GRAM NEGATIVE BACILLI

Marvi F. Dulnuan-Niog, MD. FPSP

Gram – Negative Bacilli

• Clinically Relevant Groupings:

I. Affecting gastrointestinal system

II. Of animal source - zoonotic
III. Affecting respiratory system
Scope

• Introduction
• Identification
• Diagnostic laboratory findings
• Pathogenesis and Pathology
• Clinical findings
• Treatment
• Epidemiology, Prevention and Control
GRAM NEGATIVE
ENTERIC ORGANISMS
Gram Negative Enteric Organisms

• Part of the normal gastrointestinal flora

or cause gastrointestinal diseases

• Members:
1. Family Enterobacteriaceae
2. Family Vibrionaceae
3. Family Pseudomonaceae
4. Family Bacteroidaceae
Family
Enterobacteriacea
 Family Enterobacteriacea

• Largest group of gram negative organisms

– 51 genera (National Library of Medicine’s Internet Taxonomy Database)
– 44 genera and 176 named species (Bergey’s Manual of Systematic Bacteriology, Vol. 2)

• Clinically significant
– 20-25 species

• Small gram negative, non-spore former bacilli

Family Enterobacteriacea
Genus Species Genus Species
Escherichia coli albertii Enterobacter aerogenes cloacae
blattae vulneris gergoviae hormaechei
furgosonii hermanii cancerogenus(taylorae)
Shigella dysenteriae flexneri Pantoea agglomerans
boydii sonnei Hafnia alvei
Salmonella typhi bongori Cronobacter Sakazakii
enterica Morganella morganii
Edwardsiella tarda hoshinae Proteus mirabilis vulgaris
liquifaciens ictaluri penneri hauseri
myxofaciens
Klebsiella pneumoniae subsp Serratia marcescens rubidaea
pneumoniae liquifaciens fonticola
pneumoniae subsp ozoenae Odirefera plymuthica
pneumoniae subsp Providencia alcalifciens retgerri
rhinoscleromatis Stuartii
Varicola Yersinia pestis intermdedia
arnitholytica pseudotuberculosis
Citrobacter freundii koseri enterocolitica ruckeri
amalonaticus youngae frederiksenii
brakii farmeri intermedia
Family Enterobacteriacea

Major Pathogens Representative Disease Minor

Pathogens
Escherichia spp. UTI, Traveler’s Diarrhea,
Neonatal meningitis
Shigella spp Dysentery
Salmonella spp Typhoid fever, Enterocolitis Arizona, Citrobacter,
Edwardsiella
Klebsiella spp Pneumonia, UTI
Enterobacter spp Pneumonia, UTI Hafnia
Serratia spp Pneumonia, UTI
Proteus spp UTI Providencia,
Morganella
Yersinia spp Plague, Enterocolitis,
Mesenteric adenitis
 Enterobacteriacea
General Characteristics
• Ubiquitous in nature
• Part of the normal flora
- Except: Salmonella and Shigella spp. (primary pathogens)

• Aerobic or Facultative anaerobe

• Non-sporeformer
• Complex cell wall
• Capsule, slime layer or neither)
• Possess fimbrea(pili)
• Ferment glucose - often with gas
Enterobacteriacea
General Characteristics
• Lactose Fermenters
- Except Shigella spp., Salmonella spp . (non-fermeters)
Proteus spp (Slow-fermenter)

• Motile with peritrichous flagella

– Except Klebsiella spp. and Shigella spp.
• Catalase positive(+)
- Except Shigella dysenteriae type1
• Oxidase negative (-)
- Except Pleosiomonas spp.
• Reduce nitrate to nitrite
 Enterobacteriacea
Presumptive Identification
I. Use of Differential and Selective media
Colony Color
Agar Bacteriostatic Fermentative H2S Color
Lactose Non-Lactose
Agent Sugar indicator Indicator
Fermenter Fermenter
MacConkey Crystal Violet Lactose None Neutral Red Red/Pink Colorless
Agar (MAC) Bile Salts
Eosin Eosin Y Lactose None Eosin Y Red/ Pink Colorless
Methylene Methylene Blue Methylene
Blue (EMB) Blue
Hektoen Bile Salts Lactose Sodium Bromthymol Yellow Orange Green/Blue green
Enteric Agar Salicin Thiosulfate Blue
(HEA) Ferric
Ammoniu
m Citrate
Xylose Lysine Bile Salts Lactose Sodium Phenol Red Yellow Colorless
Desoxycholat Xylose Thiosulfate
e (XLD) Sucrose Ferric
Ammoniu
m Citrate
Enterobacteriacea
Definitive Identification

1. Biochemical Reactions
2. Serologic antigenic structures
3. Molecular
Enterobacteriacea
Biochemical Reactions
1. Triple Sugar Iron (TSI) -Contains glucose, sucrose and lactose (fermentable sugars)
-Phenol red (pH indicator)
-Peptone (carbon/nitrogen source)
-Sodium thiosulfate(sulfur source)
-Ferric ammonium sulfate(hydrogen sulfide indicator)

If glucose and lactose is

A/A K/A A/A K/K
fermented  acid production in
H2S
slant and butt  Yellow slant and
butt (A/A)

If only Glucose fermented  acid

production in the butt  Yellow
butt/ and neutral slant (K/A)

If neither glucose or lactose is

fermented  Neutral slant and
butt (K/K)
 Enterobacteriacea
Triple Iron Sugar (TSI)
Enzymes Enterobacteriaceas
A. Rapid Lactose Escherichia spp.
Lactos permease Enterobacter spp
Fermenters Beta Klebsiella spp.
Galactosidase

B. Late Beta Slamonella arizonae

Lactose Galactosidase Serratia spp.
Fermenters Yersinia spp.
Shigella sonnei
Hafnia alvei
Citrobacter spp.
C. Non- None Salmonella spp.
Lactose Shigella spp.
Fermenters Proteus spp.
Morganella spp.
Erwinia spp.
Providencia spp.
Edwardsiella spp.
Enterobacteriacea
Biochemical Reactions
2. Indole -Tryptophanase enzyme produced by certain bacteria
Tryptophanase
Tryptophan in Peptone -------------------------------> indole, skatole, indoleactic acid
Paradiethylaminobenzaldehyde
Indole -----------------------------------------------------------------------------> Red
(KOVAC’S Reagent)

Enterobacter claocae Escherichia coli

Serratia marcescens Proteus vulgaris
Klebsiella penumoniae Klebsiella oxytoca
Salmonella spp. Shigella spp.
Enterobacteriacea
Biochemical Reactions
3. Methyl Red -Use Methyl Red-Voges-Proskauer Medium (MRVP)
4. Voges-Proskauer -Members of Enterobacteriaceae can be grouped based on the
product produced from glucose metabolism

Glucose Metabolism produce:

MR
-If acid -------------------> Red VP +
MR + Klebsiella spp
Escherichia coli Serratia spp.
Salmonella spp. Enterobacter spp.
Shigella spp.
Proteus spp

-If Acetoin or Apha Naphthol (VP)

Acetylmehtylcarbinol ---------------------------> Pink Red
40% KOH
 Enterobacteriacea
Biochemical Reactions
5. Citrate -Use Simmon’s Citrate agar
-Some organisms utilize citrate as source of carbon --------------> Ammonia

Ammonia + Ammonium Hydroxide -----> Alkaline pH

Bromthymol
Alkaline pH -----------------------> TORQUISE BLUE
Blue
Citrate Utilization -Negative Citrate Utilization Positive
Escherichia coli Klebsiella spp
Salmonella spp Serratia spp.
Shigella spp Entrobacter spp

6. Motility

All Motile, except:

Klebsiella spp
Shigella spp.
Enterobacteriacea
Biochemical Reactions
7. Urease -Detect the organisms ability to hydrolyze urea
Urea Agar: Christensen Agar
Rustigian and
Stuart Urea Broth
Indicator – Phenol red

Urease
Urea ----------------------> Ammonia ( pH )
Carbon Dioxide & Water
pH
Phenol Red ---------------------> PINK

Urease (--): Urease +:

Escherichia coli Proteus spp.
Klebsiella spp
 Enterobacteriacea
Biochemical Reactions
8. Lysine Deaminase
9. Lysine Decarboxylase
Lysine Iron Agar:
-Contains lysine(amino acid), glucose, Protein and Bromcresol
purple(pH indicator), Sodium thiosulfate
-Measures 3 parameters 1. Lysine deamination
2. Lysine decarboxylation
3. Hydrogen sulfide production

Deaminase
Lysine ---------------------------------> Cadaverine ( pH) -------------------------> Purple butt

Decarboxylase
Lysine ---------------------------------> alpha-ketocarobxylic acid ( pH) -------> Red Slant
 Enterobacteriacea
Biochemical Reactions

Lysine Deamination and Decarboxylation

Patterns:
1. Alkaline(purple) slant/ Alkaline (purple) butt = K/K
 Organism decarboxylates lysine but cannot deaminate
 Salmonella typhimurium

2. Alkaline(purple) slant/ Acid (yellow) butt = K/A

 Indicates that the organism fermented glucose but was
unable to deaminate or decarboxylate the lysine
 Escherichia coli (with no blackening)
 Citrobacter freundii (with blackening)

3. Bordeaux red slant and acid (yellow) butt = R/A

 Indicates that the organism deaminated lysine
but could not decarboxylate it
 Proteus mirabilis
Enterobacteriacea
Virulence
• Virulence :
– ability to adhere
– ability to colonize
– ability to produce toxin
– ability to invade tissue
Enterobacteriacea
Antigenic Factors
• Basis of Serologic Groups
Antigen Structure Characteristic
O Antigen Somatic O antigen, Cell Wall Heat-stable
H Antigen Flagellar Antigen Heat -labile
K Antigen Capsular Antigen Heat-labile
Vi Antigen Virulence
Enterobacteriacea
Categories of Clinical Infections
1. Primary pathogen
 Salmonella enterica
 Shigella spp.
 Yersinia spp.

2. Opportunistic pathogen
 Escherichia coli
Escherichia coli
Escherichia coli

 Member of the normal flora

 Most significant, recognized human pathogen
 Described by Theodore Escheric, 1885
 Primary marker of fecal contamination in water
purification
Escherichia coli
Morphology
– Gram negative bacilli
– Attributes of Pathogenecity:
1. 3 Antigenic structures
A. Cell Wall (O) Ag,
B. Flagellar (H) Ag
C. Capsular (K) Ag
2. Has pili
3. Toxins
3.1. Enterotoxins (enterotoxins)
i. Heat-labile-stimulates adenylate cyclase
ii. Heat-stable-stimulates guanylate cyclase
3.2. Endotoxin
Escherichia coli
• Isolated from Stool, Blood, Urine Body
fluids and other specimens
• Cultural Characteristics
1. MacConkey Agar
• Lactose positive colonies (pink) with
precipitated bile salts

2. Eosin Methylene Blue

• Greenish Metallic sheen
• Flat, non-viscous colonies
3. Blood Agar
• Gamma hemolytic
Escherichia coli
• Cultural Characteristics
– Positive for the following test:
 Indole A/A

 Methyl Red
 Lysine Decarboxylation (K/A on LIA)
 Lactose Fermentation (A/A on TSI)
 Produce gas from Acid
 Mannitol Fermentation
 Malonate utilized as source of carbon
 B-glucoronidase using the substrate 4-
methylkumbelliferyl-B-glucoronide(MUG)
Escherichia coli
Pathogenesis and Clinical Findings
Escherichia coli
Pathogenesis and Clinical Findings
Symptoms may occur only 1 to 7 days after exposure (within 3 to 4 days)
1. Diarrheal Disease
- classified by the characteristic of their virulence properties
Organism Virulence Factors Spectrum of Disease

Enteropathogenic Plasmid EPEC adherence factor (EAF) Diarrhea in infants in

E.coli (EPEC)  attachment to enterocytes developing, low-income nations
Chromosomal locus of enterocyte –usually self limited; can cause
effacement factor (LEE)  effacement of chronic disease; Outbreaks of
microvilli diarrhea in nurseries

Enterotoxigenic Colonization Factor Antigen(CFA)  “Traveler’s diarrhea”; and

E.coli (ETEC) adherence to epithelial cells childhood diarrhea – profuse
Heat –labile exotoxin (LT) attaches watery stools
enterocytes; beta sub-unit attachment to “Montezuma’Revenge
GM ganglioside; beta sub-unit activates
adenylyl cyclase
Heat-stable exotoxi n(ST) activates
guanylyl cyclase
Escherichia coli
Pathogenesis and Clinical Findings
Diarrheal Disease

Organism Virulence Factors Spectrum of Disease

Enteroinvasive Invades the intestinal mucosal Similar to Shigellosis, “bloody
E.coli (EIEC) epithelial cells diarrhea”
Occur most commonly in children in
developing countries and in
travelers to these countries

Enteroaggregative Heterogenous, exact mechanism not Acute and chronic diarrhea (>14
E.coli (EAEC) known days duration)
-Binding by pili Food-borne illnesses in
-ST-like and hemolysin like toxin industrialized countries ; have been
associated with traveler’s diarrhea;
persistent diarrhea in HIV patients
Escherichia coli
Pathogenesis and Clinical Findings
Diarrheal Disease

Organism Virulence Factors Spectrum of Disease

Enterohemorrhagic Cytotoxic toxin “Shiga-like” toxin Transmitted by ingestion of
E.coli (EHEC)/ 1. Shiga-like toxin 1 – identical to undercooked ground beef or raw
Shiga-toxin Shiga toxin of Shigella dysenteriae milk.
producing E.coli 2. Shiga-like toxin 2
(STEC) Associated with hemorrhagic
STEC 0157:H7 – most common colitis; Hemolytic Uremic
serotype Syndrome,; Microangiopathic
hemolytic anemia and
thrombocytopenia

STEC 0157:7 – negative on MUG Test

Escherichia coli
Pathogenesis and Clinical Findings
2. Urinary Tract Infection

 Most common cause of Community Urinary tract infection

 Accounts to 90% of first UTI among young women
 Presents with urinary frequency, dysuria, hematuria , pyuria

 Virulence:
1. O antigen types (Uropathogenic E.coli) – produce hemolysin
cytotoxicity and tissue invasion
2. K antigen (causes pyelonephritis) – elaborate P fimbrae that
binds to the P blood group antigen
Escherichia coli
Pathogenesis and Clinical Findings
3. Sepsis
- Most common cause gram negative sepsis
- Newborns are highly susceptible due to lack of
IgM antibodies
- May occur secondary to UTI

4. Meningitis
- E.coli along with group B streptococci are the
leading causes of Meningitis in infants.
- K1 antigen in seen in 75% o cases, cross reacts
with the group B capsular polysaccharide
Klebsiella-Enterobacter-
Serratia group

Usually Opportunistic
pathogens

Nosocomial infections
– UTI and Pneumonia
Klebsiella spp.

• Inhabitants of the gastrointestinal tract and

nasopharynx

• Most likely to be primary, non-opportunistic

pathogen of the group
Klebsiella spp
Morphology
• Gram negative bacilli
• Non-motile
• Virulence factors:
– Thick anti-phagocytic capsule in Klebsiella pneumonaie –
Quellung Reaction
– Endotoxin
Klebsiella spp.
• Isolated from Stool, Blood, Urine Body
fluids and other specimens

• Cultural Characteristics
1. MacConkey Agar
• K. pneumoniae Lactose positive
colonies (pink mucoid colonies)

2. Eosin Methylene Blue

• Pink colonies
Klebsiella spp.

• Cultural Characteristics A/A

– Positive for the following test
 Lysine Decarboxylation (K/A on LIA)
 Lactose Fermentation (A/A on TSI)
 Citrate utilized as source of carbon
 Voges –Proskauer

– Negative for the following test

 Indole
 Methyl Red
 Klebsiella pneumoniae
Pathogenesis and Clinical Findings
Species Disease conditions
1. Klebsiella -Infections usually occur in patients with predisposing
pneumoniae conditions : Advancing Age, Diabetes, Chronic Diseases,
Alcoholism etc
-Causes nosocomial Pneumonia and UTI
-Extensive hemorrhagic necrotizing consolidation –
“Lobar Pneumoniae” – presents Currant –Jelly Sputum
-Second most common cause of sepsis
-K1Antigens increasingly isolated from community-acquired
pyogenic liver abscess.
2. Klebsiella -Isolated in nasal mucosa – fetid progressive Atrophy of
pneumoniae Mucous Membranes “Atrophic Rhinitis”
subspecie ozonae
2. Klebsiella -Carry Heat-Labile cytotoxin , associated with self-limiting
oxytoca antibiotic associated hemorrhagic colitis.
4. Klebsiella -Causes chronic genital ulcerative diseased
granulomatis, formely “Granuloma Inguinale”
Calymmabacterium
granulomatis
Enterobacter spp

• Part of the normal flora

• Listed in the top 10 most frequently isolated health-

care associated infections -associated with
1. Contaminated medical devices such as
respirators and other instrumentation
2. Hospital acquired pneumonia and UTI
Enterobacter spp
Morphology
• Highly motile gram negative rods
• Lactose fermenter
• Virulence:
1. Contain capsule with mucoid colonies
2. Plasmids – encode for multiple antibiotic resistance
* ampC (Chomosamal B-lactamase)  intrinsically resistant to
Ampiciliin and 2nd Gen. Cephalosporis.
*mutants  hyperproduction of Beta-Lacatamase  reristant to 3rd
Gen. Cephalosporins.
• Culture:
MacConkey – Pink colonies
EMB agar – Pink “Fish-eye” colonies
 Enterobacter spp
Pathogenesis & Clinical Findings
E. aerogenes
E. claocae Cause broad range hospital-
acquired infections such as
E. sakazakii Now in the genus Cronobacter Penumeonia, Urinary Tract
Infections and Wound and Device
E. gorgoviae Strong urease producer Infectios

Hafnia alvei Formerly E. hafnia
Motile, non-lactose fermenter
Cronobacter sakazakii
- Associated with bacteremia,
meningitis and necrotizing colitis in
neonates
Associated with gastrointestinal
Infections
Often identified in patients sever
underlying illnesses (malignancies)
or after surgery or trauma
Serratia spp.
Characteristics
• Generally non-pigment
• 10% produce pigments

• Motile
• Slow lactose fermenter
• Positive for DNAase
• Positive for Orthonitrophenyl galactoside (ONPG)
• Virulence: Endotoxin
Serratia spp.
Characteristics
Species Characteristics
Serratia marcescens Produce red colonies due to prodigiosin pigment

Serratia rubidea Also Produce red pigments

Serratia odorifera Produces rancid potato-like odor
Serratia spp.
Pathogenesis and Clinical Manifestations

Species Characteristics
Serratia marcescens Ranked 12th most commonly isolated organism in
Serratia rubidea pediatric patients in north America, Latin America
and Europe
Serratia odorifera
Transmission maybe person-to-person, but is often
associated with medical devices – urinary catheters,
respirators, intravenous fluids and other medical
solutions.

Serratia marcescens are resitant to aminoglycosides and penicillin;

infection can be treated with 3rd Gen, Cephalosporins
Proteus spp. -Providencia spp.-
Morganella Group
• General Characteristics
– Motile, gram negative bacilli
– Non-lactose fermenters
– Produce phenylalanine deaminase
– Produce urease

– Pathogenecity:
1. Urease production  stone formation (Struvite)
2. Motility – H Antigen
Proteus spp. -Providencia spp.-
Morganella Group
• Normal inhabitants of the gastrointestinal tract
• “Swarming growth” on culture media
• “Chocolate cake” or “Burnt Chocolate” smell
Species H2S Indole Ornithine
production Decarboxylation
Proteus mirabilis + - +
Proteus vulgaris + + -
Providencia rettgeri - + +
(Formerly Proteus rettgeri)
Morganella morganii - + -
(Formerly Proteus morganii)

Antibodies are proteus against certain strains and used to diagnose Rickettsial
infections
Proteus mirabilis – OX-K Proteus vulgaris – OX-K, OX-2 and OX-9
Proteus spp. -Providencia spp.-
Morganella Group
Clinical Manifestations
Species
Proteus mirabilis
Proteus vulgaris
Providencia rettgeri
(Formerly Proteus rettgeri)
Morganella morganii
(Formerly Proteus morganii)
Clinical manifestation
Often associated with Urinary Tract Infection and
Struvite stone formation
Has been isolated from wound and ears
Most commonly associated with UTI and the feces
of children with diarrhea
Found ubiquitous through out the environment and
are often associated with stool specimens collected
from patients with symptoms with diarrhea
Escherichia coli, Klesiella spp., Enrerobacter
spp., Serratia spp., Proteus spp, Morganella
spp.and providencia spp.

Treatment and management

Antimicrobial agents Sulfonamides, Ampicillin, Cephalosporins,

Fluoroquinolones and aminoglycosides,
Tetracyclines
Relief of other Surgical correction
predisposing factors - relief of urinary obstruction
-closure of a perforation in the intestine
-Resection of bronchiectatic portion of lung
Restoration of Fluid and electrolyte balance
Escherichia coli, Klesiella spp., Enrerobacter
spp., Serratia spp., Proteus spp, Morganella
spp.and providencia spp.

Prevention and Control

• Proper sanitation
• Frequent Hand washing
• Rigorous asepsis, sterilization of equipment,
disinfection, restraint in intravenous therapy
 Biochemical Differentiation of Important Gram – negative Rods.
Organisms Oxi NR & Indole MR VP Citrate Ureas PD LD GL Motility
dase Catalase e
E. coli - + + + - - - - +/- - +
Klebsiella - + - +/- + + + - + - -
pneumoniae
oxytoca - + + +/- + + + + + - -

Salmonella - + - + - - - - + - +
typhi
paratyphi A - + - + - - - - - - +
cholerasuis - + - + - + - - + - +
Shigella spp. - + D + - - - - - - -
Proteus - + + + - D + + - + +
vulgaris
mirabilis - + - + +/- D + + - + +

Serratia - + - +/- + + +/- - - - +

merecenses
Enterobacter - + - - + + D - - - +
cloacae
Yersenia - + D + +/- - + - - - +
enterocolitica
Pseudomonas + - NA NA NA NA NA NA D + -
Vibrio cholera + + + +/- +/- NA NA NA + NA +
Family
Pseuodomonaceae
Pseudomonaceae
“Pseudomonads”

are found everywhere

1. Soil
2. Water (pools/hot tubs)
3. Fomites (everyday objects)
4. Skin
5. Medical instruments

Causes opportunistic infections

Pseudomonaceae

Have gone through revisions

Some have been reclassified
Burkholderia Ralstonia
Stenotrophomonas Comamonas
Acidovirax
Brevundimonas
Pseudomonas
Pseudomonaceae

Psuedomonas aeruginosa – most significant

Previous Name Current Name

Pseudomonas cepacia Burkholdaria cepacia complex
Pseudomonas pseudomallei Burkholderia pseudomallei
Pseudomonas maltophila Stenotrophomonas maltophila
Pseudomonas acidovorons/ Delftia acidovorans
Camamonas acidovorons
Pseudomonas aeruginosa
Cultural Characteristics
 Grows readily on many types of culture media
Nutrient Agar (smooth, translucent, sweet aroma & pigment)
Blood Agar (many hemolytic)
MacConkey (colorless, non-lactose fermenter)
Cetrimide (selective medium)

 Wide range of temperature 50 – 420C;

Best at 370C

 Produces odor “
Sweet” or“ Grapelike” or “Corn-taco-like”
Pseudomonas aeruginosa
Cultural characteristics
Biochemical Reactions
Positive Negative
Oxidase Indole
Catalase Methyl Red
Nitrate reduction Voges-Proskauer
Citrate Urease

O/F Test - Oxidative

Sugar fermentation
Glucose only
Lactose, Sucrose and Mannitol - Negative
 Pseudomonas aeruginosa
Clinical Manifestations

 Commonly causes nosocomial infections

 Common infections are rare
 Urinary tract infection after catheterization
 Acute purulent meningitis after lumbar puncture
 Post-tracheostomy pneumonia
 Septicemia in debilitated patients
 Wound and burn infections -“Blue pus”
 Chronic otitis externa and otitis interna -“Swimmer’s ear”
 Eye infection - Keratitis
 Acute necrotizing vasculitis -“Ecthyema gangrenosum”
Pseudomonas spp.
Clinical Manifestations

Pseudomonas cepacia/ Burkholderia cepacia Also causes infection in

Pseusomonas maltophila/ Burkhoderia maltophila immunocompromised
e.g. Sepsis, pneumonia, UTI

Pseudomonas mallei – causes Glander’s disease

Pseudomonas pseudomallei/ “Whitmore’s Bacillus”– causes melioidosis
- Vietnam Timebomb Disease
Pseudomonas spp.

Prevention
• Ensure appropriate control guidelines are followed
• Sterilization and decontamination of medical supplies
• Sensitive to:
 Heat, killed at 550C in 1 hour
 Acid, beta glutaraldehyde, Strong phenolic disinfectants

• Resistant to:
 Usual Chemical agents
 Common antiseptics – ammonium compound, hexachorophane
ATYPICAL PNEUMONIA
I. Mycoplasma spp.

• smallest (0.2 to 0.8 um in diameter)

• free – living organisms
• capable of self - replication – binary
fission
Mycoplasma spp.

• Pleomorphic

• lack cell wall, covered by

cell membrane
Mycoplasma spp.

• lack enzyme pathway for purine and

pyrimidine synthesis
- require complex media –
e.g. beef heart infusion broth
supplemented with horse
serum, yeast extract, and
nucleic acids.

• no toxins
 Differentiation of Mycoplasma spp.

Organism Clinical Laboratory diagnosis

Virulence Disease Growth Culture
requirement

Mycoplasma 1.Protein P1 Atypical 1. Cholesterol 1.Biphasic culture

pneumoniae – adherence pneumonia 2. Nucleic system
“Pleuropnuemo to respiratory “walking acids - incubated at 35 to
nia-like epithelium pneumonia” 37OC up to 3 weeks
organism 2. Hydrogen - PPLO Agar
(PPLO) or peroxide and “ fried egg”
Eaton agent Superoxide 2. Beta - hemolytic
 Differentiation of Mycoplasma spp.

Mycoplasma Pelvic inflammatory disease 1. Cholesterol 1. H broth

hominis -color change from yellow to red
2. H agar
-colonies about 200-300um dia.
“fried egg”
- pH 6-8
3. Agar incubated anaerobically or in
5 to 7% CO2
4. Sheep blood agar – “”pinpoint
colonies”

Ureaplasma Non-gonococcal urethritis 1. Cholesterol 1. U broth

urealyticum 2. Urea -color change from yellow to red
2. U agar
- 15 to16 um dia.
Stain dark brown in 5 minutes with
addition of CaCl2-urea solution.
3. Agar anerobically incubated or in 5
to 7% CO2
 Mycoplasma pneumoniae

Pathogenesis:

- Person to person transmission via

infected respiratory infections.

- M. pneumonia attaches to the

surface of the respiratory
epithelium – mediated by P1
Adhesion protein
- Remain extracellular
 Mycoplasma pneumoniae
Clinical Findings:
- Incubation period: 1 to 3 weeks
- Clinical Spectrum: Asymptomatic to serious pneumonitis, with
neurologic and hematologic involvement ; v skin lesions
- Insidious onset
 Mycoplasma pneumoniae
Diagnosis:
• Largely clinical recognition of the syndrome
• Laboratory tests is secondary
– Serologic testing – mainstay of diagnosis
Cold agglutinins
• appear by the end of 1st week or early in the
2nd week
• IgM antibodies against I antigen of human
RBC
– WBC slightly elevated
– Sputum to rule-out other caused of Pneumonia
– Culture is highly specialized
Mycoplasma pneumoniae

Cold agglutinin test

• false positive:
1. Infuenzae virus infection
2. Adenovirus infection

• diagnostic: fourfold increase of

antibody titer in CFT.
 2. Family Chlamydiaceae
• Formerly known as BEDSONIA (large virus)
• 2 genera:

1. Chlamydia trochomatis

2. Chlamydia psitacci – now Chlamydophila

psitacci
Chlamydia pneumoniae – now Chlamydophila
pneumoniae
 Chlamydiaceae
– obligate intracellular bacteria
– cannot replicate outside cells.

– extremely small organisms

– Gram staining : red

: gram
negative

– does not have peptidoglycan

and muramic acid
Chlamydiaceae

• virulence: resistance to lysozyme

• Non– motile, no pili, no exotoxins

 Chlamydiaceae
• have a replicative cycle different from all other
bacteria.
• Two Distinct Forms:
1. Elementary body (EB) – infectious form
transforms to RB 6 – 8 hours after entry to cell

2. Reticulate body (RB) – reproductive form

a. Larger and osmotically fragile
b. Can reproduce by binary fission - replication
c. Requires ATP from the host
d. Where DNA, RNA & Protein synthesis
Life cycle
 Diagnosis:
• Differentiation of Chlamydiaceae
Organism Culture Other diagnostic tests EB shape Glycogen Sulfa
staining of susceptibility
inclusion
Chlamydia Cell culture 1. Non-culture direct detection test Round + S
trochomatis 1.McCoy Cells - DFAT
2.Green Buffalo - EIA
Monkey cells - Nucleic acid hybridization and
amplication tests
2. Serologic
3. Frei test – for lymphogranuloma
venereum

Chlamydophila Rarely cell culture Serologic Round _ R

psittaci
Chlamydia Rarely cell culture Serologic Pear or _ R
pneumoniae round

Cell Culture – reference method for diagnosis

Glycogen stain – Iodine or Giemsa
Chlamydiaceae

Other tests:
• Direct Fluorescent Antibody tests (DFAT) –
detects antibodies

• Enzyme Immunoassays (EIA) – detect

chlamydial LPS with monoclonal or
polyclonal antibodies labeled with enzyme
Chlamydiaceae

• Serologic tests:
1. complement fixation
• CFT titer of 1:64 – presumptive diagnosis of
LGV
• CFT titer of >1:32 – presumptive of psittacosis
• CFT titer of >1:512 IgG or >1:16 IgM –
diagnosis of C. pneumoniae
2. Immunofluorescent tests
• Microimmunofluorescence test – for C.
pneumoniae
Clinical Differentiation of Chlamydiaceae
Organism Reservoir Transmission Disease Treatment

Chlamydia Birds & Inhalation of dried Psittacosis – viral like atypical Tetracycline
psittaci poultry bird feces pneumonia (fatal)
(Avian) (no human
transmission)
Chlamydophila Humans 1. Direct personal 1. Serotype A,B,C Tetracycline
trochomatis contact Trachoma - corneal scarring and and
2. Perinatal blindness erythro
transmission mycin
2. Serotype D to K
- Inclusion conjunctivitis in
neonates
- Infant interstitial pneumonia
- Non – gonococcal urethritis,
cervicitis and pelvic inflammatory
disease
- Non – gonoccocal urethritis,
epididymitis and prostatitis in
men.

3. Serotypes L1,L2 & L3

Lymphogranuloma venereum

Chlamydophila Humans Respiratory droplets Atypical pneumonia (self limiting) Tetracycline

pneumoniae
Strain TWAR