Enterobacteriaceae: Introduction and Classification PDF

ENTEROBACTERIACEAE GRAM-NEGATIVE BACILLI 1 Family Enterobacteriaceae --  A family of medically important gram (-) bacilli, Family Enterobacteriaceae is the largest, most heterogeneous collection; 63 genera are defined; 20-25 are clinically significant; other species ar...

ENTEROBACTERIACEAE GRAM-NEGATIVE BACILLI 1 Family Enterobacteriaceae --  A family of medically important gram (-) bacilli, Family Enterobacteriaceae is the largest, most heterogeneous collection; 63 genera are defined; 20-25 are clinically significant; other species are encountered infrequently.  Classification is based on: a. DNA homology d Susceptibility to Genus & Species-specific. bacteriophages b. Biochemical properties e. Nucleic acid hybridization & sequencing c. Antigenic structures f. Antibiotic Susceptibility Patterns  Gram (-) bacilli with rounded ends measuring 0.3 to 1.0 x 1.0-6.0 µm. (sometimes may appear as coccobacilli).  Are facultatively anaerobic, non-spore-forming rods; nonmotile or motile, some with polar flagella; catalase positive and oxidase negative; reduces nitrate to nitrite; and acid production from glucose fermentation.  Some possess pili or fimbriae; have simple nutritional requirements.  Organisms are found worldwide in soil, water and vegetation, are part of the normal intestinal flora of most animals, including humans. 2  Family Enterobacteriaceae -- (Cont’d)  These organisms are also frequently encountered in the clinical laboratory and are associated with infections of almost every area of the human body.  Some of the Enterobacteriaceae are strict pathogens, whereas others are opportunistic pathogens.  Enterobacter is named for the organism’s predominant natural habitat, the intestines of animals (from Greek enteron, meaning “intestine”).  With many harmless symbionts, includes more familiar pathogens, such as Salmonella, Escherichia coli, Yersinia pestis, Klebsiella, and Shigella.  Pathogenic Enterobacter can cause: eye and skin infections, meningitis, bacteremia, pneumonia, and urinary tract infections, etc..  Mostly, illness is associated with exposure to organisms in nosocomial settings, e.g. hospitals/nursing homes.  Identification of lactose-fermenting Gram (-) rods of the family Enterobacteriaceae (bacteria commonly referred to as coliforms) in water, used to determine if water is fecally contaminated and, therefore, may contain disease-causing pathogens transmitted by the fecal-oral route. 3  Family Enterobacteriaceae -- (Cont’d) Humans may develop enteric infections from a variety of situations: 1. Infections may be associated with lapses in personal hygiene through fecal-oral route. 2. Through poor sanitation in underdeveloped countries. 3. Colonization of the skin and respiratory tract of hospitalized patients Humans may acquire these bacteria thru: 1. Ingestion of contaminated food or water. 2. Nosocomially, through contact with patients or health care personnel or contaminated medical Instruments. 3. Endogenously, through their own normal flora. Common types of infections attributed to the Enterobacteriaceae, include: 1. Urinary tract infection 2. Bacteremia 3. Gastroenteritis  These bacteria are also known to cause disease in poultry, livestock, fish and vegetable crops, in addition to being significant human pathogens. 4 Four (4) Major Features of Enterobacteriaceae: 1. All ferment glucose – often with gas formation; they do not oxidize glucose. * 2. All reduce nitrates to nitrites except, Erwinia and Pantoea agglomirans. (Slide # 6) 3. All are Cytochrome oxidase negative except, Plesiomonas (Slide # 6)  Oxidase reaction is especially important in making a rapid distinction between the Enterobacteriaceae and the majority of the Gram (-) Non-fermenters (Pseudomonas, Burkholderia, Acinetobater, Moraxella, Eikenella…..) 4. All, except Klebsiella, Shigella and Yersinia are motile. 5 Members of the Family Enterobacteriaceae belong to one of two Major Groups: I Species that either commonly colonize the human GIT or are most notably associated with human infections. (Primarily intestinal pathogens) a. Salmonella b. Shigella c. Yersinia II Genera that may colonize humans but are rarely associated with human infections or are most commonly recognized as environmental inhabitants or colonizers of other animals (cause opportunistic infections in man) a. Escherichia coli* e. Hafnia j. Edwardsiella b. Klebsiella* g. Proteus * k. Erwinia c. Enterobacter h. Morganella l. Pectobacterium d. Serratia i. Providencia m. Citrobacter  New genera and biotypes: a Budivicia d Ewingella g Leminorella j Rahnella b Buttiauxella e Kluyvera h Moellerella k Tatumella c Cedecea f Koserella i Obesumbacteri l Xenorhabdus 7  …Family Enterobacteriaceae belonging to one of two Major Groups: Cont’d.:  Cited bacteria cause human diseases, including 30-35% of all septicemias, more than 70% of UTI and many intestinal infections. a. Salmonella, Shigella and Yersinia are always associated with disease in humans. b. E. coli, Klebsiella & Proteus are members of the normal commensal flora that cause opportunistic infections.  Infections caused by the Enterobacteriaceae can originate from: a) an animal reservoir (most Salmonella & Yersinia species infection) b) a human carrier (Shigella & Salmonella) c) through endogenous spread of the organism in a susceptible patient (E. coli - through one’s own normal flora) and can involve virtually all body sites.  For normal human colonizers (microbial flora) infection results from a) a patient’s own bacterial strain/ endogenous strain establish infection in a normally sterile body site (endogenous transmission) b) Passed from one patient to another, such as among debilitated, hospitalized patients (nosocomially acquired) 8  …Family Enterobacteriaceae belonging to one of two Major Groups: Cont’d.:  A number of Enterobacteriaceae harbor chromosomal elements that code for the production of bactericidal substances known as Colicins or Bacteriocins. Colicins - high molecular weight bactericidal protein (pore-forming toxins) that are produced by certain strains of bacteria that is active against some other strains of the same or closely related species.  It attacks different molecular sites such as DNA, RNA or protein synthesis or ATP formation. They do not attack indiscriminately but only attack certain susceptible strains.  The selectivity in action of Colicins has been used as an epidemiologic tool known as Colicin Typing.  Its production is controlled by plasmids; bacteriocin-producing strains are resistant to their own bacteriocin, thus bacteriocins can be used for “typing” of organisms. Physiology & Structure  Moderately sized - 0.3 to 1.0 µm x 1.0-6.0 µm; Gram (-) bacilli with rounded ends (sometimes may appear as coccobacilli); either motile or nonmotile - motile with peritrichous flagella, some with polar flagella; nonspore-forming.  Facultative anaerobe (grow rapidly aerobically and anaerobically on non- 9  Fimbriae are Bristle- like, short fibers occurring on the bacterial surface of both gram (+) and gram (-) Fimbriae help bacteria to attach to animals' skin or each other. The attachment occurs through adhesins produced by fimbriae. 10 Colonial Characteristics:  Morphological characteristics of culture on different selective media were used to identify members of the Family Enterobacteriaceae. E.g., Hektoen Enteric Agar (HE), Xylose Lysine Desoxycholate (XLD) A. The ability to ferment lactose were used to differentiate lactose fermenters from strains that do not ferment lactose.  Fermentation is indicated by a color change in the medium which results from a drop in pH, detected by a pH indicator incorporated in the medium.  Non-fermenting species are differentiated by lack of color change and colonies retain the original color of the medium → Non-lactose fermenter – colorless colonies Rapid Lactose fermenters: a Escherichia coli c. Enterobacter cloacae. b Enterobacter aerogenes (Genus changed d. Klebsiella. to Pantoea)  Slow Lactose Fermenter: a Edwardsiella c. Citrobacter e. Providencia. 11  Colonial Characteristics: (Cont’d.) B Resistance to Bile salts in some selective media.  Shigella & Salmonella - resist Bile salt Other species / Commensals - do not resist Bile salts & bacteriostatic agents C Presence of a prominent capsule. Klebsiella - capsulated Other members - surrounded by a loose-fitting , diffusible slime layer  Members do not liquify gelatin; gas and H2S production is variable with each species.  They are easily destroyed by heat and common germicides and disinfectants, such as - phenol, Formaldehyde, Halogen compounds & Glutaraldehyde  Relatively sensitive to drying; 39-59% G + C DNA content  The implementation of the “Matrix-assisted Laser Desorption Ionization 12  There are four stages in mass spectrometry – ionization, acceleration, deflection, and detection.  MALDI TOF/TOF mass spectrometers are used to reveal amino acid sequence of peptides using post-source decay or high energy collision-induced dissociation (further use see mass spectrometry). Besides proteins, MALDI-TOF has also been applied to study lipids.  It is used to analyze extremely large molecules. This technique directly ionizes and vaporizes the analyte from the condensed phase.  It is a form of mass spectrometry that is used to determine which elements or molecules are present within a given sample. TOF mass spectrometry relies on the relationship between a particle's mass and the speed at which it will move through the spectrometer.  In the case of MALDI-TOF, the analyzer separates molecules based on the time it takes each of them to fly through the time-of-flight tube or “drift” region to the detector. It 13 is this travel through the drift region that separates the molecules. Antigenic Structures  Enterobacteriaceae have a complex antigenic structure. They are classified by a) More than 150 different heat-stable somatic antigens b) more than 100 heat-labile capsular antigens c) More than 50 Flagellar antigens  The Kauffman-White Classification is used to classify Salmonella based on O and H antigens. More than 2000 serovars (serotypes) of Salmonella exist based on typing of O & H antigens. (Next slide)  The heat-stable LPS is the major cell wall antigen and consists of 3 components: 1. Somatic antigen (O Ag): A lipopolysaccharide (LPS), the external part of the cell wall and is the major cell wall antigen of gram (-) Enterics and is composed of -- a. O polysaccharide - antigenically variable b. Core polysaccharide - common to all Enterobacteriaceae c. Lipid A - an endotoxin - Antibodies to O antigen are predominantly IgM which is resistant to heat/alcohol. An organism may carry several O antigens. - O antigens may be associated with specific human diseases. e.g. Specific O antigen of E coli in diarrhea and UTI - LPS is also referred to as endotoxin which is common to all gram (-) bacteria - It can be detected by agglutination with specific antisera. 14  Antigenic Structures (Cont’d.) Kauffman-White Classification  This classification has been used to classify Salmonella based on the O and H antigens.  More than 2000 serovars (serotypes) of Salmonella exist based on typing for the O and H antigens.  For epidemiologic purposes, Salmonella isolates are tested with polyvalent O antisera. If the isolate is positive with polyvalent antisera, specific monovalent antigen testing is done. Once the serogroup has been determined, the particular serotype should be identified.  When agglutination does not occur with the polyvalent antisera, a suspension of the organism should be boiled for 15 minutes and then retested. (Heating destroys the capsular antigen, which may block the reactivity of the O antigen.)  Salmonella may also agglutinate with the V antisera before, but not after boiling. 15  Isolates are usually serotyped in reference laboratories because of the procedure’s complexity.  Antigenic Structures (Cont’d.) 2. Capsular Antigen (K Ag)  It is either a protein or polysaccharide.  Organisms with specific K antigen have been associated with increased virulence. The V antigen* of Salmonella typhi is the best known.  It is heat-labile and covers the O antigen, thus inhibiting agglutination with type-specific O antisera. (Thus, after determination of the K antigen, the organism must be boiled for 30 minutes and then retested to detect the O antigen.)  Klebsiella, Salmonella and E. coli possess K antigens. Example: Escherichia coli strains producing K1 antigens are prominent in neonatal meningitis.; Klebsiella pneumoniae also possess K antigen – responsible for Respiratory Tract Infections (Serotypes 1 & 2) and Urinary Tract infection (Serotypes 8, 9, 10 and 24). 3. Flagellar Antigen (H Ag); (H from Hauch – the German term for “breath” or “mist”)  Protein; heat-labile (can be removed/denatured by heat & alcohol; preserved by Formalin)  Present only in motile members of the Family Enterobacteriaceae.  This can be absent in a cell or undergo antigenic variation which is thought to be due to the difference in amino acid sequence of the particular flagella type.  H antigen can agglutinate with Anti-H antibody which is mainly IgG. H antigen on the16  Antigenic Structures (Cont’d.)  Important: Take note that there is overlapping of antigenic structures between Enterobacteriaceae and other bacteria: Examples: a) Most Enterobacteriaceae share the 014 antigen of E. coli. b) Type 2 capsular polysaccharide of type 2 Pneumococci is similar to type 2 capsular polysaccharide of Klebsiella. c) Some K antigens cross-react with capsular polysaccharide of Hemophilus influenzae or N. meningitides. d) E. coli 075:k100:H5 can induce antibodies that react with Hemophilus influenzae type B. Common virulence factors associated with Enterobacteriaceae: a) Endotoxin  A virulence factor shared among all aerobic and some anaerobic gram (-) bacilli.  The activity of the toxin depends on the Lipid A component of LPS, which is released during cell lysis.  Many systemic manifestations of gram (-) bacterial infections are initiated by endotoxin, including: a. Complement d Thrombocytopenia g. Shock activation. 17  Antigenic Structures (Cont’d.) b) Capsule  Encapsulated Enterobacteriaceae are protected from phagocytosis by the hydrophilic capsular antigens which repel the hydrophobic phagocytic cell surface.  These antigens interfere with the binding of antibodies to the bacteria and are poor immunogens or activators of complement.  The protective role of the capsule is diminished if specific anti-capsular antibodies develop. c) Antigenic Phase Variation  The expression of the capsular K and the flagellar H antigens is under the genetic control of the organism and each of these antigens can be alternately expressed or NOT expressed (Phase variation), which can protect the bacteria from antibody-mediated cell lysis. d) Sequestration of Growth Factors  In contrast with the nutrients provided to the organism in enriched media, the bacteria must become nutritional scavengers when growing in-vivo.  Iron is an important growth factor required by bacteria, but it is bound in heme proteins (e.g., hemoglobin, myoglobin) or in iron-chelating proteins (e.g., transferrin, lactoferrin). The bacteria counteract this by producing their 18  Antigenic Structures (Cont’d.) e) Resistance to Serum killing  Whereas many bacteria can be rapidly cleared from blood, virulent organisms capable of producing systemic infections are frequently resistant to serum killing.  Bacterial capsule can protect the organism from serum killing. Other poorly defined factors prevent the binding of Complement components to the bacteria & subsequent complement-mediated clearance. f) Antimicrobial Resistance  As rapidly as new antibiotics are introduced, organisms can develop resistance to them.  This resistance can be encoded on transferable plasmids & exchanged among species, genera & even families of bacteria. Determinants of Pathogenicity -- 1. Endotoxin  The lipopolysaccharide portion of the cell wall.  Toxicity is associated with the Lipid A component of the lipopolysaccharide; can be extracted by - a Phenol-water b. Trichloroacetic c. EDTA 19  Antigenic Structures (Cont’d.) 2. Enterotoxins  Bacterial substances that exert their toxic effect in the small intestine, causing a transduction of fluid into the ileum → Diarrhea  Found a. EPEC c. Shigella e. Salmonella species in: dsenteriae b. Klebsiella d. Shigella pneumoniae flexneri 3. The ability to adhere, invade and colonize tissues 20 Laboratory Diagnosis -- Specimen Collection & Transport – Since most often these bacterial species are isolated with other organisms including the fastidious pathogens, one must provide appropriate collection and transport media to ensure isolation of both opportunistic and fastidious pathogens. Isolation & Identification  Members of the Family Enterobacteriaceae are routinely isolated from stool cultures therefore, complete identification should be directed only toward true intestinal pathogens.  Generally, enteric opportunistic organisms isolated from sites that are normally sterile are highly significant.  The media for the isolation of Enterobacteriaceae depends on the source of specimen. A combination of MacConkey and BAP is usually acceptable21for stool specimens.  Laboratory Diagnosis – (Cont’d.) I. Direct Microscopic Examination  Gram-stained smears of fecal specimens for presumptive identification of Enterics are of less value since all are indistinguishable from one another; all are gram negative bacilli, short, small to moderately- sized with rounded ends.  Smears prepared from CSF, blood and other body fluids or exudates from uncontaminated sites may be examined for the presence of gram (-) bacteria.  Smears may aid the clinician in the preliminary diagnosis of the infection and appropriate therapy can be instituted immediately.  Direct smear examination of stools may reveal inflammatory cells which is helpful in determining whether the gastrointestinal disease is toxin-mediated or an invasive process. 22  Laboratory Diagnosis – (Cont’d.) II. Culture 3 General types of media are inoculated: 1. Non-selective media (BAP/ Chocolate Agar) - Sometimes omitted because colonies are generally the same for all members. 2. Selective & Differential Media 3. Enrichment Broth (Selenite F or GN Broth) (Slide # 24, 25) - some labs omit this medium  Other than stool cultures, a combination of McConkey and BAP is usually accepted. Suggested guide for the isolation of Enterics: 1. Inoculate specimen directly to McConkey (Slide #25) or EMB Agar (Differential media) for primary isolation of all species of enteric Gram(-) bacilli. (Slide # 26) 2. Directly inoculate either Hektoen Enteric Agar (HE) (Slide # 27) or Xylose Lysine Deoxycholate (XLD) Agar( Slide # 28) plates for the selective screening of pathogenic organisms - Salmonella or Shigella 23 Selenite F Broth is a selective enrichment medium that aids in the isolation of Salmonella species from various specimens. It inhibits the growth of coliforms and other competing bacteria while promoting the growth of Salmonella, allowing for their subsequent isolation on selective agar media. Growth of organisms in Selenite F Broth is indicated by turbidity in the medium. 24 GN Enrichment Broth  Laboratory Diagnosis – (Cont’d.) 3. Enrich a small portion of the specimen by heavily inoculating either Selenite F or Gram Negative Broth (GN) Broth.  If Selenite - subculture to HE agar within 8-12 hours; for the isolation of Salmonella from feces, urine or sewage that have heavy concentration of mixed bacteria.  Inhibitory to E. coli and other coliforms including Shigella If GN Broth - subculture within 4 hours; for recovery of Salmonella and Shigella when they are in small numbers in fecal specimens. 4. Incubate all plates at 350C for 24-48 hours. Pick suspicious colonies for definitive biochemical and serologic testing. 26   A selective and differential medium containing lactose, bile salts, neutral red, and crystal violet.  Bile salts & crystal violet inhibit growth of Gram-pos Lactose fermenters = red growth Lactose nonfermenters = colorless colonies Enterobacteriaceae, Salmonella typhi, Shigella dysenteriae - lactose negative  E.coli, Enterobacter, Klebsiella - lactose positive  developed by Alfred Theodore MacConkey in the McConkey Agar 20th century. 27 Escherichia coli colonies on EMB agar (Note: greenish metallic sheen) Salmonella Proteus E. coli Hektoen Enteric Agar  The figure shows three XLD agar plates. Bacteria were not cultivated on the plate in image A. On the plate in image B, Salmonella sp. has been cultivated. Note that the colonies are pink with a black centre. On plate C, E. coli has been cultivated. Note the colour change of the upper part of the plate, where E. coli (lactose fermenter) colonies appear. X ylose lysine deoxycholate agar (XLD agar) is a selective growth medium, which is used for isolation of Salmonella spp. and Shigella spp. from clinical samples and from food. The pH of XLD agar is 7.4 and it contains the pH indicator methyl red, which gives a red or pink colour. XYLOSE LYSINE DESOXYCHOLATE AGAR 30 The members of the Enterobacteriaceae most often is associated with diarrhea are Salmonella, Shigella, specific diarrheagenic E. coli and Yersinia enterolitica. MacConkey/ EMB (Differential media) Stool Hektoen Enteric Specimen agar/XLD (Selective agar) Selenite or Tetrathionate Broth or GN Broth (Enrichment Broth)  Enrichment Broths hold the normal flora coliforms, such as E. coli, in a lag period of growth while permitting the pathogenic Salmonella and Shigella to enter a logarithmic phase of growth. These broths contain a high concentration of bile salts which inhibit the multiplication of the normal flora coliforms. Enrichment broths can be re-incubated for as 31 long as 24 hours if needed.  Some important observations on BAP which are helpful in identification. (Some laboratories substitute Mac with BAP) Examples:  Members of the genus Proteus produce characteristic swarming motility on BAP (Slide # 33)  Klebsiella pneumoniae which produces capsule, appears as mucoid colonies. (Slide #34)  Organisms that are H2S positive often produce subsurface greening of BAP (Slide # 35) 32  33 Proteus organism swarming on agar Klebsiella pneumoniae - 34 mucoid colonies H2S positive - subsurface greening of Blood Agar (Alpha hemolysis) 35 Laboratory Diagnosis Laboratory Diagnosis --II. Culture (Cont’d.) --  The normal flora coliforms appear as lactose (+) colonies on differential and selective plates. (colored colonies) - Lactose Fermenter  Pathogenic Salmonella and Shigella appear as lactose (-) colonies. (colorless colonies – Non-lactose fermenter)  Sorbitol MacConkey is inoculated in suspected cases of E. coli O157:H7 (verocytotoxic E. coli); if Enterotoxigenic E. coli is suspected, BAP is inoculated.  CIN plate (Cefsulodin-Irgasan-Novobiocin) is inoculated if Yersinia interocolitica is suspected. CIN agar is incubated at room temperature (22oC – 26oC) for 24 hours to enhance recovery.  A highly selective agar plate, such as Brilliant Green Agar or Bismuth Sulfite Agar can be included for specific Salmonella, such as Salmonella typhi. For Urine Culture:  MacConkey & BAP are a suitable combination.  A colony count should be performed on all urine specimens. - If a 1µl (0.001 ml) calibrated loop is used to inoculate the plate, the number of colonies counted must be multiplied by 1000 to gain the colony count per ml 36 of urine. - For catheterized or suprapubic samples, 10µl loop may be used. In this case, the Biochemical Tests  After observation of growth on MacConkey, the oxidase test is performed. Oxidase Filter Paper Test Spot Test Test Tube Direct Plate Method Method  Any Oxidase (-) organism isolated from MacConkey can be suspected of being a member of the Family Enterobacteriaceae 37 NOTE: PROCEDURE OF THE OXIDASE TEST IS FOUND AT THE END OF THESE NOTES. Carbohydrate Fermentation Tests 1. For Lactose Fermentation & Utilization of a. TSI Carbohydrates: b. ONPG Test 2. Test for Glucose Metabolism & its Metabolic a. Methyl Red Test Products: b. Voges-Proskauer Test 3. Other Tests: a. Indole Test g. Potassium Cyanide b. Citrate Utilization Test h. Motility, Indole, Ornithine Test (MOI), (KCN) c. Urease Production Test i. Lysine Iron Agar Test (LIA) d. Motility Test j. Nitrate Reduction Test e. Malonate test k. Decarboxylase Test f. Phenylalanine Deaminase Test Triple Sugar Iron Agar Reaction (Slide # 40)  Triple Sugar Iron Agar (& Kligler Iron Agar (KIA) Illustration – (Slide # 40, 41)) contain a limiting amount of glucose and a tenfold greater lactose concentration. Enterobacteriaceae and other glucose fermenters first begin to metabolize glucose, as glucose-utilizing enzymes are present consecutively and the bacteria can gain the 38... Triple Sugar Iron Agar Reaction (Cont’d.)  Glucose-Fermenting Bacteria Glucose utilization occurs both aerobically on the slant where the oxygen is available as a terminal electron acceptor, and in the butt where conditions are anaerobic. Once the organism has reduced all of the available glucose to pyruvate, it will further metabolize pyruvate via the Krebs cycle (on the slant) to produce acid end-products. The acid in the medium causes the pH indicator, phenol red to assume a yellow color. Thus, after 6 hours of incubation, both the slant and butt of the TSIA or KIA will appear yellow. A/A Reaction  Lactose-Fermenting Bacteria After depletion of the limited glucose, an organism that is able to do so will begin to utilize lactose or sucrose. Since there are 10x as much lactose (& sucrose in TSIA) as glucose in the agar, the organism will have enough substrate to continue making acid end-products. The slant and butt will remain yellow after 24-48 hours incubation. This reaction is called acid over acid (A/A) and the organism is identified as lactose fermenter. The production of gas will cause the medium to break up or to be pushed up the tube so that a gas-producing lactose fermenter will give an A/A plus gas formation.  Non-Lactose Fermenting Bacteria If the organism cannot use the lactose in the medium, it must produce energy in a less efficient way by using the proteins and amino acids in the medium as nutrient sources. Protein metabolism occurs primarily on the surface of the slant where oxygen is plentiful. The by-products of peptone breakdown (such as ammonia) are alkaline and cause the phenol red indicator to revert back to its original red color. After 24-48 hours incubation of a non- 39 Triple Sugar Iron Agar Reaction A/A, Gas K/A K/A, K/K Legend: A - Acid; K- H2 S alkaline Kligler’s iron agar (KIA) tubes with several reaction patterns A: Acid/Acid, Gas; B: Acid/Acid, No gas; C: Alkaline/Acid; D: Alkaline /Acid, H2S+; E: Alkaline/Alkaline... Triple Sugar Iron Agar Reaction (Cont’d.)  Rapid lactose-fermenter: a. Escherichia coli c. Klebsiella b. Enterobacter  Non-lactose Fermenter: a. Salmonella c. Proteus b. Shigella  Slow Lactose Fermenter: a. Citrobacter d. Serratia b. Hafnia e. Providencia c. Edwardsiella f. Erwinia 42 Selective Differential Media 1. McConkey Agar (Slide #27)  Bile Salt & Crystal violet inhibit the growth of gram (+) bacteria and some fastidious gram (-) bacteria.  Lactose is the sole carbohydrate present in the medium  LF - Red colonies (varying shades of red) (Red below pH 6.8) NLF - Colorless or transparent 2. Eosin Methylene Blue (EMB) (Slide # 28)  Inhibits gram (+) bacteria  LF - Green-Black with metallic sheen (Slide # 28) NLF - Transparent colonies / colorless 43 3. Desoxycholate – Citrate Agar (DCA) (Slide # 45)  Contains 3x the concentration of Bile salts as the McConkey agar; sodium and Ferric Citrate retards the growth of E. coli.  Neutral Red - pH indicator (red below pH 6.8)  LF - deep red ; NLF - colorless colonies  Desoxycholate Citrate Agar is a moderately selective and differential plating medium used for isolating enteric bacilli, particularly Salmonella and many Shigella species. Desoxycholate Citrate Agar is a modification of Desoxycholate Agar formulated by Leifson. 4. Endo Agar (Slide # 47)  It is used to grow coliforms from water, milk and other foodstuffs.  Sodium sulfite & Basic fuchsin - inhibit the growth of gram (+) bacteria  LF - pink to rose red; NLF - almost colorless to faint pink 45  Salmonella typhimurium on DCA Agar SALMONELLA TYPHI, S. PARATYPHI AND SHIGELLA TYPES YIELD COLOURLESS (LACTOSE-NEGATIVE) COLONIES WHILE LACTOSE POSITIVE ORGANISMS LIKE E. COLI ARE PINK TO RED. THIS IS DUE TO THE NEUTRAL RED IN WHICH PRESENCE LACTOSE FERMENTING BACTERIA FORM RED COLONIES WHILE 46 NON FERMENTING WILL APPEAR CLEAR, WITH OR WITHOUT BLACK CENTERS. LACTOSE FERMENTING COLONIES HAVE A DESOXYCHOLATE PRECIPITATION ZONE AROUND THEM. Shigella sonnei on DCA Escherichia coli grows red colonies Escherichia coli (lactose positive) accompanied by red staining of the and Salmonella (lactose negative) on Endo agar. surrounding medium (sodium Cultivation 24 hours, 37°C in an aerobic sulphite/fuchsin reaction). atmosphere. Endo Agar Highly Selective Culture Media 1. Hektoen Enteric Agar (HE Agar) (Slide # 48, 49, 50)  A direct plating medium for fecal specimens to increase the yield of Salmonella and Shigella from the heavy numbers of normal flora.  High salt concentration inhibits growth of all gram (+) bacteria and retards the growth of many strains of coliforms.  Rapid Lactose Fermenters - moderately inhibited and produce bright Orange to Salmon pink colonies  Salmonella colonies - Blue green with black centers from H2S gas  Shigella - more green than Salmonella, with the color fading to the periphery of the colony.  Proteus - somewhat inhibited; colonies are small, transparent and more glistening or watery in appearance.  Hektoen enteric agar (HEK, HE or HEA) is a selective and differential agar primarily used to recover Salmonella and Shigella from patient Shigella flexneri & Salmonella typhimurium on Hektoen agar 49 Salmone l la Proteus E. coli Hektoen Enteric Agar  Highly Selective Culture Media (Cont’d.) 2. Xylose Lysine Desoxycholate Agar (XLD) (Slide # 53)  Less inhibitory to growth of coliforms than HE and is designed to detect Shigella in feces after enrichment in GN broth.  E. coli, Klebsiella & Proteus – Enterobacter species - Bright Yellow colonies.  Salmonella & Arizona - Red colonies, most with black centers from H2S gas  Shigella, Providencia and many Proteus species - do not utilize the carbohydrate → Translucent colonies  Citrobacter - Yellow with black center  Salmonella - Red with black centers 3. Salmonella - Shigella Agar (SSA) (Slide # 54)  Highly selective medium formulated to inhibit the growth of most coliforms and permit the growth of Salmonella and Shigella from environmental and clinical specimens.  LF - colored Red  Salmonella - colorless colonies with black center  Shigella - colorless colonies with NO blackening at the center.  Motile strains of Proteus do not swarm. Xylose Lysine Desoxycholate 53 Agar Salmonella - Shigella Agar (SSA) Salmonella will not ferment lactose, but produce hydrogen sulfide (H2S) gas. The resulting bacterial colonies will appear colorless with black centers. Shigella do not ferment lactose or produce hydrogen sulfide gas, so the resulting colonies will be colorless. Coliform bacteria such as E. coli will ferment the lactose in the media, resulting in bacterial growth with a pink color. They do not produce any hydrogen sulfide. Enterobacter and Klebsiella app ears larger than E. coli, mucoid, pale, opaque cream to pink. 55 Colony morphology of E.coli, Salmonella and Shigella in Salmonella-Shigella Agar 56  Highly Selective Culture Media (Cont’d.) 2. Bismuth Sulfite Agar  Highly selective for Salmonella typhi  Bismuth and Brilliant green are inhibitory to gram (+) bacteria and most species of gram (-) bacteria  Most lactose fermenters and Shigella are inhibited completely.  Salmonella typhi - black with metallic sheen  Salmonella interitides - black but without metallic sheen.  Salmonella gallinarium, cholerasuis & paratyphi - greenish colonies  Medium must be used on the day it was prepared. Bismuth sulfite agar (BS) is a selective as well as a differential medium for isolation and presumptive identification of Salmonella spp, especially Salmonella Typhi. Salmonella spp are the causative agent of various diseases like gastroenteritis, sepsis, and enteric fever. Salmonella can be isolated from a wide range of clinical, food, sewage, and other environmental samples. Bismuth sulfite agar is a modification of the original Wilson and Blair Medium 58 ORTHONITROPHENYL GALACTOSIDE (ONPG) TEST  Orthonitrophenyl galactoside (ONPG) is structurally similar to lactose, except that orthonitrophenyl has been substituted for glucose. Upon hydrolysis, through the action of the enzyme, beta galactoside, ONPG cleaves into two residues, galactose and orthonitrophenol. ONPG is a colorless compound; orthonitrophenol is yellow, providing visual evidence of hydrolysis. Principle: Lactose-fermenting bacteria possess both lactose permease and beta galactosidase, two enzymes required for the production of acid in the lactose fermentation test. The permease is required for the lactose molecule to penetrate the bacterial cell where the beta galactosidase can cleave the galactoside bond, producing glucose and galactose. Non-lactose fermenting bacteria are devoid of both enzymes and are incapable of producing acid from lactose. Some bacterial species appear to be non-lactose fermenters because they lack the permease but do possess β- galactosidase and give a positive ONPG test. So-called lactose fermenters may be delayed in their production of acid from lactose because of sluggish permease activity. In these instances, a positive ONPG test may provide a rapid identification of delayed lactose fermentation.  ORTHONITROPHENYL GALACTOSIDE (ONPG) TEST (Cont’d.) Procedure: Growth from KIA/TSIA is emulsified in 5 ml. NSS to produce a heavy suspension. A drop of Toluene is added to extract the enzyme from the bacterial cells. Equal amount of ONPG solution is added and placed in a waterbath at 37oC. Result: A distinct yellow color develops within 5-10 minutes. Most tests are positive within 1 hour. However, reactions should not be interpreted as negative before 24 hours. Interpretation: A positive result indicates that the organism has produced orthonitrophenol from the ONPG substrate through the action of β- galactosidase. INDOLE TEST Indole, a benzzyl pyrrole, is one of the metabolic degradation products of the amino acid, tryptophan. Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid and ammonia. Principle: Indole test is based on the formation of a red color complex when indole reacts with the aldehyde group of p-dimethylaminobenzaldehyde (the active chemical in Kovac’s and Ehrlich’s regent.) Procedure: 1. Inoculate Tryptophan Broth with test organism. 2. Incubate at 37oC for 18-24 hours. 3. Add 15 drops of Kovak’s reagent down the inner wall of the tube. (If Ehrlich’s reagent is used, add 1 ml of Chloroform first before adding Ehrlich’s reagent) Interpretation: Positive Test (+): The development of a bright fuschia red at the interface of the reagent and the broth (or the chloroform layer) within Indole Test METHYL RED TEST Methyl red is a pH indicator with a range between 6.0 (yellow) and 4.4 (red).The pH at which Methyl red detects acid is considerably lower than the pH for other indicators used in bacteriologic culture media. Thus, in order to produce a color change, the test organism must provide large quantities of acid from the carbohydrate substrate being used. Principle Methyl red is a quantitative test for acid production, requiring positive organisms to produce strong acids (lactic acid, formic acid) from glucose through the mixed acid fermentation pathway. Since many species of the Enterobacteriaceae may produce sufficient quantities of strong acids that can be detected by the Methyl red indicator during the initial phase of the incubation, only organisms that can maintain this low pH after prolonged incubation (48- 72 hours), overcoming the pH buffering system of the medium, can be called Methyl red positive Media & Reagents: MR-VP Broth (Clark Lubes Medium); Methyl Red indicator Procedure: 1. Inoculate MR-VP broth with test organism 2. Incubate at 370C for 48-72 hours. 3. Add 5 drops of Methyl red directly to the broth. Interpretation: Positive Test (+): the development of a stable red color in the surface of the medium Methyl Red Test VOGES – PROSKAUER TEST Principle The test is based on the conversion of acetoin from glucose to diacetyl through the action of Potassium hydroxide (40%) and atmospheric oxygen. Diacetyl is converted unto a red complex under the catalytic action of alpha-naphthol and creatine. Media & Reagents: MR-VP Broth Alpha naphthol (5%) Potassium hydroxide (40%) Procedure: 1. Inoculate MR-VP Broth with test 3. Add 40% KOH (.2 ml) organism. 4. Shake tube gently and stand for 10-15 2. Incubate for 24 hours at 370C. minutes. 3. Add 5% α – naphthol (.6 ml) Positive Test (+): Development of a red color after 15 minutes. (This indicates the presence of diacetyl, the oxidation product of acetoin) Positive organisms: Klebsiella- Enterobacter-Hafnia-Serratia CITRATE UTILIZATION TEST Principle The utilization of Citrate by a test organism is detected in a Citrate Medium by the production of alkaline by-products. Bacteria that can utilize Citrate can also extract Nitrogen from the Ammonium salt, with the production of Ammonia, leading to alkalinization of the medium from the conversion of Ammonia to Ammonium hydroxide (NH4OH). Bromthymol Blue, colored yellow below pH 6.0 and blue above H 7.6, is the indicator. Media & Reagent Simmon’s Citrate Slant Procedure 1. Streak the test organism into Simmon’s Citrate slant 2. Incubate at 370C for 24-48 hours. Positive Test (+): Development of a deep blue color within 24-48 hours (indicating that the organism has been able to utilize citrate in the medium, with the production of alkaline products.) Reactions of the Enterobacteriaceae in triple sugar iron agar and lysine decarboxylase broth. R = red (alkaline), Y = yellow (acid), H2S+ = hydrogen sulphide produced, H2S− = hydrogen sulphide not produced.  TSI Reaction of the Most common Members of the Family Enterobcteriacease TSI Reaction of the Most common Members of the Family Enterobcteriacease (cont’d) UREASE PRODUCTION Urea is a diamide of Carbonic acid. All amides are easily hydrolyzed with the release of ammonia and carbon dioxide. Principle Urease is an enzyme possessed by many species of microorganisms that can hydrolyze Urea. The ammonia reacts in solution to form Ammonium carbonate, resulting in alkalinization and increase in the pH of the medium. Procedure Stuart’s Urea Broth is inoculated with a loopful of a pure culture of the test organism and is incubated at 35oC for 18-24 hours. Result Red color throughout the medium indicated alkalinization and Urea hydrolysis.  Organisms that hydrolyze urea rapidly may produce positive reactions within 1-2 hours; less active species may require 3 or more days. Urease Test MOTILITY TEST TUBE MOTILITY TEST Procedure:  Using the sterile inoculating needle, remove suspicious colony of an 18-24 hour culture. Positiv Negati Inoculate motility agar medium (Peptone e ve water with 0.2% New Zealand agar, semisolid agar) carefully stabbing the needle 3-4 cm into the medium then withdrawing the needle so that a line of inoculum can be seen.  Incubate the tube aerobically at 35-37°C for 18-24 hour.  Tetrazolium salts may be added to motility media to make them easier to read.  The salt is colorless, but as the organisms grow, the dye gets incorporated into the bacterial cells, where it is reduced to an insoluble red pigment. Control: Pseudomonas aeruginosa or equivalent is  Non-motile organisms, such as B. non-motile. Motile: organisms will spread out into the medium from the site of 77 inoculation, diffuse zone Non-motile: organisms remain at the site of inoculation Motility Test 1 2 3  Motility Test WET MOUNT PROCEDURE Procedure : 1. Make suspension of a colony of test organisms in distilled water on a glass slide. Alternatively, a loop of medium from a fresh broth culture can be used. Put a cover glass on it. Examine under the microscope using hypochlorite solution as it contains live organisms.  Hanging Drop Technique HANGING DROP PREPARATION STEP 1 Concavity Slide HANGING DROP PREPARATION STEP 2 81  HANGING DROP PREPARATION STEP 3 HANGING DROP PREPARATION STEP 4 82  Examine the preparation under low power objective lens, with reduced light.  You can adjust the light of the microscope by using the diaphragm lever to maximize the visibility of the cells & to develop better contrast.  Brownian movements and passive drifting off of all the organisms (motile & non-motile) are usually visible in the Hanging drop slides but on precise and accurate examination of the Hanging drop preparation, you will be able to differentiate the Brownian movement with true motility of bacterial cell. Brownian Movement - These are the oscillatory movement at a nearly fixed point. Don’t misunderstand the passive drifting and Brownian motion of the bacterial cell with motility. Observe carefully ……. 83  Hanging Drop Method : Clean a cover slip. Apply Vaseline at its 4 corners. Put a drop of distilled water in the center and emulsify a colony of test organisms. Put the glass slide gently on it and hold it up-side down. See under microscope with 10X and then 40X objective. Margins of drops are specifically seen. Motile organisms are clearly seen moving rapidly in the field. Non-motile organisms show to-and-fro Brownian motion, but these don’t move in relation to each other.  MOTILITY TEST Some Important Motile and Non-motile Bacteria 1. All enterobacteriaceae are motile, except Shigella species. 2. Klebsiella species are non-motile. 3. Pseudomonas species are motile with the help of a unipolar flagellum. 4. Vibrio colony has a shooting star motility. 5. Campylobacter has a darting motility. 6. Giardia lamblia has a falling leaf motility. 7. Listeria monocytogenes has a tumbling motility. MALONATE TEST Malonate test is a colorimetric test of the ability of bacteria to use malonate as a source of carbon, the endpoint of which is the production of alkaline metabolites that induce a color change. Principle An organism that simultaneously can utilize sodium malonate as its carbon source and ammonium sulfate as its nitrogen source produces an alkalinity due to the formation of sodium hydroxide. This results in an alkaline reaction which in a medium containing malonate, changes the indicator (bromothymol blue) from its original green color to light blue or Prussian blue. Organisms which cannot utilize malonate and ammonium sulfate and do not ferment dextrose produce no color change. Organisms which are malonate-negative but do ferment dextrose result in the development of a yellow color due to increased acidity in the medium. An inoculum from a pure culture is transferred aseptically to a sterile tube of malonate broth. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. Abundant growth and a change from green to blue in the medium indicates a positive test for growth using malonate. Hence, if a microbe can use malonate for carbon and energy, it will grow on malonate broth. The use of malonate leads to a rise in pH of the medium, and a pH indicator changes color. Media The medium used is malonate broth. Malonate Broth, prepared according to the formula  MALONATE TEST (Cont’d.) The pH indicator is bromthymol blue, which is green at neutral pH, yellow at acidic pH 7.6. Procedure 1. Using a light inoculum from an 18-24 hour pure culture, inoculate the tube containing malonate broth. 2. Incubate the tube with loosened caps at 35ºC in an aerobic atmosphere for 24-48 hours. 3. Observe for alkalization (blue color) at 24 and 48 hours. Results A positive malonate test is indicated by the development of a blue color in the medium. A negative malonate test is indicated by the media remaining green or turning yellow due to dextrose fermentation. Limitations It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification. Some malonate-positive organisms produce only a slight alkalinity which renders difficulty in interpretation.  PHENYLALANINE DEAMINASE TEST Principle The test depends upon the detection of Phenylpyruvic acid in the test medium after growth of the test organism. The test is positive if a green color develops upon addition of a solution of 10% Ferric chloride. Procedure Phenylalanine Agar (slant) is inoculated with the test organism. After incubation at 35oC for 18-24 hours, 4-5 drops of the Ferric chloride reagent are added directly to the surface of the agar. Interpretation The immediate appearance of an intense green color indicates the presence of phenylpyruvic acid and a positive test. 90 LITMUS MILK TEST Milk is an excellent medium for the growth of microorganisms because it contains the milk protein casein, the sugar lactose, vitamins, minerals and water. Litmus milk is a milk-based medium used to distinguish between different species of bacteria. The lactose (milk sugar), litmus (pH indicator), and casein (milk protein) contained within the medium can all be metabolized by different types of bacteria. The test differentiates microorganisms based on various metabolic reactions in litmus milk, including reduction, fermentation, clot formation, digestion, and the formation of gas. Principle The major milk substrates capable of transformation are the milk sugar lactose and the milk proteins casein, lactalbumin, and lactoglobulin. To distinguish among the metabolic changes produced in milk, a pH indicator, the oxidation reduction indicator litmus, is incorporated into the medium. Litmus milk then forms an excellent differential medium in which microorganisms can metabolize milk substrates depending on their enzymatic complement. A variety of different biochemical changes result. Fermentation of lactose is demonstrated when the litmus turns pink as a result of acid production. If sufficient acid is produced, casein in the milk is coagulated, solidifying the milk. With some organisms, the curd shrinks and whey  LITMUS MILK TEST (Cont.d) Media: Powdered skim milk (100 g), litmus (0.5 g), sodium sulphite (0.5 g), per 1000 mL, pH 6.8. Method 1. Inoculate with 4 drops of a 24-hour broth culture. 2. Incubate at 35°-37°C in ambient air. 3. Observe daily for seven days for alkaline reaction (litmus turns blue), indicator reduction, acid clot, acid reaction (litmus turns pink), rennet clot, and peptonization. Positive test Acid pH: pink to red color Alkaline pH: purplish- blue color Reduction: white Acid curd: hard curd with clear supernatant (whey) Digestion: Dissolution of clot with clear, grayish, watery fluid and a shrunken, insoluble pink clot Rennet curd: soft curd followed by peptonization (alkaline pH, supernatant brown) Gas production: bubbles in coagulated milk Expected Results  LITMUS MILK TEST (Cont.d) Uses 1. The litmus milk test differentiates members of the Enterobacteriacaeae from other gram-negative bacilli based on the enterics’ ability to reduce litmus. 2. It is commonly used to differentiate members within the genus Clostridium. 3. It mainly aids in the identification and differentiation of Enterococcus, and Lactic acid bacteria. The media may also be used to grow lactic acid bacteria Limitations 1. Litmus media reactions are not specific and you should do additional tests for definitive identification of microorganisms. 2. Litmus milk is a complex medium that can produce a diversity of results. Because of this, litmus milk can give quite unreliable results. 3. A clot formation is simply recorded as “clot” and cannot clearly differentiate between a clot and curd formation in this medium. 95 Principle of Phenylalanine Deaminase Test Some bacteria have the capacity to synthesize phenylalanine deaminase enzyme, which oxidatively deaminates (removes NH2) from the amino acid phenylalanine. Upon deamination of phenylalanine by the deaminase enzyme, phenyl pyruvic acid and ammonia are released. Thus released phenyl pyruvic acid reacts with the chelating agent ferric chloride in the reagent and results in the formation of light to deep-green colored complex, indicating a positive reaction. 96 MOTILITY – INDOLE - ORNITHINE (MIO) Principle MIO Medium is prepared to provide three differentiating tests in one culture tube: motility, indole production, and ornithine decarboxylation. A. MOTILITY: Organisms are stabbed into the semisolid medium using a straight wire. If the organisms are motile, they will migrate from the stab line by means of their flagella, producing turbidity or cloudiness throughout the medium. Non-motile organisms grow only along the stab line, leaving the surrounding medium clear. B. INDOLE: Tryptophan is an ingredient contained in the medium by the inclusion of peptones. If the organism possesses the enzyme tryptophanase, with the addition of Kovacs reagent, a red color will form at the top of the medium if indole is present. This reaction occurs as a result of the indole reacting with the aldehyde to yield a quinone or quinone-type structure, resulting in a red color in the alcohol layer. A negative test results in no color change. C. ORNITHINE: The medium also tests for the presence of the enzyme ornithine decarboxylase by including L-ornithine in the agar. If the organism possesses the enzyme, it will be activated in an acid environment created by the initial fermentation of glucose. Once the amino acid is decarboxylated, the Principle of Ornithine Decarboxylase Test An inoculum from a pure culture is transferred aseptically to a sterile tube of ornithine decarboxylase broth. The inoculated tube is incubated at 35-37 C for 24 hours and the preliminary results are determined. The microbe must first use the glucose present to cause the pH to drop. This is indicated by a change from purple to yellow. Once the medium has been acidified, the enzyme ornithine decarboxylase is activated. The culture is incubated an additional 24 hours at 35-37 C to allow the microbe to now use the ornithine. The final results are then obtained by observing the tube at 48 hours. Change back to purple from yellow indicates a positive test for ornithine decarboxylase. Failure to turn yellow at 24 hours or to revert back to purple at 48 hours indicates a negative result MOTILITY – INDOLE - ORNITHINE (MIO) (Cont’d.) Media Motility Indole Ornithine Medium: Yeast extract, Peptone, L- ornithine (.5%), Dextrose (.1%), Agar (.4%), Indicator: Bromcresol Purple, Final pH +/- 2 at 25oC.  For procedure and positive results for Motility and Indole, please refer to previous lessons)  Organisms ferment dextrose to form acid, which causes the pH indicator bromocresol purple to change from purple to yellow. NITRATE REDUCTION TEST Principle Organisms demonstrating nitrate reduction have the capability of extracting oxygen from nitrates to form nitrites and other reduction products. The presence of nitrites in the test medium is detected by the addition of Alpha-naphthylamine and Sulfanilic acid, with the formation of a red diazonium dye, p-sulfobenzene-azo-alpha- naphthylamine. Media & Reagents Nitrate Broth (Nitrate Agar Slant) Reagent A (Alpha-naphthylamine + 5N HAC) Reagent B ( Sulfanilic acid +5N HAC) Procedure Inoculate the Nitrate Medium with a loopful of the test organism. Incubate at 35oC for 18-24 hours. At the end of the incubation, add 1 ml each of Reagent A and Reagent B to the test medium in that order. Interpretation The development of a red color within 30 seconds after adding the test reagents indicates the presence of Nitrites and represents a positive  NITRATE REDUCTION TEST (Cont’d.) If no color develops after adding the test reagents, this may indicate: a) that the nitrates have NOT been reduced (a true negative reaction) or, b) that the nitrates have been reduced to products other than nitrites, such as ammonia, nitric oxide, nitrous oxide and hydroxylamine. To detect “unreduced” Nitrates, add a small quantity of Zinc dust. Zinc ions reduce nitrates to nitrites. Result: Development of red color is a positive result. This indicates the presence of unreduced nitrates and confirms a true negative result. DECARBOXYLASE TEST Decarboxylases are a group of substrate-specific enzymes that are capable of attacking the carboxyl (COOH) portion of amino acids, forming alkaline-reacting amines. Each decarboxylase enzyme is specific for an amino acid. Lysine, Ornithine and Arginine are the three amino acids routinely tested in the identification of the Enterobacteriaceae. The specific amine products are as follows: Lysine ------------ Cadaverine Ornithine ------- Putrescine Arginine --------- Citrulline Principle The amino acid to be tested is added to the Moeller Decarboxylase Medium prior to inoculation with the test organism. The tube is anaerobically incubated with mineral oil. During the initial phase of incubation, the tube turns yellow owing to the fermentation of the small amount of glucose in the medium. If the amino acid is decarboxylated, alkaline amines are formed and the medium reverts to its original purple color. Interpretation Conversion of the tube to yellow color indicates that the organism is Ornithine Decarboxylase Test  _ + Ornithine Decarboxylase Test Lysine Decarboxylase Test KCN TEST The test determines the ability of the organism to live and grow in KCN. For differentiation of enteric bacilli. Principle: KCN Broth Base facilitates the recognition of enteric bacilli, similar to Citrobacter freundii, especially those that are slow fermentative but develop rapidly in the presence of cyanide. Also, it is very useful in differentiating Salmonella (including the Arizona group) from the Bethesda- Ballerup group. Proteose peptone provides essential growth nutrients. Sodium chloride provides the osmotic balance. Sodium phosphate and potassium phosphate provide minerals and ions and act as a buffer system. Procedure: Before use add 0.075 ml 0.5 % solution of potassium cyanide. Inoculate the medium lightly so that the inoculum cannot be misinterpreted as growth when cultures are examined. This may be accomplished by using a 3 mm loopful of an overnight (24 hours) broth culture or transferring a light inoculum from an agar slant culture with a straight wire. Final pH at 25°C: 7,6 ± 0.2. Incubate at 37±1°C  KCN TEST (Cont’d) Result: With Growth: Enterobacter, Klebsiella, Proteus, Citrobacter, Providencia, Hafnia No Growth: Escherichia, Arizona, Salmonella , Shigella  Citrobacter freundii grows in the medium (produces enzymes that are resistant to KCN).  Salmonella is sensitive to KCN.  Not performed in Clinical Laboratories because of its potential hazard to personnel.  Prepared tubes stored at 2 - 25°C shielded from light.  The following results were obtained in the performance of the medium from type cultures after incubation at a temperature of 37±1°C and observed 24-48 hours: (Control) Escherichia coli ATCC 25922 No Growth Citrobacter freundii ATCC 8090 Good Uninoculated selective media used for the isolation of members of the Enterobacteriaceae: XLD medium (left), brilliant green agar (top) and MacConkey agar (right). Salmonella enteritidis Proteus mirabilis Edwardsiella tarda. Escherichia coli Klebsiella Enterobacter pneumoniae. aerogenes Providencia stuartii Serratia marcescens Citrobacter diversus. Yersinia enterocolitica Yersinia pseudotuberculosis Reactions of some members of the Enterobacteriaceae on selective/indicator media Routine isolation of important members of the Enterobacteriaceae and their presumptive identification on colonial morphology and /or biochemical tests.+ = positive reaction, − = negative, ‘IMViC’ = indole, methyl red, Voges–Proskauer and citrate tests, TSI = triple sugar iron agar, lysine = lysine decarboxylase test (Cont’d on next Routine isolation of important members of the Enterobacteriaceae and their presumptive identification on colonial morphology and /or biochemical tests.+ = positive reaction, − = negative, ‘IMViC’ = indole, methyl red, Voges–Proskauer and citrate tests, TSI = triple sugar iron agar, lysine = lysine decarboxylase test 119 Antibiotic Resistance in Enterobacteriaceae  Enterobacteria are the most commonly encountered pathogenic bacteria in clinical cases and the environment. Hence, they are highly influenced by the intake of antibiotics for disease management or in agriculture and the environment.  The scenario of antibiotic resistance among the members of Enterobacteriaceae is worst.  Initially, beta-lactam antibiotics like derivatives of penicillin and the 1 st or 2nd generation cephalosporins were considered effective against them. However, in the present situation due to the development of resistance against the Beta-Lactam antibiotics, the treatment option is very costly and limited.  ESBL (extended-spectrum beta-lactamase), ABL (AmpC beta-lactamase), and other types of beta-lactamase-producing Enterobacteriaceae are responsible for several cases of human infection all around the world.  Carbapenems also referred to as the last line of antibiotics, are also slowly losing their effectiveness due to the evolution of carbapenem’s resistance Enterobacteria. Carbapenems resistance genes are frequently encountered in several pathogenic species in the family. 120 HTTPS://WWW.YOUTUBE.COM/WATCH?V=E4A8G1O72AM 121 Phages, formally known as bacteriophages, are viruses that solely kill and selectively target bacteria. They are the most common biological entities in nature, and have been shown to effectively fight and destroy multi-drug resistant bacteria. Phage typing involves the examination of the sensitivity ofmicroorganisms to the lytic action of selected phages. Phage typing is still used to identify and distinguish different strains within a given species when isolated from different origins (disease, food, water, environmental) or geographical locations. Phages may trigger the immune system to overreact or cause an imbalance. Some types of phages don't work as well as other kinds to treat bacterial infections. There may not be enough kinds of phages to treat all bacterial infections. Some phages may cause bacteria to become resistant. There are 2 types of phages: lytic and temperate. Strictly lytic phages infect their host cell and cause it to burst, thus killing 122 the bacterium. Temperate, or lysogenic, phages don't kill their bacterial prey outright—they integrate their genome (which  END https://www.youtube.com/watch?v=E4a8g1o72AM 12