Medically Important
Gram negative bacilli (Part 1)
Dr Ekta Chourasia
Lecturer, Microbiology
Representative Gram-negative Bacilli
1. Gram-negative rods
a. Family Enterobacteriaceae: Medically
important species
- Escherichia coli,
- Salmonella typhi, Shigella sp,
2. Curved rods
- Vibrio spp
- Campylobacter spp
- Helicobacter spp
- Klebsiella spp,
- Proteus spp
- Yersinia spp.
3.Miscellaneous
b. Pseudomonas sp. & related organisms
- Legionella spp
c. Bordetella pertussis
- Chlamydia spp
d. Haemophilus spp
- Rickettsia
Dr Ekta  Page 2
General Features of Enterobacteriaceae
Present in large intestine
Gram negative bacteria
Aerobic or facultative anaerobic
Motile by peritrichate flagella
Grow on ordinary media (non fastidious)
Ferments glucose with acid & gas or only acid
Catalase + ve & oxidase -ve
Dr Ekta  Page 3
Classification of Enterobacteriaceae

Based on lactose fermentation –
oldest method :
1.
Lactose fermenters e.g.
Escherichia coli, Klebsiella sps
2.
Late lactose fermenters e.g.
Shigella sonnei
3.
Non lactose fermenters e.g
Salmonella, Shigella sps other than
Shigella sonnei
-
Dr Ekta  Page 4
Commensal intestinal bacteria:
LF
Intestinal pathogens: NLF
LF
NLF
Escherichia coli

Named after Escherich, first to describe colon bacillus

Normal flora of the human & animal intestine.

Remains viable in the feces for few days.

Detection of E. coli in the drinking water – indicates recent pollution
with human or animal feces.
Dr Ekta  Page 5
Antigenic Structure of Gram –ve Bacteria

Three antigens – serotyping
of E.coli
1.
H – flagellar antigen
2.
O – somatic antigen
3.
K – capsular antigen
Majority do not possess K Ag.
Dr Ekta  Page 6
Virulence Factors
- Two types of virulence factors: Surface Ags & Toxins
1.
2.
Surface Antigens

LPS surface O Ag – endotoxic activity

Envelope or K Ag – protects against phagocytosis

Fimbriae – colonisation, found in strains causing diarrhoea and
urinary tract infections
Toxins (Exotoxins) – two types

Enterotoxins – pathogenesis of diarrhoea
- 3 types : LT (heat labile toxin),
ST (heat stable toxin) &
VT (verocytotoxin or shiga- like toxin)

Hemolysins
Dr Ekta  Page 7
Heat Labile Toxin (LT)
 Resembles cholera toxin in its
structure, function and mode of
action
 Complex of polypeptide
subunits.
 LT: one subunit of A
(action- enzymic),
five subunits of B (binding)
Dr Ekta  Page 8
Heat Labile Toxin (LT)
Escherichia coli /
Vibrio cholerae
Gut lumen
Intestinal
epithelial cell
Dr Ekta  Page 9
Pathogenicity / Clinical Infections
1.
Urinary tract infection
2.
Diarrhoea
-
Infantile diarrhoea
-
Traveller’s diarrhoea
-
Bloody/ Hemorrhagic diarrhoea
3.
4.
Pyogenic infections
-
Wound infection, especially after surgery of lower intestinal tract.
-
Peritonitis.
-
Biliary tract infection.
-
Neonatal meningitis.
Septicemia – can lead to fatal conditions like Septic shock
Dr Ekta  Page 10
Lab Diagnosis of UTI
Specimens
Microscopy
Wet mount
Urine
Mid stream urine (MSU)
Catheter specimen urine (CSU)
Supra pubic aspiration (SPA)
Pus cells / hpf
Bacteria
Gram stain
Gram negative bacteria
(1bacteria / oil field is significant)
Urine Culture
To know significant bacteriuria
Dr Ekta  Page 11
Lab Diagnosis of E. coli UTI
Significant
bacteriuria
> 105 organism / ml of MSU
Culture
BA / MAC : LF (flat)
CLED medium
Identification tests
I M Vi C test: + + - TSI agar
Dr Ekta  Page 12
Acid, no gas
Diarrheagenic E.coli
 Enteropathogenic E. coli (EPEC) – infantile diarrhea, nontoxigenic
 Enterotoxigenic E. coli (ETEC) – traveller’s diarrhea, resembles cholera
 Enteroinvasive E. coli (EIEC) – bloody diarrhea (blood, mucus &
leucocytes with stool)
 Enterohemorrhagic E. coli (EHEC) or Verocytotoxigenic E. coli
(VTEC):- O157:H7 serotype (food poisoning) - Hemorrhagic colitis,
Hemolytic uraemic syndrome
 Enteroaggregative E. coli (EAEC) : “stacked brick” appearancepersistent diarrhea in children
 Diffusely adherent E. coli (DAEC)
Dr Ekta  Page 13
Klebsiella pneumoniae

General features
-
Normal gut flora in the intestine
-
Gram negative bacilli (short & plump)
-
Capsulated, non-motile, produces mucoid
LF colonies on MAC

Pathogenicity
-
Pneumonia: hospital & community acquired
-
Meningitis & enteritis in infants
-
Urinary Tract Infection
-
Septicemia
Dr Ekta  Page 14
Proteus
Normal gut flora in the intestine
Gram negative bacilli, pleomorphic
Motile, Non lactose fermenter (NLF) on MAC
Swarms on BA, Urease +, H2S +
Species
P. mirabilis
P. vulgaris
UTI
Pneumonia
Wound infections
Urease converts urea to NH4 & CO2 causing alkalinization of
urine leading to renal calculi (stones)
Proteus antigens are used in the Weil - Felix test to
diagnose Rickettsial diseases
Dr Ekta  Page 15
Shigella
 Classification – 4 species : biochemical & serological
characteristics.
- Sh.dysenteriae
- Sh.flexneri
Non Lactose Fermenter
- Sh.boydii
- Sh.sonnei
Dr Ekta  Page 16
-
Late Lactose Fermenter
Shigella species- Mannitol fermentation
Mannitol
Non Fermentation
Fermentation
S. dysenteriae - 12
S. flexneri- 6
S. boydii - 18
S. sonnei
(Late lactose fermenter)
Dr Ekta  Page 17
Epidemiology & Clinical Syndromes
 Causes Bacillary Dysentery – frequent passage of blood stained,
mucopurulent stools.
 Incubation period: 1-7 days, usually 48 hrs
 Low Infectious dose: 10-100 bacilli
 Feco-oral transmission
 Common in pediatric age group (1-10 years) – leading cause of
infantile diarrhea.
 Sh.dysenteriae type I : most serious form of dysentery.
 Shigellosis : whole spectrum of disease caused by Shigella.
 Complication: Hemolytic Uremic Syndrome
Dr Ekta  Page 18
Pathogenesis
Two-stage disease
Early stage
Watery diarrhea attributed to the enterotoxin activity of Shiga toxin in
the small intestine
Second stage
Dysentery due to adherence and tissue invasion of large intestine
(cytotoxic activity of Shiga toxin)
Fever attributed to neurotoxic activity of toxin
Shiga toxin
Enterotoxic, neurotoxic and cytotoxic
Similar to Shiga-like toxin of Enterohemorrhagic E. coli (EHEC)
Dr Ekta  Page 19
Laboratory Diagnosis
 Specimen: fresh feces – mucus flakes (buffered glycerol saline –
transport medium)
 Microscopy: Gram–ve, nonmotile bacilli
 Culture
 MacConkey agar: NLF colonies
 Enrichment broth – Selenite F, Gram-ve broth
 Selective media – Deoxycholate agar (DCA), Salmonella-Shigella (SS) agar, XLD
(Xylose Lysine deoxycholate)
 Slide agglutination with polyvalent & monovalent sera.
Treatment
 Oral rehydration
 Antibiotics for severe & toxic cases – Nalidixic acid or Norfloxacin.
Dr Ekta  Page 20
Salmonella

Gut of domestic animals & poultry.

Divided into 2 groups :
1.
Enteric fever group – typhoid & paratyphoid bacilli.
2.
Food poisoning group – usually animal parasites, producing
gastroenteritis, septicemia or localized infections.
Dr Ekta  Page 21
Pathogenesis
 Source of infection - Carriers , Cases, Poultry, dairy
 Transmission - Ingestion of contaminated water or food
 High infectious dose - 108 CFU
 Incubation period - 7-14 days
Dr Ekta  Page 22
Infection pattern of Salmonella
Salmonella are ingested in
contaminated food or water
Organisms reach the
terminal ileum
Enteritis
Organisms invade the gut wall
& cause ulcertion, perforation
& hemorrhage
Organisms spread to intestinal
lymphatics & are phagocytosed by
macrophages
Organisms disseminate to
bones, kidneys, lungs,liver,
brain & blood
Dr Ekta  Page 23
Enteric fever or
typhoid fever
Pathogenicity
 Enteric fever – Typhoid & paratyphoid fever.
- Clinical features: nausea, vomiting, fever, bradycardia, toxemia,
splenomegaly, hepatomegaly, diarhoea alternating with constipation.
 Septicemia with or without local suppurative lesions.
 Gastroenteritis
Dr Ekta  Page 24
Lab diagnosis of Enteric fever
Specimens
Blood, Bone marrow, urine, stool, pus, CSF
1st week
Blood culture
BHI broth
2nd week
Antibody detection
(serum)
Widal test
3rd week
Urine culture
4th week
Stool culture
Dr Ekta  Page 25
Use selective & enrichment
medium
Morphological & Cultural characteristics
 Motile, gram negative bacilli
 Non lactose fermenting (NLF – pale
colored) colonies on MacConkey &
Deoxycholate citrate agar (DCA).
 Enrichment broth - Selenite F,
Tetrathionate broth
 Selective media – Wilson & Blair (jet
black colonies due to H2S), XLD, SS
agar.
XLD
Dr Ekta  Page 26
Serology - Widal Test
Tube agglutination test
To detect antibodies in patient serum
Test is performed after 2 wks
To diagnose Enteric fever
Antigens used
TO
O antigen of S typhi
TH
H antigen of S typhi
AH
H antigen of paratyphi A
BH
H antigen of paratyphi B
CH
H antigen of paratyphi C
O is group specific
Enteric fever
H is species specific
Typhoid or paratyphoid
Dr Ekta  Page 27
Prevention & Treatment
Detection of Carriers
Vaccines
 Food handlers & Cooks
 TAB
 Repeated stool cultures
 Typhoral
 Vi agglutinins indicates carrier
status
 Typhim
Treatment
 Ciprofloxacin
Dr Ekta  Page 28
Pseudomonas aeruginosa – General characteristics
 Obligate aerobe
 small gram-negative rods with a single polar flagellum,
produce oxidase & catalase
 common inhabitant of soil & water
(ubiquitous-wide spread)
 grapelike odor
 greenish-blue pigment (pyocyanin)
 resistant to soaps, dyes, quaternary ammonium
disinfectants, drugs, drying
 frequent contaminant of ventilators, IV solutions,
anesthesia equipment
 opportunistic pathogen
 multidrug resistant
Dr Ekta  Page 29
Pathogenicity
 common cause of nosocomial infections in hosts with burns, neoplastic
disease, cystic fibrosis
 Can cause: pneumonia, UTI, abscesses
 Septicemia can lead to: endocarditis, meningitis, bronchopneumonia
 Corneal ulcers from contaminated lens solutions
 Ear infections (Otitis) “swimmer’s ear”
 Skin rash (contaminated hot tubs, saunas, swimming pools)
Dr Ekta  Page 30