Gram Negative Rods
Gram Negative Rods
Gram Negative Rods
ENTEROBACTERIACEAE
A. Characteristics
Named, as well coliforms or enterobacilli
Found as normal flora in intestinal tract of humans and animals.
Gram-negative, non-spore forming, aerobic and facultative anaerobic bacteria.
Most are motile.
Grow over a wide range of temperature in ordinary media.
All ferment glucose with acid production.
Oxidase negative.
Release endotoxin from their cell wall.
Some release exotoxin.
Most of them have possessed three types of antigenes.
1. H antigen-. Flagellar protein
Possessed by motile enterobacteriaceae.
Heat labile and sensitive to alcohol
May interfere with agglutination by O antisera
2. K antigen- .Capsular polysaccharide or protein
Surroundes the cell wall.
Heat labile and may be associated with virulence
May interfere with agglutination by O antisera
3. O antigen- .Outer membrane lipopolysaccharide.
Found in the cell wall of enterobacteriaceae.
Resistant to heat and alcohol, and usually detected by bacterial agglutination
Antibodies to O ags are usually IgM
GENUS: ESCHERICHIA
Main species of medical importance is Escherichia coli.
I. Escherichia coli
A. Characteristics:
Normal flora in human and animal gastrointestinal tract.
Found in soil, water and vegetation.
Most are motile; some are capsulated.
B. Clinical features:
Urinary tract infection- cystitis, pyelonephritis
Wound infection- appendicitis, peritonitis
Neonatal septicemia and meningitis
E.coli-associated diarrheal disease
1. Enteropathogenic E.coli(EPEC)
causes outbreaks ofself-limiting infantile diarrhea
they also cause severe diarrhea in adults
antibiotic tretment shorten the duratin of illness and cure diarrhea
2. Enteroinvasive E.coli(EIEC)
Non-motile, non-lactose fermenting E.coli invade the mucosa of the ileum and colon, and causes shigellosis-like
dysentery in children in developing countries and travellers to these countries
3. Enterotoxigenic E.coli(ETEC)
Colonization factor of the organism promote adherence to epithelial cells of small intestine followed by release of
enterotoxin which causes toxin-mediated watery diarrhea in infants and young adults.
It is an important cause of traveller’s diarrhea
Antibiotic prophylaxis can be effective but may increase drug resistance (Should not be uniformly recommended)
4. Entero haemorrhagic E.coli( EHEC)
Cytotoxic verotoxin producing E.coli serotype O157:H7 causes haemorrhagic colitis (severe form of diarrhea),
hemolytic uremic syndrome characterized by acute renal failure, hemolytic anemia and low platelet count
5. Enteroaggressive E.coli( EAEC)
Adhere to human intestinal mucosal cells and
produce ST-like toxin and hemolysin, and
causes acute and chronic diarrhea in persons in developing countries
Produce food-borne illness in developed countries
C. Laboratory diagnosis:
Specimen: Urine, pus, blood, stool, body fluid
Smear: Gram-negative rods
Culture: Lactose-fermenting mucoid colonies on mac conkey agar and some strains are hemolytic on blood agar.
Biochemical reaction: Produce indole from tryptophan containing peptone water. Reduce nitrate to nitrite.
Serology: For serotyping (Epidemiologic information)
Treatment: Base on antibiotic sensitivity pattern
GENUS: KLEBSIELLA
A. Characteristics:
a. Non-motile, lactose-fermenting, capsulated, gram-negative rods.
I. K.pneumoniae
It is found as a commensal in the intestinal tract, and also found in moist environment in hospitals.
It is an important nosocomial pathogen.
It causes:
a. Pneumonia
b. Urinary tract infection
c. Septicaemia and meningitis (especially in neonates)
d. Wound infection and peritonitis
II. K. rhinoscleromatis
It causes rhinoscleroma of nose and pharynx to extensive destruction of nasopharynx (hebra nose).
III. K.ozaenae
It causes ozena manifesting with foul smelling nasal discharge leading to chronic atrophic rhinitis.
GENUS: ENTEROBACTER
It is gram-negative lactose fermenting motile rods, and found as a commensal in the intestinal tract of humans and animals
and moist environments.
Medical important species is Enterobacter aerogens.
o It produces mucoid colony resembling klebsiella on Mac Conkey agar.
o Enterobacter aerogens is associated with urinary tract infection, wound infection and septicaemia in
immunocompromised and chronically deblitated patients.
GENUS: CITROBACTER
It is gram-negative lactose fermenting motile rods, and opportunistic pathogen.
Medical important species is Citrobacter freundii.
Citrobacter freundii is associated with urinary tract infection, wound infection and septicaemia in immunocompromised
and chronically deblitated patients.
GENUS: SALMONELLA
Most isolates of salmonellae are motile
It grows readily on simple media
It never ferment sucrose or sucrose
Form acid +/- acid from glucose or mannose Species of medical importance are: S. typhi, S. paratyphi, S. enteritidis
A. Clinical features:
1. Enteric fever
It is caused by S.typhi and S.paratyphi, and
transmitted by fecal-oral route via contaminated food and drinks
Incubation period: 10-14 days
Predisposing factors:
i. Reduced gastric acidity
ii. Disrupted intestinal microbial flora
iii. Compromised local intestinal immunity
.
Paratyphoid fever is milder than typhoid fever
Complications:
o Intestinal perforation
o Lower gastrointestinal bleeding
o Dissenmination to different body organs
o including meninges and brain
Mortality rate: Untreated cases: 10-15%; Treated cases: < 1%
2. Bacteremia with focal lesions
Causative agent: S. choleraesuis
Manifests with blood stream invasion with focal lesions in lungs, bones and meninges Intestinal manifestation are
often absent
3. Gastroenteritis
It is caused by S. enteritidis & S. typhimurium
Incubation Period (IP)= 8-48 hrs
It manifests with initial watery diarrhea, and later bloody mucoid diarrhea associated with crampy abdominal pain
and tenesmus.
Bacteremia is rare (2-3 % of cases)
It usually resolves in 2-3 days
A. Laboratory diagnosis:
1. Blood, Bone marrow, stool, urine and serum for enteric fever.
Blood – 80% positive in the first week.
Stool- 70-80% positive in the second and third week.
Urine- 20% positive in the third and fourth week.
Serum for widal test- positive after the second week of illness.
2. Stool for gastroenteritis.
Gram reaction: Gram-negative rods
Culture: Bacteriologic methods for salmonella isolation
3. Serology: (wiedal test)
Tube dilution agglutination test
Used to determine antibody titers in patients with unknown illness
Method:
o Serial dilutions of unknown serum are tested against antigens from representative salmonella species.
o The highest diluted serum with positive result is taken as a titer
Interpretation of result
High or rising titer to O antigen (≥ 1:160) suggests active infection.
High or rising titer to H antigen (≥ 1:160) suggests past infection or immunization.
High titer to the Vi antigen occurs in some cases
Causes of false positive Widal test
Malria infection
Other acute febrile illness
Poor quality reagent
Causes of false negative widal test
Spcimen collted after antibiotic adminastartion
Specimen collted at early stage of diseases
B. Treatment:
For cases: Chloramphenicol, Fluoroquinolones, 3rd generation cephalosporins
For carriers: Ampicillin followed by cholecystectomy
Note: salmonellae persist in gall bladder in chronic carriers
GENUS: SHIGELLA
Species of medical importance are:
o S. dysenteriae (Subgroup A)
o S. flexneri (Subgroup B)
o S. boydii (Subgroup C)
o S. sonnei (Subgroup D)
C. Treatment: Ciprofloxacin
GENUS: PROTEUS
Proteus species are found in the intestinal tract of humans and animals, soil, sewage and water.
They are gram-negative, motile, non-capsulated , pleomorphic rods.
A. Clinical features:
P. mirabilis
o Urinary tract infection
o Septicemia
o Abdominal and wound infection
o Secondary invader of ulcer, burn, pressure sores and chronic discharging ear.
P. vulgaris
o Important nosocomial pathogen.
o Isolated in wound infection and urinary tract infection.
B. Laboratory diagnosis:
Specimen: Urine, pus, blood, ear discharge
Smear: Gram-negative rods
Culture: Produce characteristic swarming growth over the surface of blood agar.
Biochemical reaction:
o Proteus spp……….. Urease positive
o P. vulgaris………... Indole positive
o P. mirabilis……….. Indole negative
Serology: Cross react with Weil-fellix test
GENUS YERSINIA
A. General characteristics:
Animals are natural hosts of yersinia, and humans are accidental hosts of yersinia infection
Short, pleomorphic microaerophilic or facultatively anaerobic gram negative rods exhibiting bipolar staining with special
stains
I. Yersinia pestis
Plague bacillus with gram negative, non-motile, facutatively anaerobe possessing bipolar granules
A. Antigenic structure:
LPS: Endotoxic effect
Envelope protein (Fraction I): Antiphagocytic property
V-W antigens: Plasmid gene-encoded virulence factor
Coagulase (produced at 28°C; mice body Temp )
Exotoxin (lethal for mice/unknown role in humans)
Bacteriocin (pesticin)
B. Pathogenesis and clinical features:
Rat flea (Xenopsylla cheopis) gets infected by biting an infected rodent → infected rat flea bites human (accidental host) →
organism migrate to regional lymphnodes from the site of bite (bubonic plaque) and gets into the blood via lymphatics
(septicemic plaque), or Primary pneumonic plaque results from inhalation of infective droplets, usually from an infected
coughing person
IP=2-6 days
Human Plague: 3 types
i. Bubonic plague: Fever, vomiting, painful lymphadenitis(buboes) in the groin or axillae
ii. Pneumonic plague: Ip is 1-3 days; Profuse mucoid or bloody expectoration with signs of pneumonia
iii. Septicemic plague: Fever, vomiting, diarrhea, hypotension, altered mentation, renal and heart failure, intra
vascular coagulopathy
C. Lab. Diagnosis:
Specimen: Lymphnode aspirate, CSF, blood
Smears: Wright’s stain, immunofluorescence stain, methylene blue stains, basic fuchsin stain; Wayson’s stain to
demonstrate bipolar granules
Culture: Grow in blood agar or MacConkey agar
Note: All cultures are highly infectious and must be handled with extreme caution
Biochemical reaction: Catalase positive; Oxidase negative
Serology: Fluorescent antibody technique using Y. pestis antisera
Prognosis: Mortaity rate is 50% (100% for pneumonic plaque)
D. Treatment:
a. Streptomycin
b. Tetracycline
c. Steptomycin + tetracycline or chloramphenicol
A. Y. enterocolitica
50 serotypes
Y. enterocolitica Serotype 03, 08, and 09 cause human disease
Human infection occurs by contaminated food and drinks from domestic animals or rodents
B. Y. pseudotuberculosis
Six serotypes
Y. pseudotuberculosis serotype 01 accounts for most human infection
Human infection results from ingestion of food and drinks contaminated by animal feces
Antigenic structure
o Inv (invasion) locus
o AIL (attachement invasion locus)
GENUS: PSEUDOMONAS
General characteristics:
Gram-negative motile aerobic rods having very simple growth requirement.
Can be found in water, soil, sewage, vegetation, human and animal intestine.
Species of medical importance: P. aeruginosa, P. pseudomallei
I. Pseudomonas aeruginosa
Found in human and animal intestine, water, soil and moist environment
In hospitals:
o Primarily a nosocomial pathogen.
o Invasive and toxigenic, produces infections in patients with abnormal host defenses
A. Antigenic characteristic:
a. Pili: Adhere to epithelial cells
b. Exopolysaccharide: Anti-phagocytic property/ inhibit pulmonary clearance
c. Lipopolysaccharide: Endotoxic effect
d. Enzymes
i. Elastases: Digests protein (elastin, collagen, IgG)
ii. Proteases
iii. Hemolysins
iv. Phospholipases C (heat labile): Degrade cytoplasmic membrane components
. e. Exotoxin A: Cytotoxic by blocking protein synthesis
B. Clinical features:
Pathogenic only when introduced into areas devoid of normal defenses eg. Breached mucus membrane or skin, use of IV
line or urinary catheterization, neutropenia of any cause.
Urinary tract infection- chronic, complicated Urinary tract infection and associated with indwelling catheter.
Wound infection of burn sites, pressure sores and ulcers.
Septicaemia- “Ecthyma gangrenosum” skin lesion (haemorrhagic skin necrosis)
Otitis externa- Malignant external ear infection in poorly treated diabetic patients.
Pneumonia- Infection of the lung in patients with cystic fibrosis.
Eye infection- Secondary to trauma or surgery.
C. Laboratory diagnosis:
Specimen: pus, urine, sputum, blood, eye swabs, surface swabs
Smear: Gram-negative rods
Culture:
o Obligate aerobe, grows readily on all routine media over wide range of temperature(5-42°C).
o Bluish-green pigmented large colonies with characteristic “fruity” odor on culture media.
Biochemical reaction: Oxidase positive, Catalase positive, Citrate positive, Indole negative, Produce acid from carbohydrate
by oxidation, not by fermentation.
NOTE: identification of the bacteria is based on colony morphology, oxidase-positivity, characteristic pigment production and groth
at 42°C
GENUS: VIBRIOS
Actively motile, gram-negative curved rods.
Species of medical importance: Vibrio cholerae-01
I. Vibrio cholerae
A. Characteristics:
Found in fresh water, shellfish and other sea food.
Man is the major reservoir of V. cholerae-01, which causes epidemic cholera. Readily killed by heat and drying; dies in
polluted water but may survive in clean stagnant water, esp. if alkaline, or sea water for 1-2 weeks.
B. Antigenic structure:
Six major subgroups.
All strains possess a distinctive O antigen and belong to subgroup I with subdivision into three serotypes; Ogawa, Inaba,
Hikojima.
Any serotype can be either Classical or El Tor biotype.
El Tor biotype is more resistant to adverse conditions than Classical diotype of V. cholerae.
H antigen: Little value in identification
C. Clinical features:
Route of infection is fecal-oral route.
After ingestion of the V.cholerae-01, the bacteria adheres to the intestinal wall without invasion then produces an
exotoxin causing excessive fluid secretion and diminished fluid absorption resulting in diarrhea (rice water stool)
which is characterized by passage of voluminous watery diarrhea containing vibrios, epithelial cells and mucus and
result in severe dehydration.
D. Laboratory diagnosis:
Specimen: Stool flecks
Smear: Gram-negative motile curved rods
Motility of vibrios is best seen using dark-field microscopy.
Presumptive diagnosis: Inactivation of vibrios in a wet preparation after adding vibrio antiserum.
Culture:
a. TCBS (thiosulphate citrate bile salt sucrose agar) media
Selective media for primary isolation of V.cholerae.
Observe for large yellow sucrose-fermenting colonies after 18-24 hrs of incubation.
b.Alkaline peptone water: Enrichment media for V.cholerae-01
Growth on and just below the surface of peptone water with in 4-6 hours at room temperature as
well as 37°C
Biochemical Reaction:
o Oxidase-positive.
o Ferment sucrose and maltose(acid; no gas).
o Do not ferment L-arabinose.
GENUS: CAMPYLOBACTER
Characteristics:
Small, delicate, spirally curved gram-negative bacteria.
Motile bacteria with single polar flagellum.
Stricly microaerophilic bactria requiring 5-10% o2 and 10% CO2 enriched environment.
Oxidase and catalase positive.
Species of medical importance: Campylobacter jejuni & Campylobacter coli
A. Characteristics:
Gram-negative non-spore forming motile rods with comma, S or ‘gull-wing’ shapes.
Requires selective media like skirrow’s and Butzler’s media for isolation of the bacteria from faecal specimen.
C. Clinical features:
Inoculum dose: 104 organisms
Source of infection is contaminated food, drinks ,and unpasteurized milk
The organism multiply in small intestine, invade the epithellium and produce inflammation
Campylobacter enteritis manifests with fever, headache, malaise, crampy abdominal pain and bloody mucoid diarrhea, and
usually self-limited enteritis in a week period
D. Laboratory diagnosis:
Specimen: Stool
Microscopy: Typical ‘gull-wing’ shaped gram-negative rods.
Typical darting motility of the bacteria under dark field microscopy or phase contrast microscopy
Culture: Grow best at 420c on selective media but can be cultured at 37°C
Watery and spreading or round and convex colonies on solid media at low oxygen tension.
Biochemical reaction:
o C jejuni ……………….. hydrolyzes hippurate.
o C. coli ……………… does not hydrolyze hippurate.
E. Treatment: Erythromycin
a. Shorten the duration of fecal shedding of bacteria
General characteristics:
Spiral-shaped gram negative, microaerophilic, motile rods with polar flagella
C. Lab. Diadnosis:
Specimen: Gatric biopsy, serum
Smear: Giemsa or silver stain
Culture: Skirrow’s media
Tanslucent colonies after 7 days of incubation
Biochemical reaction: Catalase positive; Oxidase positive, Urease positive
Serology:
o Detection of antibodies in the serum specific for H. pylori
o Detection of H. pylori antigen in stool specimen
Special tests: Urea breath test
D. Treatment:
Triple or quadruple therapy: Amoxicillin + clarithromycin/ metronidazole + Proton pump inhibitors (PPI (Omeprazole or
lansoprazole))
Metronidazole + Bismuth subsalicylate/ Bismuth subcitrate + Amoxicillin / Tetracycline + PPI
GENUS: LEGIONELLA
General characteristics:
Fastidious, aerobic, gram negative intracellular rods
Ubiquitous in warm moist environment
A. Antigenic structure:
Complex surface antigens
>10 serogroups
L.peumophila serogroup 1 is the most common serogroup isolated in humans
Proteases, Phosphatases, Lipases, DNase, RNase
Major secretory protein (Metalloprotease): Possess cytotoxic and hemolytic property
1. Legionnaires disease: Pneumonic presentation with high fever, chills, dry cough, hypoxia, diarrhea, and altered mentation
2. Pontiac fever: Fever, chills, malaise, headache, malaise, altered mentation
C. Laboratory diagnosis:
Specimen: Bronchial washing, Lung biopsy, Blood
Smears: DFA (direct flourescent antibody) staining
Silver staining
Cuture: Grow in BCYE (buffered charcoal-yeast extract) agar media
Biochemical tests: Catalase positive, Oxidase positive Hydrolyse hippurate
Serologic testing: Useful in the diagnosis of retrospective outbreaks of legionella infection
D. Treatment:
Erythromycin, Rifampin