ENTEROBACTERIACEAE basics Chapter I Dr.T.V.Rao MD4/16/2012 Dr.T.V.Rao MD 1

Bacteria Gram Gram positive negative Cocci Bacilli Cocci Rods4/16/2012 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.4/16/2012 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) 4/16/2012 Dr.T.V.Rao MD 4

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.Rao MD 5

Classification of Enterobacteriaceae Enterobacteriaceae Lactose fermenters Non-lactose fermenter E. coli, Citrobacter, Salmonella, Shigella Klebsiella, Enterobacter Proteus, YersiniaThere are several selective and differential media used to isolate distinguishes between LF & LNFThe most important media are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella Shigella (SS) agar In addition to Triple Sugar Iron (TSI) agar 4/16/2012 Dr.T.V.Rao MD 6

Differentiation between LF & NLF by Growth on MacConkey agar MacConkey agar is selective & differential medium for Enterobacteriaceae MacConkey Agar ContainsBile salts Crystal violet Lactose Neutral red pH indicatorInhibit growth of G+ve bacteria Cause of differential Acidic: PinkCause of selectivity Lactose feremnters Lactose non feremnters 4/16/2012 Dr.T.V.Rao MD colorless colonies Pink colonies

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 Bacilli4/16/2012 Dr.T.V.Rao MD 8

Enterobacteriaceae Taxonomical Tribe I Escherichia Genus 1 Escherichia, 2 Edwardsville 3 Citrobacter 4 Salmonella 5 Shigella4/16/2012 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 . Providencia4/16/2012 Dr.T.V.Rao MD 10

Classification of Enterobacteriaceae ( Contd ) Tribe IV; Erwinieae Genus 1. Erwinia4/16/2012 Dr.T.V.Rao MD 11

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 1 2 3 4 Flame & Cool 5 4/16/2012 Dr.T.V.Rao MD 12 Flame & Cool

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.Rao MD 13

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.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.4/16/2012 Dr.T.V.Rao MD 16

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.4/16/2012 Dr.T.V.Rao MD 17

E.coli on Blood Agar Many pathogenic strains are haemolytic on blood agar.4/16/2012 Dr.T.V.Rao MD 18

E.coli Biochemical Characters, Glucose,Lactose,Mannitol,Maltose fermented. with A/G Indole + Methyl Red + Voges Proskauer I,M,Vi,C tests. Citrate Urease not produced.4/16/2012 Dr.T.V.Rao MD 19

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.Rao MD 20

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.Rao MD 21

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 enterotoxins4/16/2012 Dr.T.V.Rao MD 22

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 toxins4/16/2012 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:4/16/2012 Dr.T.V.Rao MD 24

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.Rao MD 25

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.Rao MD 26

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 cells4/16/2012 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).4/16/2012 Dr.T.V.Rao MD 28

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 Diarrhea4/16/2012 Dr.T.V.Rao MD 29

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 toxin4/16/2012 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).4/16/2012 Dr.T.V.Rao MD 31

Classification of E.coli1.Enteropathogenic EPEC2.Enterotoxigenic ETEC3.Enteroinvasive EIEC4.Enterohemorrhagic EHEC5.Enteroaggresive EAEC4/16/2012 Dr.T.V.Rao MD 32

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 Institutions4/16/2012 Dr.T.V.Rao MD 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 Institutions4/16/2012 Dr.T.V.Rao MD 34

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 border4/16/2012 Dr.T.V.Rao MD 35

Laboratory Diagnosis EPEC Confirm with Polyvalent sera Test Sero groups with polyvalent and monovalent sera. HEp2 adherence.4/16/2012 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 antigens4/16/2012 Dr.T.V.Rao MD 37

Enterotoxigenic E.coliProduce Heat stable /Heat labile toxinsAdheres to epithelium of small intestine.Present with Nausea, Vomiting and Lose stoolH L like cholera toxinCauses accumulation of fluidsAdhesive factors Fimbriae specific receptor in the intestinal epithelium CFAMortality in children < 5 years4/16/2012 Dr.T.V.Rao MD 38

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.4/16/2012 Dr.T.V.Rao MD 39

Treatment and Prophylaxis in Travelers diarrhea Doxycycline, Trimethoprim, Norfloxacillin Fluroquinolones Avoid contaminated food, Safe protected water ,prefer bottled water, Hot foods, Hot Drinks, Boiled milk4/16/2012 Dr.T.V.Rao MD 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 milk4/16/2012 Dr.T.V.Rao MD 41

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 test4/16/2012 Dr.T.V.Rao MD 42

Entero invasive E.coli Some are non motile strains, Atypical resembles like Shigella. Clinically mild diarrhea Sereny test positive animal Rabbit. ELISA4/16/2012 Dr.T.V.Rao MD 43

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 cooking4/16/2012 Dr.T.V.Rao MD 44

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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 464/16/2012 Dr.T.V.Rao MD humans or animals

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.Rao MD 47

EHEC ( E.coli ) Culture DNA detection methods. Cytotoxic effects on Vero cells. Detection with monovalent sera O157/H74/16/2012 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 enterotoxin4/16/2012 Dr.T.V.Rao MD 49

Enteroaggresive E.coli EAEC Stool Culture methods Detection of Enterotoxin4/16/2012 Dr.T.V.Rao MD 50

Treatment E.coli Gastrointestinal disease Fluid replacement Antibiotics not used usually unless systemic infections prevails e.g. hemolytic-uremia4/16/2012 syndrome Dr.T.V.Rao MD 51

E. coli Clinical significance Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis4/16/2012 (kidney Dr.T.V.Rao MD 52 infection).

Ascending urinary tract infection4/16/2012 Dr.T.V.Rao MD 53

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.Rao MD 54

Facts on UTI Women suffer more than males Short urethra Pregnancy, Sexual intercourse /Honey moon cystitis. Other factors Urethral obstruction, Urinary stones Congenital malformations Neurological disorders, Catheterization , CystoscopyUsually cystitis is produced from fecal strains entering urethra4/16/2012 Dr.T.V.Rao MD 55

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.Rao MD 56

Other Bacteria and UTI Majority of UTI are caused by E.coli Other agents which cause UTI,1 Staphylococcus2.Proteus.3.Pseudomonas,4.Klebsilella spp,Citrobacter,5.Enteococcus.4/16/2012 Dr.T.V.Rao MD 57

Other infection with E.coli Pyogenic infections. Intraabdominal infections Peritonitis. Abscess. Septicemias Produce Drug resistant infections.4/16/2012 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 positive4/16/2012 Dr.T.V.Rao MD 60

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 pneumonia4/16/2012 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 Klebs (18341913). Frequent human pathogens, Klebsiella organisms can lead to a wide range of disease states, notably pneumonia, urinary tract infections, septicaemia, and Dr.T.V.Raotissue infections. 624/16/2012 soft MD

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 ornithinolytica4/16/2012 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.4/16/2012 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 antigens4/16/2012 Dr.T.V.Rao MD 65

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 pneumonia4/16/2012 Dr.T.V.Rao MD 66

Klebsiella species (contd) Significant biochemical reactions Lactose positive Most are urease positive Non-motile4/16/2012 Dr.T.V.Rao MD 67

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 meningitis4/16/2012 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 positive4/16/2012 Dr.T.V.Rao MD 69

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 antibiotics4/16/2012 Dr.T.V.Rao MD 70

Serratia species (contd) 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.Rao MD 71

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 characteristic4/16/2012 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 -4/16/2012 Dr.T.V.Rao MD 73

Proteus, Morganella & Providencia species All are normal intestinal flora Opportunistic pathogens Deaminate phenylalanine All are lactose negative4/16/2012 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 positive4/16/2012 Dr.T.V.Rao MD 75

Proteus species (contd) A exhibits characteristic swarming B shows urease positive on right4/16/2012 Dr.T.V.Rao MD 76

Morganella species Morganella morganii is only species Documented cause of UTI Isolated from other anatomical sites Urease positive Phenylalanine deaminase positive4/16/2012 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 positive4/16/2012 Dr.T.V.Rao MD 78

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.Rao MD 79

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