1 (OVERVIEW) DR. AMADIN A. OLOTU LECTURER/CONSULTANT MEDICAL MICROBIOLOGIST BOWEN UNIVERSITY/BOWEN UNIVERSITY TEACHING HOSPITAL OGBOMOSO OUTLINE

• INTRODUCTION, • DEFINITION • SCIENTIFIC CLASSIFICATION • SOME CLINICALLY IMPORTANT GENERA • MORPHOLOGY • BIOCHEMICAL CHARACTERISTICS INTRODUCTION

•A large family of heterogenous enteric gram negative bacilli (GNB). •They are normal inhabitants of the gut in humans and animals •They constitute most of the aerobic normal flora of the gut and many do not cause disease in the normal gut. •They are common at other body sites and are the most common group of GNB isolates in the clinical microbiology laboratory DEFINITION AND CHARACTERISTICS

• The enterobacteriaceae are a family that are • Enteric gram negative bacilli • oxidase negative • positive • ferment glucose to produce acid • motile by peritrichous flagella or immotile • reduce nitrates to nitrites • are aerobes or facultative anaerobes • may or may not ferment lactose • They can grow on basic nutrient media without supplements. • 1 to 3 µm in length and 0.5 µm in diameter • Their mean generation time is 20–30minutes • They show resistance to various chemicals (bile salts), • this is the principle behind selective culturing on agar such as MacConkey. • Some species such as show on the surface of solid agar media with no inhibitory substances, this phenomenon is known as “swarming”. • Some strains produce bacteriocins or colicins which are toxic to other strains SCIENTIFIC CLASSIFICATION

• Kingdom: Bacteria • Phylum: Protobacteria • Class: Gamma • Order: Enterobacteriales • Family: Enterobacteriaceae • Over 50 genera have been defined. • >95% of the clinically significant strains fall into 10 genera and about 25 species SOME CLINICALLY IMPORTANT GENERA (LACTOSE FERMENTERS) • Escherichia • Klebsiella Klebsiella • Enterobacter spp Enterobacter aerogenes Enterobacter agglomerans • Citrobacter Citrobacter diversus NON- LACTOSE FERMENTERS

• Proteus • Providencia alcalifaciens • Morganella Morganella morganii NON- LACTOSE FERMENTERS • Salmonella • • Serratia Serratia marcesans Serratia liquifaciens • Yersinia Yersinia pseudotuberculosis MORPHOLOGY

• 1. Flagella Enterobacteriaceae are motile via peritrichous flagella except Shigella and Klebsiella which are non-motile. • Constitute the H antigens -used for typing. -Non motile organisms lack flagellar H antigens. • The flagella are maximal in young cultures 2. Capsule

• The capsule is a thin layer of surface polysaccharide. • Constitutes the K antigen. • Formation enhanced by sugar-containing media. • Capsules are particularly heavy in Klebsiella, it creates mucoid colonies. • Capsule -inhibits phagocytosis -may cross react with capsular antigens of other bacteria. 3. Fimbriae (Pili)

• Hair-like projections of protein on cell surface • Promote adhesion and are developed in old (24-48 hours) broth cultures. • The ability to agglutinate red cells, inhibition by mannose and the width differentiate the types. • Fimbriae may interfere with H agglutination. • They are preserved by formalin and destroyed by heat. 4. Cell wall

• Consists of 2 layers: an outer layer and an inner layer. • Outer layer • Is a complex of lipopolysaccharide (LPS) • The side chains of repeating sugar units project from the outer LPS layer constituting the O (somatic) antigen -used for typing. • Core glycolipids form the basal layer to which side chains are attached (The enterobacterial common antigen) • Phospholipid membrane similar in structure to the cell membrane (and therefore termed the ‘outer membrane’) • Proteins, outer membrane proteins, (OMPs) are present in the phospholipid membrane. They include those responsible for solute transport (‘porins’) and structural lipoproteins. • Lipid A forms a layer between the lipopolysaccharide and phospholipids. Is the toxic moiety of ‘endotoxin’. Biologic effects; • induction of host febrile response by production of IL-1 and prostaglandins, • activation of complement, • induction of interferon production, • production of tumour necrosis factor, • production of colony stimulating factor • activity as a B-cell mitogen. Inner layer of cell wall • Consists of peptidoglycan that maintains the cell wall rigidity. • In between the 2 layers of the cell wall lies the periplasmic space which contains enzymes Virulence Factors

• A number of factors are known to play a role in the pathogenicity of various Enterobacteriaceae . The most important are: • Adhesion factors. Attachment fimbriae, attachment pili, colonizing factor antigens( CFAs). • Invasivefactors. Proteins localized in the outer membrane (invasins) that facilitate the invasion of target cells. • Exotoxins. • —Enterotoxins disturb the normal functioning of enterocytes. Stimulation of adenylate or guanylate cyclase; increased production of cAMP This results in the loss of large amounts of electrolytes and water. • —Cytotoxins exert a direct toxic effect on cells (enterocytes, endothelial cells). • Endotoxin. Toxic effect of lipoid A as a component of LPS • Capsular resistance to phagocytosis. • Cumulation of Fe2+ Active transport of Fe2+ by siderophores in the bacterial cell BIOCHEMICAL CHARACTERISTICS

• The metabolic properties of the enterobacteriaceae are applied in the design of the battery of biochemical tests utilized in medical microbiology laboratories for identification of the organisms. Fermentation of

• Monosaccharide and disaccharides are used. • The Kligler’s iron agar (glucose and lactose in 1:10 ratio) and Triple sugar agar (glucose, lactose and sucrose in 1:10:10 ratio) are mostly used. • Organisms which ferment glucose produce acid which initially turns the whole medium yellow. • Degradation of protein in the medium leads to the production of alkaline amines, but since this requires the presence of oxygen, it occurs only on the slant which is exposed to air and not the butt. • After 24 hours, organisms which ferment only glucose produce an acid butt and an alkaline (red) slant. • Lactose (and sucrose) if fermented produce acid that can turn the whole medium yellow, regardless of amine production. • The production of H2S will turn the medium black. • Gas production manifests as bubbles in the agar, separation of the agar from the wall of the tube or complete disruption of the medium. Typical reactions are as follows: • Alkaline slant/Alkaline butt • No fermentation • Non fermenters • E.g.

• Alkaline slant/ Acid butt • Glucose fermented • Lactose (or sucrose for TSIA) not fermented • Non-lactose-fermenting bacteria • E.g. Shigella spp • Alkaline slant/Acid (Black) butt Glucose fermented Lactose not fermented H2S produced Non-lactose fermenting- H2S producing bacteria E.g. Salmonella spp and Proteus spp • Acid slant/ Acid butt Glucose and Lactose (Sucrose for TSIA) fermented Lactose fermenting coliforms E.g. E. coli and the Klebsiella-Enterobacter spp.

Fermentation of glucose

• Ferments glucose to produce acid Fermentation of lactose

• Lactose is a disaccharide composed of glucose and galactose. • The lactose plus indicator is incorporated into commonly used selective media e.g. MacConkey, deoxycolate citrate agar. Fermentation of lactose

ONPG (o-nitrophenyl-B-D-galactopyranoside) Test

• The enzyme B- galactosidase mediates lactose fermentation. Bacteria that rapidly ferment lactose also have an enzyme called permease that speeds up the reaction. Late lactose fermenter however lack permease and so fermentation is slower (2-10days). True nonlactose fermenters do not have either enzyme. • The test is a rapid (4 hour) test that detects B- galactosidase. • It distinguishes some strains of; E. coli from species of Shigella; Citrobacter spp and Salmonella sar, and Arizonae (ONPG-positive) from Salmonella spp (ONPG-negative). o-nitrophenyl-B-D galactopyranoside (ONPG) test

B-galactosidase breaks down ONPG into o-nitrophenyl, a yellow compound and galactose A yellow color indicates a positive test while a negative test is indicated by no color change IMViC • Indole production from tryptophan E.g. E. coli, Citrobacter, Edwardsiella, Klebsiella oxytoca, Morganella morganii, Proteus vulgaris/hauseri, Providencia, some Shigella, Serratia and Yersinia. • ; test identify bacteria that produce strong acids from glucose. E.g. Citrobacter, Edwarsiella, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Serratia, Shigella, Yersinia. IMViC contd

• Voges-Proskauer test; Production of acetyl methyl carbinol-Acetoin: Is a fermentation product of Klebsiellae-Enterobacter-Serratia-Hafnia group. NB; Most species of Enterobacteriaceae that are Voges-Prokauer positive are Methyl red negative and vice versa

• Citrate utilization. E.g. Klebsiella, Proteus, Providencia, Serratia.

• NB: IMViC (Indole, Methyl red, VP and Citrate) were used to detect faecal contamination of food and water. They differentiate E. coli (I+M+ VP-C - ) from Enterobacter (I-M-VP+C+)

Gas production from glucose.

• E.g. E.coli, Klebsiella, Enterobacter, Citrobacter, Proteus spp, Morganella, Salmonella spp. Motility (360C):

• Motility distinguishes two non-motile genera (Shigella and Klebsiella). Yersinia spp (motile at 220C). H2S

• Hydrogen sulphide H2S production: Edwardsiella tarda, Salmonella spp, Citrobacter freundii, Proteus spp. Deamination of phenylalanine;

• Is characteristic of Proteus, Morganella and Providentia species. production

• Urease production is characteristic of Proteus species. • Organisms that produce less urease include Klebsiella, Enterobacter, Brucella and Bordetella bronchiseptica. Urease production Decarboxylation of arginine, lysine and ornithine

• Lysine decarboxylation test differentiates lactose negative Citrobacter from Salmonella. • Ornithine decaboxylase distinguishes Shigella species; S. sonnei (98%), S. boydii (2.5%), S. dysenteriae and S. flexneri are negative. • Arginine decarboxylation e.g. Citrobacter, Enterobacter, E. coli. Biochemical properties of selected Enterobacteriaceae • API 20E strips give accurate identifications based on extensive databases and are standardized, easy-to-use test systems. The kits include strips that contain up to 20 miniature biochemical tests. It is manufactured by Biomerieux

BIBLIOGRAPHY AND REFERENCES • Mandel, Douglas and Bennetts’ principles and practice of infectious diseases. 9th ed.Philadelphia. Churchhill Livingstone Elsevier • Jawetz, Melnink and Adelberg’s medical microbiology. 28th edition THANK YOU FOR YOUR ATTENTION ANY QUESTIONS