week NON FERMENTER GRAM NEGATIVE BACILLI

What are they?

Large groups, many different genera
Saprophytes: found on soil, water
Some commensals (1 gain, another no gain nor harm)
Infections: Immunocompromised patients
Entering sterile sites

Why called non-fermenting

Breakdown carbohydrates by oxidative pathway ( aerobically by
using Oxygen), cannot metabolize glucose thus unable to ferment.
Test: Oxidative fermentative test O-F test
Method of test:
Media: 1% glucose broth with a pH indicator
2 tubes: one overlayed with oil (Why?)
Incubate: 37oC up to 4 days
Change: red to yellow if glucose is metabolized to produce acid

Results of OF test
Oxidative organisms: glucose used only in aerobic tube, need
oxygen to grow and oxidize substrate
Fermentative organisms: Use glucose with or without oxygen,
week
Common Non-Fermenting genera
3 Pseudomonas
Stenotrophomonas
Burkholderia
Alcaligenes Flavobacterium Acinetobacter
Agrobacteria

Pseudomonas
1. Pseudomonads are Gram-negative, aerobic, rod-
shaped bacteria with widespread occurrence in nature
2. Occurs in damp biotopes
3. The most important species from a medical point of
view is Pseudomonas aeruginosa

Pseudomonas aeruginosa
Occurrence, significance:
1. Widespread in nature
2. They are regularly found in soils, surface water, including the ocean,
on plants and, in small numbers, in human and animal intestines

Pseudomonas aeruginosa
1. Pseudomonas infections occur only in patients with weakened
immune defense systems,
2. Pneumonias in cystic fibrosis, colonization of burn wounds,
endocarditis in drug addicts, postoperative wound infection,
urinary tract infection, sepsis.
3. Multiple resistance to anti-infective agents presents a
therapeutic problem
week
3 Morphology and culture

*P. aeruginosa are plump,

*2–4 μm long rods with one to *sveral polar flagella.
*Some strains can produce a viscous extracellular slime layer.
*Motile (by single or multiple polar flagella) gram-negative rods
*Obligate (strict) aerobes
*Oxidase (usually) and catalase positive
*Nonfermentative chemoheterotrophic respiratory metabolism
*Minimal nutritional reqts.; Many organic compounds used as C and N
sources, but only a few carbohydrates by oxidative metabolism
*Glucose used oxidatively
*Lactose negative on MacConkey’s agar *Some strains produce diffusible
pigments:
*Pyocyanin (blue); fluorescein (yellow); pyorubin (red)
*P. aeruginosa produces characteristic grape-like odor and blue-green pus
& colonies

Pathogenesis and clinical pictures

-P. aeruginosa usually enters body tissues through injuries
-It attaches to tissue cells using specific attachment fimbriae
-The most important virulence factor is exotoxin A which blocks
-translation in protein synthesis
-A cytotoxin damages cells by creating transmembrane pores

Virulence
Factors
week Pathogenesis and clinical pictures
3 -Opportunistic organisms
-UTI in catheterized patients
-Wound infections: in burn patients, ulcers
-Septicemia and meningitis in neonates
-Chest infections: ventilated patients
Cystic fibrosis patients
-Major cause of cross-infection in hospital
( easily multiplies in any moist environment)
Infections are exceptionally difficult to treat because of antimicrobial
resistance.
Although pseudomonads are easy to identify in the laboratory,
antimicrobial susceptibility testing is indicated.

Treatment:AGGRESSIVE! Use combination of: Aminoglycoside &

New generation Cephalosporin Fluoroquinolone

Difficulty: Need to use high doses and aminoglycosides are toxic

(ototoxicity, nephrotoxicity)

Burkholderia mallei

- B mallei is a small, non motile, non pigmented, aerobic

gram- negative rod
-Grows readily on most bacteriologic media
-It causes glanders, a disease of horses, mules, and donkeys
transmissible to humans.

In horses, the disease has prominent

• pulmonary involvement
• subcutaneous ulcerative lesions
• lymphatic thickening with nodules
week
• Human infection can be fatal
3 • Begins as an ulcer of the skin or mucous membranes
followed by
lymphangitis and sepsis.
• Inhalation of the organisms may lead to primary
pneumonia.

Burkholderia pseudomallei

-B pseudomallei is a small, motile, aerobic gram-negative

bacillus
-It grows on standard bacteriologic media, forming colonies
that vary from mucoid and smooth to rough and wrinkled
and in color from cream to orange
-It grows at 42 °C and oxidizes glucose, lactose, and a variety
of other carbohydrates
-B pseudomallei causes melioidosis of humans,
primarily in Southeast Asia and northern Australia
-The organism is a natural saprophyte that has been
cultured from soil, fresh water, rice paddies, and
vegetable produce

-Human infection probably originates from these sources by

contamination of skin abrasions and possibly by ingestion or
inhalation.
-Epizootic B pseudomallei infection occurs in sheep, goats, swine,
horses, and other animals, though animals do not appear to be a
primary reservoir for the organism.
week
-A localized suppurative infection can occur at the inoculation site
3 where there is a break in the skin.
-This localized infection may lead to the acute septicemic form of
infection with involvement of many organs
-The most common form of melioidosis is pulmonary infection
-The patient may have fever and leukocytosis, with consolidation of
the upper lobes

Diagnose
1-The diagnosis of melioidosis should be considered for a patient from
an endemic area who has fulminant upper lobe pulmonary or
unexplained systemic disease
2-A Gram stain of an appropriate specimen will show small gram-
negative bacilli; bipolar staining (safety pin appearance) is seen with
Wright's stain or methylene blue stain

Treatment
*Melioidosis has a high mortality rate if untreated *Surgical drainage
of localized infection may be
necessary.
*Antibiotic susceptibility testing is an important guide for treatment
*B pseudomallei usually is susceptible to a variety of antibiotics,
including tetracycline, sulfonamides, trimethoprim-sulfamethoxazole,
chloramphenicol, amoxicillin or ticarcillin with clavulanic acid,
piperacillin, imipenem, and third- generation cephalosporins

1- The duration of antimicrobial therapy should be at least 8 weeks

2-There is no vaccine or preventivemeasure
3- Relapses are common
Patients with severe specific
infections should be treated parenterally (eg, trimethoprim-
sulfamethoxazole or a third-generation cephalosporin such
as ceftazidime);
week
Burkholderia cepacia
3
Burkholderia cepacia is an environmental organism that is able
to grow in:
-water,
-soil,
-plants,
-animals, and
-decaying vegetable materials
B. cepacia is a major pathogen in Cystic Fibrosis patients
The cause of pneumoniaå
week GRAM POSITIVE BACILLI
4
Many genera (saprophytes, commensals , pathogens)

Classified based on
Cell morphology and arrangement
Atmospheric (aerobic/anaerobic)
Catalase (+/-)
Spore formation (sporing/non-sporing)

Bacillus
Aerobic spore former
Habitat saprophytes, soil , dust, water
Species:human infections
- B.anthracis
- B.cereus
- B.subtilis

Morphology
Large gram positive bacilli (Streptobacilli)
May appear gram variable
All species motile ( except B.anthracis)
Culture
dry beta-hemolytic colonies on blood ( except B.anthracis)
-

B.anthracis : dry irregular colonies non-hemolytic

- e n e

Pathogenicity
B.anthracis anthrax
B.cereus Food poisoning
B.subtilis infections in immunosupressed

a
week Anthrax
*disease of animals
4 *Organism in soil, form spore, remain viable for long time
*Human infected after contact with spores
=

Types of Anthrax
* b b

Cutaneous : through Enteric: Ingestion of Pulmonary: Inhalation

damaged skin- can contaminated meat- of spores ( Wool
travel to blood fatal sorter's disease)
(septicemia)

Cutaneous Anthrax Infection of

the Hand and Cheek

Incubation period 1-7 days

Mode of transmission Contact with spores,spore contaminated materials or

infected lesions.

Clinical features Itchy papule like ‘insect bite’ , ulcerates, discharge+,

surrounded by swelling. “Painless swelling”: Cardinal Feature D/D Cellulitis.

‘Black Eschar’ Ulcer with depressed black center in 2-6 days.

Untreated progresses to Septicemia or meningitis.
Case fatality 20% untreated.
A

Responds well to antibiotics.

week Inhalational anthrax

4 Min.Infective dose: 8,000- 10,000 (US Dept of defense: lethal dose

for 50% subjects)
Particle size: <5 microns.
I.P: 1-7 days.
Mild & nonspecific flu like symptoms
Second phase: Acute Respiratory distress, sepsis & hemorrhagic
mediastinitis causing mediastinal widening.
Untreated ~ 97% mortality

Lab diagnosis
Risk group 3
Gram stain:
Specimen: large capsulated gram pos bacilli From culture: gram
• •
positive, spore , Streptobacilli,
• no capsule =

Capsule detection
McFadyean reaction
Stain: Loeffler’s Polychrome Methylene
Blue stain
Result: Organism (blue)
Capsule (red)
week Laboratory diagnosis of Anthrax
4 Specimens: Blood culture, Sputum, swabs from cutaneous lesions,
CSf, dry nasal swab*
Environmental: air samples & surface swabs.
Transport with extreme care!
Stains: Gram’s, Spore,capsular
Culture on BA/PEA.
PCR, DFA, Strain typing
(Ames, Vollum,Sterne) & Serum ELISA for antibodies
Bacillus anthracis Vegetative Cells and Spores.
A shows a Gram's stain of B. anthracis vegetative bacteria.
B shows an electron photomicrograph of a B. anthracis spore
(arrowhead) partially surrounded by the pseudopod of a cultured
macrophage (x137,000).

HANDLING LABORATORY SPECIMEN

If B. anthracis is suspected, these precautions should be followed:

1- Wear gloves and protective gowns when handling clinical

specimens & Wash immediately with soap and water if there is direct
contact with a clinical or lab specimen- Avoid splashing or creating
aerosols

2- Perform lab tests in an annually certified Class II Biological Safety

Cabinet & use standard lab protective eyewear and a mask

3- Blood cultures should be maintained in a closed system (blood

culture bottles)

4- Keep culture plates covered at all times; minimize exposure when

extracting specimens for testing

5- Work on a smooth surface that can be cleaned easily and wipe

with bleach regularly.
 Lab or clinical specimen material spill or splash
week * Remove outer clothing carefully while still in the area and place in
4 a labeled, plastic bag.
* Remove rest of clothing in the changing room and place in a labeled
plastic bag
* Shower thoroughly with soap and water in the changing room
* inform your supervisor and Infection Control team

If exposure to contaminated sharps occurs

* Follow standard reporting procedures for sharps exposures
* Thoroughly irrigate site with soap and water and apply a
• hypochlorite solution. DO NOT
disinfectant solution such as a 0.5%
SCRUB AREA.
* Promptly begin prophylaxis for cutaneous anthrax*

DECONTAMINATION
- Effective sporicidal decontamination solutions: Commercially-
available bleach, 0.5% hypochlorite (a 1:10 dilution of household bleach)

Accidental spills of material known or suspected to be contaminated

with B. anthracis
For contamination involving fresh clinical samples:
-Flood with a decontamination solution
- Soak five minutes before cleaning up

For contamination involving lab samples, such as culture plates or blood

cultures, or spills occurring in areas that are below room temperature

-Gently cover spill, then liberally apply decontamination solution

-Soak for one hour before cleaning up
-Any materials soiled during the clean-up must be autoclaved or
incinerated
 Treatment of Anthrax
week
Inhalation & Ingestion Cutaneous
4
-Ciprofloxacin 500mg BD Ciprofloxacin 500mgBD for 7
-Doxycycline 100mg BD days.
-Amoxycillin 500mg TDS Upto 60 days if concurrent
All for 60 days inhalational suspected

Dealing with a suspicious package

-Do not open the letter.If the letter has already been opened and
powder spills out, do not clean it up.
-Keep others away from the area.Double bag the letter; plastic is
best (use plastic/rubber gloves and a mask if available).
-Immediately wash your hands with soap and water.
-Notify your supervisor & Infection Control team.
-Evacuate the area & Ensure that all persons who have handled
the letter wash their hands.
-Make a list of names of all persons who have handled the letter
-Place all clothing items worn when in contact with the letter
into plastic bags & Keep these bags with you, so that they are
available for law enforcement officials.
-As soon as possible shower with soap and water
week Gram Positive Bacilli Corynebacterium
5
General characteristic
- Corynebacteria are Gram-positive
- catalase positive,
- non-spore-forming,
- non-motile,
- rod-shaped bacteria that are straight or slightly cur ved
- Metachromatic granules are usually present representing
stored phosphate regions
It is also known as the Klebs-Löffler bacillus
* Genus has many species
* Most commensals called Diptheroids

Human pathogens
nd#
1. C.diphtheriae- Diphtheria
(3 biotypes) gravis, mitis, and intermedius
2 .C.hemolyticum sore throat, ulcers
3 .C.jeikium or Corynebacteria JK systemic infections in
immunosuppressed, heart valve infections (endocarditis)
commensal
4. C.ulceranspharyngitis like diphtheria
Diphtheria
Respiratory diphtheria- more serious
Cutaneous diphtheria –skin infection, wound
contaminated with C.diphtheria,
rarely systemic
 Respiratory Diphtheria
week
• Serious and fatal, if untreated
5 • Begins upper resp. tract
• Organism inhaled
• Exotoxin( neurotoxin) spreads via blood to other organs (heart,
muscle, kidney, liver)
• Throat acute inflammation – white membrane over the throat ,
can spread down and cause death from asphyxia (Laryngeal
diphtheria)
• Clinical diagnosis : presence of membrane
• Major infection in the past prior to anti-toxin and vaccination

Cutaneous diphtheria

Bullous neck
White pseudomembrane
of Diphtheria
Lab diagnosis
Specimen: nasophyrangeal swab , wound swab
Gram stain: pleomorphic gram pos bacilli,gram variable,
coryneform (Chinese letter arrangement)

Special stain Albert’s stain(KLB stain)

* Stains volutin (metchromatic polyphosphate
granules found in cytoplasm).
- Malachite Green: Bacilli green,
- Toludine blue :granules (dark red spot)
week Culture
5 Selective media used to differentiate
commensal species from C.diphtheriae

1) Hoyle’s Tellurite blood agar (Hoyle's Agar is a selective media

that uses tellurite to differentially select Corynebacterium
diphtheriae from other upper respiratory tract flora)

2) Tindsdale medium (production of H2S)

The dark halo is due to the production of H2S from cystine,
interacting with the telluride salt

Lab Diagnosis
Identification by
• Biochemical tests: Hiss’s Serum sugars.
• Glucose & maltose +, Sucrose –
• Urease + : C. ulcerans.
• Gelatin liquefied.(C.ulcerans+ C.diphtheriae)
Virulence testing of C.diphtheriae

1. ELEK’s test ( Ag-Ab test)

Method of test-
• Horse serum agar used
• Filter paper with antitoxin
• Test organism
• Known positive and negative control
• Check for precipitation

2.Guinea pig inoculation.

week Treatment of Diphtheria
5 • Mainstay of treatment is Antidiphtheritic
serum (antitoxin).
• Antibiotics used for
1. Treatment of carriers
2. Synergistic with antitoxin for patients.
• Sensitive to Erythromycin, Penicillin & Clindamycin.
week Listeriosis
6 Listeria monocytogenes
• Gram-positive rod, non-spore forming, non-
branching, aerobic.
• Catalase positive
• Grows well at refrigerator temperature
(4 – 10 oC) : Cold enrichment
• May look like diphtheroids, cocci, diploccocci.
• Tropism for nervous system
• Facultative intracellular organism
• Grows well on Blood Agar, narrow zone of
hemolysis .
• Characteristic motility:
• ‘Tumbling motility’:end - over- end at room
temp.(25oC)
• Umbrella motility seen in stab cultures of semisolid agar.
• Biochemical tests to confirm id: Glucose, VP & Esculin positive

Umbrella motility of
Listeria monocytogenes on SBA
Listeria in semisolid agar

Listeria selective agar

week
Listeria Epidemiology
6
• Important zoonoses.
• Of the seven Listeria species, only L. monocytogenes affects humans
• Common in soil, decaying vegetation, fecal flora of many mammals
(including 15% of healthy adults)
• Foods: raw vegetables, raw milk, fish, poultry, meats, esp. deli meats
(15-70%)

Listeria hysteria
Several reported food borne Listeria outbreaks:
• Coleslaw, milk, soft cheeses (Brie,camembert,blue vein)
• Deli ready meats, especially chicken, had highest rates of
contamination
• CDC study:
– 11% of all refrigerator food samples were
contaminated
– 32% of sporadic cases can be attributed to soft cheeses or deli
meats
– 64% of pts had at least one contaminated food

Populations at risk

Impaired cell-mediated immunity:

• Infants < 1 mo old (10% of patients)
• Pregnant women (30% of patients; 60% of cases bet ween
10-40 years old)
• Adults > 60 years old
• Hematologic malignancy patients
• HIV patients
• Organ transplantation recipients
• Corticosteroid therapy recipients
week
Listeria epidemiology
6
• Human-to-human infection not documented except in:
– Vertical transmission mother to fetus
– Cross-contamination in delivery room
– Nosocomial infection in newborn nursery
• Contaminated mineral oil
• Most cases foodborne

Listeria mechanism of pathogenesis

1-Ingestion of Listeria from food

2-Intestine
3-Mean incubation period: 31 days
4-L. monocytogenes crosses mucosal barrier
5-bloodstream
6-Hematogenous dissemination
7-Brain, placenta and other sites

Listeria virulence factors

• Internalin: allows attachment to host
epithelial cells
• Listeriolysin O: enables listeriae to escape from host cell
phagosome
• Act A: allows recruitment of host cell actin filaments
• Siderophores: allow scavaging of iron from transferrin

mechanism of pathogenesis
• Listeria’s surface protein “internalin” interacts with
E- cadherin, a receptor on epithelial cells.
• Phagocytosis induced.
week
7 • Listeriolysin O lyses the phagolysosomal membrane.
• Bacterium escapes into cytoplasm and avoids intracellular
I killing.
• In cytoplasm, bacteria divides.
• Doubling time ~ 1 hr
• Listeria uses virulence factor Act A to induce host cell actin
polymerization.
• Actin filaments propel the bacteria to the host cell
membrane.
• Listeria has successfully hijacked the contractile system of
the host cell for cell to cell migration.

• Bacteria push against cell membrane to form

“filopods”.
• Filopods are ingested by adjacent cells.
• The life cycle begins again in the new cell.
• Listeria has moved from cell to cell without being exposed
to antibodies, complement, or neutrophils.

Listerial infections

Infection in Pregnancy
• 26-30 weeks gestation: decline in cell- mediated immunity
• Listeria may proliferate in placenta – Acute febrile illness
– Arthralgia
– Headache
– Backache
week
6 • Result of infection in pregnancy:
– Stillbirth, neonatal death (22%)
– Premature labour
– Spontaneous abortion if amnionitis
• Untreated bacteremia self-limited
• Early antimicrobial therapy results in healthy infant
• CNS infection rare in pregnancy

Neonatal infection
1. Granulomatosis infantiseptica
• Disseminated listerial infection
• Caused by in utero infection
• Infant stillborn or dies within hours of birth
• Widespread microabscesses and granulomas, especially in liver and
spleen
• Listeria visible in meconium

2. Early-onset sepsis syndrome

• Acquired in utero
• Associated with prematurity
• Bacteria found everywhere, but highest concentrations in
newborn lung and gut, suggesting acquisition from infected amniotic
fluid rather than hematogenous spread.

3. Late-onset meningitis
• Acquired from birth canal at delivery
• Occurs at ~2 weeks of age in term infants
week
CNS Infection
6
• Listeria has a tropism for the brain, especially
brainstem, and meninges
• Meningitis most frequent listerial infection
• 5th most common cause of bacterial meningitis, but
has highest mortality (22%)
• 3rd major cause of neonatal meningitis
• 2nd major cause of meningitis in elderly
• Most common cause bacterial meningitis in pts with
immuno-compromise

Unique features of Listeria meningitis

• Presentation may be subacute
• Neck rigidity less common (only 80% of adults)
• Movement disorders more common (ataxia, tremors,
myoclonus) (15-20%)
• Seizures more common (25%)
• Fluctuating mental status common
• Positive blood cultures more likely (75%)
• CSF findings:
– Negative gram stain (60%)
– Glucose level not low (normal in >60%)
– Mononuclear cells predominate (30% cases)

CT brain of a normal 3 week old

Plain CT of a 2 week old baby
with Listeria meningitis
week
Other Listeria infections
6
• Endocarditis
– Affects both native and prosthetic valves – 7.5% of adult listerial
infections
– Rarely seen in children
• Febrile gastroenteritis
– In healthy, nonpregnant pts: self-limited diarrhea, nausea,
vomiting, fever
• Local infections
– Seen in vets and farmers handling poultry

Consider Listeria in:

• Neonatal sepsis or meningitis

• Meningitis/ parenchymal brain infection in: – Pt with HIV,
hematologic malignancy, transplant, steroids – Subacute
presentation
– Adults >50 years
– Gram-positive bacilli or “diptheroids” in CSF or blood
• Subcortical brain abscess
• Fever during pregnancy
• Foodborne outbreak of febrile gastroenteritis in which
routine cultures fail to yield a pathogen

Listeriosis Diagnosis
• Isolation of L. monocytogenes from CSF, blood
• MRI superior to CT for identifying parenchymal brain
involvement, especially in the brainstem
week
Listeriosis Treatment
6
• Meningitis:
– Ampicillin + gentamicin x 21 days
– or TMP-SMX (Trimethoprim/sulfamethoxazole) x 21 days
– Avoid corticosteroid therapy (?) to avoid impairment of
cellular immunity
• Bacteremia only:
– Amp + gent (or TMP-SMX) x 14 days
• Rhombencephalitis or brain abscess:
– treat minimum 6 weeks
– Follow with serial MRI or CT scans
• Listerial endocarditis:
– treat 4-6 weeks
• Always include ampicillin in empiric tx for
septicemia or meningitis in infants < 3 mo.
• Withhold iron replacement tx during listeriosis infection
• ? Combination of TMP-SMX + amp may have lower rate of
tx failure with fewer neurologic sequelae than amp + gent.

No cheese please!

Listeriosis prevention
For everyone:
• Thoroughly cook raw food from animals
• Thoroughly wash raw vegetables
• Keep uncooked meats separate from other foods
• Avoid raw milk
• Wash hands, knives, cutting boards after handling uncooked
foods
week In immunocompromised, elderly, pregnancy
6 • Avoid soft cheeses (feta, Brie, Camembert)
• Reheat left-overs and ready-to-eat foods (e.g. hot dogs) until
steaming hot before eating
• +/- avoid deli foods and reheat cold cuts before eating
week CLOSTRIDIA
7
General characteristics

-The genus Clostridium consists of relatively large, Gram-

positive, rod- shaped bacteria

-All species form endospores and have a strictly fermentative

type of metabolism

-Most clostridia will not grow under aerobic conditions and

vegetative cells are killed by exposure to O2, but their spores are
able to survive long periods of exposure to air

-The clostridia are ancient organisms that live in virtually all of

the anaerobic habitats of nature where organic compounds are
present, including soils, aquatic sediments and the intestinal
tracts of animals

-Clostridia are able to ferment a wide variety of organic

compounds. They produce end products such as butyric acid, acetic
acid, butanol and acetone, and large amounts of gas (CO2 and H2)
during fermentation of sugars.

-Most of the clostridia are saprophytes, but a few are

pathogenic for humans, primarily Clostridium perfringens, C.
difficile, C. tetani and C. botulinum.

-Clostridium tetani and Clostridium botulinum produce the most

potent biological toxins known to affect humans
week Infections 4 important species that cause human disease
7 1) Cl. tetani – tetanus ( Trismus or lockjaw)
2) Cl. perfringens– Gas gangrene, food poisoning
3) Cl. difficile – PMC (Pseudomembranous colitis)
4) Cl. botulinum- Botulism

Tetanus
• Caused by Clostridium tetani
• Gram-positive, endospore-forming, obligate anaerobe
• Grows in deep wounds
• Tetanospasmin released from dead cells blocks relaxation pathway in
muscles
• Prevention by vaccination with tetanus toxoid and booster
• Treatment with tetanus immune globulin
• Acute infectious disease is due to the toxin of tetanus
bacillus Clostridium tetani
• Characterized by convulsions and intermittent tonic
spasms of voluntary muscle.
• Characteristic symptom is stiffness of the jaw
('lockjaw').
• Difficulty opening their jaws; spasm of the facial muscles produce a
characteristic expression with a fixed smile and elevated eyebrows (risus
sardonicus).
• Rigidity or spasm of the abdominal, neck and back muscles-even
opisthotonus- may occur.
• High mortality
C.tetani
Gram stain
Infant with Tetanus
week C.Perfringens-Gas gangrene
7 • Begins as wound infection – accident or surgery
• Spores germinate and produce toxin
• Metabolism of bacteria- produce a lot of gas
• Cure: - amputation
- Intensive antibiotic & antitoxin.
Gas Gangrene

Cl.perfringens food poisoning

• Ingestion of contaminated meat proudcts
• Spores survive cooking and germinate
• Enterotoxin produced that cause diarrhoea and vomiting

Cl. difficile
Pseudomembranous colitis(PMC) Antibiotic
Associated Diarrhoea(AAC)

• Acute and fatal intestinal disease

• Profuse diarrhoea , ulcers
• Fatal-in old patients
Occurs:
Post antibiotic therapy
Hospital outbreaks – nosocomial infections
week Cl. botulinum-Botulism
7 • Caused by Clostridium botulinum
• Gram-positive, endospore-forming, obligate
anaerobe
• Intoxication comes from ingesting botulinal toxin
• Botulinal toxin blocks release of neurotransmitter,
causing flaccid paralysis
• Prevention
– Proper canning
– Nitrites prevent endospore germination in sausages

• Rare and v. serious illness

• Ingestion of the toxin
• Implicated in food poisoning from canned foods (preformed toxin)
• Toxin causes- Flaccid PARALYSIS and death

• 3 forms of Botulism:
1. Food borne botulism (which is usually caused by eating home-
canned foods that contain the toxin)
2. Wound botulism (when the bacteria infect a wound and produce
the toxin inside of it)
3. Infant botulism (Bacterial multiplication in the gut)

Infant botulism
• This illness usually affects babies who are
bet ween 3 weeks and 6 months old, but all babies are at risk for it until
their first birthdays.
• So if an infant ingests them, the bacteria germinate, multiply, and
produce a toxin. That toxin interferes with the normal interaction
bet ween the muscles and ner ves and can hamper an infant's ability to
move, eat, and breathe.
Also know that honey is a known
source of the bacteria spores that
cause botulism. For this reason,
honey shouldn't be given to babies
younger than 1 year old.
week Laboratory diagnosis of Clostridia
7 • Microscopy: Thick gram pos bacilli, with square ends
• Culture: anaerobic
Identification:
- Cell morphology
- Colonial morphology
- BBL crystal/API anaerobes
- Toxin tests -- eg Nagler test

C.perfringens-Nagler test
• Principle: toxin neutralization test
• Detects toxin A -The toxin is lecithinase which hydrolyses the
phospholipid lecithin (a component of cell membranes)
• Method:
-Egg yolk agar (source of lecithin)
- Antitoxin spread half of the plate
- Organism spread over the whole plate
- Incubated overnight

Results of Nagler test

 Mycobacteria

• (The Acid and Alcohol Fast Bacilli. AAFBs)

• This is a large family of bacteria containing many
species.
• Some are human pathogens others commensal and
saprophytes

Cell morphology.
• Shape: Bacilli, some are short others long.
• Some species are arranged in a serpentine cording

Cell wall structure:

• They have a very thick waxy cell wall containing a large
amount of the fatty acid called Mycolic acid.
• This dense cell wall does not allow the Gram stain to
penetrate easily so the bacilli either are not stained at all or
are very pale Gram positive bacilli.
• Because of the poor Gram stain reaction special stains are
needed for their detection. There are 2 routinely used:
• 1. Ziehl - Neelson stain.
• 2. Auramine stain.
Ziehl - Neelson stain ( ZN stain)
• This stain gave these bacteria the name of Acid and Alcohol
Fast Bacilli.
• Acid Fast because the bacilli are not decolorized by acid
alcohol.
• Staining theory:
• The slide is flooded completely with Carbol fuchsin (red stain)
• The slide is then flooded with a 1% solution of hydrochloric
acid in isopropyl alcohol (or methanol) to remove the carbol
fuchsin, thus removing the stain from cells that are
unprotected by a waxy lipid layer
• After decolorizing the slide is flooded with a counter stain.
Either Methylene blue or Malachite green

Auramine stain
This has the same principle as the Z.N. Stain but the
stain used in place of Carbol fuchsin is the fluorescent
stain Auramine. The counter-stain is potassium
permanganate. The smear is examined using a
fluorescent microscope.
Species of Mycobacteria
 Opportunistic Mycobacteria

• Other species cause opportunistic infections and low grade

infections, these are called the opportunistic Mycobacteria
• Most of these species are grouped together and called the
MOTT species.
• MOTT stands for Mycobacteria Other Than Tuberculosis

Avium Intracellulare group

• An important group within the MOTT are the Avium

Intracellulare group.
• These species cause a variety of diseases, most commonly
in children, and
the immune suppressed.
• Recently there has been a very large increase of these
infections in AIDs
patients
• These infections are particularly severe as many are
caused by multiple resistant species

Infections by MTB
• One third of the world is infected
• 8 million have active disease
• 3 million deaths annually, 98% in Africa/Asia. • 50%
mortality, if untreated.
• One person infected every second
• 14-30% of TB cases, HIV-coinfected
• Can occur in different organs
 Mycobacteria tuberculosis
• At the present time there is a huge increase in the number of
tuberculosis infections, many of which are caused by strains that
are highly resistant to many antibiotics.
• Some years ago with better living standards and immunization
it was thought that this disease would become less important but
today there are more cases than ever

Pathogenesis of tuberculosis
• Infections start as respiratory infections, person-person
spread- during coughing, sneezing, talking or singing!!
• If the infection is not contained in the lungs it may spread via
the blood to other organs.
• When several organs are affected at the same time the
infection is regarded as systemic and is called Miliary TB

Pulmonary tuberculosis

•Tubercle bacilli reach alveoli.

•Ingested by alveolar
macrophages
•Multiply intracellularly & form granulomas
• “Caseation” & cavity formation.
•Spread through lyamphatics to
regional Lymph nodes.
•Through blood stream to distant organs- bones, kidneys, brain
etc.
 Pulmonary TB
• Latent TB:
Persons who have the infection but no disease.
• Active TB:
• TB bacilli overcome the defense mechanisms & multiply with
tissue destruction –Cough & hemoptysis (Blood in sputum)
• Systemic symptoms like fever, loss of weight, loss of appetite,
night sweats etc.

Tuberculin skin test

• Test for Delayed Hypersensitivity to TB antigens.

• Intradermal test
• Inject 0.1ml of 5 Tuberculin units of PPD (tuberculin purified
protein derivative)
• Produce wheal 6-100mm
• Read after 48-72 hours.
• Measure only induration (Thickening) in mm > 10mm =
Positive.

TB skin test
Mantoux test

Diagnosis
• Microscopy.
• Prepared Smears are stained with the Z.N or
Auramine stain,
• It is essential to include a known positive smear
as a positive control.
• Most diagnoses are made by microscopic
examination of the stained smear so a well-
prepared and examined smear is important.
Specimens in which commensals are found need to be decontaminated
before culture.
• This involves killing the commensals before the specimen is
inoculated on to the media.
• This is usually by adding 4% NaOH to the specimen and incubating
for 10-15 minutes. This kills all other bacteria and spores but not
the Mycobacteria

Culture
There are both manual and automated methods used for culture of
Mycobacteria.
Manual methods
• M. tuberculosis and other pathogenic Mycobacteria are
slow growing fastidious organisms that will not grow on
routine lab media. All cultures are incubated for a minimum
of 6-8 weeks at 37oC

Special selective enrichment media are used, these are:

• 1. Lowenstein -Jenson agar slopes. There are several formulas of
this but all are enriched with egg, glycerol and other nutrients.
• They are made selective by the addition the stain Malachite
Green and some antibiotics.

M. tuberculosis colonies appear, usually after 3 to 6

weeks, as irregular raised pale yellow colonies with a hard
texture. Usually described as Rough, Tough, and Buff
• Kirschner’s Broth. This enrichment fluid media is used to isolate
M. tuberculosis from fluid specimens e.g.,CSF, bone fluid, etc.
• ColoniesofM.tuberculosisgrowaswhiteflakesinthemedium.
• Reduce the turn around time to approx 10 days.
 Automated culture methods

• Radioactive Detection: An example of this is the BACTEC system. This is

a commercial method developed for detecting the early growth of
Mycobacteria.
• After decontamination the specimen is diluted and added to a
specialized selective enriched liquid medium and incubated.
• The medium contains radioactive C14 Palmitic acid, which is a
substrate that is only used by the Mycobacterium. If growth occurs
radioactive 14CO2 is produced which collects in the air above the liquid
medium.
• This is called the head space gas.

The head space gas is tested automatically at fixed intervals

and the amount of radioactivity is measured and recorded.
• If Mycobacteria are growing the level of radiation is above
the accepted normal value so the culture is then considered
positive and further tests done.
• These automated methods can detect growth more rapidly,
growth can be detected in 7 to 12 days instead of up to 6 weeks.

• Fluorescent Detection:
• There are now newer automatic methods that do not use
radioactivity but other products of metabolism which produce
fluorescent metabolites & are detected using a fluorescent
sensor.(MGIT®)

Identification of Mycobacterium species

• Once organism has been cultured it is identified
either manually or by the automated methods.
1. Rate of Growth:
• Slow growers (up to 6 weeks or more)
• Fast growers( within 1 week)
2. Optimal temperature:
Thermophiles,
Mesophiles
Psychrophiles 3. Colony pigment:
a) Photochromogen
b) Scotochromogen
c) Non-chromogen
4 . Biochemical tests ( niacin, Nitrate, Heat resistant catalase, Urease,
Tellurite, Arylsulfatase etc)
5. Gene probe methods
M. tuberculosis
• Slow grower
• Mesophile
• Non-chromogen

Automated identification method

• Bactec can differentiate bet ween MTB and other
Mycobacteria
• NAP test ( Nitro-acetylamino-propiophenone) –inhibits
growth of MTB
• Mycobacteria + Media ( with NAP) and C14 Palmitic acid

NAP test results

• BACTEC reading falls as MTB fails to grow in
presence of NAP
RESULTS:
• Growth reading falls----- MTB
• Growth reading increase---- Other Mycobacteria
 Treatment
• Specialized antibiotics are needed for the treatment of
Mycobacterial infections(anti-tubercular drugs). These include:
• First Line drugs like:
• Isoniazid
• Rifampicin
• Ethambutol
• Streptomycin.
• Second Line Drugs like Kanamycin, Clofazimine, Doxycycline,
Ciprofloxacin.
• These are used for Treatment of M tb infections by bacteria
resistant to the first line drugs

Sensitivity Testing by manual methods.

• Because of the slow growth of many of these species routine
sensitivity tests cannot be used.
• Automated sensitivity tests.
• The antibiotics are put into a specialized culture media, this is
then inoculated with the test organism. Growth is monitored
regularly. Results are usually available within a week.

TB lab
• Remember Risk Group 3 organism.
• Strict Biosafety precautions.
• Biosafety cabinets
• HEPA filters
• Remember Universal precautions + Masks & Gowns while
handling cultures
• Important cause of lab acquired infections among
technologists...
week Microbial Diseases of the Respirator
13
14
The Upper Respiratory System
Nose
Pharynx (throat)
Middle ear
Eustachian tubes

The Lower Respiratory System

Larynx
Trachea
Bronchial tubes
Alveoli
Pleura

Normal Microbiota of Respiratory System

- Suppress pathogens by competitive inhibition in upper

respiratory system
-Lower respiratory system is sterile
-Coarse hairs in the nose filter large particles from air entering
the respiratory tract.
-The ciliated mucous membranes of the nose and throat trap
airborne particles and remove them from the body.
-Lymphoid tissue, tonsils, and adenoids provide immunity to
certain infections.

The lower respiratory system consists of the larynx, trachea,

bronchial tubes, and alveoli.
- The ciliary escalator of the lower respiratory system helps
prevent microorganisms from reaching the lungs.
- Microbes in the lungs can be phagocytized by alveolar
macrophages.
- Respiratory mucus contains IgA antibodies.
week Upper Respiratory System Diseases
13
14 -Pharyngitis
-Laryngitis
- Tonsillitis
- Sinusitis
-Epiglottitis: H. influenzae type b

Streptococcal Pharyngitis
-Also called strep throat
- Streptococcus pyogenes
-Resistant to phagocytosis
-Streptokinases lyse clots
-Streptolysins are cytotoxic
-Diagnosis by enzyme immunoassay (EIA)
Scarlet Fever
-Streptococcus pyogenes
-Pharyngitis
-Erythrogenic toxin produced by
lysogenized S. pyogenes

Diphtheria
-Corynebacterium diphtheriae: Gram-positive rod
-Diphtheria toxin produced by lysogenized C.
diphtheriae
Diphtheria membrane: Fibrin, tissue, bacterial cells
Prevented by DTaP vaccine
-Diphtheria toxoid
-Cutaneous diphtheria
-Infected skin wound leads to slow-healing ulcer
week Otitis Media
13
14 -S. pneumoniae (35%)
- H. influenzae (20–30%)
- M. catarrhalis (10–15%)
- S. pyogenes (8–10%)
- S. aureus (1–2%)
- Incidence of S. pneumoniae reduced by vaccine

The Common Cold

- Rhinoviruses (50%)
- Coronaviruses (15–20%)
week Lower Respiratory System Diseases
13
14
Bacteria, viruses, and fungi cause
Bronchitis
Bronchiolitis
Pneumonia
1- Pertussis (Whooping Cough)
-Bordetella pertussis Gram-negative coccobacillus
-Capsule
-Tracheal cytotoxin of cell wall damaged ciliated cells
- Pertussis toxin
-Prevented by DTaP vaccine (acellular Pertussis cell
fragments)

Stage 1: Catarrhal stage, like common cold

Stage 2: Paroxysmal stage—violent coughing sieges
Stage 3: Convalescence stage

2-Tuberculosis d
Mycobacterium tuberculosis
Acid-fast rod; transmitted from human to human
-M. bovis: <1% U.S. cases; not transmitted from human
- -
F

to human
-

-M. avium-intracellulare complex infects people with

late-stage HIV infection
- -

Treatment of Tuberculosis 6561ax1

Treatment: Prolonged treatment with multiple antibiotics
Vaccines: BCG, live, avirulent M. bovis; not widely used in
United States
week Diagnosis of Tuberculosis
13 - Tuberculin skin test screening
14 -Positive reaction means current or previous
infection
-Followed by X-ray or CT exam, acid-fast staining of
sputum, culturing of bacteria

3-Pneumococcal Pneumonia ->

SS31 s
05D1
- Streptococcus pneumoniae
- Gram-positive encapsulated diplococci
j-,
Symptoms: Infected alveoli of lung fill with fluids; interferes
with oxygen uptake
Diagnosis: Optochin-inhibition test or bile solubility test
serological typing of bacteria
- . . .

Treatment: Penicillin, fluoroquinolones

Prevention: Pneumococcal vaccine
4-Haemophilus influenzae Pneumonia
Gram-negative coccobacillus
-Predisposing factors: Alcoholism, poor nutrition,
cancer, or diabetes
-Symptoms: Resemble those of pneumococcal pneumonia
-Diagnosis: Isolation; special media for nutritional
requirements
-Treatment: Cephalosporins
 5-Mycoplasmal Pneumonia
week
13 Primary atypical pneumonia; walking pneumonia
-Mycoplasma pneumoniae
14
-Pleomorphic, wall-less bacteria
-Common in- children and O
young adults
Symptoms: Mild but persistent respiratory low
fever, cough, headache
Diagnosis: PCR and serological testing
Treatment: Tetracyclines

Legionellosis
-Legionella pneumophila
-Gram-negative rod
-Found in water
-Transmitted by inhaling aerosols; not transmitted from
human to human
Symptoms: Potentially fatal pneumonia that tends to
affect older men who 00drink or smoke heavily
Diagnosis: Culture on selective media, DNA probe
Treatment: Erythromycin

Psittacosis (Ornithosis) - -1
-Chlamydophila psittaci
-Gram-negative intracellular bacterium
-Transmitted toO humans by elementary bodies from bird
-

droppings
-

-Reorganizes into reticulate body after being

phagocytized

Symptoms:if any, are fever, headache, chills

Diagnosis: Growth of bacteria in eggs or cell culture
Treatment: Tetracyclines
week Chlamydial Pneumonia
13 -Chlamydophila pneumoniae
14 -Transmitted from human to human
Symptoms: Mild respiratory illness common in young
people; resembles mycoplasmal pneumonia
Diagnosis: Serological tests
Treatment: Tetracyclines

Q Fever
Causative agent: Coxiella burnetii
Reservoir: Large mammals
Tick vector
Can be transmitted via unpasteurized milk
Symptoms: Mild respiratory disease lasting 1–2 weeks;
occasional complications such as endocarditis occur
Diagnosis: Growth in cell culture
Treatment: Doxycycline and chloroquine

Melioidosis
Causative agent: by Burkholderia
pseudomallei
Reservoir: Soil
Mainly in southeast Asia and northern
Australia
Symptoms: Pneumonia, or tissue
abscesses and severe sepsis
Diagnosis: Bacterial culture
Treatment: Ceftazidime
week Types of sputum specimens & processing
13 4 terms commonly used
14 1.Mucopurulent Thickgreenishyellowmucus
(indicates infection)
2. Purulent Yellow-Green mucus thinner consistency
( indicates infection)
3. Mucoid Clear colorless white mucus.
4. Blood-tinged Hemoptysis (indicates Tuberculosis,
pneumonia etc.

Sputum Vs Saliva
Gram stain
Count Epithelial cells and WBCs (pus cells)
Greater than 25 epithelial cells = saliva
Greater than 25 pus cells = sputum
10-25 equivocal, interpret with culture report.
week Infectious Diseases Affecting the
15 Genitourinary System
Structure and Function of Urinary System
Urinary system:
Two kidneys
Two ureters
One urinary bladder
One urethra
Infection prevented by:
Valves prevent backflow to kidneys
Acidity of urine
Mechanical flushing

Female Urinary System

Predisposition to infection
Short urethra
Proximity to anus
The Female Reproductive System
Two ovaries
Two uterine (fallopian) tubes
The uterus, including the cer vix; the vagina
External genitals
The male reproductive system consists of
t wo testes,
a system of ducts,
accessory glands,
and the penis
week
15

Urinary tract infections typically occur when bacteria

enter the urinary tract through the urethra and begin
to multiply in the bladder.

Cystitis
An inflammation of the urinary bladder
Urethritis
An inflammation of the urethra
Pyelonephritis
An inflammation of one or both kidneys

-Urinary system infections are most frequently

initiated by an inflammation of the urethra, or urethritis.
-Because of the proximity of the anus to the urinary opening,
intestinal bacteria predominate in urinary tract infections.
-Most infections of the urinary tract are caused by Escherichia
coli.
week Cystitis
Usual causative agents:
15 E. coli
Staphylococcus saprophyticus (normal flora of the
female genital tract)
Symptoms:
Dysuria (difficult or painful urination)
Pyuria (pus in urine)
Diagnosis: >100 CFU/ml potential pathogens and + LE
(leukocyte esterase) test
Treatment: Trimethoprim-sulfamethexazole

Why women are predispose for

cystitis?
-The female urethra is less than 2 inches long, and
microorganisms traverse it readily.
-It is also closer than the male urethra to the anal opening and
its contaminating intestinal bacteria.
-These considerations are reflected in the fact that the rate of
urinary tract infections in women is eight times that of men.

Cystitis
Interestingly, daily ingestion of cranberry
juice prevents E. coli from adhering to epithelial cells

Pyelonephritis
In 25% of untreated cases, cystitis may progress to
pyelonephritis.
Causative agent: Usually E. coli
Symptoms: Fever; back or flank pain
Diagnosis: 104 CFUs/ml and + LE (leukocyte esterase) test
Treatment: Cephalosporin
week
15 Pyelonephritis
-f pyelonephritis becomes chronic, scar tissue forms in the
kidneys and severely impairs their function.
-Because pyelonephritis is a potentially life- threatening
condition, treatment usually begins with intravenous,
extended term administration of a broad-spectrum
antibiotic

Leptospirosis
Causative agent: Leptospira interrogans
Reser voir: Dogs and rats
Transmission: Skin/mucosal contact from urine-
contaminated water
week
Sexually Transmitted Infections (STIs) that can affect
15 urinary tract
-Prevented by condoms
-Bacterial infections are treated with antibiotics

Gonorrhea
Caused by Neisseria gonorrhoeae (also known as gonococcus)
If left untreated, may result in -Endocarditis
-Meningitis
-Arthritis
-Ophthalmia neonatorum

Symptoms:
Men: Painful urination and discharge of pus; most of the
cases are limited to the distal urogenital tract but can
spread to the prostate
Women: Few symptoms but possible complications, both
genital and urinary tract can be infected, pelvic
inflammatory disease (infertility)
Diagnosis: Gram stain, ELISA, PCR
Treatment: Fluoroquinolones

Ophthalmia neonatorum
Transmitted from an infected mother to an infant
during its passage through the birth canal, 1 day after
birth
week
15

Nongonococcal Urethritis (NGU)

Nonspecific urethritis
Chlamydia trachomatis
Symptoms: Painful urination and watery discharge; in
women, possible complications, such as PID
Diagnosis: Culturing, PCR
Treatment: Doxycycline, azithromycin

Chlamydia
Most common reportable infectious disease in the U.S. at
least t wo times more common than gonorrheia.
Majority of cases are asymptomatic
symptoms in males
-Inflammation of the urethra (nongonococcal gonorrheia)
-Symptoms mimicking gonorrhea
-Untreated infections may lead to epididymitis
Symptoms in females
Cer vicitis
Discharge
Salpingitis
may lead to pid
week
Conjunctivitis (trachoma)
15
Chlamy'dia trachomatis
produces conjunctivitis after day three post birth, but
may occur up to t wo weeks after delivery,
Less inflamed, more watery