Transcript pht313 part 2 gram -ve

PHT313
Dr. Hesham Radwan
1. Neisseria species
1. Primary pathogens:
a. N. gonorrhoeae (Gonococcus)
• ALWAYS pathogenic
b. N. meningitidis (Meningococcus)
• May be carried as commensal in nasopharynx flora
(10-25% carrier)
–Grow on chocolate agar at 370C under 5-10% CO2
2. Non-pathogenic (Commensals)
• Example N. lactamica
–Grow on ordinary medium at room temperature
–Habitat of non-pathogenic
• Upper respiratory, Genitourinary and Alimentary tract
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Pathogenic Neisseria
General characteristics
◦ Gram-negative diplococci with
adjacent sides flattened
(like coffee beans)
◦ Non motile
◦ Oxidase and Catalase positive
◦ Aerobic, capnophilic (5% CO2) and oxidative
◦ Requires complex media pre-warmed to 370C
◦ Susceptible to cool temperatures, drying and fatty acids
• Soluble starch added to neutralize fatty acid toxicity
◦ Intracellular human-specific pathogen
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a. Neisseria gonorrhoeae
Virulence factors
1. Fimbrae (pili)
•To adhere to host cells and to each other
•>100 serotypes known according to pilus protein
–Has not polysaccharide Capsule
2. Outer membrane proteins (formerly Proteins I, II, & III)
Involved in adherence to host cells
3. IgA protease- cleaves IgA on mucosal surfaces
IgA blocks the ability of bacteria to adhere
IgA protease involved in successful colonization
4. Lipopolysaccharide
Prevents phagocytosis
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Pathogenicity
• Pyogenic infection of columnar and transitional epithelial cells
– Urethral, endocervix, anal canal, pharynx, conjunctiva
1. Venereal (Sexual Transmitted Disease (STD))
A. Genital infections
– Gonorrhea
B. Extragenital infections
– Pharyngitis and Anorectal infections
2. Non-venereal
• Ophthalmia neonatorum
• Vaginitis in small girls
–Contaminated toilet seats and contaminated towels
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Gonorrhea
• Gonorrhea transmitted by sexual intercourse
• Gonorrhea is the second most common venereal disease
• Incubation period: 2 to 7 days
Females
Males
50% risk of infection after single exposure
20% risk of infection after single exposure
Symptomatic infections frequently diagnosed
Most initially symptomatic (95% acute)
Major reservoir is asymptomatic carriage
Major reservoir is asymptomatic carriage
Genital infection primary site is cervix (cervicitis), but vagina, Genital infection generally restricted to urethra
urethra, rectum can be colonized
(urethritis) with purulent discharge and dysuria
Ascending infections in 10-20% including salpingitis (Fallopian Rare complications may include epididymitis,
tubes), tubo-ovarian abscesses, PID, chronic infections can prostatitis, and periurethral abscesses
lead to sterility
Disseminated infections more common, including septicemia, Disseminated infections are very rare
infection of skin and joints (1-3%)
Bacteremia and Gonococcal arthritis occurs as a result of
disseminated gonococcal bacteremia
Can infect infant at delivery (opthalmia neonatorum)
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Ophthalmia Neonatorum
• Contamination of infant’s eye during labour through the birth
canal of mother
• In infancy, an eye infection (ophthalmia neonatorum) may
occur during vaginal delivery
• Conjunctivitis with mucopurulent discharge
• May cause blindness if not treated
• Infection is preventable with the application of erythromycin
eye drops at birth (chlamydia & Neisseria)
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Laboratory Diagnosis
1. Clinical specimens
– Gonnorhea
• Female
–Cervical discharge in acute and chronic
–Cervical swab may give positive results
• Male
–Acute: Urethral purulent discharge after urinating
–Chronic: The morning drop or prostatic massage
– Ophthalmia neonatorum
• Mucopurulent discharge
2. Direct microscopy (Gram stain)
◦ Small, gram-negative diplococci in presence of (WBCs)
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Laboratory Diagnosis
3. Culture
 Inoculate specimen on non selective (chocolate agar) or selective
media [Thayer-Martin]
 Incubated at 350 C in 5% CO2
 The suspected colonies are Gram-negative diplococci with
adjacent sides flattened (like coffee beans)
4. Biochemical tests
 Oxidase + & produce acid from glucose only
 Fresh growth must be used for testing, because
N. gonorrhoeae produces autolytic enzymes
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Prevention and control
 Penicillin no longer drug of choice due to:
Continuing rise in the MIC
Beta-lactamase production (some strains)
 Ceftriaxone, cefixime or fluoroquinolone combined with
doxycycline or azithromycin for dual infections with Chlamydia
 Chemoprophylaxis against gonorrhea is of little value
 Chemoprophylaxis against ophthalmia neonatorum with 1%
silver nitrate 1% tetracycline or 0.5% erythromycin eye ointment
 Measures to limit epidemic include education, detection, and
follow-up screening of sexual partners, use of condoms
 No vaccine is available
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b. Neisseria meningitidis (Meningcoccus)
Encapsulated small, gram-negative diplococci
Similar to Neisseria gonorrhoeae
The main differences are
Portal of entry
N. gonorrhea
N. meningitidis
Genital tract
Respiratory tract
Polysaccharide capsule Absent
Present
Beta-lactamase
Some
None
Acid from maltose
Negative
Positive
Vaccine
Not available
Available
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Pathogenicity:
Pili-mediated receptor-specific colonization of cells of nasopharynx
Antiphagocytic polysaccharide capsule
Allows systemic spread in absence of specific immunity
13 serogroups six of which (A, B, C, W135, X, Y) can cause epidemics
Serogroups A, B, C, Y, W135 account for 90% of all infections
Serotypes A, B and C are the most common worldwide
Serotype A is common in epidemics in Africa
Toxic effects mediated by hyperproduction of LPS (endotoxin)
Other virulence factors as in N. gonnorhea
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Clinical Infections
1. Meningitis (30-50%)
2. Meningitis with meningococcemia (40%), or
3. Meningococcemia without obvious meningitis (7-10%)
 Second most common cause of community-acquired meningitis
 Person-to-person transmission by respiratory droplets
 Commonly colonize nasopharynx of healthy individuals;
highest oral and nasopharyngeal carriage rates in school-age
children, young adults and lower socioeconomic groups
 Requires close contact with infectious person in crowded
conditions (e.g. military barracks, prisons, Hajj and other)
 Requires lack of specific antibody (susceptibility)
 Incubation period is 2-10 days
 Symptoms: Fever, headache, stiff neck, nausea, vomiting and
purulent meningitis with increased WBCs
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Laboratory Diagnosis:
 Specimens
 CSF appears turbid (meningitis)
 Blood (meningecoocemia)
 Nasopharyngeal swabs (carrier)
 Large numbers (e.g. >107cells/ml) of encapsulated, small,
gram-negative diplococci and PMN’s can be seen
microscopically in CSF
 Extracellular and intracellular
 Culture
 Transparent, non-pigmented nonhemolytic colonies on
chocolate, TM agar with enhanced growth in 5% CO2
 Biochemical and Immunologic tests
 Oxidase-positive
 Acid production from glucose and maltose
 Immunologic methods are available for sero-grouping
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Prevention
and
control
Prophylaxis
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1. Antibiotic prophylaxis is recommended for certain close contacts
2. Avoid crowdedness
3. Vaccination
• Several vaccines are available to control the disease
• They are designed to prevent serogroups A, C, Y, and W-135
• They are lacking serogroup B antigen
1. Tetravalent Meningococcal Polysaccharide vaccine (MPSV4)
– Available since 1981 and used for people >55 years
2. Meningococcal Conjugate Vaccine (MCV)
• When polysaccharides are conjugated to carrier proteins, the
polysaccharide antigens become immunogenic in infants and
prime for memory anticapsular antibody responses
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Treatment
Penicillin (drug of choice)
Other options: rifampin or sulfonamide (Chemoprophylaxis)
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Enterobacteriaceae
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Coliforms (lactose ferementers)
Normal inhabitants of GIT of human and animals
Source of noscomial infections
Opportunistic or cause secondary infections of
wounds, urinary and respiratory tracts and the
circulatory system e.g. E. coli, Klebsiella
 E. coli used as biological indicator in water pollution
2. True pathogens (Lactose non-fermenters)
 Salmonella spp., Shigella spp., Yersinia spp.
 Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)
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Media for isolation
 Selective differential media for enteric pathogens
 MacConkey agar
 EMB agar
 SS agar
 Selective by incorporation of dyes and bile salts
 Differential by incorporation of lactose and/or Fe+3
 Fe+3 is incorporated to detect H2S
 Classified as lactose fermenters & non-lactose fermenters
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I- Escherichia coli
 General characteristics:
 Gram-negative Motile rods
 Non-spore forming,Facultative anaerobic, Oxidase -ve
 Ferment glucose and lactose
 Normal flora of intestine
 Opportunistic pathogens
 E. coli may be pathogenic inside or outside Intestine
 Some strains (Pathogenic) cause various forms of
gastroenteritis
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Virulence factors
1.
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Adhesions (Colonization factors)
Pili or fimbriae & nonfimbrial factors
Host defense
Capsule
OMPs are involved in helping the organism to
invade by helping in attachment and in initiating
endocytosis
3. Exotoxin production (Enterotoxin)
• Heat-Labile (LT) & Heat-Stable Toxin (ST) (ETEC)
• Shiga-like toxin (Verotoxin) (EHEC)
4. Endotoxin (Pyrogen)
– Lipid A of LPS causes fever and endotoxic shock
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Diseases caused by E. coli
A. Intestinal: Diarrhea (Toxin and/or adhesion)
A. Enterotoxegenic E. coli (ETEC)
Acquired by
B. Enteropathogenic E. coli (EPEC)
ingestion of
C. Enteroinvasive E. coli (EIEC)
contaminated
D. Enterohemorrhagic E. coli (EHEC)
food and water
E. Enteroaggregative E. coli (EAEC)
B. Extraintestinal:
1. Urinary Tract Infections [UTI] (Pili)
2. Neonatal meningitis (K1 antigen)
3. Sepsis (commonly in debilitated hospitalized patients)
 Endotoxic shock
 Due to lipid A (Pyrogen)
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 Fever and sudden hypotension
Summary of E. coli gastroenteritis
M.O.
Site
Pathogenesis
Invasion
Diseases
Symptoms
ETEC Small
LT + STcAMP + cGMP Non
intestine  Fluid + electrolyte loss
invasive
•Traveler's
Watery
diarrhea,
diarrhea
cramps, nausea, low
•Infant diarrhea grade fever
EIEC
Dysentery
Large
nonfimbrial adhesin (NFA): Invasive
intestine
OMP
EPEC Small
NFA:
intimin
intestine EPEC adherence factor
Some reports of shiga-like
toxin
EHEC Large
Cytotoxic shiga-like toxin
intestine (verotoxin)
Poorly
invasive
Infantile
diarrhea
Poorly
invasive
Bloody
diarrhoea and
haemolytic
uraemic
syndrome
EAEC Small
Production of enterotoxin Non
intestine that similar to ETEC
invasive
Aute
and
persistent
diarrhoea in
children and
adults
dysentery-like
diarrhea,
severe
inflammation, fever
Watery diarrhea
With mucous
without blood or pus
With fever & vomiting
•Hemorrhagic colitis
with sever abdominal
cramps,
watery
diarrhea followed by
blood, no fever
•HUS
•Watery diarrhea
•Vomiting
•Abdominal pain
•Without inflammation
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or fever
Urinary Tract Infection (UTI)
 E. coli is the most common organism causing UTI
◦ Community acquired 90%
◦ Hospital acquired (50%)
◦ UTI is the disease of female (Short urethra)
 Fecal E. coli acquires pili to colonize mucosa of UT
 Travel up urethra & infect balder (Cystitis)& sometimes
move further up to infect kidney (pyelonephritis)
 Symptoms:urinary frequency, dysuria, hematuria, pyuria
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Diagnosis of UTI
• Specimen
– MSU (Mid-Stream Urine)
• Culture
• On MacConkey agar
– Gram negative, Lactose
colonies)
• Viable count
– ≥100,000 (105) cfu/ml urine
• IMViC + + - -
fermented
(Pink
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Neonatal meningitis
• E. coli is the second most common cause
• S. galactaiae (Group B) is the first
• Occur during the first month of life
• Lab diagnosis
• Specimen: CSF
• Culture: Pink colonies on MacConkey agar (LF)
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II- Salmonella
 General characteristics:
 Gram-negative
rods
belonging
to
Enterobacteriaceae
 Do not ferment lactose and H2S positive
 Salmonellae live in the intestinal tracts of animals
 Not
considered part of normal intestinal flora in human
 Always PATHOGENIC to human
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Classification of Salmonella
A. Kaufman-White- Le Minor Classification
Based on O and H antigen serotyping
64 O and 114 H variants identified
Classified into 9 groups (A-I) according to O antigen
Each group can be classified into subgroups
according to H Ag
• Salmonella can be detected by its group O antigen
and then by its type specific H antigen
• >2500 known serovars
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Classification of Salmonella
B. US CDC (Center for Disease Control)
1. S. enterica
• Subdivided into 6 subspecies enterica, salamae,
arizonae, diarizonae, indica, houtanae
• Of these six subspecies, only subspecies enterica is
associated with disease in warm-blooded animals
• S. enterica subsp. enterica serovar Typhi or S. Typhi
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S. enterica subsp. enterica ser.Typhimurium or S. Typhimurium
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S. enterica subsp. enterica ser. Enteritidis or S. Enteritidis
2. Salmonella bongori (Subspecies V)
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Species of Salmonella
• Two important members of Salmonella causing
diseases:
A. Salmonella causing enteric fever
– Salmonella Typhi or Salmonella Paratyphi A,B, and C
– These organisms penetrate intestinal mucosa
– Detected in blood, urine and stool
B. Salmonella causing food poisoning
– Salmonella Enteritidis and Salmonella Typhimurium
– These organisms do not penetrate intestinal mucosa
– Detected in stool only
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Enteric Fever “Typhoid”
• Enteric fevers are severe systemic forms of salmonellosis.
• Caused by S. typhi whereas a milder form “Paratyphoid” caused by S.
paratyphi A, B or C
• Mode of Transmission
• Via fecal-oral route through fecally-contaminated food or water from
either chronic carrier or case
• A few individuals continue to harbour Salmonella in their gall-bladders
and intermittently excrete organisms in their faces
• Temporary excretors: Patients who excrete bacteria for <year
• Chronic carrier: Patients who excrete bacteria for > year
• Incubation Period
• 10-14 day during which bacteria multiplies in Peyer’s patches
• Pass to blood via lymphatics resulting in bacteriaemia in 1st week
• In 2nd week M.O. passes to different organs including peyer’s patches
causing ulcers, gall bladder, liver, kidney & rarely menings
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Symptoms of Enteric Fever
• Symptoms begin after an incubation period of 2 weeks
• Enteric fevers may be preceded by gastroenteritis,
which usually resolves before the onset of systemic
disease
• The symptoms of enteric fevers are nonspecific and
include
• Fever, headache, delirium (sustained fever), malaise
and tender abdomen
• Complications include intestinal hemorrhage and
perforation
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Laboratory diagnosis
A. Direct diagnosis:
1. Specimen: Blood during 1st week, urine during 2nd week
and stool during 3rd week
2. Isolation of microorganism:
I. From blood using blood culture
– Five to 10 ml of blood is taken during the 1st week of
infection,
– Add to 50-100 ml sterile nutrient broth and incubate at
370C for 24 hrs.
– Subculture is done on MacConkey's agar which shows
colorless colonies in positive case
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Laboratory diagnosis
II. From stool
• By culture on enrichment medium such as selenite F or
tetrathionate broth which inhibits the growth of coliform and
allow the growth of Salmonella and Shigella
• Subculture on MacConkey, SS or DCA agar
• On MacConkey's agar they give colorless colonies
• On SS agar they give colorless colonies with black edges
due to H2S production
3. Biochemical Reactions:
•
The suspected colonies were subjected to biochemical reactions
•
Oxidase negative, not ferment lactose and sucrose, H2S positive
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Laboratory diagnosis
A. Indirect: Serological diagnosis (Widal Test):
•
In the 2nd week of the disease, antibodies against
Salmonella are present in the patient's serum and can be
detected serologically by Widal test (agglutination test)
•
Widal test is positive and valid during 2nd week
•
Serial dilutions of patient's serum are added to an equal
volume of common O and specific H antigens
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Agglutination of O- antigen and one only of the H-antigens
at a titer 1/80 or above is diagnostic
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Salmonella causing food poisoning
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The causative agent : S. typhimurium & S. enteritidis
Mode of infection:
Consumption of contaminated food
Food as cakes, pastries and various milk and egg dishes
Cattle, sheep, hens, ducks and turkeys are often infected and
the organism may contaminate meat and meat products
Infective dose 100,000 bacteria
Gastric juice is the an important host defense and decreased
acidity is a predisposing factor
Incubation period is 12-24 hrs and recovery within 4-7 days
It is self limiting disease
Manifestations include
Nausea, vomiting, and abdominal discomfort, non-bloody
diarrhea and slight fever
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I- Shigella
General Characteristics
Gram negative rods
Non motile
Non spore-forming
Non capsulated
Oxidase negative
Ferment glucose with acid only
Non lactose fermentating organism
H2S negative
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Shigella species
Disease:
Bacillary dysentery (Shigellosis)
S. sonnei is the most common, followed by S. flexneri
Mode of Transmission
Oral-fecal transmission
<200 bacilli are needed for infection in health individuals
Incubation periods
It varies between 1-3 days
Symptoms
Ranges from asymptomatic to severe bacillary dysentery
Watery diarrhea changing to dysentery with frequent small
stools with blood, pus and mucus
Fever, tenesmus and abdominal cramps
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Stages of shigellosis
A. Early stage:
1. Ingestion of contaminated food or water
2. Noninvasive colonization and cell multiplication
3. Production of the enterotoxin in the small intestine
4. Watery diarrhea attributed to enterotoxic activity of
Shiga toxin (similar to LT of ETEC)
5. Fever attributed to neurotoxic activity of toxin
B. Second stage:
1. Adherence to and tissue invasion of large intestine
2. Typical symptoms of dysentery
3. Cytotoxic activity of Shiga toxin increases severity
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Laboratory identification
• Specimen:
– Stool (mucous bloody part of stool) or rectal swap
• Stool culture
– Specimen is inoculated in Selenite broth at 370C for 24 h
– Then subculture on MacConkey or SS
– On MacConkey agar: they give colorless colonies
– On SS agar: they give colorless colonies
• Gram stain
– Gram negative bacilli, NON MOTILE
• Biochemical reactions
– Oxidase negative, ferment glucose, non ferment lactose
and H2S NEGATIVE
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COMMENSAL ENTEROBACTERIAE
• Opportunistic pathogens and are common
cause of nosocomial infections
1. Klebsiella
Lactose Fermnters
2. Enterobacter
3. Serratia
4. Proteus
Non-Lactose Fermnters
5. Morganella
6. Providencia
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Klebsiella-Enterobacter-Serratia group
• General characteristics:
– These organisms are very similar
– All are motile except Klebsiella
– MR negative; VP positive
– Simmons citrate positive
– H2S negative
– Some weakly urease positive
– Phenylalanine deaminase negative
• These organisms belonging to Enterobacteriaceae
• Frequently in large intestine but also found in soil and water
• Usually opportunistic pathogens
• Wide variety of infections: primarily pneumonia, wound and UTI
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Klebsiella pneumoniae
 Virulence factors
1. Polysaccharide capsule
 Protects against phagocytosis and antibiotics
 Makes the colonies moist and mucoid
2. Adhesions
 Clinical significance
 Major cause of nosocomial infections
 Nosocomial Pneumonia
 Important respiratory tract pathogen outside hospitals
 3% of bacterial pneumonia
 Bloody sputum (50%) (Thick, Jelly and Red Sputum)
 Septicemia, Meningitis and UTI
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Proteus-Providencia-Morganella
• These organisms belonging to Enterobacteriaceae
• All are normal intestinal flora but also found in soil and water
• Opportunistic pathogens
• Non-lactose fermenters
• Phenylalanine deaminase and urease positive
• Urease positive after 2-6 hrs (urea → NH3+ CO2)
• All are highly motile
• Proteus sp. swarming on blood agar
• Swarming characterized by expanding waves (rings) of
organisms over the surface of blood agar
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Proteus species
 P. mirabilis and P. vulgaris are widely recognized human pathogens
 Isolated from urine, wounds and ear and bacteremic infections
 They found in colon and able to colonize urethra especially in female
1.
P. mirabilis causes urinary tract infections (UTI):
Proteus
sp.
produces
urease
which
alkalinizes
urine
→precipitation of calcium and magnesium salts → stone
formation →renal epithelium damage
2.
P.
vulgaris
causes
nosocomial
infections
(pneumonia,
bacteremia) and UTIs
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Laboratory diagnosis
 Specimen:
 Urine or Stool.
 Culture:
 On MacConkey agar →non lactose fermenters→ colorless
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colonies
On SS agar →non lactose fermenters → colorless colonies
with black center
On ordinary media, such as nutrient agar, blood agar, show
swarming (successive waves on the surface) due to high
motility of Proteus
Biochemical reactions: Urease , phenyldeaminase and
H2S positive
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Pseudomonas aeruginosa
Gram-negative bacilli belonging to Pseudomonadaceae
Motile by single polar flagellum, non spore
Non fastidious- Minimal nutritional requirements
Produces grape-like odor and blue-green pus and colonies
Two types of soluble pigments:
1. Pyoverdin : Yellow-green pigment & fluorescent
2. Pyocyanin: Blue-green pigment and non-fluorescent
 Strict aerobic, O+/F-, Oxidase and catalase positive
 Optimum temperature is 37 0C & able to grow at 42 0C
 Resistant to dyes, weak antiseptics and many antibiotics
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Epidemiology
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The most important pathogenic is Ps. aeruginosa
Opportunistic pathogen
Important agent in causing nosocomial infections
Ubiquitous in moist environment, hospital, in soil and water
Colonized in the intestine in10% of human
Represents 10-20% of hospital-acquired infections
Transiently colonize respiratory & GIT of hospitalized
patients:
1. Treated with broad-spectrum antibiotics
2. Exposed to respiratory therapy equipment
3. Hospitalized for long periods
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1. Adhesins
Pathogenesis
 Fimbriae, Pilli, Alginate (mucoid exo-polysaccharide)
 Alginate slim forms biofilm that protect from antibodies,
complement, phagocytosis & antibiotic
2. Invasins
a. Proteases - Inactivate IFN and TNF
i. Elastase
 Break down of elastin-containing tissues e.g. blood
vessels, lung tissue, skin
 Cleaves collagen, IgG, IgA and complement)
 Produce hemorrhagic lesions associated with
disseminated infection
ii. Alkaline protease (lyses fibrin)
b. Hemolysins (phospholipase & lecithinase) & Leukocidin
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Pathogenesis
3.Toxins
 Exotoxin A & S
 Inhibit protein synthesis (Like diphtheria toxin)
 Immunosuppressive
 Causes dermatonecrosis in burn wounds, corneal
damage in ocular infections, and tissue damage in chronic
pulmonary infections
 Pyocyanin – inhibits mitochondrial enzymes, impairs
ciliary function, mediates tissue damage
 LPS (endotoxin)
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Clinical Finding
1. UTIs (Catheterized patients)
2. Wound, burn & other skin & soft tissues infections
 Echtyma Gangrenosum
3. Pneumonia (Cystic Fibrosis)
4. Eye infection- Contaminated contact lens clearing fluids
5. Chronic otitis media & otitis externa (swimming pool)
6. Meningitis- following lumbar puncture
7. Systemic infections- Septicemia in debilitated patients
8. Pseudomonal Endocarditis
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Echtyma Gangrenosum
• Causative agent: Ps. aeruginosa
• Under risk: immunocompromised, burn patients, and
other critically ill patients
• Round or oval (1-15cm) severe invasive cutaneous
ulceritic single or multiple lesion with halo of erthryma
• Pseudomonas exotoxin: Tissue destruction
• Elastase degrades elastin in blood vessels wall
• Phospholipase C degrades phospholipids in cell
membranes
• Pyocyanin generates reactive oxygen species
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Typing methods
1. Bacteriocin typing
 Three types of bacteriocins are produced-R,F,S
 Pyocin produced by test strain is employed to assess the
growth inhibition of 13 indicators strains
 Depending upon the growth inhibition of 13 indicators
strains,105 types are recognized
 Most popular method used
2. Phage typing
3. Serotyping-based on O & H,17 serotypes are recognized
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Laboratory diagnosis
 Specimen
 Urine, pus, sputum, CSF, blood, skin swap
 Microscopical Examination
 Gram-negative rods and motile
 Cultural Characteristics
 On Nutrient agar:
 Colonies are surrounded by bluish green coloration
 On selective media "Cetermide"
 Pigments are more obvious
 On Blood agar: -hemolytic colonies
 On MacConkey agar
 Pale yellow colonies i.e. non lactose fermenters
 Ps. aeruginosa able to grow at 42 0C for 3 days
 Pyocin typing: Identification for epidemiological purpose
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Vibrionaceae
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General characteristics
Gram negative, curved, comma shaped bacilli
Motile by single polar flagella
Non spore, Non capsulated
Facultative anaerobes, Fermentative
Oxidase and catalase positive
Most vibrios have relatively simple growth factor
requirements and grow well in alkaline pH
Natural inhabitants : Aquatic environment
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Vibrio cholerae
• >200 serogroups based on O-antigen
• V. cholerae O1 (classical and El Tor biotypes) and
O139 are primarily responsible for cholera outbreaks
– V. El Tor biotype causes cholera-like but milder
• Recently, V. cholerae O75 and O141 strains has
been associated with cholera-like diarrhea
• Some O1 strains do not produce cholera enterotoxin
(atypical or nontoxigenic O1 V. cholerae)
• Cholera is endemic in southern Asia (India, Pakistan
and Bangladesh), Latin America
57
Pathogenesis
• Mode of Transmission
– Consumption of contaminated water or food
• Incubation period:1-4 days
 High infectious dose: >108 CFU
 V. cholerae attach to mucosa of small intestine
 V. cholerae multiply & secrete enterotoxin (cholargen)
 Toxin binds to specific receptor on the intestinal
mucosal cell
 Toxin stimulates activity of cAMP, resulting in active
secretion of chloride & secondary loss of Na and H2O
 The patient loss 20 Litre fluids/day
 V. cholerae cause same disease as ETEC but more severe
58
Diagnosis of V. cholerae
 Specimen: Rice watery stool or rectal swab
 Culture on Alkaline Peptone Water (APW) then
subculture on selective differential medium
Thiosulfate Citrate Bile Salts Sucrose agar
 Selective due to alkalinity pH9 & contains bile salts
 Differential because contains sucrose
 Sucrose fermenting V. cholerae yellow colonies
 Sucrose non fermenting V. parahemolyticus appears
as blue to green colonies
 Sucrose fermentation is gold standard in identification
59
Diagnosis of V. cholerae
• Any sucrose fermenting colonies were subjected to
Gram stain and oxidase test
• Gram stain
– Gram negative short rods, comma shaped, motile
• Biochemical reactions:
–Oxidase positive
–O+/F+
–Cholera red reaction
• Serology:
–Diagnosis confirmed & serotyping done by
agglutination with specific antisera (O1, O139)
60
V. parahaemolyticus
 It is the cause of acute gastroenteritis following
ingestion of contaminated sea-food such as raw fish
 Marine organism which live in high salt conc.
 So it requires 2% Na Cl in the isolation medium
◦ It is invasive affecting the colon
◦ V. cholerae is noninvasive, affecting the small
intestine through secretion of an enterotoxin
Allied Vibrios are large group of organisms; some are
saprophytic while others cause disease in animals
61
Campylobacter
Campylobacter, belong to Campylobacteriaceae,
It was formerly grouped with vibrios why?
Gram-negative curved, S-shaped or spiral short bacilli
Motile by single polar flagella (Cork screw)
Non-spore forming ,Microaerophilic, Oxidase and catalase positive
15 species; 6 subspecies 12 associated with human infection
Divided into 2 groups on the basis of growth temperature:
Thermotolerant species which grow at 420C
– C. jejuni (poultry; humans) C. coli (porcine; human)
– C. jejuni accounts for >80% of all Campylobacter infection
• Non-thermotolerant - grow at 250C (C. fetus; veterinary pathogen)
• Primarily zoonotic with variety of animals (reservoirs) for infection
• Also, food animal such as poultry, cattle, sheep and pigs
•
•
•
•
•
•
•
•
62
Campylobacter
• Campylobacter is the main causative agent of diarrhoea
(enteritis ), mainly children, in developing countries
• C. jejuni, E. coli and Rotavirus are the three most common
cause of infantile diarrhoea in the world
• Infections usually sporadic, occurring in summer
• Mode of transmission
 Acquired by the fecal-oral route via ingestion of;
 Improperly handled or cooked food primarily poultry
 Also, drinking of un-pasteurized milk
 Children is the most commonly affected worldwide
 Incubation period
 The illness appears to last from 2-7 days
63
Pathogenesis and Virulence factor
1. C. jejuni infection vary considerably from asymptomatic to
severe bloody diarrhea, high fever, and prostration
2. Enterocolitis begins as watery, foul smelling diarrhoea
followed by bloody stools accompanied by fever,
headache and sever abdominal cramps
3. Systemic infection: bacteremia (very rare)
• Rarely C. jejuni spreads systematically (bacteremia)
This organism multiply in the small intestine, invade
the epithelium and produce gastroenteritis
 Then C. jejuni secretes
◦ Enterotoxin similar to cholera toxin and LT of E. coli
◦ Cytotoxin that destroy mucosal cell
64
Diagnosis of C. jejuni
 Specimen
◦ Stool culture is done on:
◦ Selective medium (Skirrow’s medium, Blood agar +
cefoperazone, vancomycin, amphotericin B)
◦ Incubation temperature at 420C in microaerophilic
condition (5%CO2, 10%O2 & 85%N2) for 48-72 hrs
 Gram-stain
◦ Gram stain examination of the colony should be
performed along with oxidase test
◦ Oxidase +ve colony exhibiting characteristic Gram
stain appearance can be reported as Campylobacter
65
Diagnosis & Treatment
• Special test for C. jejuni
• Hippurate hydrolysis is the major test for
distinguishing between C. jejuni (positive) and other
Campylobacter spp.
 Treatment
 Campylobacter jejuni isolates have variable
susceptibilities to a variety of antimicrobials
◦ Azithromycin and erythromycin (Macrolides) are
the drug of choice
66
4. Helicobacter
•
•
•
•
•
•
•
•
•
•
•
•
Helicobacter is closely resemble to Campylobacter
Gram-negative spiral bacilli
Non-spore forming
Motile by multiple polar flagella
Microaerophilic
Grow at 370C and slow growing organism (7-10 days)
Requires enriched media with blood, hemin
Such supplements protects the organism from oxygen free
radicals, H2O2 and fatty acids present in the media
Oxidase and catalase positive
Helicobacter is urease +ve while Campylobacter -ve
It survives in acidic environment of stomach & duodenum
It hides in mucus & neutralizes acid in its local environment
67
Diseases Associated with H. pylori
1. Gastritis (irritation & inflammation of lining of stomach)
 Symptoms include nausea, vomiting and frequent
complaints about pain in the abdomen
2. Peptic and duodenal ulcer
 Sores that form in the stomach or the duodenum
 The common symptom is burning pain in abdomen
 Ulcers that bleed, causing hematemesis (bloody vomit or
vomit that looks like coffee grounds) or melena (stool
that's black, bloody or looks like tar)
3. Stomach cancer later in life
No bacteremia or disseminated diseases occurs
68
Mode of transmission
• Scientists suspect that H. pylori infection may be
contagious
• The infection seems to run in families
• More common in crowded or unsanitary conditions
• Infection may be passed from person to person??
• How spread happens isn't really known
• Transmission believed to be by fecal-oral route
69
Pathogenesis
•
•
•
•
Most bacteria killed in environment of gastric lumen
H. pylori proliferates in mucus layer by aid of flagella
Then reach epithelial cells underneath- more neutral pH
H. pylori survives because of virulence factors that
contribute to gastric inflammation, alter gastric acid
production and cause tissue destruction
• Virulence Factors
A. Initial colonization facilitated by:
1. Acid inhibitory protein - blocks acid secretion from
parietal cells during acute infection
2. Urease - neutralizes gastric acids due to NH3 production
• Urease stimulates monocytes and neutrophil chemotaxis;
stimulates production of inflammatory cytokines
70
3. Heat shock protein - enhances urease expression; coexpressed with urease on bacterial surface
4. Flagella - allows penetration into gastric mucous layer
5. Adhesins - mediate binding to host cells
B. Localized tissue damage mediated by:
1. Mucinases and phospholipases - disrupt gastric mucus
2. Vacuolating cytotoxin - induces vacuolation in epithelial
cells that results in epithelial cell damage
3. SOD and catalase - prevent from phagocytosis and
intracellular killing
71
Diagnosis
• H. pylori is diagnosed by non-culture method such as
1. Blood antibody tests
2. Urea breath tests (UBT)
3. Endoscopic biopsies
4. Stool antigen tests
– H. pylori isolated by culture of gastric biopsy on
non-selective media enriched with blood or serum
for 7-10 day
– Skirrow’s media supplemented with blood or serum
72
Diagnosis
1. Blood antibody test
– Blood tests for the presence of antibodies to H. pylori can
be performed easily and rapidly
2. Urea Breath Test (UBT)
• UBT is a safe, easy & accurate test for detection of H. pylori
• UBT relies on ability of H. pylori to hydrolyse urea into CO2
• CO2 is absorbed from stomach & eliminated in the breath
• 10-20 minute after swallowing a capsule containing a minute
amount of radioactive urea, a breath is collected
• The presence of radioactive CO2 in the breath (a positive test)
means that there is active infection
73
3. Endoscopic biopsies
• Endoscopy is an accurate test for diagnosing H. pylori as
well as the inflammation and ulcers that it causes
• For endoscopy, the doctor inserts a flexible viewing tube
(endoscope) through the mouth, down the oesophagus,
and into the stomach and duodenum
• During endoscopy, small tissue samples (biopsies) from
the stomach lining can be removed
• A biopsy is placed on a special slide containing urea
• If the urea is broken down, this means that there is an
infection with H. pylori
Test kit showing negative and positive results for Helicobacter
74
Treatment of H. pylori
• The regimen most commonly recommended
for first line treatment of H. pylori is triple
therapy with a PPI (lansoprazole 30 mg twice
daily, omeprazole 20 mg twice daily,
pantoprazole 40 mg twice daily, rabeprazole
20 mg twice daily, or esomeprazole 40 mg
once daily), amoxicillin (1 g twice daily), and
clarithromycin (500 mg twice daily) for 7 to 14
days. We suggest treatment for 10 days to two
weeks.
Haemophilus
•
•
•
•
•
•
•
General characteristics:
Belonging to the Pasteurellaceae
Non intestinal pleomorphic Gram negative coccobacilli
Microaerophilic
Non motile, Non spore forming
Usually capsulated
Fastidious
– Dependent on the growth promoting substances
• X (haematin) and V (NAD) which present in blood
• Stellatism = Growth of Heamophilus on a culture of
Staph. aureus.
• Oxidase and catalase positive
76
Differentiation of Species
Hemolysis
Growth
Factor
X
V
H. influenzae
-
+
+
H. aegyptius
-
+
+
H. ducreyi
-
+
-
H. parainfluenzae
+
-
+
77
H. influenzae
Opportunistic pathogen
Exclusively human pathogen
Not pathogenic for animals
Diseases due to H. influenzae considered under 2
groups –invasive and non invasive
1. Nonencapsulated strains (Non invasive)
• Part of normal flora of the respiratory tract
• Colonize in nasopharynx of 80% of healthy individuals
• Causes pneumonia, otitis media and sinusitis
•
•
•
•
78
H. influenzae
2. Encapsulated strains (Invasive)
• Typing based on capsule polysaccharide a → f
• Type b is the most pathogenic & colonized in 5%
• Polyribose-ribitol phosphate (PRP) capsule (type b)
• Penetrate nasopharynx and invade blood directly
• Causes meningitis, pneumonia, bacteremia
• 95% of invasive disease caused by type b
• Vaccination with Hib conjugate vaccine
• Given IM at 2,4,6 month & poster dose at 12-18 month
79
Virulence factors of H. influenzae
• Polysaccharide capsule
– Antiphagocytic and major pathogenesis factor
• Fimbriae the adherence to human mucosal cells
• LPS- lipid A -major role in non capsule strains
• All virulent strains produce neuraminidase and IgA protease
• Many respiratory pathogens, including H. influenzae, S.
pneumoniae and P. aeruginosa, express neuraminidases
– Bacterial neuraminidase facilitates mucosal infection by
participating in biofilm production
• No exotoxins
80
H. aegypticus
• Cause of acute mucopurulent conjunctivitis (Pinkeye)
• The disease is common in Egypt
• It is spread very easily, especially among children
• Pinkeye is transmitted mechanically by common
towels or by flies
• Diagnosis is made from the conjunctival discharge
81
H. ducreyi
• This is the cause of chancroid (Soft chancre) which
is venereal disease
• Chancroid is characterized by painful genital ulcers
• This organism needs only X factor
• Diagnosis is made from the discharge of the ulcer
82
Diagnosis of Haemophilus
• Specimen: CSF, blood, sputum
• Diagnosis is confirmed when the organism is isolated
from a sterile body site (Blood & CSF)
• Diagnosis is not confirmed when it is isolated from
nasopharyx or sputum
• Stain: Gram negative coccobacilli
• Culture:
• On chocolate agar: A 24 h colony of H. influenzae is
larger than that observed on blood agar
• On nutrient agar plate with added X & V factors at
37°C in an enriched CO2 incubator
83
Diagnosis of Haemophilus
• Satellitism:
•
•
•
•
•
•
•
Blood agar contains much X factor and little V factor
S. aureus produces V factor
A lawn of test bacteria is plated onto a blood agar plate
S. aureus is placed on plate & the culture is incubated
H. influenzae will grow in the hemolytic zone of S. aureus
The hemolysis of cells by S. aureus releases nutrients
H. influenzae will not grow outside the hemolytic zone of S.
aureus due to the lack of nutrients in these areas
• Capsule swelling:
• Specific antiserum added to a slide of the organism allows
swelling of the bacterial capsule thus permitting rapid
84
diagnosis of H. influenzae in sputum
Bordetella
• Small capsulated Gram negative coccobacillus
• Strict aerobe, oxidative, oxidase variable?
• Three important species
1. B. pertussis causes WHOOPING COUGH (Pertussis)
• Strictly human pathogen: infant & young children
2. B. parapertussis–causes mild form of whooping cough
3. B. bronchoseptica
• Widespread in animals (dog) & causes kennel cough
• Rarely causes respiratory or wound infection in humans
85
B. pertussis Pathogenesis
1. Respiratory Colonization
 Attaches to ciliated epithelium of URT VIA
a. Filamentous hemagglutinin (FHA)
– Protein on pili
– Antibodies against FHA inhibit colonization
– Fimbriae are NOT involved
b. Pertussis Toxin
◦ Do not invade underlying lung tissues
◦ Decreased cilia activity & epithelial cell death occur
◦ 7-10 days, NO symptoms
◦ Positive cultures toward the end of this stage
86
Toxin-mediated disease
1.
•
•
•
•
2.
•
•
3.
•
4.
Pertussis toxin
Responsible for adherence
↑Adenylate cyclase ↑ intracellular cAMP
This causes cellular dysfunction
Inhibition of host phagocyte oxidative responses
and the inhibition of natural killer cell activity
Dermonecrotic toxin (lethal toxin)
Released upon cell lysis causing strong
vasoconstriction
Results in O2  convulsion
Tracheal cytotoxin
Prevents ciliated epithelial cells from beating
LPS –endotoxin-irritation and damage of cell
87
Clinical significance
• Pertussis is Toxin-mediated disease
• Very contagious & can cause serious
illness―especially in babies too young
• MODE OF TRANSMISSION: droplet inhalation
1. INCUBATION PERIOD:1-2 weeks
• Organism multiply & start to liberate toxins
• Pertussis lasts for 6 weeks and appear in 4 stages
3. CATARRHAL STAGE occurs - rhinorrhea, mild
cough, sneezing & fever whereby a lot of organisms
are spread through respiratory secretions
• This last ~ 1-2 weeks
88
4. PAROXYSMAL STAGE that lasts 2-4 weeks
• Patient has rapid, consecutive cough with a rapid
intake of air between coughs (has a whooping sound)
– Ciliary action of RT has been compromised
– Mucous has accumulated
– Patient is trying to cough up mucous accumulations
– Coughs are strong enough to break ribs!
5. Finally there is a CONVALESCENT STAGE during
which symptoms gradually subside
• This can last for months
89
Diagnosis
• Specimen is taken during PAROXYSMAL STAGE
– Nasopharyngeal swabs
– Cough plate method
• Highly fastidious & requires special media+additional
nutrients for growth+absorbents to remove toxic
substances found in complex media e.g. fatty acids
• Culture on Bordet-Gengou medium
 Contains glycerol, potato infusion, albumin (binds
fatty acids), and up to 50% defibrinated SRBCs
• Charcoal agar +10% horse blood ± Cephalexin
• 3-7 days for growth, hemolytic colonies
• A characteristic pearl like colonies are observed
• Direct fluorescent antibody testing, PCR, Slide agglutination
90
Differentiation of Bordetella species
Urease
Oxidase?
Motility
-
+
-
+
+
-
-
+
+
+
+
Growth on
Species
Sheep blood agar
and MacConkey
BordetGengou agar
B. pertussis
-
+
B. parapertussis
B. bronchiseptica
91
Prevention
1.
•
•
2.
•
•
Pertussis Whole cell vaccine
Merthiolate-killed bacterial cell suspension
Part of the DTP vaccine P in DTP for Pertussis
Pertussis Acellular vaccines
Consist of 1–5 purified, detoxified toxins & adhesins
Introduced in developed regions ∼15 years ago to
replace Killed vaccines
• Has fewer side effects than the whole cell vaccine
• Diphtheria-Tetanus-Pertussis
vaccine
using
acellular pertussis is known as DTaP
• 2, 4, 6, 12-18 months and 4-6 years of age.
92
Legionellae
•
•
•
•
•
•
•
•
•
•
•
Gram negative rod belonging to Legionellaceae
Stains poorly with gram stain
Facultative intracellular pathogen
> 57 species, ½ of species implicated in human disease
L. pneumophila is the most important human pathogen
Recently became opportunistic human pathogen
~ 90% of all cases of legionellosis (Legionnaires' disease)
15 serogroups, 88.6% of cases are caused by serogroup 1
Serogroup 1 seems to be more virulent
Not grow on Sheep Blood Agar
Requires specialized media to grow
• Cysteine and iron are essential for growth
93
L. pneumophila
• Reservoir principally aquatic
• L. pneumophila is not a free-living organism but an
intracellular parasite of amoebae
• Natural environment
– Lakes and rivers
• Artificial environment
– Showers, taps, air conditioning system
– Normally found in hot water (up to 500C) tanks
– Multiply in pipes in sediment & live many years
– Sediment provides shelter and nutrition for other
bacteria that can supply cysteine
• Important to detect L. pneumophila serogroup1 in water
94
Legionnaires disease
• The portal of entry is the respiratory tract via inhalation
• Legionellae are associated with water-based aerosols
1. Air conditioning cooling towers
2. Sauna
•
Person to person spread does not occur
• Nosocomial infections
– Due to presence of bacteria in water taps, sinks & showers
95
Legionnaires disease
• In general, healthy people rarely get disease
• People under Risk
• People with defect in cell mediated immunity
1. Typically occurred in older man who smokes and
consumes substantial amount of alcohol
2. Patients with AIDS, cancers, Renal transplants, patient
treated with corticosteriods or elderly
96
L. pneumophila
• Initial host response: acute inflammatory response of
aveoli & then bronchioles (similar to pneumococcal infection)
• Neutrophils accumulate followed by macrophage
• Different from pneumococcal infection
1. Organisms located inside of macrophage
2. Inhibit lysosomal fusion & acidification of phagocyte
97
Clinical Presentations
• Pontiac fever
–
–
–
–
Pontiac, Michigan in 1968
Incubation period 1-2 days
Flu-like, Milder (no mortality) and self-limiting
Persists for 2-5 days, then spontaneously resolves
• Legionnaire's disease
–
–
–
–
–
Incubation period 2-10 days
Atypical pneumonia
Most cases resolve spontaneously in 7-10 days but
In older or immunocomprmised may be fatal
15-75% mortality
98
Laboratory Diagnosis of Legionella
• Specimen: sputum and blood
• Culture: on Buffered Charcoal Yeast Extract Agar (BCYE)
– Grow after 3-5 days
– Appear as small colonies with ground glass appearance
• Microscopy -Difficult because of
• Lack of staining
• Intracellular nature
• Require large number of organisms to detect
• Detection of antigen in respiratory secretion or urine
• Seroconversion: ≥4 fold rise in specific serum antibody titer
• Direct fluorescent antibody (DFA) staining
99
Yersinia, Francisella and Brucella
• Small Gram-negative rods
• Zoontic diseases
• True pathogens: isolation always associated with disease; i.e.,
always clinically significant
• NOTE: Previously studied nonfermenters were all opportunistic
pathogens
• Very virulent and able to penetrate any body area they touch
– Skin through insect bite, animal bite or direct contact with animals
– Lung after inhalation of infected aerosolized matter
• Non-fermentative
• Facultative intracellular pathogens
• Common treatment: Aminoglycosides and/or Doxycycline which
must be given for prolonged period so as to reach the hidden
intracellular bacteria
100
1. Brucella
• Brucella are small gram-negative coccobacilli, strict aerobic, nonfermentative lacking a capsule, flagella, endospores
• Oxidase and catalase are positive
• Some species require 5-10% CO2 (B. abortus ) for primary isolation
in Lab.
• Requires specialized media and prolonged incubation for growth in
culture
• Facultative Intracellular pathogen
• Brucella spp. is the causative agent of brucellosis
– Brucellosis is a zoonotic infection transmitted to humans
– The disease is rarely, if ever, transmitted between humans
– Brucellosis also called Undulant fever, Malta fever,
Mediterranean fever
101
Human Brucellosis & Associated Species
Severe
102
Epidemiology
Brucellosis occurs worldwide; major endemic areas include
countries of the Mediterranean basin, Arabian Gulf, Indian
subcontinent and Mexico, Central and South America
Major in Saudi Arabia especially Aseer
B. melitensis is the species that infects humans most frequently
The incubation period ranges from a few days to a few months
The disease is manifested as fever accompanied by a wide array of
other symptoms
103
• Human brucellosis is mostly found among farmers, Vet and others
who work with infected animals
• The organism enters the body through
1. Ingestion of contaminated food such as raw milk, cheese made
from unpasteurized milk or raw meat
2. Direct inoculation through skin abrasions from handling animal
carcass, placenta or contact with animal vaginal secretions
3. Inhalation of infectious aerosols
• Localize on reticule-endothelia system (Lymph nodes, liver,
spleen, bone marrow)
• Many organisms are killed by macrophage but some survive
where they are protected from antibodies
104
Clinical Signs of Human Brucellosis
Multi-systemic disease with a broad spectrum of symptoms
Human brucellosis is manifested by intermittent fever
Patient has 3-4 weeks fever and then 3-4 weeks without fever
Incubation Period:1-6 weeks
Asymptomatic infections are common
The disease is extremely variable and the clinical signs may appear
abruptly in symptomatic cases
 After incubation period, an acute febrile illness with non-specific flulike signs occur
 Fever, malaise, chills, night sweats, fatigue, weakness,
myalgias, weight loss, arthralgias and nonproductive cough
 Splenomegaly, hepatomegaly and chest pain
 Anorexia, nausea, vomiting, diarrhea and constipation
105






Clinical Signs of Human Brucellosis
 In many patients, the symptoms last for 2-4 weeks and are
followed by spontaneous recovery
 Others develop an intermittent fever
 Most with this undulant (Rising and falling) form recover
completely in 3-12 Months
 A few become chronically ill
 Relapses (10%) can occur months after the initial symptoms
 Chronic disease and recurrence are common. Why?
 Because it can survive in phagocytes & multiply to high
concentrations
 Complications are seen occasionally, particularly in the undulant
and chronic forms
 The most common complications are arthritis, spondylitis,
epididymo-orchitis and chronic fatigue
106
Brucellosis in Animals
 Brucella cause abortion in animals
 Brucella infects organs rich in erythritol
 Breast, uterus, placenta and epididymis
 Does Brucella cause abortion in humans?
 Brucella does not cause abortion in Human. Why?
 Because of absence of erythritol in human placenta and fetus
 Asymptomatic carriage, sterility or abortions
 Transmitted between animals in aborted tissues
107
Laboratory Diagnosis
A. Culture
• When brucellosis is suspected, Lab. should be informed to
maintain cultures for minimum of 4 weeks
• Blood culture is done into liver infusion broth during febrile attack
• Inoculate 2 tubes, one of them in 10-20% CO2 and the other in the
normal atmospheric conditions
• Rate of isolation from blood ranges from 15-70% depending on
methods used and incubation period
• Bone marrow or lymph node cultures can be done if the blood
culture is negative
• Bone marrow cultures have higher yield than blood
• Most Labs. use rapid isolation techniques, (e.g. BACTEC)
• PCR used for rapid diagnosis of Brucella in blood specimens
108
Laboratory Diagnosis
Serological Tests
• Most serological studies for diagnosis of Brucellosis are
based on antibody detection
• Antibodies appear in the patient’s serum 7-10 days after
the onset of clinical features
• These include:
1. Serum agglutination (Standard tube agglutination)
2. ELISA
3. Complement fixation
4. Indirect Coombs
109
Serum agglutination
• This test is insensitive and should not be relied on for diagnosis
• A titer of >1:160 supports the diagnosis of brucellosis
• A fourfold increase in agglutinating antibodies over 4 to 12 weeks
provides even stronger evidence for the diagnosis
• A titer of 1:100 may be met in normal people
– Due to previous subclinical infection
• False-negative reactions
– Result from prozone phenomenon (High serum concentration)
• False-positive reactions
– Result from cross-reaction with antibodies to Yersinia, Cholera and
Tularemia
• These can be avoidable by routinely diluting serum beyond 1:320
• Must be done using wide range of dilutions of patient’s serum
• In certain conditions antibodies are blocked giving no visible
agglutination, so Coomb’s test must be done
110
Prevention and Control
• Brucellosis can be eradicated from animals by testing them
and slaughtering the positive
• Immunization of animals with a live attenuated vaccine
• Pasteurization of milk and dairy products
• Use of proper safety techniques in clinical laboratory
working with Brucella
111
Treatment
• Intracellular localization of brucellae, believed to offer some
protection against antimicrobials, thus drugs with good intracellular penetration are necessary for cure
• Tetracyclines among most active drugs for treating brucellosis
• Relapse rate unacceptably high with single-drug therapy
• Combination therapy is recommended
• There are two major regimens:
• Regimen A: Doxycycline 100 mg orally twice daily for 6 weeks
+ Streptomycin 1 gm IM once daily for the first 14 - 21 days
• Regimen B: Doxycycline 100 mg orally twice daily plus
rifampin 600-900 mg (15 mg/kg) orally once daily for 6 weeks
112
Yersinia Species
Yersinia formerly classified in the Pasteurellaceae family
So Yersinia formerly called Pasteurella
Yersinia reclassified in the Enterobacteriaceae family
Three species of Yersinia cause disease in humans
Y. pseudotuberculosis
Y. enterocolitica
– Both are animal pathogens
– Y. pseudotuberculosis causes pseudotuberculosis in animals
– Y. enterocolitica sometimes infects man causing Yersiniosis
– Fever, diarrhea, abdominal pain and arthritis
– Yersiniosis is usually self-limiting and does not require treatment
– Both are enteric food and water borne pathogens
– Acquired by ingestion of contaminated food
3. Y. pestis
113
•
•
•
•
1.
2.
Y. pestis (formerly Pasteurella pestis)
•
•
•
•
•
•
•
•
•
•
•
•
•
Discovered in 1894 in Hong Kong by Alexandre Yersin
Gram negative short bacilli (coccobacilli)
Exhibit bipolar staining (special stain e.g. Giemsa stain)
Dark blue bioplar and the remaining appears light blue
Facultative anaerobes, Fermentative, non-motile, non-spore forming
Oxidase negative (Pasteuralla and Brucella are oxidase positive)
Have capsule when freshly isolates
Loss of their capsule when passed in laboratory
Found in low frequency in wild rodent populations (rat)
Rat flea is prime transmitter of disease
Disease:
Plague (Black death)
Disease has an extremely important place in human history 114
Virulence Factors
• Pathogenic Y. pestis produce two antiphagocytic components;
• Fraction 1 (F1) capsular antigen and the VW antigens
• Both are required for virulence
• They are only produced when the organism grows at 37°C
• They not expressed at temperatures < 37°C
• Yersinia is not virulent in fleas Why?
– Because their body temperature normally levels around 25°C
• The bacteria are capable of surviving and multiplying within
monocytes, but not PMNs, and upon emerging from the monocytic
host, the bacteria possess their F1 and VW antigens
115
Transmission Cycles
116
Transmission of Y. pestis
• Bubonic and Septicemic plague
– Disease endemic to rat species
– Flea bites rat and then Y. pestis replicates in flea’s digestive tract
– A solid mass forms, Obstructs the fleas gut, Transfers to the flea’s
mouth and then Introduced to human by flea bits
– Bubonic and Septicemic can occur directly when rat bites human
• Bubonic and Septicemic can not be transferred human to human
• Pneumonic – primary:
– Contracted when infected droplets are directly inhaled
– This can be passed from person to person
• Secondary: develops when bubonic or septicemic goes untreated 
moves to lungs and then can be spread to someone else
117
Types of Plague
• Incubation period 2-6 days
1. Bubonic Plague
– Most common
– Infection of the lymph system (attacks immune system)
– Fever, headache, chills, weakness, swollen and tender lymph
glands
2. Pneumonic Plague
– Most serious type of plague
– Infection of the lungs leading to pneumonia
– Fever, headache, weakness, rapid onset of pneumonia
(accompanied by: shortness of breath, chest pain, cough, bloody
or watery sputum)
3. Septicemic Plague
– Bacteria reproduces in the blood
– Can be contracted like bubonic plague but is most often seen as
a complication of untreated bubonic or pneumonic plague
– Fever, chills, weakness, abdominal pain, shock, bleeding
underneath skin or other organs
118
Stages of Disease
• Bacteria travel through the blood to the nearest lymph nodes
• In lymph nodes, Y. pestis is ingested by fixed macrophages
– Y. pestis grows in macrophages but not PMNs and replicates
– Elicits an inflammatory response (the bubo)
– Bubo is swelling of the lymph nodes
• Bacteria from the bubo leak into blood stream (septicemic plague)
• Lysis of the bacteria releases LPS, which causes septic shock
• Eventually bacteria reach the lung, where they parasitize the lung
macrophages (pneumonic plague)
• At the pneumonic stage, the bacteria can be spread to others via
aerosols (respiratory droplets)
• Direct inhalation at this point of the disease, induces more rapid
development (than flea)
– At this stage the bacteria have well developed virulence factors
119
needed to colonize the human body
Lab diagnosis
• Diagnosis is based primarily on clinical suspicion
• Specimen
– Blood (Septicemic)
– Sputum or (Pneumonic)
– Pus from the bubo (Bubonic)
• Direct smear
– By Giemsa stain shows the characteristic morphology of Y.
pestis
• Smear is the best diagnostic procedure
• Culture
• Blood agar: non-hemolytic
• or enteric media (MacConkey agar): non-lactose ferment
– After 24-48 hrs the colonies were pinpoint grey to greyish white, and
slightly mucloid
120
Treatment
• Without treatment, fatality rates: up to 90% for bubonic
plague, 100% for Septicemic or pneumonic plague
• Treatment, fatality rate= (5-20%)
• Rapid treatment is critical to improved survival
• No major antibiotic resistances have developed
• Plague is usually treated with Streptomycin (Drug of choice)
• Other antibiotics
fluoroquinolones
:
tetracyclines,
chloramphenicol,
– Course of treatment: 10-14 days
121
Prevention and Control
•
•
•
•
•
Keep rodent population down by proper disposal of garbage
Anti-rats, anti fleas measures
Eliminate crowded living conditions of substandard housing
Strict Isolation of cases (Quarantine)
Vaccines
– Effective but protects for less than a year
– Heat killed vaccine consists of whole killed Y. pestis cells
– Requires a series of injections over a 6 month period
– Live attenuated vaccines: Injection of non-pathogenic mutant,
derived from a fully virulent strain
• Chemoprophylaxis
– Sulfonamides and tetracyclines during epidemics
122
Francisella tularensis
• Small gram-negative coocobacilli (0.2 by 0.2-0.7 µm)
• Nonmotile, non-spore forming, strict aerobic, and non-fermentative
• Fastidious and slow-growing
 Requires cysteine-supplemented specialized media
 Blood-glucose-cysteine agar
 Requires prolonged growth
• Facultative intracellular bacterium
• Survive up to weeks at low temperatures in water, soil, and animal
carcasses
• Major target organs are lymph nodes, lungs, pleura, spleen, liver
and kidney
• One of the most infectious pathogenic bacteria known
• Substantial capacity to cause illness and death
123
Francisella tularensis
• Disease:
– Tularemia Also Known As…
– Rabbit fever, Deer-fly fever, Glandular tick fever
– It resembles bubonic plague
• Virulence Factors
– Antiphagocytic capsule
– Thin lipid capsule present in pathogenic strains
– Facultative intracellular parasite that can survive in
macrophages of the reticuloendothelial system
124
Clinical Presentation of Tularemia
NOTE: Also Gastrointestinal & Pneumonic forms of disease
125
Two subspecies (biovars)
Characteristics
Jellison Type A
Jellison Type B
Geographic distribution North America
Europe, Asia
Source of infection
Rabbit and ticks
Contaminated water or
rodents
Severity
Highly virulent (Sever)
Relatively virulent (Mild)
Mortality
5-6% in untreated <0.5% in untreated
cutaneous disease
cutaneous disease
Biochemical properties
•Glycerol fermentation Positive
•Citrulline ureidase
Positive
Negative
Negative
126
Infection
• Incubation period
– 1-21 days (average=3-5 days)
• Infective dose
– 10-50 organisms
• Duration of illness
– 2 weeks
• Mortality
– treated: low
– untreated: moderate
– Ulceroglandular and glandular tularemia are rarely fatal
(mortality rate < 3%)
– Pulmonic or Septicemic tularemia is more acute form of
disease (mortality rate 30-60 %)
• Vaccine efficacy
– good, ~80%
127
Route of Transmission
Skin or conjunctiva
Skin
GI tract
Respiratory tract
Mode of Transmission
Handling of infected animals
Bite of arthropod (vector) which has fed on an
infected animal (Reservoir)
Ingestion of improperly cooked meat or
contaminated water
Aerosol inhalation
• Very small number of organisms to become infected (10-50)
• Humans cannot transmit infection to others
• Reservoirs
– Rabbits, Aquatic Rodents, Rats, Squirrels, Lemmings, Mice
• Vectors
– Ticks, Mosquitoes, Biting Flies
• Ulceroglandular Tularemia (Most common)
– Transmitted through a bite from an arthropod vector which
has fed on an infected animal (Reservoir)
128
Diagnosis of F. tularensis
 Because this bacterium so virulent, most labs will not
culture it from pus or blood
 It is not advisable to drain infected lymph nodes
 Fastidious and slow-growing
 Requires cysteine-supplemented specialized media
 Requires prolonged growth
 Diagnosis rests on
• Clinical picture
• Skin test similar to tuberculin and brucellin
• Measurement of antibodies titer
129
Prevention
• Avoidance of reservoirs and vectors
• Protective clothing and gloves
• Laboratory personnel should be made aware of potential for Fransicella in
clinical specimens
• Best Immunity (Permanent)
– Previous infection with avirulent strain
• Live Vaccine Strain (LVS)
– Best prophylactic
• Only effective vaccine against tularemia
• Doesn’t provide 100% immunity
• Possibility of varying immunogenicity between different batches
• Possibility of a spontaneous return to virulence
• Foshay’s Vaccine (killed bacteria)
– Provides lesser immunity towards systemic and fatal aspects of disease
than LVS
130
Antibiotics to Treat Tularemia
• Antibiotics are effective for treatment after exposure
– Antibiotic treatment must begin several days post-exposure to prevent relapse
• Streptomycin and gentamicin
– Effective against tularemia
– Require intramuscular or intravenous administration
– High toxicity profile
– Can be relapses of tularemia on aminoglycosides
– There exist streptomycin-resistant strains of F. tularensis
• Tetracyclines and chloramphenicol
– Effective against tularemia
– Can be administered orally
– Higher relapse rate than aminoglycosides
• Quinolones (including ciprofloxacin)
– Generally works well and Low relapse rate
– Can be administered orally
– Has not been used extensively for treatment
131
Anaerobic Gram negative bacilli
• Gram negative rods, Anaerobic, Non-spore forming
• Over 24 genera of Gram-negative anaerobic bacilli
• The most important genera:
– Bacteroides, Prevotella, Fusibacterium and Porphyromonas
– B. fragilis, B. melaningenicus (now P. melaningenicus) and B.
corrodens are the most important species
• They predominant components of the florae of mucous membranes
• B. fragilis group are the predominant in the colon (1011/g of feaces)
• Anaerobes outnumber aerobes by 1000:1 in the large intestine
– They play an important role in almost all intra-abdominal infections
• 60% of female carry it in their vagina
• P. melaningenicus and B. corrodens occur primarily in oral cavity
132
•
•
•
•
•
•
•
•
•
•
•
Anaerobic Gram negative bacilli
Opportunistic pathogens
Cause serious infections
Infections are generally polymicrobial
Transmission
Endogenous spread to normally sterile tissues or fluids
Bacteroides infections can develop in all body sites, including the
CNS, the head, the neck, the chest, the abdomen, the pelvis, the skin,
and the soft tissues
Predisposing factors
Any condition predisposing to anaerobic infections such as diabetes
Surgical/Trauma patients and chronic diseases
Human and animal bites
Decreased redox potential: Local tissue necrosis, impair blood supply
and growth of facultative anaerobes (E. coli) at the site contribute to
anaerobic infection
133
– E. coli utilize O2 therefore reduce it to level allow anaerobic to grow
Anaerobic Gram-Negative Infections
• CNS Infections
• Brain abscess and meningitis
– Brain abscesses are commonly caused by adjacent chronic infections in
ears, mastoids, sinuses, oropharynx, teeth or lungs
• Head and neck Infections
• Chronic otitis media; sinusitis; mastoiditis; tonsillar, peritonsillar, and
retropharyngeal abscesses; cervical lymphadenitis
• Dental infections, include periodontal disease, gingivitis, localized
periodontitis, pericoronitis, endodontitis, periapical and dental
abscesses and post-extraction infection
• Pleuropulmonary Infections
• 90% of patients with community-acquired aspiration pneumonia
• B. fragilis is found in 25% of lung abscesses
• Intra-abdominal Infections
• Secondary peritonitis, Appendicitis and Abdominal abscesses 134
Anaerobic Gram-Negative Infections
• Gynecoloical Infections
• Bacterial vaginosis; soft tissue perineal, tubo-ovarian, pelvic and
vulvar abscesses; endometritis; salpingitis; pelvic cellulitis;
intrauterine device–associated infection; septic abortion and
postsurgical obstetric and gynecologic infections
• Skin and Soft Tissues Infections
• Infected cutaneous ulcers, secondary diaper rash
• Cutaneous and subcutaneous abscesses, breast abscesses
• Necrotizing fasciitis, necrotizing synergistic cellulitis, gas gangrene
• Osteomyelitis and septic arthritis
• Bacteremia (5-15%)
135
Bacteroides Virulence Factors
 Polysaccharide capsule
◦ Antiphagocytic and adhesive
 Lipopolysaccharide
◦ Stimulates leucocytes migration and chemotaxis
 Agglutinins
◦ Enhance adherence
 Histolytic enzymes
◦ Tissues destruction, inactivates Immunogloublins
 Oxygen tolerance
◦ Catalase and superoxide dismutase
 Beta-lactamases
 Inactivates penicillins
136
• Diagnosis
• Direct-needle aspiration is the best method of obtaining a
culture
• Specimens obtained from normally sterile sites, such as blood
or spinal, joint, or peritoneal fluids
• Bacteroides can be isolated anaerobically on blood agar
containing kanamycin and vancomycin to inhibit unwanted
bacteria and incubated anaerobiacally
• Treatment
• Directed at all major aerobic and anaerobic pathogens
• Clindamycin, cefoxitin and cefotetan
• Almost
sensitive
to
metronidazole,
carbapenems,
chloramphenicol and combinations of penicillin/β-lactamase
137
inhibitors
• Prevention and Control
• Avoiding conditions that reduce the redox potential of the
tissues
• Preventing the introduction of anaerobes of the normal flora to
wounds, closed cavities, or other sites prone to infection
• Surgical intervention, abscesses drainage and antibiotic
treatment
• Prophylactic therapy are used when medical procedure disrupt
the mucosa or if disruption of mucosa causing trauma
• No vaccines are available
138
General characteristics
Spirochetes
• Tiny (0.2 W x 6-20 m L), thin walled, flexible,
spirals (helical) that like corkscrew
• They are coiled into regular helices (6-14/cell)
• Move in a unique spinning fashion via 6 thin
endoflagella called axial filaments
• Flagella lie between outer membrane & PDG layer
• Gram negative
• Too small to be seen using the light microscope
139
•Special procedures are required to view spirochetes
– Dark field microscopy
– Immunofluorescence
– Silver stain (Fontana’s silver or Giemsa’s stain)
•Pathogenic species do not grow on artificial media
•While non-pathogenic species can be grow
•Can be propagate some strains of spirochetes by
inoculating it into the testes of the living rabbit
140
Classification of Spirochetes
• Human pathogens belong to following 3
genera:
1. Treponema
2. Leptospira
3. Borrelia
• Others (saprophytes) are found in water,
sewage & in mouth & genital tracts of humans
141
Treponema pallidium
• The causative agent for STD “syphilis”
• Syphilis is either
1. Acquired (STD, Venereal)
• Transmitted from infected skin or mucous membrane
(genitalia, mouth, rectum) to other person by intimate contact
2. Congenital
• Transmitted from pregnant women to their fetus
3. Blood transfusion
• Rarely reported
• Men are more susceptible to contract syphilis than women
• Marked increase in syphilus in homosexual men
142
Stages of venereal syphilis
A. Primary syphilis
•
Primary lesion is Hard chancre
Develop on genitals in 10-90 day
Chancre heals spontaneously in 3-12 week without scar
Enlargement of lymph nodes on genital organs
Firm, painless, indurated & ulcerated lesion; covered
with a thick exudate rich in spirochetes
Highly infectious
•
Spirochetes spread widely in tissue leading to 2ry syphilis
•
•
•
•
•
143
Stages of venereal syphilis
B. Secondary syphilis
•
•
•
•
•
•
•
•
Most infectious stage
Untreated patient enters bacteremic or 2ry syphilis
1-3 months later, secondary lesions may appear
T. pallidium multiply and spread via blood
This stage is systematic and characterized by
Generalized lymphadenopathy
Maculopapular skin rashes on the body & condylom
latum (wart) at mucocutaneous junctions
Lesions heal spontaneously over 6 week and yet the
disease may progress latency & then to tertiary stage
144
Stages of venereal syphilis
• Latent Syphilis
•
•
•
•
•
•
•
•
Most patients are asymptomatic during this period
Early latent period
Last for 1-2 years after the 2nd stage
25% of patient develop symptoms of 2ry syphilis
Patient can infect other
Late latent period
Last for many years
No symptoms occur and patients are not infectious
• 30% of untreated patients slowly progress to 3ry syphilis
• The rest will remain asymptomatic
145
Stages of venereal syphilis
C. Tertiary syphilis
• Develops over 6-40 year with slow inflammatory
damage to organs, blood vessels & nerve cells
• It can be grouped into 3 general categories
1. Gummatous syphilis
• Occurs 10 year after 1ry stage in 10% of untreated
• May show localized granulomatous lesions (gumma)
which eventually necrosis and become fibrotic
• Noninfection lesion are found mainly in skin & bones
146
Stages of venereal syphilis
2. Cardiovascular syphilis
• 3-10 year after 1ry stage in 15% of untreated patients
• Characterized by aortic aneurysm & necrosis in aorta
3. Neurosyphilis (Late tertiary or quaternary syphilis)
• Occurs 10-20 year in 8% of untreated patients
• Asymptomatic neurosyphilis, subacute meningitis, brain
infarction, mental deteriorations & psychiatric symptoms
• In 3ry lesion , T. pallidium are rarely seen
147
Congenital syphilis
• Transplacental spread of T. pallidium after the 1st
trimester of pregnancy may result in
– Fetal death
– Prematurity or
– Congenital syphilis
• The early syndrome may not present at birth, only
developing during the subsequent 4 weeks
• They are characterized by
– Generalized rash resemble 2nd acquired syphilis
– Enlargement of the liver, spleen and lymph nodes
• Congenital syphilis leaves residual characteristic sign
– Hutchinson’s teeth
– Saddle nose or nose deformity
148
Immunity of syphilis
Immunity of T. pallidium is incomplete
Antibodies do not stop the progression of the disease
Treated syphilitic Patient can contract syphilis again
Late syphilitic patient is relatively resistant to reinfection
The antigens of T. pallidium induce
Specific antibodies (non-treopenemal); detected by
immuno-fluorescence or hemagglutination tests
• Nonspecific antibodies (reagin): detected by the
flocculation of cardiolipin extracted from beef heart tissues
• Both specific and non-specific antitreponemal antibodies
are used in serological diagnosis of syphilis
•
•
•
•
•
•
149
Diagnosis of Syphilis
• Syphilis is confirmed by
– Finding T. pallidium in exudates from the lesions
– Or by detection of antibodies in the serum
• During t 1ry and 2ry stages spirochetes are
demonstrated in the early lesions in wet preparation
by dark field or immunfluorescense microscopy
• Stained with either Fontana or Giemsa stain
• Not seen on a Gram-stained smear
• During 2ry & 3ry syphilis can be detected serologically
150
Serological Tests of Syphilis
Patients develop 3 types of antibodies
1st is known as a lipoidophil antibody called regain
Tests depend on the detection of regain
Non-specific test/Standard Tests for Syphilis (STS)
STS used for routine screening
STS involves use of non-treponemal antigens
(Cardiolipin) which react with regain in patient’s serum
• Can be demonstrated by CFT (Wassermann test) or by
Flocculation Tests such as Venereal Disease Research
Laboratory (VDRL) or Rapid Plasma Regain (RPR) test
•
•
•
•
•
•
151
Serological Tests of Syphilis
• 2nd antibody called Reiter which react with a protein
component found in non-pathogenic T. reiter
• This test called Group specific test
• Demonstrated by Reiter Protein CFT
• This test is more specific than the STS but gives
fewer false positive results
152
Serological Tests of Syphilis
• 3rd type of antibody reacts directly with T. pallidium
• These tests are specific
• Using Nichol’s strain
• It can be demonstrated by T. pallidium
Immobilization (TPI) test, Fluorescent Treponemal
Antibody (FTA) test and T. pallidium
Haemagglutination (TPHA) test
• This test is the most specific tests
153
Prevention, Control & Treatment
• There is no vaccine against syphilis
• This disease is controlled by
– Early diagnosis and appropriate treatment
– Use of condoms
– Follow-up of infected individuals & their contacts
• This disease is treated by Penicillin
• Tetracycline or Erythromycin can be used in allergic
patients to Penicillin
154
Non-venereal Treponema
• There are three subspecies of T. pallidium
–T. pallidium subspecies endemicum
• It causes endemic syphilis (Bejel)
–T. pallidium subspecies pertenue
• It causes yaws
–T. pallidium subspecies carateum
• It causes pinta
155
Borrelia causing diseases
Borellia
Causing
Relapsing Fever
Endemic (European)
Longer febrile period
Fewer relapses
Lyme
Endemic (Africa)
Shorter febrile period
More relapses
B. recurrentis
Transmitted by Lice
B. burgdorferei
B. duttoni
Transmitted by Ticks
156
Main differences between the two types of
relapsing fever
European or louse-borne type West African or Tick-borne type
Caused by B. recurrentis
B. duttoni
Transmitted by body louse Tick
Often appears in epidemics Endemic
Long febrile period and fewer
relapses
Shorter febrile and more relapses
Spirochetes not transmitted Transmitted to tick progeny
to louse progeny
157
Rickettsiae and Chlamydiae
• Are Rickettsiae & Chlamydiae Bacteria or Virus?
• They share a few characteristics of viruses
a. Their size relative to virus 300-500 nm
b. Obligate intracellular parasites
– Lack of ATP-generating ability
– Need to obtain ATP from the host cell
c. Grow in living media (yolk sac of chick embryo)
• They similar to bacteria
A. They have both DNA and RNA
B. They have ribosomes
C. Sensitive to antibacterial agents
158
Rickettsiae and Chlamydiae
• Similarity between Rickettsiae & Chlamydiae
• Rickettsiae are similar to Chlamydia in that
 Both are obligate intracellular energy parasites Share
with
 Both are similar in size as large viruses
viruses
 Both grow in living media (yolk sac of chick embryo)
 They have both DNA and RNA
Share with
 They have ribosomes
bacteria
 Both are replicate by binary fission
 Both are sensitive to antibacterial agents
159
Rickettsiae and Chlamydiae
• Rickettsiae are different to Chlamydia in that
 Require arthropod vector (except for Q fever)
 Replicates
freely in cytoplasm while Chlamydia
replicates in endosomes
 Ricketsia has tropism for endothelial cell
that line blood
vessels while Chlamydia like columnar epithelium
160
Comparison of Chlamydiae & Rickettsiae
with Bacteria & Viruses
Bacteria
Chlamydia & Rickettsia Viruses
300-3000
350
15-350
Obligatory intracellular No
Yes
Yes
Nucleic acids
DNA & RNA
DNA & RNA
DNA OR RNA
Reproduction
Fission
Complex cycle with fission
Synthesis & assembly
Antibacterial sensitivity Yes
Yes
No
Ribosomes
Yes
Yes
No
Metabolic enzymes
Yes
Yes
No
Size (nm)
Energy production
Yes
No
No
161
Rickettsiae
Taxonomy of Rickettsia
 Family: Ricketsiaceae
 Genus: Rickettsia, Coxiella, Bartonella
 Species
1. Rickettsia prowazekii (epidemic typhus)
2. Rickettsia typhi (endemic typhus)
3. Rickettsia rickettsii (spotted fever)
4. Bartonella quintana (trench fever)
5. Bartonella henselae (cat scratch fever)
6. Coxiella burnetii (Q fever)
162
Rickettsiae
Gram-negative, coccobacilli, non-motile
 Best stained by Giemsa or Machiavello stains
Small (0.3-0.5 x 1-2 m) prokaryotic cells
 Contain both RNA & DNA & Multiply by binary fission
Obligate intracellular energy
 Except Bartonella quintana
They grow in tissue culture or yolk sac
 Except B. quintana: grow on blood agar under 5%CO2
All Rickettsial diseases are zoonotic
 Except Epidemic typhus and Trench fever
163
Rickettsiae
• Transmitted through arthropod vector from reservoir
• Arthropods serves as both vector and reservoir
– Except C. burnetii does not transmitted through vector
• Cause skin rash, fever, headache & malaise
• ESCHAR, black ulcer developed at site of inoculation
– Except C. burnetii cause pneumonia, slow fever &
hepatitis
• Rickettsiae are susceptible to antiseptics, dryness & heat
– Coxiella resist pasteurization at 600C for 30 m
• Rickettsiae are sensitive to chloramphenicol & doxycycline
164
Pathogenic Mechanism
Virulence factors：
Mechanism：
Endotoxin, Phospholipase A, slime layer
Bites or faeces of arthropod
Local lymph or micro blood vessels
(1st bacteremia)
Endothelial cells, micro blood vessels
in whole body
(2nd bacteremia)
Fever, rash, headache, etc
Targets:
Endothelial cells, micro blood vessels
165
Rickettsial Diseases: 1-Typhus Group
Organism
Reservoir
Vector
Epidemic Typhus
Endemic Typhus
Rickettsia prowazekii
Human and flying squirrels
Lice borne
Rickettsia typhi
Rats and small rodent
Flea borne
Transmission Infected louse feces rubbed into
Inc. period
broken skin
8 days
Infected flea feces
rubbed into broken skin
7-14 days
Distribution
Worldwide
worldwide
Clinical
Sudden onset of fever & headache Gradual onset of Fever,
Rashes which spares the palms,
Headache, Rash
soles and face
Delirium/ stupor
Gangrene of hands or feet
166
Rickettsial Diseases:2- Spotted fever Group
Rocky Mountain Spotted
Fever
Causative agent
Reservoir
Vector
Transmission
Geographic
distribution
Clinical
Mediterranean Sea
Spotted Fever
Rickettsia rickettsii
Dogs, rabbits, & wild rodents
Tick borne
Rickettsia conorii
Rodent
Tick borne
Wild and dog ticks bite
USA
Tick bite
Southern Europe,
Africa, Middle East
•Fever
•Severe Headache
•Rash
•Fever
•Conjunctival redness
•Sever headache
•Rash on wrests, ankles, soles,
palms initially and becomes
more generalized later
167
Rickettsial Diseases: 3. Trench Fever
 Also called Five-day fever
 Causative agent: Bartonella quintana
 Reservoir: Human only
 Vector: Body human louse
 Transmission: Via feces of infected body lice being scratched
into the skin
 Geographic distribution:
 Temperate regions and high elevations in the tropics, including
South America
 Clinical
◦ Fever, Headache and Back pain
◦ It lasts for 5 days and recurs at 5 day intervals
168
Rickettsial Diseases: 4- Q Fever
 Coxiella burnetii is the causative agent of Q fever. Q from Query
 Geographic distribution: Worldwide
 Clinical: Fever, Headache, Viral-like pneumonia, No Rash
– Complication of Q fever are Hepatitis and Endocarditis
 It can grow at pH 4.5 within phagolysosomes
 Coxiella burnetii is unique to the Rickettsiae Why???
 Because like Gram-positive spore formers
 This endospore confers properties to the bacteria that differ
from other Rickettsiae:
 This make the organism resistance to heat and drying
 Also extracellular existence
169
Q Fever
Q fever is zoonotic and occupational disease
Reservoir: Cattle, sheep and goats
Non-arthropod vector transmission due to
Coxiella burnetii grows in Ticks and Cattle
The spores remain viable in;
– Dried tick feces deposited in cattle hide and
– Dried cow placentas following the birthing
• So Pneumonia occurred via inhalation of Spores via
– Airborne transmission of spore from hide or dried placenta or
– Via consumption of spore contaminated unpasteurized milk
•
•
•
•
•
170
Laboratory diagnosis
•
•
•
•
•
Specimen: Blood and/or autopsy (Tissue)
Staining
Gram stain: gram negative coocobacilli (poorly stain)
Giemsa stain: purple
Isolation: difficult and dangerous
– The organism can be inoculated into tissue culture and grown over 4-7
days but this is very hazardous to personnel
• Serodiagnosis
1. Complement Fixation test (CFT)
Complement
Complement
Ag-Ab
Ag + Ab
Indicator system
RBCs-anti-RBCs
If the complement is free
Ag-Ab
+
Indicator system
RBCs-anti-RBCs
No lysis
Ag-Ab
Lysis
171
Laboratory diagnosis
2.
Indirect Immuno-fluorescence reaction (IIFR)
• Rckettsial antigen + Patient’s serumn
Ag-Ab complex
• Add anti-antibody (Fluorescence) Ab-F
• Result in Ag-Ab-Ab-F which detected under fluorescence microscope
• The use of immunofluorescent antibodies to examine a biopsy can be diagnostic
3.
Weil-Fleix Reaction
• Some Rickettsia share antigenic characteristics with non-motile Proteus vulgaris
strain
• P. vulgaris strains that share these common antigens are designated OX-2, OX19, OX-K
• This is done by mixing patient’s serum (Antibody) and P. vulgaris strains (Antigen)
• Agglutination means positive test
• Results are shown in the Table
172
Weil-Fleix Reaction
Disease
Weil-Fleix
OX-19
OX-2
OX-K
Rocky Mountain spotted fever +
Mediterranean Sea fever
+
+
+
-
Rickettsial pox
-
-
-
Epidemic typhus
Endemic typhus
+
+
-
-
-
-
Trench fever
-
-
-
Q fever
-
-
-
-
173
Control
 Sanitary:
 Arthropod and rodent control
 Immunological:
 No vaccines are currently available
 Chemotherapeutic:
 Tetracycline or chloramphenicol are drugs of choice
174
Chlamydiae
• Chlamydia is extremely tiny
• Gram-negative cocci bacteria, non-motile
• Have rigid cell wall
– Have outer membrane & Cytoplasmic membrane
– Lacking PDG and muramic acid
• Basophilic because stained blue by Giemsa stain
– Elementary bodies stain purple
– Reticulate body stain blue
– Inclusion body stain dark purple
• All share common complement fixing antigen
175
Special Growth developmental Cycle
•Elementary body
•Infects host epithelial cell
•Taken by endocytosis
•Reticulate body
•Formed 1-2 hr latter
•Larger than EB
•Divided by Binary fission
•Inclusion body
•Aggregates of small particles
•Formed within 24-48 hrs
•Host cell rupture releasing EB
•Infects again new host cell
176
Characteristics of elementary and
reticulate bodies of Chlamydia
ELEMENTARY BODY (EB)
RETICULATE BODY (RB)
Size 0.3 um (300 nm)
RNA:DNA content = 1.1
Infectious
Adapted for extracellular survival
Hemagglutinin present
Induces endocytosis
Metabolically inactive
Size 0.5 - 1.0 um (500-1000 nm)
RNA:DNA content = 3.1
Not infectious
Adapted for intracellular growth
Hemagglutinin absent
Does not induce endocytosis
Metabolically active
177
Classification and Differentiation of Chlamydiae
Subgroup A
Subgroup B
Mammalian parasites
Bird parasites
Compact inclusions
Diffuse inclusions
Transmitted by contact
Transmitted by inhalation
Glycogen synthesized
Glycogen not synthesized
Folates synthesized (Sensitive)
Folates not synthesized (resistant)
Sensitive to D-cycloserine
Resistant to D-cycloserine
Restricted host range
Broadening of host range
• Chlamydia trachomatis
• Chlamydia psittaci
• Infects
non-ciliated
columnar • Chlamydia pneumoniae
epithelial cells (EYE and GENITAL)
Both infect LUNG
178
SubgroupA: Chlamydia trachomatis
 Three biovars (biological variants):
1. Biovar Trachoma (15 Serologic types (A-K)
– A, B, Ba, C, D, Da, E, F, G, H, I ,Ia, J, Ja, K
– Serotypes A, B & C cause Trachoma
– Serotypes D-K cause inclusion conjunctivitis (Newborn),
Non-Gonococcal Urethritis, Cervicitis, Infant pneumonia
2. Biovar lymphogranuloma venereum, LGV (4 serologic
types) (L1, L2, L3,L4)
3. Biovar mouse
 Infects human non-ciliated columnar epithelial cells: Eye and
Genitals Except Biovar mouse
179
a. Trachoma
 Severe form of chronic conjunctivitis
 Caused by C. trachomatis biovar Trachoma Serotypes A, B & C
 Transmission occurs by hand-to-hand transfer of infected
secretions to eye by infected articles (Towels)
 Can be also transmitted by droplets, contaminated clothing,
flies and by passage through an infected birth canal
 Infections occur most commonly in children
 Incubation period of 5 to 12 days
180
a. Trachoma
 Conjunctivitis, pink eye, Eye discharge, Swollen eyelids,
Swelling of lymph nodes in front of the ears, corneal ulcer
 Producing scarring and deformity of the eyelids and corneal
vascularization and opacities which may lead to blindness
 Blindness develops slowly over 10-15 years
 Occurs worldwide primarily in areas of poverty & overcrowding
 500 million people are infected worldwide
 7 - 9 million people are blind as a consequence
181
b. Inclusion conjunctivitis
 Collection of initial bodies in cytoplasm of conjunctival cells
 STD (Sexual Transmitted Disease) caused by C. trachomatis
biovar Trachoma Serotypes D-K
 Infection derives from mother to neonates during birth
 Mucopurulent yellow discharge and swelling of eyelids
 Develops 5-14 days after birth
 Newborns are given Erythromycin eye drops prophylactically
182
c. Infant pneumonia
• Spread of Biovar Trachoma serotypes D-K through
nasolacrimal duct
• Common in babies
• Develops between 4-11 weeks of life
• Initially, the baby develops upper respiratory symptoms
followed by rapid breathing, cough and respiratory distress
• Diagnosed by presence of Chlamydial anti-IgM and/or
demonstration of C. trachomatis in clinical specimen
• Treated with oral erythromycin
183
d. Urethritis (Male/Female)
 Caused by C. trachomatis Biovar Trachoma Serotypes D-K
 Urethritis –One cause of Non-Gonococcal Urethritis (NGU)
◦ NGU are most common caused by C. trachomatis & U. urealyticum
 STD and Majority (>50%) asymptomatic
 Symptoms : mucoid or clear urethral discharge, dysuria
 Incubation period unknown (5-10 days in symptomatic infection)
 Clinically, no difference between NGU and gonococcal urethritis
 Urethritis usually occurs as mixed infections
 Empirical therapy for urethritis
 Single dose of ceftriaxon (against Gonococci) followed by days
course of doxycycline or azithromycin (against NGU agents) 184
e. Cervicitis and Pelvic Inflammatory Disease (PID)
 Cervicitis
•
•
•
•
•
◦ Majority (70%-80%) are asymptomatic
◦ Local signs of infection, when present, include:
 Mucopurulent endocervical yellow discharge
 Edematous cervical ectopy with erythema and friability
Infection can spread upwards to involve uterus, fallopian tubes
(Salpingitis) and ovaries
PID develops abnormal vaginal discharge or uterine bleeding,
pain with sexual intercourse, nausea , vomiting and fever
The most common symptom is lower abdominal pain
Infection by both N. gonorrhoeae & C. trachomatis is called PID
Treated by ceftriaxon followed by 14-days doxycycline
185
f. Lymphogranuloma Venereum (LGV)
• STD
• Caused by C. trachomatis biovar lymphogranuloma venereum
L1, L2, L3 serotypes
• Starts with painless papule or ulceration on the genitals that heal
spontaneously
• The bacteria migrate to regional lymph nodes which enlarge over
the next 2 months
• These nodes become increasingly tender and may break open
and drain pus
• Fever, skin rash, nausea, and vomiting are often found
186
Lab diagnosis
• Specimen:
– Sputum, Conjunctival scrapping, urethral discharge
or pus in LGV
• Satin:
– Giemsa’s stain
• Culture
– Cell culture treated with cycloheximide
• Non-culture tests
– Nucleic Acid Amplification Tests (NAATs)
– Non-Nucleic Acid Amplification Tests (Non-NAATs)
– Serology (Complement Fixation Test)
187
Subgroup B: C. psittaci
• It infects > 130 species of birds
• It is the causative agent of;
– Psittacosis (parrots; parrot fever) or
– Ornithosis (Pigeons, chicken, ducks and turkey)
• Occupational disease for poultry workers
• Zoonotic disease (animal diseases)
• Infection occurred by inhalation of respiratory discharge,
feather or contaminated fecal material of birds
• Occurs after 1-3 weeks after exposure
• Infection results in atypical pneumonia
188
Subgroup B: C. pneumoniae
• First recognized in 1983 as a respiratory pathogen,
after isolation from a student with pharyngitis
• Pneumonia or bronchitis, gradual onset of cough with
little or no fever. Less common presentations are
pharyngitis, laryngitis, and sinusitis
• Person-to-person transmission by respiratory secretions
• All ages at risk but most common in school-age
children
189
Mycoplasma & Ureplasma
 Smallest free-living organisms (150-250 nm)
 Pass through some bacterial filters
 Lack of a cell wall
 Three layer membranes
◦ Outer and inner: proteins and saccharide
◦ Middle: 1/3 cholesterol
 Multiple shapes: round, pear shaped & even filamentous
 Require complex media containing sterol
 Require sterols for growth and for membrane synthesis
 Grow slowly (3 weeks) by binary fission and produce "fried
egg" or “T strain” (tiny strain) colonies on agar plates
190
• Two genera that infect humans: Mycoplasma and Ureaplasma
• They belong to Mycoplasmataceae
• There are many species of mycoplasmas
• Only four are recognized as human pathogens;
1. Mycoplasma pneumoniae
– Upper respiratory
pneumonia
tract
disease,
Tracheobronchitis,
atypical
2. Mycoplasma hominis
– Pyelonephritis, pelvic inflammatory disease, postpartum fever
3. Mycoplasma genitalium
– Nongonococcl urethritis (NGU)
4. Ureaplasma urealyticum
– NGU and may play a role in male fertility
– Isolation pathogen cultured in pH 6.0 media
– Requires 10% urea for growth
191
Pathogenesis
 Adherence factors
 Adherence proteins are one of the major virulence factors
 Adhesin localizes at tips of the cells and binds to sialic acid
residues on host epithelial cells
 Toxic Metabolic Products




Intimate association provides an environment in which toxic
metabolic products accumulate and damage host tissues
Products of metabolism : hydrogen peroxide and superoxide - oxidize host lipids
Inhibit host cell catalase
Immunopathogenesis



M. pneumoniae is a superantigen
Activate macrophages and stimulate cytokine production and
lymphocyte activation
192
Host factors contribute to pathogenesis
M. pneumoniae
Need 10-20% Serum to culture in pH 7.8-8.0
Pathogenesis: P1 protein, capsule and saccharide
Spread by close contact via aerosolized droplets
Incubation period: 1-3 weeks
Cause tracheobronchitis, primary atypical pneumonia
– Fever, headache, sore throat and cough
– Initially cough is non-productive but occasionally
paroxysmal
• Antibodies play a role in controlling infection, particularly IgA
• Delayed type hypersensitivity
•
•
•
•
•
193
Medical Mycology
Myco = Fungi
Ology = Science
Mycology is the science deals with fungi
Mycoses = Fungal infection
Dermatophytosis - "ringworm" disease of the
nails, hair, and/or stratum corneum of the skin
caused by fungi called dermatophytes
Dermatomycosis - more general name for any
skin disease caused by a fungus
194
What is the FUNGUS?
Eukaryotic → a true nucleus
Do not contain chlorophyll
Organoheterotrophic
Cannot photosynthesize their own food
Live either as saprophytes or parasites
Have chitin cell walls
Produce filamentous structures
Reproduce by spores (sexual & asexual)
Are aerobic life forms
195
What are Actinomyces?
True Bacteria (Prokaryotic)
Similar to fungi (fungi-like bacteria) WHY?
1. Clinical infection resembles mycoses
2. Grow on mycotic media
3. Grow slowly >48 h
4. Gross colonies resemble fungi
 (rough, heaped, short aerial filaments)
5. Resemble mycelia microscopically,
branched mycelia in tissue and smears
with
196
Fungi vs. Bacteria
Bacteria
Fungi
Nucleus
Prokaryotes
Eukaryotes
Cell Wall
Peptidoglycan
*Chitin
Nutrition
Auto- or Heterotrophs Heterotrophs
Reproduction Binary fission
Example
E. coli
Sexually & asexually
Aspergillus
•Chitin is not found in any other microorganisms
197
Types of fungi
1. Multicellular: Molds- filamentous
Penicillium, Aspergillus
2. Unicellular: Yeasts
 Candida, Cryptococcus
3. Dimorphic Fungi
 Dermatophytes, Histoplasma
198
YEAST
Facultative Anaerobes
Fermentation=ethanol and CO2
Non-filamentous unicellular fungi
Spherical or oval
Reproduction by
Fission or budding
199
DIMORPHIC FUNGI
 Growth as a mold or as a yeast
 Most pathogenic fungi are dimorphic fungi
 At 37o C yeast-like
 At 25o C mold-like
 Can also occur with changes in CO2
 Fungi grow differently in tissue
nature/culture; often dictated by temp
vs
200
Basic structure of fungi
1. Hyphae
Main body of most fungi is made up of fine,
Cylindrical, branching threads called hyphae
Tubular cell wall filled with cytoplasm & organelles
Most hyphae are 2-10 m diameter
2. Mycelia
When formed of many cells, cellular units connect
together (intertwining) to form long filamentous
A. Aerial mycelium
Part projects above the surface of medium
B. Vegetative mycelium
Part penetrate into medium and absorb food
201
Basic structure of fungi
• Septa—regular cross-walls formed in hyphae
• Septate: Hyphae with septa - or
• Aseptate or coenocytic: Lacking septa except to
delimit reproductive structures & aging hyphae
• Primary septa: Formed as a process of hyphal
extension & generally have a septal pore, which
allows for cytoplasmic & organelle movement
• Secondary
or
adventitious
septa
are
imperforate, formed to wall off ageing parts of
the mycelium
202
Reproduction
• Propagate via formation of spores
• Sexual and asexual spores
• The shape and type of spores are
– Different from one type of fungi to another
– Important
in
the
classification
&
identification of different species of fungi
203
Asexual spore
• Conidiospore
• Multiple (chains) or single spores formed at the
end of an aerial hypha
• Not enclosed by a SAC
– Aspergillus spp.
– Penicillium spp
• Conidiophore: filament that forms Conidospore
204
ConidioSpores
Arthrospores
Cells in hyphae develop thick wall & separate by
disarticulation
Coccidioides- genus of dimorphic Ascomycete
Blastospores
Thickened wall
Bud from the parent cell
Candida, Cryptococcus
205
Chlamydospores
• Spore contained within hypha
• Rounded & Thick cell wall
• Chlamydophores
– Aerial hypha with chlamydospores
• e.g. Candida albicans
206
Sporangiospore
Spore contained in a sporangium at the end of a
sporangoiphore
Sporangium:
A sac or cell containing spores produced asexually
Sproangiophore- aerial hypha with sporangium
Rhizopus spp
207
Sexual spores
• Sexual spores - exhibit fusion of nuclei
• Ascospore
– Formed in sac-like cell (ascus)
– Often 8 spores formed
– Ascomycetes
• Basidiospore
– Produced on a specialized club-shaped structure
(basidium)
– Basidiomycetes
• Zygospore
– Thick-walled spore formed during sexual
reproduction
– Zygomycetes/ Mucor and Rhizopus
208
Classification of Fungi
Comprised of over 100,000 species
Classified into 4 orders according to the
presence or absence of sexual reproductive cycle
and
the
nature
of
sexual
spores
Orders of Fungi
1. Zygomycetes
2. Ascomycetes
Mycelium
Reproduction
Non-septate Sexual
Septate
Sexual
3. Basidomycetes
Septate
4. Deutromycetes (Fungi Septate
imperfecti)
Sexual
No sexual
209
ZYGOMYCETES
• Phylum Zygomycota/Sexual/Non-septate
• Lower fungi
• < 1000 species
• Produce zygospore, or sporangiospores
• Include the common bread molds and other fungi
that cause food spoilage
• Mucor and Rhizopus are most familiar example
210
Ascomycetes
• Phylum Ascomycota, Septate/Sexual/Higher fungi
• Certain yeasts & some fungi that causes plant disease
• Contains more than 30,000 species of unicellular
and multi-cellular fungi
• Produce sexual spores (ascospores)
• Produce asexual exospores (conidia)
• Unicellular e.g. Saccharomyces, Candida
• Multicellular e.g. Penicillum, Asperigullus, Claviceps,
Dermatophytes, Sporothrix schenckii, Histoplasma
211
Basidomycetes
• Phylum Basidiomycota/Septate/Sexual
• Higher Fungi-Over 30,000 different species
• Sexual spores borne on clublike stalks (basidia)
• Mushrooms
(Agaricus
bisporus,
Agaricus
campestris) are the most familiar members
• Among
Basidiomycota,
only
Malassezia
& Cryptococcus are frequent human pathogens
212
Septate mycelium: Deutromycetes:
• Fungi imperfecti
• Sexual life cycle is either unknown or absent
• Reproduce by various types of asexual spores
including budding
• Have an abundant mycelium at times while at other
times they grow as yeast-like cells
• Includes majority of pathogenic to man & animal
• Resemble Ascomycetes in morphology
• Examples:
Trichophyton,
Epidermophyton,
Microsporon, Candida, Cryptococcus, Histoplasma
213
Fungal cell wall composition
Consists of
 80% of polysaccharides
Polysaccharide fibrils provide rigidity/integrity of wall
 20% of the wall consists of Proteins
1. Structural components (Fibrous)
A. Chitin microfibrils
Polymers of ß (1-4)-linked N-acetylglucosamine
B. Chitosan (in Zygomycota only)
De-acetylated chitin
C. ß-linked glucans
Polymers of β-1,3-linked glucose residues with
short β -1,6-linked side chains
214
Fungal cell wall composition
2. Matrix components (Gel-like components)
 Structural polymers are contained in gel-like
matrix
A. Mannoproteins
 Glycoproteins (form matrix throughout wall)
B.  (1,3) glucan
215
Structure of cell membrane
Semi-permeable
Phospholipid bilayer
Involved in uptake of nutrients
Anchorage for enzymes/proteins, e.g., chitin synthase,
glucan synthase, etc.
Signal transduction
Differs in that it contains ERGOSTEROL
Site of action for certain antifungal drugs
 Human cell doesn't contain ergosterol
 Human cell contains cholesterol
216
Medical Effect of Fungi
•
•
•
•
•
•
•
•
I. Mycotoxicoses:
Mould produces secondary metabolite (MYCOTOXINS)
Highly toxic to humans
Ingestion of toxic fungi or their metabolites
Ergotism is caused by eating bread prepared from rye
infected with Claviceps purpurea
Historically, several large scale outbreaks of madness in
populations have been attributed to ergotism
Ergot are -adrenergic blockers inhibits response to
adrenaline vasoconstriction necrosisgangaren
Symptoms consisted of inflammation of infected tissue,
followed by necrosis and gangrene
217
Medical Effect of Fungi
Natural occurrence:
Food products contaminated with AFLATOXINS include
cereal (maize, rice & wheat), oilseeds (groundnut,
soybean & cotton), spices (black pepper, coriander &
zinger), tree nuts (almonds, and coconut) & milk
Physical and chemical properties:
Aflatoxins are potent toxic, carcinogenic, mutagenic,
immunosuppressive agents, produced as secondary
metabolites by Aspergillus flavus and A. parasiticus
218
Medical Effect of Fungi
II. Hypersensitivity Disease:
Fungal spores are inhalated
They can be an antigenic  elicit immune
response  production of Ig or sensitized
lymphocyte
Example is hypersensitivity pneumonitis
219
Medical Effect of Fungi
III. Colonization and resultant disease:
They may attack:
1. Outermost layers of Skin, hair and/or mucous
membrane  superficial mycoses
2. Epidermis as well as nail and hair  cutaneous
mycoses
3. Dermis, subcutaneous tissues, muscle and face 
Subcutaneous mycoses
4. The internal organs as the lungs, CNS, bones etc. 
systemic mycoses
5. Opportunistic - cause infection only in the
immunocompromised
220
A. Superfacial mycosis
1. Tinea versicolor (Pityriasis versicolor)
2. Tinea nigra
• They are extremely superficial mycoses
• Primary Manifestation is pigment change of the
skin
• Both are named for their respective skin
manifestation
221
Tinea versicolor
(Pityriasis versicolor)
• Causative agent: Malassezia globosa
– Less common-Malassezia furfur
• Lipophilic Yeasts belongs to Basidomycota
• Normal flora of skin and scalp
• Superficial opportunistic pathogens of the skin
• Associated with seborrheic dermatitis, dandruff
(Pityriasis capitis) and atopic dermatitis
• Pityriasis versicolor is chronic superficial mycoses
• Characterized by a blotchy discoloration of skin
which may itch
• With sunlight exposure the skin around patches will
tan, but patches remains white
222
Pityriasis versicolor
• Chronic superficial mycoses
• Characterized by hyperpigmented lesions
– Well-demarcated white, pink, or brownish lesions,
often coalescing and covered with thin furfuraceous
scales
• The colour varies according to;
– The normal pigmentation of the patient
– exposure of the area to sunlight
– the severity of the disease.
• Lesions occur on the trunk, shoulders and arms,
• Rarely on the neck and face
• Fluoresce a pale greenish colour under Wood's ultraviolet light.
223
B. cutaneous Mycoses
Dermatophytes attack keratinous structure of
skin, hair and nail and cause the group of disease
known as Ringworms or Tinea
Candida can attack the skin, the mucous
membranes and rarely the internal organs
Non-dermatophyte moulds e.g. Hendrsonula
toruloidea, Scytalidium hyalium, Scopulariopsis
brevicaulis
224
i. Dermatophytes
• Confined to the outer layers of skin, hair & nails
• Do not invade living tissues
• Called dermatophytes (keratinophilic fungi)
• Produce extracellular keratinas  hydrolyze keratin
• Utilize keratin for their nutrition
• Keratin is the chief protein in skin, hair and nail
• They caused dermatophycosis“Ringworm" or “Tinea"
• Dermatophycosis refers to the characteristic central
clearing that often occurs in dermatophyte infections225
I. dermatophytes
1. Trichophyton (19 species)
Affect hair, skin & nails
Infect both children & adults
2. Epidermophyton
Infect skin, nails (rarely hair)
Infect adults, rarely in children (ringworm)
Epidermophyton floccosum
3. Microsporum (13 species)
Affect hair, skin (rarely nails)
Frequently in children, rarely in adults
M. canis is the most common infect man
226
Ecology and Mode of transmission
• To determine the source of infection
• Anthrophilic
– Some Dermatophytes affect man only
– Person -to-person transmission through
contaminated objects (comb, hat, etc.)
– T. rubrum and T. mentagrophytes
• Zoophilic
– Other affect animal mainly
– Direct transmission to humans by close
contact with animals
– M. canis and T. verrucosum
227
Ecology and Mode of transmission
• Geophilic
– Other live in soil and can affect man
– Transmitted to humans by direct exposure
– M. gypseum
• Each geographic locality has its own
dermatophyton
– T. violacium is the prevalent causative
organism of Tinea capitis in Egypt
– M. audouini is prevalent cause in England
228
Tinea corporis
Small lesions occurring anywhere on the body
Causative agent:
Trichophyton rubrum, T. mentagrophytes, M.
canis, and M. audouinii
Live on the skin surface (opportunistic)
Reservoir: Humans, soil & animals
Acquired by person-to-person transfer usually
via direct skin contact with infected individual
Animal-to-human transmission is common
229
Tinea pedis "athlete's foot"
Infection of toe webs and soles of feet
Causative agent:
Trichophyton rubrum, T. mentagrophytes and
Epidermophyton floccosum
Requires warmth and moisture to survive and grow
Causes scaling, flaking & itch of affected areas
Reservoirs: Humans
Athlete's foot is a communicable disease
Transmission: when people who regularly wear
shoes go barefoot in a moist environment
230
Tinea capitis
Infection of the scalp
Causative agent:
Trichophyton and Microsporum invade the hair shaft
Trichophyton infection prevail in Central America to
USA & in parts of Western Europe
Microsporum infection are in South America, Southern
& Central Europe, Africa & Middle East
Reservoirs: Humans & animals (dogs, cats and cattle)
Transmitted by humans, animals or objects that harbor
231
the fungus
Dermatophytes
4. Tinea cruris - "jock itch"
Infection of the groin, perineum or perianal area
Trichophyton rubrum
Some other contributing fungi are Candida albicans, T.
mentagrophytes and Epidermophyton floccosum.
5. Tinea barbae
Ringworm of the bearded areas of the face and neck
 Trichophyton mentagrophytes and T. verrucosum
232
Dermatophytes
6. Tinea unguium (onychomycosis)
– Infection of nails
– Common - Trichophyton rubrum
– Less common- T. interdigitale, Epidermophyton
floccosum, T. violaceum, Microsporum gypseum, T.
tonsurans, T. soudanense
– Reservoirs: Humans and rarely animals or soil
233
Treatment of Dermatophytes
All are sensitive to grisofulvin
Tolfnatate available over the counter – Topical
Terbinifine (Lamisil) - oral, topical.
Ketoconazole seems to be most effective for
tinea versicolor and other dermatophytes
Itraconazole - oral
Echinocandins (caspofungin)
234
Lab diagnosis of Dermatophytes
• Specimen collection
– Skin Scrapings, nail scrapings and epilated hairs
• Direct Microscopy
– Specimens should be examined using 10% KOH
and Parker ink or calcofluor white mounts
– Characteristic hyphae can be seen
235
Lab diagnosis of Dermatophytes
•
•
•
•
Culture
Specimens should be inoculated onto
Sabouraud's dextrose agar (General purpose)
Selective – Mycosel agar
– Gentamicin: inhibits normal bacterial flora
– Cycloheximide: inhibits saprophytic fungi
– containing cycloheximide and incubated at
250C for 4 weeks
– The growth of any dermatophyte is significant
236
II. Candida
• Yeast-like organism that lives as a commensal in oral mucosa,
throat, skin, scalp, vagina, fingers, nails, bronchi, lungs, or
intestine and vagina
• Opportunistic organism
• The causative agent of candidiasis
– C. albicans, C. tropicalis & C. glabrata
• Candidiasis encompasses infections that
from superficial such as thrush and vaginitis
range
• Rarely become systemic in immunocompromised patients
– Septicaemia, endocarditis and meningitis
237
Symptoms of Candida albicans
• Thrush appears as creamy-white or bluish-white
patches on the tongue - which is inflamed and
sometimes-beefy red - and on the lining of the mouth, or
in the throat.
• Diaper rash caused by candida is an inflammation of the
skin, usually red and sometimes scaly.
• Vaginitis is characterized by a white or yellow discharge.
– Inflammation of the walls of vagina and of the vulva
(external genital area) causes burning and itching.
• Infections of the fingernails and toenails appear as red,
painful swelling around the nail.
• Later, pus may develop.
238
Laboratory diagnosis of candida
• Specimen:
– A scraping or swab of the affected area or blood
(candidemia) is placed on a microscope slide
• Microscopic examination
– A drop of 10% KOH solution is added to the specimen.
– KOH dissolves the skin cells, but leaves Candida intact,
permitting visualization of pseudohyphae and
budding yeast cells typical of many Candida species.
• Culture
– Swab/blood is streaked on a culture SDA medium
– The culture is incubated at 37°C for several days
– The characteristics of colonies may allow initial
diagnosis of organism causing disease symptom 239
Laboratory diagnosis of candida
• A germ tube test is a diagnostic test in which a
sample of fungal spores are suspended
in serum and examined by microscopy for the
detection of any germ tubes
• It is particularly indicated for colonies of white or
cream color on fungal culture, where a positive
germ tube test is strongly indicative of Candida
albicans
240
Treatment of candidacies
– Oral drugs: Amphotericin B, fluconazole, and
ketoconazole, are the drugs most commonly
used to treat candidiasis
– Topical administration of antifungal drugs such
as clotrimazole, miconazole , tioconazole, and
nystatin
– The drug of choice is nystatin
241
c. Subcutaneous Mycoses
Rare
Confined to subcutaneous tissue & rarely spread
systemically
Confined mainly to tropical regions
Include heterogeneous group of soil fungal infections
Introduced into the extremities by trauma/wound
Tend to be slow in onset and chronic in duration
The main subcutaneous fungal infections include
Sporotrichosis, chromoblastomycosis, MYCETOMA,
lobomycosis,
rhinosporidiosis,
subcutaneous
zygomycosis, & subcutaneous phaeohyphomycosis242
C. Subcutaneous Mycoses
1. Sporotrichosis caused by Sporothrix schenckii
The fungus is saprophyte on dead plant material
Dimorphic fungi
Colonies of media at 250C have delicate radiating forms
that appear as white at first but turned black with
prolonged incubation
Microscopic examination reveals branched hyphae with
numerous conidia
243
Sporotrichosis
• It was once common in Europe but cases are now rare
• Most prevalent in Americas, South Africa & Australia
• Infection usually follows and insect bite, thorn pricks or
scratches from a fish spine.
• Certain occupation groups appear to have increased risk
from infection
• These include florists, farm workers and others who
handle hay and moss
• The most common symptom is an ulcerative lesion that
may develop into lymphangitis
• Treated by saturated solution of KI and Amphotericin B
244
2. Chromoblastomycosis
 Chronic, localized, slowly progressive infection
of the subcutaneous tissue caused by several
species of dematiaceous fungi
 Tissue proliferation occurs around the area of
inoculation producing crusted, verrucose, wartlike lesions
 Causative agent
Most common: Cladophialophora carrionii &
Fonsecaea pedrosoi
Less common: Fonsecaea compactum, Phialophora
verrucosa
245
Chromoblastomycosis
• Mode of Transmission
• Traumatic implantation of fungal elements into skin
• The infection occurs in tropical or subtropical
climates, often in rural areas.
• Traetment
• Chromoblastomycosis
treated
removal and 5-flurocytosine
by
surgical
246
Mycetoma
Also called Maduromycosis or Madura foot
Madura foot referring to first case seen in Madura
region of India which was in the foot of patient
Syndrome involving cutaneous & subcutaneous
tissues, fascia, and bone
Infection focused in one area of the body (Foot)
Distribution: World-wide but most common in barefooted populations in tropical sub- or regions
Characterized by draining sinuses, granules (vary in
size, colour & degree of hardness) & tumefaction
247
Mycetoma
• Mode of transmission
– Traumatic implantation of causative agent
• Causative agent (50% bacteria & 50% fungi)
• Soil-inhabiting
bacteria
(actinomycotic
mycetoma or actinomycosis)
– Actinomadura madurae, Actinomyces israelii
and Nocardia brasiliensis
• Soil-inhabiting fungi (eumycetoma)
Madurella mycetomatis & Madurella grisea
248
Treatment
Treatment for eumycetoma is less successful than
for actinomycetoma
EUMYCETOMA
surgical treatment is still usually required
Ketoconazole 400mg daily, itraconazole 300 mg
daily & IV amphotericin B 50 mg daily
Therapy is suggested for 1-2 years
Flucytosine, Topical nystatin & potassium iodide
ACTINOMYCOTIC MYCETOMA
Penicillin, gentamicin & co-trimoxazole for 5 weeks
Followed by maintenance therapy with amoxicillin &
co-trimoxazole
249
Systemic Mycoses
Invasive infections of the internal organs with the
organism gaining entry by;
Lungs, GIT or through intravenous lines
They may be caused by:
1. Primary (TRUE) pathogenic fungi
2. Opportunistic fungi that are of marginal
pathogenicity
but
can
infect
the
immunocompromised host
250
I. Primary Pathogenic Fungi
• Histoplasma capsulatum (Histoplasmosis )
• Blastomyces dermatidis (Blastomycosis )
• Coccidioides immitis (Coccidiomycosis)
• Paracoccidioides brasiliensis (paracoccidioidomycosis)
• Dimorphic fungi normally found in soil
• Infection occurs in previously healthy persons
• Arises through the respiratory route (inhalation)
251
Histoplasmosis
• Caused by dimorphic Histoplasma capsulatum
• H. capsulatum is endemic in many parts of the
world including North and South America
• It is found in the soil and growth is enhanced by
the presence of bird and bat excreta
• Environments containing such material are often
implicated as sources of human infection
252
Histoplasmosis
• Lungs are the main site of infection but
dissemination to liver, heart & CNS can occur.
• Pulmonary infection can resemble symptoms
seen in tuberculosis
• Treatment:
– Amphotericin B
– Ketoconazole and other new azoles
253
II. Opportunistic Fungi
• Patients usually have some serious immune or
metabolic defect, or have undergone surgery
• The
diseases
Candidiasis,
include aspergillosis, systemic
cryptococcosis,
Zygomycosis,
Pneumocystis carinii
• Exceptionally, other fungi that are normally not
pathogenic, such as Trichosporon, Fusarium or
Penicillium, may cause systemic infections.
254
Cryptococcosis
• Systemic infection caused by encapsulated yeast Cryptococcus neoformans
•
•
•
•
•
•
•
•
Inhabits soil around pigeon roosts
Host defense is CMI
Affects both healthy & immunosuppressed individuals
Common infection of AIDS, cancer or diabetic
Primary infection in lungs via inhalation
Pulmonary infection leads to cough, fever & lung nodules
Polysaccharide capsule resists phagocytosis
Cryptococcal meningitis is most common disseminated
manifestation
255
• Can spread to skin, bone and prostate
Cryptococcosis
Lab. Diagnosis
Lumbar puncture and microscopic examination
of cerebrospinal fluid is diagnostic
India Ink for CSF
Culture on SDA
White mucoid colonies within 48hours
Cryptococcal antigen
Serum and CSF are 99% sensitive in AIDS
Serum is less sensitive in normal hosts
Treatment
Amphotericin B and fluconazole
256
Aspergillosis
• Aspergillus is found in soil, on plants & in decaying
organic matter.
• 600 species, A. fumigatus is one of the most
ubiquitous of the airborne saprophytic fungi
• A. fumigatus is the main causative agent of
Aspergillosis
• Diseases of the soil fungus called Aspergillus
• Major portal of entry is the respiratory tract
– Via inhalation of conidia
• Conidia are eliminated in immunocompetent host
by innate immune mechanism
• For people with weakened immune systems,
breathing in conidia can lead to infection
257
Aspergillosis
• Most commonly affects the sinuses or lungs
• Symptoms of sinus infections include fever,
headache, and sinus pain
• Lung infections can cause fever and cough
• In the immunosuppressed host, Aspergillus can
disseminate throughout the body.
• Treatment
• Amphotericin B and nystatin
• Voriconazole is currently the first-line treatment
for invasive aspergillosis
258
Candidiasis
• In severely immunocompromised patients, C.
albicans
can
proliferate
and
disseminate
throughout the body.
• An infection in the bloodstream can affect the
kidneys, heart, lungs, eyes, or other organs
causing high fever, chills, anemia, and sometimes
a rash or shock
259
Symptoms of Candida albicans
• Candida can cause the following problems
depending upon the organ infected:
– in kidneys can cause blood in the urine
– in heart can cause murmurs & valve damage
– in the lungs can cause bloody sputum
– in the eyes can cause pain and blurred vision
– in the brain can cause seizures and acute
changes in mental function or behavior
260