E.coli | Dr Ruhul Alam

Family Enterobacteriaceae

Gram – negative rods up to 3 μ m in length,oxidase – negative, have peritrichous flagella
non – sporing
Grow well on MacConkey agar because they are not inhibited by the bile salts in the medium
Mostly strains are opportunistic pathogens, occasionally causing clinical disease in locations other than the alimentary tract.
Usual habitat- Intestinal tract
Enterobacteria can be arbitrarily grouped into three categories: major pathogens, opportunistic pathogens and non – pathogens.
The major animal pathogens E. coli, Salmonella species and Yersinia species can cause both enteric and systemic disease.
Opportunistic pathogens: – Proteus species, Enterobacter species, Klebsiella species Some other members of the Enterobacteriaceae.
Klebsiella is the only NON_MOTILE under this family
Lactose fermenters form a pink colony due to acid production from Lactose.
Non-lactose fermenters have pale colonies and are alkaline due to utilisation of peptone.
Differentiation of Salmonella and E. coli in growth media:

Salmonella: Brilliant green (BG) agar and xylose – lysine deoxycholate (XLD) agar, are used to differentiate Salmonella from other enteropathogens.

On BGA , Salmonella colonies and the surrounding medium show a red alkaline reaction.
On XLD medium the colonies of most Salmonella serotypes are red (alkaline reaction) with black centers due to hydrogen sulfide (H2S) production.

E.coli:

Eeosin – methylene blue (EMB) agar on which the colonies of some isolates of E. coli have a unique metallic sheen.
Harlequin, which can be used for identification and enumeration of E. coli , is based on β – glucuronidase activity, an enzyme which is highly specific for E. coli. In this medium colonies develop a blue-green colour.
Colony morphology of different bacteria:
Klebsiella: Mucoid colony
Enterobacter: Mucoid colony
Proteus: Swarming growth on Blood agar
Serratia: Red pigment production
Triple Sugar Iron (TSI) agar: Triple sugar iron agar contains 0.1% glucose, 1% lactose and 1% sucrose and chemicals to indicate H2S production. Phenol red is used as an indicator for pH change [ (red at pH 8.2(Alkaline) , yellow at pH 6.4(Acidic) ] . A black precipitate of ferrous sulfide is indicative of H2S production.

Serotyping: Detection of Antigens of pathogens to distinguish the organism from other closely related organisms. Slide agglutination is one of those tests.

sOmatic antigen = “O” → Lipo-polysaccharide
Flagellar antigen = “H” → Protein
Capsular antigen = “K” → Polysaccharide / Polypeptide*

* Polypeptide in Bacillus anthracis capsule only

Fimbrial antigen = “F” → Protein

Serotyping allows identifi cation of the organisms involved in disease outbreaks and has applications in epidemiological investigations.

Members of the Enterobacteriaceae of veterinary importance:

Escherichia Coli

Bacteria belonging to the family Enterobacteriaceae
Gram – negative rods up to 3 μ m in length, have peritrichous flagella,usually motile
non – spore – forming facultative anaerobes
Grow well on MacConkey agar (Pink colony) because they are not inhibited by the bile salts in the medium.
Some strains produce colonies with a metallic sheen when grown on eosin – methylene blue(EMB) agar.
Escherichia coli, ferment lactose.This fermented lactose……Pink……????

*Salmonella don’t ferment lactose

Mostly strains are opportunistic pathogens, occasionally causing clinical disease in locations other than the alimentary tract.
Usual habitat- Intestinal tract
Some strains of E. coli , produce haemolysis on blood agar
Somatic (O), flagellar (H) and sometimes capsular (K) antigens are used for serotyping E. coli.
- sOmatic antigen = “O” → Lipo-polysaccharide
- Flagellar antigen = “H” → Protein
- Capsular antigen = “K” → Polysaccharide / Polypeptide*
- Fimbrial antigen = “F/H” → Protein in some strains of E.coli
- Most strains of E. coli can be regarded as commensal organisms and are of low virulence but may cause opportunistic infections in extraintestinal locations such as the mammary gland and urinary tract. Strains of E. coli which produce extraintestinal disease frequently colonize the intestinal tract of normal animals also.

Strains that cause enterocolitis are not usually part of the normal flora of healthy animals and infection results from direct contact with clinically or subclinically infected animals or by ingestion of contaminated food or water.

- Pathogenic strains of E. coli possess virulence factors which allow them to produce disease.

In recent years, E. coli O157:H7 and other enterohaemorrhagic serotypes have emerged as major food – borne, zoonotic pathogens in humans, responsible for the haemorrhagic colitis – haemolytic uraemic syndrome.

Pathogenesis and pathogenicity:

The virulence factors of pathogenic strains of E. coli include capsules, endotoxin, Fimbriae ,enterotoxins and other secreted substances.

Capsule (K) → Capsular polysaccharides ,which are weakly antigenic, interfere with the phagocytic uptake of these organisms.Also interferes with the antibacterial effectiveness of the complement system.

Endotoxin (O) → Lipopolysaccharide (LPS) component of the cell wall of Gram – negative organisms, is released on death of the bacteria.

It is composed of –

O- Side chain polysaccharide
A- Lipid-A
C- Core polysaccharide

The role of LPS in disease production includes pyrogenic activity, endothelial damage leading to disseminated intravascular coagulation, and endotoxic shock. These effects are of greatest significance in septicaemic disease.

Fimbriae (F/H) → Fimbrial adhesins which are present on many strains of E. coli allow attachment to mucosal surfaces in the small intestine and in the lower urinary tract. The most significant adhesins in strains of E. coli producing disease in domestic animals are K88 (F4), K99 (F5), 987P (F6), F18 and F41.Both K88 and K99 adhesins are encoded by plasmids.

Enterotoxin / Exotoxins ? → E. coli produces enterotoxins, shigatoxins or verotoxins & cytotoxic necrotizing factors.

Enterotoxins which affect only the functional activity of enterocytes.
Shigatoxins/verotoxins and cytotoxic necrotizing factors can produce demonstrable cell damage at their sites of action.

Enterotoxins: Two types of enterotoxins, heat – labile (LT) and heat – stable (ST) with two subtypes of each.(LT1,LT2,STa & STb)

- LT1 which induces hypersecretion of fluid into the intestine through stimulation of adenylate cyclase activity ↑.
- STa toxin induces increased guanylate cyclase activity↑ in enterocytes, and the resultant increase in intracellular guanosine monophosphate stimulates fluid and electrolyte secretion into the small intestine and inhibits fluid absorption from the intestine.
- STb also causes secretion of chloride and bicarbonate ions as well as inhibition of absorption of sodium ions through mechanisms which differ from those of STa & LT1
Enteroaggregative heat stable toxin 1 (EAST1) acts through the stimulation of cyclic GMP.

Verotoxins: Verotoxins (VT), also known as Shigatoxins (ST), are similar structurally, functionally and antigenically to the Shigatoxin of Shigella dysenteriae .These toxins are heat – labile and lethal for cultured Vero cells. Shigatoxigenic E. coli (STEC) colonizing the intestines can damage enterocytes and, when shigatoxin is absorbed into the bloodstream, it exerts a deleterious effect on endothelial cells in relatively defined anatomical locations such as the central nervous system in pigs. Vascular damage can lead to oedema, hemorrhage and thrombosis. The shigatoxin ST2e is implicated in oedema disease of pigs

iii. Cytotoxic necrotizing factors: CNF1 and CNF2, and recently CNF3 (Orden et al., 2007 ), have been demonstrated in extracts of strains of E. coli isolated from cases of extraintestinal E. coli infections in animals and humans. It is known that CNF1 is encoded chromosomally whereas CNF2 is encoded by a transmissible plasmid known as Vir.

Alpha – haemolysin & SIderophores

ETEC: Entero-toxigenic E.coli
EPEC: Entero-pathogenic E.coli
EHEC: Entero-hemorhagic E.coli
EAggEC: Entero-aggregative E.Coli
STEC: Shiga-toxigenic E.coli

Clinical infections

Enteric
Systemic

Enteric colibacillosis:

Post – weaning diarrhea of pigs:

Colisepticemia:

Coliform mastitis

Urogenital tract infections

Identification criteria for isolates:

On blood agar, colonies are grayish, round and shiny with a characteristic smell. Colonies may be haemolytic or non – haemolytic.
On MacConkey agar, the colonies are bright pink.
The colonies of some E. coli strains have a metallic sheen on EMB agar.
Slide agglutination tests for O and H antigens are employed for serotype identification.
For expression of fimbrial antigens, isolates should be subcultured on Minca medium.

Control:

Vaccination of pregnant cows with purified E. coli K99 fimbriae or whole – cell preparations, often combined with rotavirus antigen, can be used to enhance colostral protection.
A commercial vaccine is available for protection against E. coli mastitis in cows (see Chapter 93 ).

Salmonella

Salmonellae are usually motile and do not ferment lactose.
The genus Salmonella contains more than 2,500 serotypes, based on a system devised by Kaufmann and White in which somatic (O) and flagellar (H) antigens are identified.Occasionally, capsular (Vi) antigens may be detected.
A phage typing system has been developed for a small number of serotypes such as Typhimurium and Enteritidis and is used for epidemiological investigation of isolates.
In a modification of this scheme, two species were proposed, –

enterica (6 sub-species)
bongori.

The majority of salmonellae of veterinary importance belong to S. enterica subspecies enterica.

Salmonella serotypes of clinical importance and the consequences of infection: