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The document discusses the Enterobacteriaceae family of bacteria. It notes that they are commonly found in the large intestine of humans and are gram-negative rods that ferment glucose with acid production and reduce nitrates. The document provides classifications of Enterobacteriaceae and describes some characteristics and pathogenic members like Escherichia coli. It summarizes methods for identifying lactose fermenters versus non-fermenters and discusses virulence factors and toxins produced by pathogenic E. coli strains.

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ENTEROBACTERIACEAE basics Chapter I Dr.T.V.Rao MD 4/16/2012 Dr.T.V.RaoMD 1
Bacteria Gram positive Cocci Bacilli Gram negative Cocci Rods 4/16/2012 Dr.T.V.RaoMD 2
4/16/2012 Dr.T.V.RaoMD 3 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.
4/16/2012 Dr.T.V.RaoMD 4 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)
Basic Tests …… • Basic characters • Catalase + • Oxidase - • Reduce nitrates, • All are Gram negative and non spore forming. • Wide diversity / Antigenic heterogeneity, 4/16/2012 Dr.T.V.RaoMD 5
Classification of Enterobacteriaceae Enterobacteriaceae Lactose fermenters E. coli, Citrobacter, Klebsiella, Enterobacter Non-lactose fermenter Salmonella, Shigella Proteus, Yersinia 4/16/2012 Dr.T.V.RaoMD 6 There are several selective and differential media used to isolate distinguishes between LF & LNF The most important media are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella Shigella (SS) agar In addition to Triple Sugar Iron (TSI) agar
Differentiation between LF & NLF by Growth on MacConkey agar MacConkey Agar Contains 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 differential pH indicator Acidic: Pink Lactose non feremnters colorless colonies 4/16/2012 Lactose feremnters Pink colonDier.Ts.V .RaoMD
4/16/2012 Dr.T.V.RaoMD 8 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 medium Many are commensals Lactose fermenters Also called as coli forms , Enteric Bacilli
4/16/2012 Dr.T.V.RaoMD 9 Enterobacteriaceae Taxonomical • Tribe I Escherichia Genus – 1 Escherichia, 2 Edwardsville 3 Citrobacter 4 Salmonella 5 Shigella
4/16/2012 Dr.T.V.RaoMD 10 Enterobacteriaceae. • Tribe II; Klebsiella Genus 1. Klebsiella 2.Enterobacter, 3.Hafnia 4 Serratia Tribe III ; Proteeae Genus 1,Proteus 2,Morganella 3 . Providencia
Classification of Enterobacteriaceae ( Contd ) • Tribe IV; Erwinieae Genus 1. Erwinia 4/16/2012 Dr.T.V.RaoMD 11
Identification of Enterobacteriaceae Differentiation 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 1 2 3 4 5 4/16/2012 Flame & Cool Dr.T.V.RaoMD Flame & Cool 12
4/16/2012 Dr.T.V.RaoMD 13 Highly Pathogenic Enterobacteriaceae • Salmonella • Shigella • All are Lactose – non fermenters, • Produce colorless colonies on MacConkey medium • LF also called as Para colons,
4/16/2012 Dr.T.V.RaoMD 14 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.
4/16/2012 Dr.T.V.RaoMD 15
4/16/2012 Dr.T.V.RaoMD 16 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.
4/16/2012 Dr.T.V.RaoMD 17 E.coli Cultural 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.
E.coli on Blood Agar • Many pathogenic strains are haemolytic on blood agar. 4/16/2012 Dr.T.V.RaoMD 18
4/16/2012 Dr.T.V.RaoMD 19 E.coli Biochemical Characters, • Glucose,Lactose,Mannitol,Maltose fermented. with A/G Indole + Methyl Red + Voges Proskauer – Citrate – Urease not produced. I,M,Vi,C tests.
E.coli Antigenic 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 ) 4/16/2012 Dr.T.V.RaoMD 20
4/16/2012 Dr.T.V.RaoMD 21 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 .
4/16/2012 Dr.T.V.RaoMD 22 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
4/16/2012 Dr.T.V.RaoMD 23 Toxins and E.coli • E.coli produce Exotoxins • Hemolysins, Enterotoxins causes Diarrheas, • Important toxins produces. • Heat labile HL Heat stable HS Vero toxins VT Like Shigella toxins
4/16/2012 Dr.T.V.RaoMD 24 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:
4/16/2012 Dr.T.V.RaoMD 25 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.
4/16/2012 Dr.T.V.RaoMD 26 Toxins • Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.
4/16/2012 Dr.T.V.RaoMD 27 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 cells
E. coli toxins • Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity). 4/16/2012 Dr.T.V.RaoMD 28
4/16/2012 Dr.T.V.RaoMD 29 Toxins E.coli Labile 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
4/16/2012 Dr.T.V.RaoMD 30 Toxins of E.coli Stable 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 toxin
4/16/2012 Dr.T.V.RaoMD 31 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).
4/16/2012 Dr.T.V.RaoMD 32 Classification of E.coli 1.Enteropathogenic 2.Enterotoxigenic 3.Enteroinvasive EPEC ETEC EIEC 4.Enterohemorrhagic EHEC 5.Enteroaggresive EAEC
4/16/2012 Dr.T.V.RaoMD 33 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
Enteropathogenic E.coli r. 34 • 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 facto • Frequent in summer months • Poor hygiene predisposes. • Out breaks in Institutions 4/16/2012 Dr.T.V.Rao MD
4/16/2012 Dr.T.V.RaoMD 35 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
4/16/2012 Dr.T.V.RaoMD 36 Laboratory Diagnosis EPEC • Confirm with Polyvalent sera • Test Sero groups with polyvalent and monovalent sera. • HEp2 – adherence.
4/16/2012 Dr.T.V.RaoMD 37 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
4/16/2012 Dr.T.V.RaoMD 38 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 CFA Mortality in children < 5 years
4/16/2012 Dr.T.V.RaoMD 39 ETEC • Causes travelers diarrhea • Water contaminated with Human and Animal feces predisposes. • Laboratory Diagnosis Demonstration of Enterotoxin LT and ST Tissue culture tests, ELISA Passive agglutination tests. Animal experiments in Rabbit ileal loop test.
4/16/2012 Dr.T.V.RaoMD 40 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
4/16/2012 Dr.T.V.RaoMD 41 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
4/16/2012 Dr.T.V.RaoMD 42 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
Entero invasive E.coli • Some are non motile strains, • Atypical resembles like Shigella. • Clinically mild diarrhea • Sereny test positive animal Rabbit. • ELISA 4/16/2012 Dr.T.V.RaoMD 43
4/16/2012 Dr.T.V.RaoMD 44 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
Dr.T .V.Rao MD 45 4/16/2012
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 animals 4/16/2012 Dr.T.V.RaoMD 46
4/16/2012 Dr.T.V.RaoMD 47 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.
4/16/2012 Dr.T.V.RaoMD 48 EHEC ( E.coli ) • Culture • DNA detection methods. • Cytotoxic effects on Vero cells. • Detection with monovalent sera O157/H7
4/16/2012 Dr.T.V.RaoMD 49 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
Enteroaggresive E.coli EAEC • Stool Culture methods • Detection of Enterotoxin 4/16/2012 Dr.T.V.RaoMD 50
Treatment – E.coli Gastrointestinal disease 4/16/2012 Dr.T.V.RaoMD 51 • Fluid replacement • Antibiotics – not used usually unless systemic infections prevails –e.g. hemolytic-uremia syndrome
E. coli – Clinical significance • Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney 4/16/2012 Dr.T.V.RaoMD infection). 52
Ascending urinary tract infection 4/16/2012 Dr.T.V.RaoMD 53
4/16/2012 Dr.T.V.RaoMD 54 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,
4/16/2012 Dr.T.V.RaoMD 55 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 , Cystoscopy Usually cystitis is produced from fecal strains entering urethra
4/16/2012 Dr.T.V.RaoMD 56 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.
4/16/2012 Dr.T.V.RaoMD 57 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.
4/16/2012 Dr.T.V.RaoMD 58 Other infection with E.coli • Pyogenic infections. • Intraabdominal infections • Peritonitis. Abscess. • Septicemias • Produce Drug resistant infections.
4/ . 59
4/16/2012 Dr.T.V.RaoMD 60 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
4/16/2012 Dr.T.V.RaoMD 61 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 pneumonia
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, 4/16s /20 e 12pticaemia, andD s r.T o .V.f Rt aotM iD ssue infections. 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) 4/16K /201 l2 ebsiella ornithin D o r.Tl.V y .R taio c Ma D 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 4/16i /2n 012 fections. Dr.T .V.Rao MD 64
4/16/2012 Dr.T.V.RaoMD 65 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
4/16/2012 Dr.T.V.RaoMD 66 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 pneumonia
Klebsiella species (cont’d) – Significant biochemical reactions • Lactose positive • Most are urease positive • Non-motile 4/16/2012 Dr.T.V.RaoMD 67
4/16/2012 Dr.T.V.RaoMD 68 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 meningitis
4/16/2012 Dr.T.V.RaoMD 69 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
4/16/2012 Dr.T.V.RaoMD 70 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 antibiotics
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 → 4/16/2012 Dr.T.V.RaoMD 71
4/16/2012 Dr.T.V.RaoMD 72 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 characteristic
4/16/2012 Dr.T.V.RaoMD 73 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 -
4/16/2012 Dr.T.V.RaoMD 74 Proteus, Morganella & Providencia species • All are normal intestinal flora • Opportunistic pathogens • Deaminate phenylalanine • All are lactose negative
4/16/2012 Dr.T.V.RaoMD 75 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
Proteus species (cont’d) • A exhibits characteristic “swarming” • B shows urease positive on right 4/16/2012 Dr.T.V.RaoMD 76
4/16/2012 Dr.T.V.RaoMD 77 Morganella species • Morganella morganii is only species • Documented cause of UTI • Isolated from other anatomical sites • Urease positive • Phenylalanine deaminase positive
4/16/2012 Dr.T.V.RaoMD 78 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
4/16/2012 Dr.T.V.RaoMD 79 Citrobacter species • Citrobacter freundii associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses) • Ferments lactose and hydrolyzes urea slowly • Resembles Salmonella sp.
4/16/2012 Dr.T.V.RaoMD 80 • Programme Created by Dr.T.V.Rao MD for benefit of Medical and Paramedical Students as e-learning Resource • Email • doctortvrao@gmail

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enteroteaching-120416210800-phpapp02.pptx

  • 1.
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  • 3.
    4/16/2012 Dr.T.V.RaoMD 3 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.
  • 4.
    4/16/2012 Dr.T.V.RaoMD 4 Charactersof 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)
  • 5.
    Basic Tests …… •Basic characters • Catalase + • Oxidase - • Reduce nitrates, • All are Gram negative and non spore forming. • Wide diversity / Antigenic heterogeneity, 4/16/2012 Dr.T.V.RaoMD 5
  • 6.
    Classification of Enterobacteriaceae Enterobacteriaceae Lactosefermenters E. coli, Citrobacter, Klebsiella, Enterobacter Non-lactose fermenter Salmonella, Shigella Proteus, Yersinia 4/16/2012 Dr.T.V.RaoMD 6 There are several selective and differential media used to isolate distinguishes between LF & LNF The most important media are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella Shigella (SS) agar In addition to Triple Sugar Iron (TSI) agar
  • 7.
    Differentiation between LF& NLF by Growth on MacConkey agar MacConkey Agar Contains 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 differential pH indicator Acidic: Pink Lactose non feremnters colorless colonies 4/16/2012 Lactose feremnters Pink colonDier.Ts.V .RaoMD
  • 8.
    4/16/2012 Dr.T.V.RaoMD 8 BasicClassification • I Lactose fermenters E.coli, Escherichia Klebsiella, • II Late Lactose fermenters, Shigella sonnei, • III Non Lactose fermenters, Salmonella Shigella, Commonly tested with MacConkey medium Many are commensals Lactose fermenters Also called as coli forms , Enteric Bacilli
  • 9.
    4/16/2012 Dr.T.V.RaoMD 9 Enterobacteriaceae Taxonomical •Tribe I Escherichia Genus – 1 Escherichia, 2 Edwardsville 3 Citrobacter 4 Salmonella 5 Shigella
  • 10.
    4/16/2012 Dr.T.V.RaoMD 10 Enterobacteriaceae. •Tribe II; Klebsiella Genus 1. Klebsiella 2.Enterobacter, 3.Hafnia 4 Serratia Tribe III ; Proteeae Genus 1,Proteus 2,Morganella 3 . Providencia
  • 11.
    Classification of Enterobacteriaceae (Contd ) • Tribe IV; Erwinieae Genus 1. Erwinia 4/16/2012 Dr.T.V.RaoMD 11
  • 12.
    Identification of Enterobacteriaceae Differentiationbetween 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 1 2 3 4 5 4/16/2012 Flame & Cool Dr.T.V.RaoMD Flame & Cool 12
  • 13.
    4/16/2012 Dr.T.V.RaoMD 13 HighlyPathogenic Enterobacteriaceae • Salmonella • Shigella • All are Lactose – non fermenters, • Produce colorless colonies on MacConkey medium • LF also called as Para colons,
  • 14.
    4/16/2012 Dr.T.V.RaoMD 14 Escherichiacoli • 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.
  • 15.
  • 16.
    4/16/2012 Dr.T.V.RaoMD 16 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.
  • 17.
    4/16/2012 Dr.T.V.RaoMD 17 E.coli Culturalcharacters • 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.
  • 18.
    E.coli on BloodAgar • Many pathogenic strains are haemolytic on blood agar. 4/16/2012 Dr.T.V.RaoMD 18
  • 19.
    4/16/2012 Dr.T.V.RaoMD 19 E.coli BiochemicalCharacters, • Glucose,Lactose,Mannitol,Maltose fermented. with A/G Indole + Methyl Red + Voges Proskauer – Citrate – Urease not produced. I,M,Vi,C tests.
  • 20.
    E.coli Antigenic Structure • Somatic0 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 ) 4/16/2012 Dr.T.V.RaoMD 20
  • 21.
    4/16/2012 Dr.T.V.RaoMD 21 VirulenceFactors • 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 .
  • 22.
    4/16/2012 Dr.T.V.RaoMD 22 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
  • 23.
    4/16/2012 Dr.T.V.RaoMD 23 Toxins andE.coli • E.coli produce Exotoxins • Hemolysins, Enterotoxins causes Diarrheas, • Important toxins produces. • Heat labile HL Heat stable HS Vero toxins VT Like Shigella toxins
  • 24.
    4/16/2012 Dr.T.V.RaoMD 24 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:
  • 25.
    4/16/2012 Dr.T.V.RaoMD 25 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.
  • 26.
    4/16/2012 Dr.T.V.RaoMD 26 Toxins •Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.
  • 27.
    4/16/2012 Dr.T.V.RaoMD 27 Toxinsin 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 cells
  • 28.
    E. coli toxins •Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity). 4/16/2012 Dr.T.V.RaoMD 28
  • 29.
    4/16/2012 Dr.T.V.RaoMD 29 ToxinsE.coli Labile 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
  • 30.
    4/16/2012 Dr.T.V.RaoMD 30 Toxinsof E.coli Stable 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 toxin
  • 31.
    4/16/2012 Dr.T.V.RaoMD 31 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).
  • 32.
    4/16/2012 Dr.T.V.RaoMD 32 Classificationof E.coli 1.Enteropathogenic 2.Enterotoxigenic 3.Enteroinvasive EPEC ETEC EIEC 4.Enterohemorrhagic EHEC 5.Enteroaggresive EAEC
  • 33.
    4/16/2012 Dr.T.V.RaoMD 33 EnteropathogenicE.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
  • 34.
    Enteropathogenic E.coli r. 34 • Causesdiarrheal 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 facto • Frequent in summer months • Poor hygiene predisposes. • Out breaks in Institutions 4/16/2012 Dr.T.V.Rao MD
  • 35.
    4/16/2012 Dr.T.V.RaoMD 35 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
  • 36.
    4/16/2012 Dr.T.V.RaoMD 36 LaboratoryDiagnosis EPEC • Confirm with Polyvalent sera • Test Sero groups with polyvalent and monovalent sera. • HEp2 – adherence.
  • 37.
    4/16/2012 Dr.T.V.RaoMD 37 EnterotoxigenicE.coli • Common and causes endemics in developing countries in all age groups • May be mild watery diarrhoea to fatal conditions • Fimbrial colonization factor antigens
  • 38.
    4/16/2012 Dr.T.V.RaoMD 38 EnterotoxigenicE.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 CFA Mortality in children < 5 years
  • 39.
    4/16/2012 Dr.T.V.RaoMD 39 ETEC •Causes travelers diarrhea • Water contaminated with Human and Animal feces predisposes. • Laboratory Diagnosis Demonstration of Enterotoxin LT and ST Tissue culture tests, ELISA Passive agglutination tests. Animal experiments in Rabbit ileal loop test.
  • 40.
    4/16/2012 Dr.T.V.RaoMD 40 Treatmentand Prophylaxis in Travelers diarrhea • Doxycycline, • Trimethoprim, • Norfloxacillin • Fluroquinolones • Avoid contaminated food, • Safe protected water ,prefer bottled water, • Hot foods, Hot Drinks, • Boiled milk
  • 41.
    4/16/2012 Dr.T.V.RaoMD 41 Treatmentand Prophylaxis in Travelers diarrhea • Doxycycline, • Trimethoprim, • Norfloxacillin • Fluroquinolones • Avoid contaminated food, • Safe protected water ,prefer bottled water, • Hot foods, Hot Drinks, • Boiled milk
  • 42.
    4/16/2012 Dr.T.V.RaoMD 42 EnteroinvasiveE.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
  • 43.
    Entero invasive E.coli •Some are non motile strains, • Atypical resembles like Shigella. • Clinically mild diarrhea • Sereny test positive animal Rabbit. • ELISA 4/16/2012 Dr.T.V.RaoMD 43
  • 44.
    4/16/2012 Dr.T.V.RaoMD 44 EnterohemorrhagicE.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
  • 45.
  • 46.
    Enterohemorrhagic E.coli • Theprimary 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 animals 4/16/2012 Dr.T.V.RaoMD 46
  • 47.
    4/16/2012 Dr.T.V.RaoMD 47 EnterohemorrhagicE.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.
  • 48.
    4/16/2012 Dr.T.V.RaoMD 48 EHEC( E.coli ) • Culture • DNA detection methods. • Cytotoxic effects on Vero cells. • Detection with monovalent sera O157/H7
  • 49.
    4/16/2012 Dr.T.V.RaoMD 49 EnteroaggresiveE.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
  • 50.
    Enteroaggresive E.coli EAEC • Stool Culture methods •Detection of Enterotoxin 4/16/2012 Dr.T.V.RaoMD 50
  • 51.
    Treatment – E.coli Gastrointestinaldisease 4/16/2012 Dr.T.V.RaoMD 51 • Fluid replacement • Antibiotics – not used usually unless systemic infections prevails –e.g. hemolytic-uremia syndrome
  • 52.
    E. coli – Clinical significance •Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney 4/16/2012 Dr.T.V.RaoMD infection). 52
  • 53.
  • 54.
    4/16/2012 Dr.T.V.RaoMD 54 UrinaryTract 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,
  • 55.
    4/16/2012 Dr.T.V.RaoMD 55 Factson 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 , Cystoscopy Usually cystitis is produced from fecal strains entering urethra
  • 56.
    4/16/2012 Dr.T.V.RaoMD 56 Culturingfor 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.
  • 57.
    4/16/2012 Dr.T.V.RaoMD 57 OtherBacteria 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.
  • 58.
    4/16/2012 Dr.T.V.RaoMD 58 Otherinfection with E.coli • Pyogenic infections. • Intraabdominal infections • Peritonitis. Abscess. • Septicemias • Produce Drug resistant infections.
  • 59.
  • 60.
    4/16/2012 Dr.T.V.RaoMD 60 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
  • 61.
    4/16/2012 Dr.T.V.RaoMD 61 Klebsiellaspecies • 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 pneumonia
  • 62.
    Klebsiella • Klebsiella isa 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, 4/16s /20 e 12pticaemia, andD s r.T o .V.f Rt aotM iD ssue infections. 62
  • 63.
    Klebsiella • The genuswas 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) 4/16K /201 l2 ebsiella ornithin D o r.Tl.V y .R taio c Ma D 63
  • 64.
    Klebsiella • K pneumoniaeis 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 4/16i /2n 012 fections. Dr.T .V.Rao MD 64
  • 65.
    4/16/2012 Dr.T.V.RaoMD 65 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
  • 66.
    4/16/2012 Dr.T.V.RaoMD 66 Klebsiellaspecies • 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 pneumonia
  • 67.
    Klebsiella species (cont’d) –Significant biochemical reactions • Lactose positive • Most are urease positive • Non-motile 4/16/2012 Dr.T.V.RaoMD 67
  • 68.
    4/16/2012 Dr.T.V.RaoMD 68 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 meningitis
  • 69.
    4/16/2012 Dr.T.V.RaoMD 69 Enterobacterspecies • 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
  • 70.
    4/16/2012 Dr.T.V.RaoMD 70 Serratiaspecies • 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 antibiotics
  • 71.
    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 → 4/16/2012 Dr.T.V.RaoMD 71
  • 72.
    4/16/2012 Dr.T.V.RaoMD 72 Hafniaspecies • 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 characteristic
  • 73.
    4/16/2012 Dr.T.V.RaoMD 73 OtherEnterobacteriaceae • 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 -
  • 74.
    4/16/2012 Dr.T.V.RaoMD 74 Proteus,Morganella & Providencia species • All are normal intestinal flora • Opportunistic pathogens • Deaminate phenylalanine • All are lactose negative
  • 75.
    4/16/2012 Dr.T.V.RaoMD 75 Proteusspecies • 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
  • 76.
    Proteus species (cont’d) •A exhibits characteristic “swarming” • B shows urease positive on right 4/16/2012 Dr.T.V.RaoMD 76
  • 77.
    4/16/2012 Dr.T.V.RaoMD 77 Morganellaspecies • Morganella morganii is only species • Documented cause of UTI • Isolated from other anatomical sites • Urease positive • Phenylalanine deaminase positive
  • 78.
    4/16/2012 Dr.T.V.RaoMD 78 Providenciaspecies • 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
  • 79.
    4/16/2012 Dr.T.V.RaoMD 79 Citrobacterspecies • Citrobacter freundii associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses) • Ferments lactose and hydrolyzes urea slowly • Resembles Salmonella sp.
  • 80.
    4/16/2012 Dr.T.V.RaoMD 80 •Programme Created by Dr.T.V.Rao MD for benefit of Medical and Paramedical Students as e-learning Resource • Email • doctortvrao@gmail