inhibits the growth and virulence of
the fungal pathogen Candida albicans in vitro& in
vivo. In vitro studies have also shown
that Lactobacillus sp. reduce the pathogenicity of C.
albicans through the production of organic acids and
certain metabolites. Both the presence of metabolites,
such as sodium butyrate, and the decrease in
environmental pH caused by the organic acids reduce
the growth of hypha in C. albicans, which reduces its
pathogenicity.
Lactobacillus sp. also reduce the pathogenicity of C.
albicans byreducing C.albicans biofilm formation. On
the other hand, following antibiotic therapy,
certain Candida species can suppress the regrowth
of Lactobacillus sp. at body sites where they
cohabitate, such as in the gastrointestinal tract.
In addition to its effects on C.
albicans, Lactobacillus sp. also interact with other
pathogens. For example, Lactobacillus reuteri can
inhibit the growth of many different bacterial species
by using glycerol to produce the antimicrobial
substance called reuterin. Another example
is Lactobacillus salivarius, which interacts with many
pathogens through the production of salivaricin B, a
bacteriocin.
Probiotics
Lactobacillus species administered in combination
with other probiotics benefits cases of irritable bowel
syndrome (IBS), although the extent of efficacy is still
uncertain. The probiotics help treat IBS by returning
homeostasis when the gut microbiota experiences
unusually high levels of opportunistic bacteria. In
addition, Lactobacillus species can be administered
as probiotics during cases of infection by the ulcer-
3
causing bacterium Helicobacter pylori. Helicobacter
pylori is linked to cancer, and antibiotic resistance
impedes the success of current antibiotic-based
eradication treatments. When Lactobacillus probiotics
are administered along with the treatment as
an adjuvant, its efficacy is substantially increased and
side effects may be lessened. Also, Lactobacillus is
used to help control urogenital and vaginal infections,
suchas bacterial
vaginosis (BV). Lactobacillus produce bacteriocins to
suppress pathogenic growth of certain bacteria, as
well as lactic acid and H2O2 (hydrogen peroxide).
Lactic acid lowers the vaginal pH to around 4.5 or
less, hampering the survival of other bacteria, and
H2O2 reestablishes the normal bacterial flora and
normal vaginal pH. In children, Lactobacillus strains
such as L. rhamnosus are associated with a reduction
of atopic eczema, also known as dermatitis, due to
anti-inflammatory cytokines secreted by this probiotic
bacteria.
Oral health
Dental caries
Some Lactobacillus species have been associated
with cases of dental caries (cavities). Lactic acid can
corrode teeth, and the Lactobacillus count in saliva
has been used as a "caries test" for many years.
Lactobacilli characteristically cause existing carious
lesions to progress, especially those in coronal
caries. The issue is, however, complex, as recent
studies show probiotics can allow beneficial
lactobacilli to populate sites on teeth, preventing
streptococcal pathogens from taking hold and
inducing dental decay. The scientific research of
lactobacilli in relation to oral health is a new field and
only a few studies and results have been published.
Some studies have provided evidence of

4
certain Lactobacilli which can be a probiotic for oral
health. Some species, but not all, show evidence in
defense to dental caries. Due to these studies, there
have been applications of incorporating such
probiotics in chewing gum and lozenges. There is also
evidence of certain Lactobacilli that are beneficial in
the defense of periodontal disease such as gingivitis
and periodontitis. Food production
Some Lactobacillus species are used as starter
cultures in industry for controlled fermentation in the
production
of yogurt, cheese, sauerkraut, pickles, beer, cider, kim
chi, cocoa, kefir, and other fermented foods, as well
as animal feeds. The antibacterial and antifungal
activity of Lactobacillus species rely on production of
bacteriocins and low molecular weight compounds
that inhibits these microorganisms. Sourdough bread
is made either spontaneously, by taking advantage of
the bacteria naturally present in flour, or by using a
"starter culture", which is a symbiotic culture
of yeast and lactic acid bacteria growing in
a water and flour medium. The bacteria metabolize
sugars into lactic acid, which lowers the pH of their
environment, creating a signature "sourness"
associated with yogurt, sauerkraut, etc.
In many traditional pickling processes, vegetables are
submerged-in brine,&salt-
tolerant Lactobacillus species feed on natural sugars
found in the vegetables. The resulting mix of salt and
lactic acid is a hostile environment for other microbes,
such as fungi, and the vegetables are thus
preserved—remaining edible for long periods.

5
 Fermented Foods
• Micro-organisms cause changes in the foods which:
– Help to preserve the food,
– Extend shelf-life considerably over that of the
raw materials from which they are made,
– Improve aroma and flavour characteristics,
– Increase its vitamin content or its digestibility
compared to the raw materials.
 Lactic Acid Bacteria
• The lactic acid bacteria can be divided into two groups based on
the end products of glucose metabolism.

• Those that produce lactic acid as the major or sole product of

glucose fermentation are designated homofermentative.

• Those that produce equal amounts of lactic acid, ethanol and

CO2 are termed heterofermentative.

• The homolactics are able to extract about twice as much energy

from a given quantity of glucose as the heterolactics.
 Lactic Acid Bacteria - Growth
• The lactic acid bacteria are mesophiles:
– they generally grow over a temperature range
of about 10 to 40oC,
– an optimum between 25 and 35oC.
– Some can grow below 5 and as high as 45 oC.

• Most can grow in the pH range from 4 to 8. Though some as

low as 3.2 and as high as 9.6.
Fusobacteria ( Lecture by Assist. Prof.
Ali Fakhriddeen )
Fusobacteria are non-sporing, anaerobic, non-motile, non-
or weakly fermentative, spindle-shaped bacilli (with fused
ends: hence the name). They are normal inhabitants of the
oral cavity, colon and female genital tract and are sometimes
isolated from pulmonary and pelvic
abscesses. Fusospirochaetal infections, which they cause in
combination with spirochaetes, are
noteworthy. Fusobacterium nucleatum (the type
species), Fusobacterium periodontium and Fusobacterium
simiae are isolated mainly from periodontal disease sites,
and others such as Fusobacterium alocis and Fusobacterium
sulci are sometimes found in the healthy gingival sulcus.
Non-oral species include Fusobacterium
gonidiaformans, Fusobacterium russii and Fusobacterium
ulcerans.
Fusobacterium nucleatum

Habitat and transmission

Several subspecies of F. nucleatum have been identified in
different habitats. These include F.
nucleatum subsp. polymorphum, found in the healthy
gingival crevice, and F. nucleatum subsp. nucleatum,
recovered mainly from periodontal pockets. A third
subspecies is F. nucleatum subsp. vincentii. Infections are
almost invariably endogenous.
Characteristics
Gram-negative, strictly anaerobic, cigar-shaped bacilli with
pointed ends (Fig. 18.1). Cells often have a central swelling.
A Gram-stained smear of deep gingival debris obtained
from a lesion of acute ulcerative gingivitis is a simple method
of demonstrating the characteristic fusobacteria, together
with spirochaetes and polymorphonuclear leukocytes (Fig.

1
18.2). These, together with the clinical picture, confirm a
clinical diagnosis of acute ulcerative gingivitis.

Fig. 18.1
A photomicrograph of fusobacteria showing characteristic
Gram-negative, cigar-shaped cells with pointed ends.

Fig. 18.2 A
Gram-stained smear obtained from deep gingival plaque of a
patient with acute ulcerative gingivitis

Culture and identification

Grows on blood agar as dull, granular colonies with an
irregular, rhizoid edge. Biochemical reactions and the acidic
end products of carbohydrate metabolism help
identification. As fusobacteria can remove sulphur from
cysteine and methionine to produce odoriferous hydrogen
2
sulphide and methylmercaptan, they are thought to be
associated with halitosis.
Pathogenicity
The endotoxin of the organism appears to be involved in the
pathogenesis of periodontal disease. It possesses remarkable
adherence properties and the Fusobacterium adhesion A
(FadA), which confers this property has recently been
isolated. F. nucleatum is usually isolated from polymicrobial
infections; it is rarely the sole pathogen. Thus, in
combination with oral spirochaetes (Treponema vincentii and
others), it causes the classic fusospirochaetal infections.
These are:
• acute (necrotizing) ulcerative gingivitis or trench mouth )
• Vincent’s angina, an ulcerative tonsillitis causing tissue
necrosis, often due to extension of acute ulcerative gingivitis
• cancrum oris or noma: a sequela of acute ulcerative
gingivitis with resultant gross tissue loss of the facial region.
As fusobacteria coaggregate with most other oral bacteria,
they are believed to be important bridging organisms
between early and late colonizers during plaque formation
Antibiotic sensitivity and prevention
Fusobacteria are uniformly sensitive to penicillin and, being
strict anaerobes, are sensitive to metronidazole. Regular oral
hygiene and antiseptic mouthwashes are the key to
prevention of oral fusobacterial infections in susceptible
individuals.
Leptotrichia

Leptotrichia spp. are oral commensals previously thought to

belong to the genus Fusobacterium. They are Gram-
negative, strictly anaerobic, slender, filamentous bacilli,
usually with one pointed end. Leptotrichia buccalis, present
in low proportions in dental plaque, is the sole
representative of this genus.

3
Leptotrichia

Leptotrichia is a small genus closely related

to Fusobacterium and comprises slow-growing, gram-
negative, filamentous, anaerobic bacterial flora of the oral
cavity and genital tract. Species included in the genus
are L. buccalis, L. trevisanii, L. sanguinegens, and L.
amnionii . All Leptotrichia species are extremely fastidious
and cannot be grown easily on conventional microbiologic
media or by conventional methods. As evidenced by
sequences available in the GenBank database, most of
the 16S rRNA gene sequences are from cloned DNA from
complex flora but not from bacterial
isolates. Leptotrichia species has been suspected to play
a role in periodontal disease.
However, Leptotrichia species have only been associated
with serious systemic disease, usually in
immunocompromised patients. Bacteremia caused by L.
sanguinegens in pregnant women has also been reported.
 Lecture by Assist. Prof.
Ali Fakhriddeen
Order: Spirochaetales
Family: Spirochaetaceae
Genus: Treponema
Borrelia
Family: Leptospiraceae
Genus: Leptospira
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
 Spirochaetales Associated
Human Diseases
Genus Species Disease
Treponema pallidum ssp. pallidum Syphilis
pallidum ssp. endemicum Bejel
pallidum ssp. pertenue Yaws
carateum Pinta
Borrelia burgdorferi Lyme disease (borreliosis)
recurrentis Epidemic relapsing fever
Many species Endemic relapsing fever
Leptospira interrogans Leptospirosis
(Weil’s Disease)
 Nonvenereal
Treponemal Diseases
 Bejel, Yaws & Pinta
 Primitive tropical and subtropical
regions
 Primarily in impoverished children
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.
Venereal Treponemal
Disease
 Syphilis
 Primarily sexually transmitted disease
(STD)
 May be transmitted congenitally
Darkfield Microscopy of
Treponema pallidum
 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
 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
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)
 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
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
 Generalized
Mucocutaneous
Rash of
Secondary
Syphilis
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
 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
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)
 Prophylactic treatment of contacts identified
through epidemiological tracing
 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.
Summary of
Treponema
Infections
(cont.)

REVIEW
Actinomycosis: etiology, clinical
features, diagnosis, treatment, &
management
Assistant Professor Ali Fakhriddeen

Actinomycosis is an infrequent invasive bacterial disease that

has been recognized for over a century. Actinomyces spp. are
filamentous Gram-positive bacilli, mainly belonging to the
human commensal flora of the oropharynx, gastrointestinal
tract, and urogenital tract. To date, multiple different clinical
features of actinomycosis have been described, as various
anatomical sites (such as face, bone and joint, respiratory tract,
genitourinary tract, digestive tract, central nervous system, skin,
and soft tissue structures) can be affected. Of note, in any site,
actinomycosis frequently mimics malignancy, tuberculosis, or
nocardiosis, as it spreads continuously and progressively, and
often forms a cold abscess.
Overview of the different species
of Actinomyces bacteria that cause actinomycosis
Bacteria of the genus Actinomyces belong to the Actinobacteria
phylum and Actinomycetales Actinomyces israelii is the most
prevalent species isolated in human infections and is found in
most clinical forms of actinomycosis. Actinomyces
viscosus and Actinomyces meyeri are also often reported in
typical actinomycosis, although they are less common, and A.
meyeri is considered to have a great propensity for
dissemination. Some species, including Actinomyces naeslundii,
Actinomyces odontolyticus, Actinomyces
gerencseriae (formerly A. israelii serotype 2), Actinomyces
neuii, Actinomyces turicensis, and Actinomyces radingae, have
been associated with particular clinical syndromes. Thus, A.
israelii and A. gerencseriae are responsible for about 70% of
orocervicofacial infections. Hematogenous dissemination of
1
actinomycosis is extremely rare and has mainly been associated
with A. meyeri, A. israelii, and A. odontolyticus. Of note, most
of the Actinomyces spp. are present in polymicrobial
flora. Actinomyces infections could be polymicrobial and
associated with other bacteria, named “companion microbes”,
which contribute to initiation and development of infection by
inhibiting host defenses or reducing oxygen tension.
Microbiological diagnosis of actinomycosis
Rapid transport to the laboratory and/or transport in an
anaerobic transport medium and anaerobic growth conditions
should be used for primary isolation. The most appropriate
clinical specimens are tissue from surgical biopsy or pus; swabs
must be avoided. Moreover, the identification of Actinomyces in
mucosa, where these bacteria are normal inhabitants, is of little
significance in the absence of sulfur granules (see “Pathology of
actinomycosis”) or a typical clinical syndrome, highlighting the
importance of microbiological investigations in combination
with histologic analysis.
Actinomyces are non-spore-forming Gram-positive rods. Except
for A. meyeri, which is small and nonbranching, all the other
species are branching filamentous rods.
Growth of Actinomyces is slow; it appears within at least 5 days
and may take up to 15–20 days. Thus, incubation of at least 10
days is required before conclusion of a negative culture.
Actinomyces can be initially suspected by colony morphology
and biochemical profiling. For example, A. israelii forms a
“molar tooth” colony on agar and grows as clumps within broth,
whereas A. odontolyticus forms rust-brown or red-colored
colonies.
MALDI-TOF should be a quicker and accurate tool
for Actinomyces identification in the future.
Pathology of actinomycosis
Gram staining of pus and pathology of infected tissue is of great
interest for the diagnosis of actinomycosis, as it is usually more
2
sensitive than culture, which remains sterile in more than 50%
of cases. Once Actinomyces spp. have invaded tissues, they
develop a chronic granulomatous infection characterized by the
formation of tiny clumps, called sulfur granules because of their
yellow color. These findings are highly suggestive of the
diagnosis but are not specific, as they can be encountered in
other pathogenic conditions such as nocardiosis or chronic
cervicofacial fungal infections. However, Gram staining can
additionally show Gram-positive filamentous branching bacteria
at the periphery of the granule that is highly suggestive of
actinomycosis. Immunofluorescence techniques have poor
sensitivity but are highly specific in the diagnosis.
Drug susceptibility of Actinomyces spp
Drug resistance is not considered a problem in actinomycosis.
Indeed, Actinomyces spp. are usually extremely susceptible to
beta-lactams, and especially penicillin G or amoxicillin. As a
consequence, penicillin G or amoxicillin are considered drugs of
choice for the treatment of actinomycosis. Third-generation
cephalosporins are less frequently used even if they are
considered to be active on A. israelii; however, it is important to
note that some species are resistant to ceftriaxone (Actinomyces
europaeus and Actinomyces graevenitzii) . Piperacillin–
tazobactam, imipenem, and meropenem are considered to be
active, but the use of these broad-spectrum antibiotics should be
limited to avoid acquisition of resistant flora. Oxacillin,
cloxacillin, and cephalexin, a first-generation cephalosporin, are
not considered to be active. Metronidazole and aminoglycosides
have no in vitro activity against Actinomyces. Fluoroquinolones
(ciprofloxacin and moxifloxacin) are usually considered to be
inactive, but data are limited and controversial. Doxycycline is
considered to have a poor activity on Actinomyces spp., but
clinical successes have been reported with this drug. Macrolides
and clindamycin have been used successfully as alternatives.
As Actinomyces spp. do not produce beta-lactamases, it is not
useful to combine amoxicillin with beta-lactam inhibitors such

3
as clavulanic acid, except if co-pathogens such as
Enterobacteriaceae are involved in the disease.
Clinical features of actinomycosis

Respiratory tract actinomycosis

Epidemiology and pathogenesis
Respiratory tract actinomycosis includes pulmonary, bronchial,
and laryngeal actinomycosis. Pulmonary actinomycosis is the
third most common type of actinomycosis, after that occurring
in cervicofacial and abdominopelvic locations. In children,
pulmonary involvement is uncommon. The peak incidence is
reported to be in the fourth and fifth decades of life. Males are
more often affected than women, with a 3:1 ratio.16 Pulmonary
actinomycosis results mainly from aspiration of oropharyngeal
or gastrointestinal secretions. Consequently, individuals with
poor oral hygiene, preexisting dental disease, and alcoholism
have an increased risk for developing pulmonary
actinomycosis. Otherwise, patients with chronic lung disease
such as emphysema, chronic bronchitis, and bronchiectasis, and
patients with pulmonary sequelae following tuberculosis, are
considered to also be at risk for pulmonary actinomycosis. The
mechanism of immune response in actinomycosis remains
unclear, but some factors, by altering this response, probably
promote the disease. Human immunodeficiency virus infection,
steroid use, infliximab treatment, lung and renal transplantation,
and acute leukemia during chemotherapy have been described as
risk factors, despite few data being available in such patients
Bronchial actinomycosis is rare. It may occur after disruption of
the mucosal barrier, especially in patients with endobronchial
stent, or with a bronchial foreign body aspiration (for example,
of a fish bone).
Concerning laryngeal actinomycosis, various different forms
have been described. Vocal cord actinomycosis may mimic
primary carcinoma or papilloma, whereas in patients with past
history of laryngeal carcinoma and radiotherapy, actinomycosis

4
may mimic laryngeal cancer relapse, as it may present as an
ulcerative lesion, most often without abscess or sinus tract.
Case
A 77-year-old woman with past history of breast cancer was
admitted 7 years after radiotherapy for left mandibular
metastasis with left mandibular pain, buccal-sided bone
exposure, and sinus tract (Figure ).

Figure

Left mandibular osteomyelitis with bone exposure (A) and sinus

tract (B) following left mandibular radiotherapy in a patient
receiving long-term bisphosphonate therapy. Panoramic dental
X-ray shows mandibular lucencies (C).
Actinomycosis is a rare chronic disease caused
by Actinomyces spp., anaerobic Gram-positive bacteria that
normally colonize the human mouth and digestive and genital
tracts. Physicians have to be aware of typical clinical
presentations (such as cervicofacial actinomycosis following
dental focus of infection, pelvic actinomycosis in women with
an IUD, and pulmonary actinomycosis in smokers with poor
dental hygiene), but also must be aware that actinomycosis may
mimic the malignancy process in various anatomical sites.
Bacterial cultures and pathology are the cornerstones of
diagnosis and require particular attention to prevent
misdiagnosis. Prolonged bacterial cultures in anaerobic
conditions are necessary for identification of the bacterium, and
5
typical microscopic findings include necrosis with yellowish
sulfur granules and filamentous Gram-positive fungal-like
pathogen. Patients with actinomycosis require prolonged (6- to
12-month) high doses of penicillin G or amoxicillin, but the
duration of antimicrobial therapy could likely be reduced (3
months) for patients in whom optimal surgical resection of
infected tissues has been performed. Specific preventive
measures (reduction of alcohol abuse, dental hygiene, change of
IUD every 5 years) may limit the occurrence of actinomycosis.

6
Anaerobic bacterial pathogens

Non-sporeforming anaerobes
Bacteroides spp., Fusobacterium spp.,
Veillonella spp., Actinomyces spp.,.

Sporeforming anaerobes
Clostridium spp.
 Bacteroides fragilis
Pleomorphic in size and shape; capsulated.
Aerotolerant; growth is stimulated in 20% bile.
Constitutes less than 10% of Bacteroides species in the
normal colon, however, is the most common isolate of
anaerobes from infections (intra-abdominal, gynecologic,
and skin and soft tissue infections; bacteremia.)
Major virulence factor: capsular polysaccharides, which
may cause abscess formation when injected into the rat
abdomen.
Its LPS lacks endotoxin activity. The clinical signs of
sepsis (fever & shock) could be due to other components.
Resistant to penicillin.
Clinically Predominant Anaerobic Gram-Negatives
Anaerobic Gram-Neg. Virulence Factors
Epidemiology of
Bacteroides
Bacteroides Gram Stain
 Classification of Medically
Important Anaerobes
• Gram positive cocci
• Gram negative cocci
– Veillonella
• Gram positive bacilli
– Clostridium perfringens, tetani, botulinum, Actinomyces
– Lactobacillus
• Gram negative bacilli
– Bacteroides fragilis, thetaiotaomicron
– Fusobacterium
 Bacteroides
• Epidemiology
– B. fragilis associated with 80% of intra-abd infx
• Pathogenesis
– Polysaccharide capsule
• Increases adhesion to peritoneal surfaces (along with
fimbriae)
• Protection against phagocytosis
• Differs from LPS of aerobic GNR(gram negative & positive)
– Less fatty acids linked to Lipid A component
– Less pyrogenic activity
– Superoxide dismutase and catalase
– Elaborate a variety of enzymes
 Bacteroides
• Infections
– Intra-abdominal infections (peritonitis,
abscess); bacteremias; decubitus and
diabetic ulcers
• Treatment
– Drainage of abscess and debridement of
necrotic tissue
– Antibiotics
T Veillonella parvula
The genus Veillonella was first isolated by Veillon and Zuber in 1898.
Veillonella parvula is a gram negative, strict anaerobic, non-spore-forming coccus-shaped bacterium. It is found in
the gut of humans and dental plaque. While considered non-pathogenic, it has been linked with rare cases of
meningitis, osteomyelitis, and periodontal disease.
V. parvula is significantly involved in biofilms where it co-aggregates with other organisms, most notably with
Streptococcus mutans, onto dental plaque. This is a synergistic relationship as V. parvula cannot adhere to the
surface of teeth by itself alone. It also utilises the lactate product formed by S. mutans for its metabolism and the
biofilm has been found to be more resistant to antibiotics than either species of bacteria alone.
The cells of V. parvula are coccus shaped, non-motile, roughly 0.4 µm in diameter, and predominantly occur in
chains. Like other gram negative bacteria, V. parvula has an outer layer of lipopolysaccharide which is a known
virulence factor.

Page 1 of 2
 V.parvula Gram Stain

V. parvula is usually not considered a pathogen but it has been implicated with rare cases of meningitis,
osteomyelitis, and periodontal disease, especially in the biofilm with S. mutans, and therefore V. parvula could be
indirectly involved with the pathogenesis of other microbes.

Questions
1. With which diseases has V. parvula been implicated?
2. With which microbe does V. parvula form a synergistic relationship?
3. Describe the growth conditions of V. parvula.
• Veillonella - hydrogen sulfide producer in mouth
• Veillonella are Gram-negative cocci that are the
anaerobic counterpart of Neisseria. These non-motile
diplococci are part of the normal flora of the mouth. The
reason this organism is important is not due to its
pathogenicity. Instead, we include Veillonella because it
can be and is often mistaken for the more serious
gonococcal infection. The most common species
isolated from humans is V. parvula. Veillonella species
are negative for almost every biochemical test with the
exception of an occasional strain being positive for
catalase.