Medically Important Gram negative bacilli (Part 1)

Dr Ekta ChourasiaLecturer, Microbiology

Dr Ekta Page 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

Dr Ekta Page 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

Dr Ekta Page 4

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

- Commensal intestinal bacteria: LF

- Intestinal pathogens: NLF

NLFLF

Dr Ekta Page 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.

Dr Ekta Page 6

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 7

Virulence Factors

- Two types of virulence factors: Surface Ags & Toxins

1. 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

2. 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 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)

Dr Ekta Page 9

Heat Labile Toxin (LT)

Escherichia coli / Vibrio cholerae

Escherichia coli / Vibrio cholerae

Gut lumen

Intestinal epithelial cell

Dr Ekta Page 10

Pathogenicity / Clinical Infections

1. Urinary tract infection

2. Diarrhoea

- Infantile diarrhoea

- Traveller’s diarrhoea

- Bloody/ Hemorrhagic diarrhoea

3. Pyogenic infections

- Wound infection, especially after surgery of lower intestinal tract.

- Peritonitis.

- Biliary tract infection.

- Neonatal meningitis.

4. Septicemia – can lead to fatal conditions like Septic shock

Dr Ekta Page 11

Lab Diagnosis of UTI

Specimens Urine Mid stream urine (MSU)Catheter specimen urine (CSU)

Microscopy Wet mount

Bacteria

Gram negative bacteria

Pus cells / hpf

Gram stain

Supra pubic aspiration (SPA)

Urine Culture To know significant bacteriuria

(1bacteria / oil field is significant)

Dr Ekta Page 12

Significant bacteriuria

> 105 organism / ml of MSU

Culture BA / MAC : LF (flat)

CLED medium

Identification tests

Lab Diagnosis of E. coli UTI

I M Vi C test: + + - -

TSI agar Acid, no gas

Dr Ekta Page 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)

Dr Ekta Page 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

Dr Ekta Page 15

Proteus

Normal gut flora in the intestine

Gram negative bacilli, pleomorphic

Motile, Non lactose fermenter (NLF) on MAC

Species P. mirabilis P. vulgaris

Proteus antigens are used in the Weil - Felix test to diagnose Rickettsial diseases

UTI Pneumonia

Urease converts urea to NH4 & CO2 causing alkalinization of urine leading to renal calculi (stones)

Swarms on BA, Urease +, H2S +

Wound infections

Dr Ekta Page 16

Shigella

Classification – 4 species : biochemical & serological characteristics.

- Sh.dysenteriae

- Sh.flexneri Non Lactose Fermenter

- Sh.boydii

- Sh.sonnei - Late Lactose Fermenter

Dr Ekta Page 17

Mannitol

Non Fermentation Fermentation

S. dysenteriae - 12 S. flexneri- 6

S. boydii - 18

S. sonnei (Late lactose fermenter)

Shigella species- Mannitol fermentation

Dr Ekta Page 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: 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 19

Pathogenesis Two-stage disease

Early stage

Second stage

Fever attributed to neurotoxic activity of toxin

Dysentery due to adherence and tissue invasion of large intestine (cytotoxic activity of Shiga toxin)

Watery diarrhea attributed to the enterotoxin activity of Shiga toxin in the small intestine

Enterotoxic, neurotoxic and cytotoxicShiga toxin

Similar to Shiga-like toxin of Enterohemorrhagic E. coli (EHEC)

Dr Ekta Page 20

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 21

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 22

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 23

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

Infection pattern of Salmonella

Dr Ekta Page 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

Dr Ekta Page 25

Lab diagnosis of Enteric fever

1st week Blood culture BHI broth

2nd week Antibody detection (serum)

Widal test

3rd week Urine culture

4th week Stool culture Use selective & enrichment medium

Specimens Blood, Bone marrow, urine, stool, pus, CSF

Dr Ekta Page 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

Dr Ekta Page 27

Serology - Widal Test

Tube agglutination test To detect antibodies in patient serum

Test is performed after 2 wks

Antigens used TO

To diagnose Enteric fever

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 28

Prevention & Treatment

Detection of Carriers

Food handlers & Cooks

Repeated stool cultures

Vi agglutinins indicates carrier status

Vaccines

TAB

Typhoral

Typhim

Treatment

Ciprofloxacin

Dr Ekta Page 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

Dr Ekta Page 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)