ENTEROBACTERIACEAEbasicsChapter I

Dr.T.V.Rao MD

04/07/2023 Dr.T.V.Rao MD 1

04/07/2023 Dr.T.V.Rao MD 2

Enterobacteriaceae• Commonly present in large intestine• Non sporing , Non Acid fast, Gram –

bacilli.• A complex family of organisms, • Some are non pathogenic • A few are highly Pathogenic,• Some commensals turn out to be

pathogenic. as in UTI after catheterization.04/07/2023 Dr.T.V.Rao MD 3

Characters of Enterobacteriaceae

• All Enterobacteriaceae– Gram-negative rods– Ferment glucose with acid production – Reduce nitrates into nitrites– Oxidase negative

• Facultative anaerobic• Motile except Shigella and Klebsiella • Non-capsulated except Klebsiella• Non-fastidious• Grow on bile containing media (MacConkey agar)

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Basic Tests ……• Basic characters • Catalase +• Oxidase -• Reduce nitrates,• All are Gram negative and non spore

forming.• Wide diversity / Antigenic heterogeneity,

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Classification of Enterobacteriaceae

Enterobacteriaceae

Lactose fermentersE. coli, Citrobacter,

Klebsiella, Enterobacter

Non-lactose fermenterSalmonella, Shigella

Proteus, Yersinia

There are several selective and differential media used to isolate distinguishes between LF & LNFThe most important media are:

MacConkey agarEosin Methylene Blue (EMB) agarSalmonella Shigella (SS) agar In addition to Triple Sugar Iron (TSI) agar

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Differentiation between LF & NLF by Growth on MacConkey agar

MacConkey AgarContains

Bile salts Crystal violet Lactose Neutral red

MacConkey agar is selective & differential medium for Enterobacteriaceae

Inhibit growth of G+ve bacteria

Cause of selectivity

Cause of differentialpH indicatorAcidic: Pink

Lactose feremntersPink colonies

Lactose non feremnterscolorless colonies04/07/2023 Dr.T.V.Rao MD 7

Basic Classification• I Lactose fermenters E.coli, Escherichia Klebsiella,• II Late Lactose fermenters, Shigella

sonnei,• III Non Lactose fermenters, Salmonella Shigella,Commonly tested with MacConkey mediumMany are commensals Lactose fermentersAlso called as coli forms , Enteric Bacilli04/07/2023 Dr.T.V.Rao MD 8

EnterobacteriaceaeTaxonomical

• Tribe I Escherichia Genus – 1 Escherichia, 2 Edwardsville 3 Citrobacter 4 Salmonella 5 Shigella 04/07/2023 Dr.T.V.Rao MD 9

Enterobacteriaceae.• Tribe II; Klebsiella Genus 1. Klebsiella 2.Enterobacter, 3.Hafnia 4 Serratia Tribe III ; Proteeae Genus 1,Proteus 2,Morganella 3 . Providencia

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Classification of Enterobacteriaceae ( Contd )

• Tribe IV; Erwinieae

Genus 1. Erwinia

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Identification of EnterobacteriaceaeDifferentiation between LF & NLF by Growth on MacConkey agar• Method:

– MacConkey agar is inoculated with tested organism using streak plate technique

– Incubate the plate in incubator at 37 C/24 hrs • Results:

– LF organism appears as pink colonies (e.g. E. coli)– NLF organism appears as colorless colonies (e.g.

Shigella)

Flame & Cool

Flame & Cool

Flame & Cool

1 23

45

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Highly Pathogenic Enterobacteriaceae

• Salmonella • Shigella• All are Lactose – non fermenters,• Produce colorless colonies on

MacConkey medium• LF also called as Para colons,

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Escherichia coli• Named by Escherichia• Wide group of bacteria on basis of Bio typing and Serotyping Produce infections in Humans and

Animals Detection of E.coli in water indicates

pollution and contamination.04/07/2023 Dr.T.V.Rao MD 14

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E.coli• Morphology Gram - ve Straight rods,• 1-3 X 0.4 -0.7 microns,• Appear in singles or in pairs,• Motile by peritrichate flagella.• Very few strains non motile• Not spore forming, Non acid fast.

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E.coliCultural characters

• Aerobic / Facultative Anaerobic• Grows between 10 – 40 c optimal at 37 c• Grown in simple medium• Produce Large grayish ,Thick white , moist

smooth opaque colonies • May contain capsule.• On MacConkey medium Produce Bright

pink Lactose fermenters.04/07/2023 Dr.T.V.Rao MD 17

E.coli on Blood Agar• Many

pathogenic strains are haemolytic on blood agar.

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E.coliBiochemical Characters,

• Glucose,Lactose,Mannitol,Maltose fermented. with A/G Indole + Methyl Red + Voges Proskauer – I,M,Vi,C tests. Citrate – Urease not produced.

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E.coliAntigenic Structure

• Somatic 0 170 • Capsular K 100• Flagella H 75• Virulence factors Surface Antigens Toxins O Endotoxic activity K protects against the phagocytosis Fimbriae promote virulence ( important in UTI )

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Virulence Factors • Two types of virulence factors in

E.coli• Surface antigens and Toxins• The somatic lipopolysaccharide

surface O antigen has endotoxic activity and protects from phagocytosis and bactericidal effects of complement .

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Fimbriae• Fimbriae also promote virulence• Present in large numbers causing

mannose sensitive Haemagglutination• Colonisation factor antigens is

enterotoxigenic E.coli• E.coli produce two kinds of Exotoxins

hemolysins and enterotoxins

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Toxinsand E.coli

• E.coli produce Exotoxins• Hemolysins, Enterotoxins causes

Diarrheas,• Important toxins produces.• Heat labile HL Heat stable HS Vero toxins VT Like Shigella

toxins04/07/2023 Dr.T.V.Rao MD 23

Toxins

• Enterotoxins – produced by enterotoxigenic strains of E. coli (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two types of enterotoxin:

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Toxins

• LT – is heat labile and binds to specific Gm1 gangliosides on the epithelial cells of the small intestine where it ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP.

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Toxins

• Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.

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Toxins in E.coli• Produce Enterotoxin L T and S T• Labile toxin 1956 De experiments in

Rabbit ileal loop causes outpouring of fluids

• E.coli Labile toxin like Cholera toxin• L T contains component A and B• A = Active B= Binding• B causes Binding with Gm I Ganglioside

receptor on Intestinal epithelial cells04/07/2023 Dr.T.V.Rao MD 27

E. coli toxins• Both enterotoxins

are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity).

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Toxins E.coliLabile toxin

• Component A Activated to A1 and A2• A1 Activates adenyl cyclase in the

enterocytes to form cyclic adenosine 5 monophosphate

• Causes to increase outflow of water and electrolytes in the gut lumen causes Diarrhea

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Toxins of E.coliStable Toxin

• ST A and ST B• ST A Acts by activation of Cyclic

guano sine monophosphate.( C GMP )• Causes fluid accumulation in Intestine.• E.coli ( Some ) produce

Verocytotoxin causes cytotoxicity to Vero cells.

• Acts like Shigella dysentery toxin04/07/2023 Dr.T.V.Rao MD 30

E. coli infections• Neonatal meningitis – is the leading cause of

neonatal meningitis and septicemia with a high mortality rate.

– Usually caused by strains with the K1 capsular antigen.• Gastroenteritis – there are several distinct types of E.

coli that are involved in different types of gastroenteritis:

– enterotoxigenic E. coli (ETEC), – enteroinvasive E. coli (EIEC), – enteropathogenic E. coli (EPEC) ,– enteroaggregative E. coli (EAEC), and– enterohemorrhagic E. coli (EHEC).

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Classification of E.coli1.Enteropathogenic EPEC2.Enterotoxigenic ETEC3.Enteroinvasive EIEC4.Enterohemorrhagic EHEC5.Enteroaggresive EAEC

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Enteropathogenic E.coli• Causes diarrheal disease in children,• EPEC O26/O11• Produce Verocytotoxin• Infantile enteritis, Involves upper part of Intestine• Brush border of the intestine is lost• Intimacin – EPEC adhesion factor.• Frequent in summer months• Poor hygiene predisposes.• Out breaks in Institutions

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Enteropathogenic E.coli• Causes diarrheal disease in children,• EPEC O26/O11• Produce Verocytotoxin• Infantile enteritis, Involves upper part of

Intestine• Brush border of the intestine is lost• Intimacin – EPEC adhesion factor.• Frequent in summer months• Poor hygiene predisposes.• Out breaks in Institutions

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EPEC• EPEC are identified by serotyping by

their O and B antigens• Diagnosis is difficult during sporadics• Routine culture is done for isolation• The EPEC fails to ferment sorbitol• Causes the disruption of brush border

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Laboratory Diagnosis EPEC

• Confirm with Polyvalent sera

• Test Sero groups with polyvalent and monovalent sera.

• HEp2 – adherence.04/07/2023 Dr.T.V.Rao MD 36

Enterotoxigenic E.coli

• Common and causes endemics in developing countries in all age groups

• May be mild watery diarrhoea to fatal conditions

• Fimbrial colonization factor antigens

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Enterotoxigenic E.coli• Produce Heat stable /Heat labile toxins• Adheres to epithelium of small intestine.• Present with Nausea, Vomiting and Lose stool • H L like cholera toxin• Causes accumulation of fluids • Adhesive factors Fimbriae specific receptor in the intestinal

epithelium CFAMortality in children < 5 years

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ETEC• Causes travelers diarrhea • Water contaminated with Human and Animal

feces predisposes.• Laboratory Diagnosis Demonstration of Enterotoxin LT and STTissue culture tests,ELISAPassive agglutination tests.Animal experiments in Rabbit ileal loop test.

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Treatment and Prophylaxis in Travelers diarrhea

• Doxycycline,• Trimethoprim,• Norfloxacillin• Fluroquinolones• Avoid contaminated food,• Safe protected water ,prefer bottled

water,• Hot foods, Hot Drinks,• Boiled milk

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Treatment and Prophylaxis in Travelers diarrhea

• Doxycycline,• Trimethoprim,• Norfloxacillin• Fluroquinolones

• Avoid contaminated food,• Safe protected water ,prefer bottled water,• Hot foods, Hot Drinks,• Boiled milk

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Enteroinvasive E.coli• Resembles Shigella in many respects • Non lactose fermenter and non motile• They invade the intestinal epithelium • Penetrate HeLa cells in tissue culture • May produce mild diarrhoea to frank

dysentery• Sereny test

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Entero invasive E.coli• Some are non motile

strains,• Atypical resembles

like Shigella.• Clinically mild

diarrhea • Sereny test positive

animal Rabbit.• ELISA

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Enterohemorrhagic E.coli• Produce Verocytotoxin or shiga like toxin• Mild diarrhea - can be fatal hemorrhagic

colitis. and uremic syndrome.• Present in Human and Animal feces.• Hemorrhagic complication with O157 in

Japan and USA.• Salads vegetables, Radish Proper cooking

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Dr.T.V.Rao MD 4504/07/2023

Enterohemorrhagic E.coli• The primary target for VT appear to be

vascular endothelial cells• This may contribute to HUS with

characteristic renal lesion is capillary micro angiopathy

• The typical EHEC serotype is 0 157 ; H7• The disease may manifest as food

poisoning• Occurs due to contamination of feces of

humans or animals04/07/2023 Dr.T.V.Rao MD 46

Enterohemorrhagic E.coli can cause HUS

• HUS develops when the toxin from E. coli bacteria, known as Shiga-like toxin (SLT) enters the circulation by binding to special receptors. These Shiga-toxin receptors, known as Gb3 receptors , are probably heterogeneously distributed in the major body organs allowing disparate thrombotic (blood clotting) impacts in different HUS victims, although the greatest receptor concentration appears to be in the kidneys, especially in children.

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EHEC ( E.coli )• Culture• DNA detection methods.• Cytotoxic effects on Vero

cells.• Detection with monovalent

sera O157/H704/07/2023 Dr.T.V.Rao MD 48

Enteroaggresive E.coli• They appear aggregated in s stacked

brick formation Hep-2 cell• They produce persistent diarrheal • They produce weight heat stable

enterotoxin called as low molecular heat stable enterotoxin

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Enteroaggresive E.coliEAEC

• Stool Culture methods

• Detection of Enterotoxin

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Treatment – E.coli Gastrointestinal disease

• Fluid replacement• Antibiotics

– not used usually unless systemic infections prevails

–e.g. hemolytic-uremia syndrome

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E. coli– Clinical

significance• Is the leading

cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection).

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Ascending urinary tract infection

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Urinary Tract Infections• E.coli produce urinary tract infection.• Majority of UTI s are produce by E.coli.• Instrumentation, Prostatic enlargement,

Urinary caliculi,Pregnancy,• Asymptomatic Bacteriuria in pregnant

women,• Pyelonephritis,

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Facts on UTI• Women suffer more than males Short urethra

Pregnancy, Sexual intercourse /Honey moon cystitis.

• Other factors Urethral obstruction, Urinary stones Congenital malformation's Neurological disorders, Catheterization , CystoscopyUsually cystitis is produced from fecal strains

entering urethra

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Culturing for E.coli• Mid stream sample/semi quantitative

culturing (Kass et al ) >_ 1.00,000/ml of urine. ( significant Bacteriuria )

• Urine should not be kept in wards for > 2 hours and to be preserved at 4 c

• Culture by standard loop method. • Fixed volume cultured on MacConkey

agar Lactose fermenters I M Vi C• Antibiotic sensitivity tested.04/07/2023 Dr.T.V.Rao MD 56

Other Bacteria and UTI• Majority of UTI are caused by E.coli• Other agents which cause UTI,1 Staphylococcus 2.Proteus.3.Pseudomonas,4.Klebsilella spp,Citrobacter,5.Enteococcus.04/07/2023 Dr.T.V.Rao MD 57

Other infection withE.coli

• Pyogenic infections.• Intraabdominal infections• Peritonitis. Abscess.• Septicemias • Produce Drug resistant

infections.04/07/2023 Dr.T.V.Rao MD 58

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Klebsiella, Enterobacter, Serratia

& Hafnia sp.• Usually found in intestinal tract• Wide variety of infections, primarily

pneumonia, wound, and UTI• General characteristics:

– Some species are non-motile– Simmons citrate positive– H2S negative– Phenylalanine deaminase negative– Some weakly urease positive– MR negative; VP positive

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Klebsiella species• Usually found in GI tract• Four major species• K. pneumoniae is mostly commonly

isolated species– Possesses a polysaccharide capsule,

which protects against phagocytosis and antibiotics AND makes the colonies moist and mucoid

– Has a distinctive “yeasty” odor– Frequent cause of nosocomial

pneumonia04/07/2023 Dr.T.V.Rao MD 61

Klebsiella • Klebsiella is a genus of non-motile, Gram-

negative, oxidase-negative, rod-shaped bacteria with a prominent polysaccharide-based capsule. It is named after the German microbiologist Edwin Kleb's (1834–1913). Frequent human pathogens, Klebsiella organisms can lead to a wide range of disease states, notably pneumonia, urinary tract infections, septicaemia, and soft tissue infections.04/07/2023 Dr.T.V.Rao MD 62

Klebsiella

• The genus was originally divided into 3 main species based on biochemical reactions. Today, 7 species with demonstrated similarities in DNA homology are known. These are (1) Klebsiella pneumoniae, (2) Klebsiella ozaenae, (3) Klebsiella rhinoscleromatis, (4) Klebsiella oxytoca, (5) Klebsiella planticola, (6) Klebsiella terrigena, and (7) Klebsiella ornithinolytica04/07/2023 Dr.T.V.Rao MD 63

Klebsiella

• K pneumoniae is the most medically important species of the group. K oxytoca and K rhinoscleromatis have also been demonstrated in human clinical specimens. In recent years, klebsiellae have become important pathogens in nosocomial infections.04/07/2023 Dr.T.V.Rao MD 64

Klebsiella • Klebsiella

– NF of GI tract, but potential pathogen in other areas

– TSI A/A + gas– LIA K/K– Urea +– Citrate +– MR-, VP+– Motility -– Has both O and K antigens

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Klebsiella species• Usually found in GI tract• Four major species• K. pneumoniae is mostly commonly

isolated species– Possesses a polysaccharide capsule,

which protects against phagocytosis and antibiotics AND makes the colonies moist and mucoid

– Has a distinctive “yeasty” odor– Frequent cause of nosocomial

pneumonia04/07/2023 Dr.T.V.Rao MD 66

Klebsiella species (cont’d)

–Significant biochemical reactions• Lactose positive• Most are urease positive• Non-motile

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Klebsiella– Virulence factors

• Capsule• Adhesions• Iron capturing ability

– Clinical significance• Causes pneumonia, mostly in

immunocompromised hosts. – Permanent lung damage is a frequent occurrence (rare

in other types of bacterial pneumonia)• A major cause of nosocomial infections such as

septicemia and meningitis04/07/2023 Dr.T.V.Rao MD 68

Enterobacter species• Comprised of 12 species; E. cloacae

and E. aerogenes are most common• Isolated from wounds, urine, blood

and CSF• Major characteristics

– Colonies resemble Klebsiella– Motile– MR negative; VP positive

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Serratia species• Seven species, but S. marcescens is

the only one clinically important• Frequently found in nosocomial

infections of urinary or respiratory tracts

• Implicated in bacteremic outbreaks in nurseries, cardiac surgery, and burn units

• Fairly resistant to antibiotics04/07/2023 Dr.T.V.Rao MD 70

Serratia species (cont’d)• Major characteristics

– Ferments lactose slowly– Produce characteristic pink pigment,

especially when cultures are left at room temperature

S. marscens on nutrient agar →

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Hafnia species• Hafnia alvei is only species• Has been isolated from many

anatomical sites in humans and the environment

• Occasionally isolated from stools• Delayed citrate reaction is major

characteristic04/07/2023 Dr.T.V.Rao MD 72

Other Enterobacteriaceae• Proteus, Providencia, and Morganella

– Are all part of the NF of the GI tract (except Providencia).

– All motile, with Proteus swarming– PA +– Lysine deamination + (LIA R/A)– Urea + for most, strongly + for Proteus– TSI variable (know the reactions for each in

the lab!)– Indole – only P. mirabilis is -

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Proteus, Morganella & Providencia species

• All are normal intestinal flora

• Opportunistic pathogens• Deaminate phenylalanine

• All are lactose negative04/07/2023 Dr.T.V.Rao MD 74

Proteus species• P. mirabilis and P. vulgaris are widely

recognized human pathogens• Isolated from urine, wounds, and ear and

bacteremic infections• Both produce swarming colonies on non-

selective media and have a distinctive “burned chocolate” odor

• Both are strongly urease positive• Both are phenylalanine deaminase positive

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Proteus species (cont’d)

• A exhibits characteristic “swarming”• B shows urease positive on right

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Morganella species• Morganella morganii is only

species• Documented cause of UTI • Isolated from other

anatomical sites• Urease positive• Phenylalanine deaminase

positive04/07/2023 Dr.T.V.Rao MD 77

Providencia species• Providencia rettgeri is pathogen of

urinary tract and has caused nosocomial outbreaks

• Providenicia stuartii can cause nosocomial outbreaks in burn units and has been isolated from urine

• Both are phenylalanine deaminase positive

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Citrobacter species• Citrobacter freundii

associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses)

• Ferments lactose and hydrolyzes urea slowly

• Resembles Salmonella sp.04/07/2023 Dr.T.V.Rao MD 79

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Students as e-learning Resource• Email

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