1. Medically Important Gram negative bacilli (Part 1)
Dr Ekta Chourasia
Lecturer, Microbiology

2. Representative Gram-negative Bacilli
1. Gram-negative rods
a. Family Enterobacteriaceae: Medically important species
Escherichia coli,
Salmonella typhi, Shigella sp,
Klebsiella spp,
Proteus spp
Yersinia spp.
b. Pseudomonas sp. & related organisms
c. Bordetella pertussis
d. Haemophilus spp
2. Curved rods
Vibrio spp
Campylobacter spp
Helicobacter spp
3.Miscellaneous
Legionella spp
Chlamydia spp
Rickettsia
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3. 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
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4. Classification of Enterobacteriaceae
Based on lactose fermentation – oldest method :
Lactose fermenters e.g. Escherichia coli, Klebsiella sps
Late lactose fermenters e.g. Shigella sonnei
Non lactose fermenters e.g Salmonella, Shigella sps other than Shigella sonnei
Commensal intestinal bacteria: LF
Intestinal pathogens: NLF LF
NLF
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5. 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.
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6. Antigenic Structure of Gram –ve Bacteria
Three antigens – serotyping of E.coli
H – flagellar antigen
O – somatic antigen
K – capsular antigen
Majority do not possess K Ag.
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7. Virulence Factors
- Two types of virulence factors: Surface Ags & Toxins
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
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8. 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)
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9. Escherichia coli / Vibrio cholerae
Heat Labile Toxin (LT)
Escherichia coli / Vibrio cholerae
Gut lumen
Intestinal
epithelial cell
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10. Pathogenicity / Clinical Infections
Urinary tract infection
Diarrhoea
Infantile diarrhoea
Traveller’s diarrhoea
Bloody/ Hemorrhagic diarrhoea
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
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11. Lab Diagnosis of UTI Specimens Urine Mid stream urine (MSU)
Catheter specimen urine (CSU)
Supra pubic aspiration (SPA)
Microscopy Wet mount
Pus cells / hpf
Bacteria
Gram stain
Gram negative bacteria
(1bacteria / oil field is significant)
Urine Culture
To know significant bacteriuria
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12. 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
Acid, no gas
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13. 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” appearance- persistent diarrhea in children
Diffusely adherent E. coli (DAEC)
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14. 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
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15. 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
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16. Shigella
Classification – 4 species : biochemical & serological characteristics.
- Sh.dysenteriae
- Sh.flexneri Non Lactose Fermenter
- Sh.boydii
- Sh.sonnei Late Lactose Fermenter
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17. Shigella species- Mannitol fermentation
Non Fermentation
Fermentation
S. dysenteriae - 12
S. flexneri- 6
S. boydii - 18
S. sonnei
(Late lactose fermenter)
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18. Epidemiology & Clinical Syndromes
Causes Bacillary Dysentery – frequent passage of blood stained, mucopurulent stools.
Incubation period: 1-7 days, usually 48 hrs
Low Infectious dose: 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
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19. Pathogenesis Early stage Second stage Two-stage disease
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)
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20. Laboratory Diagnosis Treatment
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.
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21. Salmonella Gut of domestic animals & poultry. Divided into 2 groups :
Enteric fever group – typhoid & paratyphoid bacilli.
Food poisoning group – usually animal parasites, producing gastroenteritis, septicemia or localized infections.
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22. Pathogenesis Source of infection - Carriers , Cases, Poultry, dairy
Transmission - Ingestion of contaminated water or food
High infectious dose CFU
Incubation period days
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23. 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
Enteric fever or typhoid fever
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24. 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
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25. Lab diagnosis of Enteric fever
Specimens
Blood, Bone marrow, urine, stool, pus, CSF
Blood culture
BHI broth
1st week
Antibody detection (serum)
2nd week
Widal test
3rd week
Urine culture
Use selective & enrichment medium
4th week
Stool culture
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26. 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
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27. 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
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28. Prevention & Treatment
Detection of Carriers
Food handlers & Cooks
Repeated stool cultures
Vi agglutinins indicates carrier status
Vaccines
TAB
Typhoral
Typhim
Treatment
Ciprofloxacin
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29. 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
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30. 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)
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