Microbiology Lecture 6: MacConkey (+), Oxidase (-) Gram (-) Rods PDF

COURSE OUTLINE ➔ Reduce Nitrate to Nitrite 9 ◆ EXCEPT Pholorhabdus, Xenorhabdus, and some strains of...

COURSE OUTLINE ➔ Reduce Nitrate to Nitrite 9 ◆ EXCEPT Pholorhabdus, Xenorhabdus, and some strains of MacConkey (+), Oxidase(-), Gram (-) Rods (Lec #6) Enterobacter and Yersinia Lecturer: Ms. Nida C. Gelig, RMT ➔ Catalase (+) 10 ◆ EXCEPT S. dysenteriae type I and a sp. of Xenorhabdus MACCONKEY (+), OXIDASE (-), GRAM (-) RODS ➔ First group under Gram (-) bacilli MacConkey Agar ➔ Differential medium FAMILY ENTEROBACTERIACEAE ➔ Free living in nature ➔ Found in intestines of man and animals ➔ Collectively known as “Enterics” Fig. Blood Agar ◆ Also used for other bacteria in intestines (e.g. Right: Pure culture of one Enteric colony Pseudomonas and anaerobic bact.) Left: 2 types of colonies (Large=Enteric, Medium=Staph) CLINICALLY RELEVANT MEMBERS OF ENTERICS ANTIGENIC STRUCTURES Antigen Description Group Description Examples ➔ O Antigen ➔ Coliforms ➔ Escherichia ➔ Possessed by ALL Enterics ◆ E. coli ➔ Part of normal flora of the GIT of ➔ Klebsiella ➔ Found in the polysaccharide side chains of LPS or Endotoxin man ➔ Enterobacter ◆ A component of the outer membrane ➔ Serratia Somatic Opportunistic ➔ Common causes of: ➔ Citrobacter Antigen ➔ Heat-stable Pathogens ◆ Pneumonia ➔ Proteus ◆ Cannot be destroyed by heating ◆ Meningitis ➔ Providencia ◆ Septicemia ➔ Morganella ➔ Identified/detected with specific O antiserum through ◆ UTI Agglutination Tests ◆ Wound infections ➔ K Antigen, V Antigen ➔ Cause disease in GIT of man ➔ Salmonella ◆ Enterics: K Antigen ➔ Shigella ◆ Salmonella: V Antigen ➔ NOT part of normal flora ➔ Yersinia ◆ Present during infxn ➔ Covers the O Antigen Overt ◆ Found in the bacterial capsule surrounding cell MOT: Fecal-Oral Route Pathogens ➔ Ingestion of food and water Capsular ➔ Heat-labile contaminated with bacteria Antigen ◆ Destroyed by heating in boiling water bath for 10 mins ◆ EXCEPT Y. pestis (through bite of infected rat flea) ◆ When capsular antigen is destroyed, O Antigen is exposed BIOCHEMICAL AND MORPHOLOGIC PROPERTIES ◆ O antigen retesting is then performed, using specific ➔ Gram (-) coccobacilli O antiserum 1 ➔ Oxidase (-) ➔ H Antigen ◆ EXCEPT Plesiomonas sp. (new member of the family; ◆ Found in flagellin 2 Flagellar belongs to another group) Antigen ➔ Heat labile Grow well on ◆ Can be destroyed by (1) heating and with (2) ethanol ➔ MacConkey Agar ➔ Eosin Methylene Blue (EMB) Agar ★ Serotyping should be done using colonies from a nonsugar containing 3 medium to prevent false (+) results. ★ NOTE: BOTH are differential media. ○ E.g. BA, TS, Chocolate Agar, Nutrient Agar, TS Agar ➔ Ferment glucose MEDIA FOR ISOLATION ◆ Produce acid OR acid and gas from glucose I. Differential Mildly Selective Type Products ➔ Contains carbohydrates, pH indicators, chemicals ➔ Only ACID ◆ Inhibiting Gram (+) bacteria 4 Anaerogenic ➔ Both ACID and GAS Medium Carbohydrate Aerogenic (1) MacConkey Agar Lactose (2) Eosin Methylene Blue Agar (EMB) Lactose and sucrose (3) Leifson Deoxycholate Agar Lactose ➔ Facultatively anaerobic ★ All three contain Lactose 5 ➔ Can be motile or nonmotile: ➔ Thus dividing the Enterics into two groups: Motility Description Lactose Fermenters (LF) Nonlactose Fermenters (NLF) ➔ Has peritrichous flagella ◆ EXCEPT Tatumella ptyseos (polar Motile flagellated) 6 Examples: ➔ Shigella ◆ Non-motile, overt pathogen Nonmotile Fig. (Left to right) MacConkey Agar; MacConkey Agar Mixed Culture (LF and NLF); Eosin ➔ Klebsiella Methylene Blue Agar* ◆ Coliform Most Coliforms Most enteropathogens Produce acid… Do NOT produce acid Grow well on routine media, just like cocci: Reacting with pH indicators present in Do NOT react with pH indicators present ➔ Blood Agar (BA) medium in medium ➔ Chocolate Agar (CA) 7 ◆ Same as BA but cooked after blood addition Show colored colonies Show colorless colonies = ➔ Brain Heart Infusion Broth when colonies have the same color as ➔ Thioglycollate Broth the medium *Pink colony on MacConkey Agar ➔ Asporogenous *Pink colony on mixed culture *Colorless colony on mixed culture 8 ◆ Do NOT form spores Escherichia coli on Eosin Methylene Blue (greenish metallic sheen colonies) 1  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT ★ Blue colonies on CHROMagar plate are NOT salmonella, but II. Differential Moderately Selective could be coliforms ➔ Carbohydrates, pH indicators, chemicals ◆ Inhibiting Gram (+) bacteria IV. Enrichment Media & Technic (4) ◆ AND most Coliforms ➔ For stool culture to isolate enteropathogens ◆ NOT coliforms since coliforms are normally present in ➔ For isolation of Salmonella and Shigella stool Media Description ➔ Inhibits growth of coliforms Colorless colonies ➔ Promotes growth of enteropathogens (1) Salmonella Shigella Black centers ➔ Stool or rectal swab is inoculated 1st into enrichment media Agar (SSA) ➔ H2S producers ◆ Incubated ◆ Before subculturing on selective media Media Description ➔ Isolates S. typhi and Shigella sp. (1) Selenite F Broth ➔ Isolates Salmonella sp. (2) Tetrathionate Broth ◆ OTHER than S. typhi Figs. Cultures on SSA (1) E. coli Pink colonies on SSA (3) Gram (-) Broth (GN ➔ Isolates S. typhi and Shigella sp. (2) Salmonella spp. colorless colonies with black centers broth) (3) Shigella spp. colorless colonies ➔ Isolates = Yersinia enterocolitica Colorless colonies Procedure ➔ Shigella ➔ Stool specimen/ rectal swab is inoculated to 1 5 mL phosphate buffer (2) Xylose Lysine Red colonies Deoxycholate Agar ➔ Salmonella (4) Cold Enrichment ➔ Refrigerate for 3 weeks Technic 2 (XLD) Black centers ➔ Subculture on CIN medium ➔ H2S producers ◆ Every 4 days 3 Colorless colonies ➔ Until characteristic bullseye colonies of Y. (3) Deoxycholate ➔ Both Salmonella and Shigella enterocolitica are isolated Citrate Agar (DCA) Bluish Green Colonies ➔ Both Salmonella and Shigella BIOCHEMICAL TESTS (4) Hektoen Enteric ➔ After colonies have been grown on plated media Agar (HE) Black centers ➔ H2S producers ➔ Cocci = Perform 2-5 biochemical tests to ID organism ➔ Enterics = A battery (many) of biochemical tests Primary Characteristics Product Description (1) Ability to ferment Acid Anaerogenic = glucose, lactose, or sucrose only acid is produced Fig. Culture on HE Aerogenic = (2) Ability to produce gas H2 and CO2 both acid and gas produced Shigella and Salmonella produce bluish green colonies Only enterics that have enzyme Uninhibited coliforms produce salmon orange color (3) Ability to produce H2S H2S cysteinase are capable of producing H2S MEDIUM: Triple Sugar Iron Agar (TSI) III. Highly Selective Media ➔ 1% lactose (10x more than glucose) ➔ Promotes growth of a specific bacterium or a group of bacteria ➔ 1% sucrose (10x more than glucose) Components ➔ 0.1% glucose ➔ Peptone Media Description ➔ Selective for Salmonella typhi (1) Bismuth Sulfite Agar ➔ Phenol red ➔ Produces jet black colonies (BSA) ◆ Acidic medium = yellow pH Indicator ◆ Basic medium = pink ➔ Isolation of Salmonella species (2) Brilliant Green Agar ➔ Produces white to pink or red colony ➔ Ferrous sulfate (BGA) ◆ Surrounded by a bright halo Indicators for H2S ➔ Sodium thiosulfate production ➔ Selective for Yersinia enterocolitica ➔ In test tube w/ a slant/slope & a butt ➔ Produce “bullseye” colonies (3) Cefsulodin Irgasan ➔ Covered with a loose cover ◆ Dark red centers Novobiocin Agar (CIN) Preparation ◆ Ideal to cotton plug or loose screw ◆ Translucent borders/ Pale periphery cap to maintain aerobic condition ➔ Differentiates E.coli O157:H7… ◆ Does not ferment sorbitol ◆ No acid is produced ◆ Colorless colonies ★ O = antigen (O antigen 157) (4) MacConkey Sorbitol ★ H = flagellar antigen (H7) Agar (MAC-SOR/Smac) ➔ … From other strains of E. coli which ferment sorbitol ◆ Produce acid ◆ React with indicator ◆ Produces pink colonies Fig. (Left to right) TSI medium with slant and butt; Method of inoculation ➔ Isolation of Salmonella species (5) CHROMagar Salmonella ➔ Produce mauve-colored colonies ➔ Uninoculated medium is orange in color Procedure 1 ➔ Sterilize wire needle 2 ➔ Touch a colony growing on the plate ➔ Make a line on the slant, then stab the butt 3 ◆ Until a few mm from the bottom Fig. (1) BSA plate: jet black S. typhi colonies ➔ Streak across the line on the slant (2) CIN plate: bullseye colonies Y. enterocolitica ★ Only one inoculum for both the slant and butt (3) Smac plate: colorless E. coli O157:H7 colonies and pink E.coli fermenting strains 4 (4) CHROMagar plate: mauve salmonella colonies 2  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT ➔ After inoculation, incubate TSI medium at 35°C or 37°C ◆ In ambient air for 18-24 hrs I. Indole Production 5 ➔ Detects tryptophanase which splits tryptophan into indole. ➔ Early/delayed readings of results = incorrect interpretation ➔ Incubated at 35°C for 18-24 hours. ➔ Medium: tryptone broth ➔ Reagent: Kovac’s rgt or Erlich’s rgt (5 drops into the broth) Reading & Interpretation ➔ How to read 1st column: ◆ Reading on the left is for the slant ◆ Reading on the right is for the butt ➔ I.e Alk/A ◆ Alk (alkaline) = reading for slant ◆ A (acid) = reading for butt Fig. Reagent reactions Reading Interpretation Left: Kovac’s rgt reaction (red ring is + for indoles, yellow is -) ➔ Only glucose is fermented. Right: Erlich’s rgt reaction (purple ring is +, yellow is -) Alk/A ➔ Slant = pink II. Methyl Red Test & Voges Proskauer Test ➔ Butt = yellow Methyl Red Test ➔ Glucose, lactose, &/or sucrose are fermented. ➔ Detects strong acids from glucose in the bacteria such as: ◆ Acetate A/A ◆ Formate ➔ Slant and butt = yellow ➔ Glucose, lactose, and sucrose are not fermented. Procedure Inoculate one (1) loopful of test organisms into the MRVP broth and ➔ Slant and butt = pink 1 incubate. Alk/Alk ➔ Peptone is utilized 2 After incubation, add five (5) drops of methyl red indicator into the broth. ◆ Aerobically = In the slant ➔ Slant = pink Results ➔ Butt = orange (retains color of medium) Positive (+) Negative (-) ➔ Glucose, lactose, and fructose are not fermented. Color Red medium Yellow medium Alk/NC pH pH 4.5 or less pH > 5.6 ➔ Peptone is utilized aerobically only. (very acidic) (still acidic) ◆ In the slant, not the butt ★ NC = no change ★ For enterics, the TSI reactions produced are only Alk/A and A/A since they ferment glucose. Gas production (CO₂ and H₂) ➔ Presence of cracks or bubbles. Voges Proskauer Test ➔ Add “G” or encircle butt reaction when observed. ➔ Detects acetoin or acetylmethylcarbinol production from glucose Example: A/AG or A/Ⓐ ➔ Medium: H₂S production ◆ Methyl Red-Voges Proskauer (MRVP) Broth ➔ Blackening in the butt. ➔ 2 step reaction: ➔ Reagent: a. Bacterium (cysteinase) + Na thiosulfate = H₂S↑ ◆ 6 drops α-naphthol b. H₂S + ferrous sulfate = FeS↓ ◆ 2 drops 40% KOH ➔ Add “with H₂S” after the butt reaction when observed. Example: A/AG with H₂S Procedure 1 Inoculate a second MR-VP broth, then incubate. 2 After incubation, add six (6) drops of α-naphthol, followed by two (2) drops 40% KOH. Results Positive (+) Negative (-) Red ring on top of medium Fig. Kligler Iron Agar or KIA (same components as the TSI but lacks Color after addition of indicators NO red ring formed sucrose) From L to R: A/AG, Alk/AG (gas in the right side of the media), Alk/A with H₂S (butt can be read with Acetoin and Is produced from glucose Is NOT produced from glucose fluorescent lamp light), Alk/Alk) Acetylmethylcar binol TSI Reaction III. Citrate Utilization Test ➔ Detects whether the enteric can utilize citrate Fig. TSI Reaction ◆ The only source of carbon for metabolism ➔ Sugars are oxidized in the slant, but are fermented in the butt. ➔ Only those Enteric organisms with the enzyme Citritase can ➔ Fermentation yields more acid than oxidation. utilize citrate ➔ Less glucose is present. ➔ Incubated at 35°C in ambient air for 18-24 hours. ➔ Prepared with a slant only ➔ Uninoculated medium is green in color Incubation Time Lactose Fermenters (LF) Non-lactose Fermenters ➔ Medium: (NLF) ◆ Simmons Citrate Agar Slant (SCA) ➔ Media becomes A/A. 6-8 hours ➔ Oxygen is depleted after 6-8 hours. ➔ Reagent: ➔ LFs utilize lactose to ➔ NLFs utilize peptone, ◆ Sodium (Na) Citrate produce more acids. producing amines ◆ Ammonium Salts 18-24 hours ◆ Remains A/A. (basic). ◆ Becomes Alk/A. ◆ Bromothymol blue (pH indicator) ➔ LFs will now utilize ➔ NLFs continue to Procedure peptones, producing produce amines. 48-72 hours Inoculate one (1) loopful of the test organism as a slant by simple streaking amines (basic). ◆ Becomes Alk/Alk. 1 or with a wire needle.. ◆ Becomes Alk/A. ★ To obtain the correct reaction, record results at 18-24 hours of 2 Incubate incubation time. 3  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT VI. Nitrate Reduction Test Results ➔ Detects nitratase (enzyme) or nitrate reductase, Positive (+) Negative (-) ◆ Which reduces nitrate to nitrite Color Blue color of slant Green medium Growth on slant With growth on slant (NaHCO3 WITHOUT growth on slant) Nitrate Agar Slant and NH3) ➔ Medium used ➔ Reagents Sodium bicarbonate and ◆ 2 drops sulfanilic acid ammonia – ◆ 2 drops α-naphthylamine Production of Which produces an alkaline Procedure reaction 1 Using a wire loop or needle, get the inoculum and do simple streaking across the slant. 2 Incubate 3 Add 2 drops sulfanilic acid 4 Add 2 drops α-naphthylamine Result IV. Motility Test Positive (+) Hanging Drop Method ➔ Rapid test True Motility Brownian Motion Formation of red azo compound Motile Non-motile (p-sulfobenzene-azo-α-naphthylamin e) on the slant ➔ Motility = also tested through bacteria growth in semisolid medium in butt tubes ◆ Medium = SIM SIM (Sulfide, Indole, Motility) ➔ Detects three parameters in one medium VII. Phenylalanine Deaminase Test ◆ (1) H2S production ➔ Detects deamination of phenylalanine to phenylpyruvic acid ◆ (2) Indole production ◆ With the action of the enzyme phenylalanine ◆ (3) Bacteria motility deaminase ➔ A wire needle is used to take the inoculum ◆ The butt is stabbed until a few mm from the bottom… Phenylalanine Agar Slant ◆ Then incubate ➔ Medium used Phenylalanine Agar ➔ Alternative medium Results ➔ FeCl3 is already incorporated Description ➔ Result: (+) Positive = Brown slant ➔Hazy (after incubation) Motile ◆ Growth spreading in the medium Reagent ➔Clear surrounding medium ➔ Added after incubation: 5 drops of 10% FeCl3 Nonmotile ◆ Growth confined only in the line of the stab Procedure Using a wire loop or needle, get the inoculum and do simple streaking ➔ Black precipitate in the medium H2S production 1 across the slant. ➔ After adding 5 drops of Kovac’s reagent: Indole 2 Incubate ◆ [+] red ring formed production 3 Add 5 drops of 10% FeCl3 ◆ [-] yellow ring formed Results Positive (+) Negative (-) Green slant after addition of FeCl3 NO green slant formation Positive only for Proteus, Morganella, Yellow color on slant due to Dark and Providencia yellow FeCl3 Fig. [L] Motile and Nonmotile, [R] H2S Production Third Tube (Control): Uninoculated phenylalanine agar V. Urease Test ➔ Requires heavy inoculum ◆ 5 loopfuls of the test organism ➔ Detects enzyme urease ◆ that splits urea into NH3 and CO2 Fig. [Left to Right] (+), (-), (ctrl) Urea Broth ➔ Medium used containing: VIII. ONPG Test (o-nitrophenol-β-D-galactopyranoside) ➔ Rapid test that detects presence of enzyme β-galactosidase Phenol Red (Indicator) ◆ Hydrolyzes ONPG and liberates o-nitrophenol, a In Alkaline Medium In Acidic Medium yellow compound Turns pink Turns yellow Results Two (2) enzymes involved in lactose fermentation: Positive (+) Negative (-) ➔ Permease Dark Pink NO color change ◆ Allows lactose into bacterial cell Presence of urease Retains butt color of medium (orange) ➔ β-galactosidase (Producing alkaline pH) ◆ Splits lactose into glucose and galactose Two (2) Types of Lactose Fermenters Fast Lactose Fermenters Slow Lactose Fermenters Have both permease and Only having β-galactosidase β-galactosidase (24 hours of incubation is inadequate) ONPG Broth ➔ Medium used 4  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT Procedure 1 Inoculate one loopful of test organism into the broth. 2 Incubate for 3-6 hours. Results Result Picture Indication β-galactosidase is present O-nitrophenol is produced Positive (o-nitrophenol is produced) Bacterium is a lactose Fig. LIA Reactions fermenter [Left to Right]: (ctrl), (P/Y), (R/Y), (P/P), (H2S) Negative Colorless medium XI. ORNITHINE DECARBOXYLATION (o-nitrophenol is not ➔ Detects ornithine decarboxylase produced) Motility, Indole, Ornithine (MIO) ★ Both SLFs and FLFs give a positive (+) result ➔ Semisolid Medium ➔ Contains: X. LYSINE DECARBOXYLATION & DEAMINATION ◆ Glucose, Ornithine ◆ Bromcresol purple (pH indicator) ➔ Detects (2) two enzymes: ◆ Lysine decarboxylase ➔ Detects (3) three properties: ◆ Lysine deaminase ◆ Motility of enterics ◆ Indole production Lysine Iron Agar (LIA) ◆ Ornithine decarboxylation ➔ Medium used that contains (1) glucose, (2) lysine ◆ Enterics ferment glucose ➔ MIO is prepared in deep butt tubes ◆ No slant ➔ And (3) bromcresol purple (pH indicator) ◆ Yellow in acidic medium Procedure ◆ Purple in alkaline medium 1 Get inoculum using wire needle, stab butt all the way to the bottom ➔ LIA is prepared with a shorter slant & deeper butt 2 Incubate 6-8 hours ◆ Compared to TSI Results ➔ Uninoculated = purple throughout the medium Positive (+) Negative (-) ➔ After incubation, originally purple medium becomes yellow Purple butt Bright yellow butt throughout ◆ Because of the acid from glucose With slight yellow tinge (from Sometimes with thin narrow rim glucose) of purple at the top H2S production Color/ End Product ➔ LIA can also detect H2S production but… Product: putrescine (alkaline) ◆ it is less sensitive compared to TSI ◆ (can be seen in the figure below, tube #4 with black ppt) Procedure 1 Get inoculum using wire needle, stab butt twice all the way to the bottom Streak across the slant 2 (different method of inoculation compared to TSI) 3 Incubate 6-8 hours Lysine Decarboxylation Lysine Deamination Butt = red Slant = red Hazy medium Motile enteric Non-motile (anaerobic process) (aerobic process) Clear medium Lysine decarboxylase acts upon Lysine deaminase acts upon lysine... Growth confines only in line of stab at the top of lysine… Producing a-ketocarboxylic acid medium Producing cadaverine (acidic) (alkaline) XII. MALONATE UTILIZATION Results ➔ Detects malonate Lysine Decarboxylation Lysine Deamination ◆ The only source of carbon for metabolism Purple butt Malonate Broth Positive Red slant Result ➔ Contains bromthymol blue as pH indicator Usually with light yellow tinge (from ◆ Like Simmons Citrate Agar glucose) Negative Yellow butt Purple slant Result Procedure 1 Get one loopful of the organism Possible LIA Reactions End Product 2 Suspend in malonate broth 3 Incubate P/P (-) Lysine deaminase ➔ Lysine Results Purple slant/ (+) Lysine decarboxylase decarboxylase Purple butt Positive (+) Negative (-) Blue broth Retains green color of medium P/Y (-) Lysine deaminase ➔ Both enzymes (light blue → deep prussian blue) Purple slant/ (-) Lysine decarboxylase absent NaOH Yellow butt R/Y (+) Lysine deaminase ➔ Lysine (-) Lysine decarboxylase deaminase Red slant/ Yellow butt Observed in: Proteus, Providencia, & Morganella ➔ Enterics can NO R/P N/A never have both enzymes 5  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT GENUS ESCHERICHIA E. coli O7K1 ESCHERICHIA COLI ➔ Somatic antigen O7 ◆ Agglutinates with O7 antiserum ➔ Predominant facultatively anaerobic sp. in the large intestine of Example man ➔ Has K1 antigen ◆ There are stricter anaerobes in the GIT ◆ Agglutinates with K1 anti-serum ➔ Index of fecal contamination in water bacteriology ◆ Presence of E. coli in H2O sample = fecally II. Extraintestinal Pathogens contaminated ➔ E. coli is a coliform ◆ Generally, does NOT cause disease in the GIT of man ➔ In differential media, E. coli will produce colored colonies ◆ Since it is a lactose fermenter 5 strains which can cause serious GIT infections: ➔ Enterotoxigenic E. coli (ETEC) ➔ Most E. coli strains are motile (peritrichous flagella) ➔ Enteroinvasive E. coli (EIEC) ➔ Enterohemorrhagic E. coli (EHEC) ➔ Most common cause of urinary tract infection (UTI), nosocomial ➔ Enteropathogenic E. coli (EPEC) infection, and Gram (-) bacteremia ➔ Enteroaggregative E. coli (EAEC) E. coli Colonies on Different Media Mode of Transmission: Media Colony Characteristics ➔ Fecal-oral route by contaminated food and water Greenish, metallic sheen EMB Flat, dry, pink colonies MacConkey **Please refer to last page for differentiation of the five strains Some are β-hemolytic (Complete hemolysis) Blood Agar LABORATORY DIAGNOSIS OF PATHOGENIC E. COLI **See summary tables for differentiation of the five strains E. coli in Different Reactions I. ETEC (Enterotoxigenic E. coli) Test Description Reading LT AND/OR ST Enterotoxins ➔ Can ferment lactose ➔ Yellow slant ➔ Can induce water and electrolytes into the lumen of the intestines ➔ Aerogenic ➔ Yellow butt ◆ Causing watery diarrhea TSI with cracks ➔ Entire medium is pushed up ➔ Heat-labile at 65 C for 30 min ◆ Agar is split into sections. ➔ Acid over Acid ➔ Antigenic - elicit antibody production in animals Gas Indole Production (+) dilation of loop because of fluid ➔ Tryptone broth with a red ring. accumulation I ➔ Positive rxn Positive ◆ enzyme Tryptophanase LT Rabbit Ileal Loop Test Methyl Red Test Cell death Chinese Hamster Ovary Cell Assay ➔ Medium is colored red after addition of the reagent. Reverse Passive Latex Agglutination Serologic test Positive Test M ➔ Positive rxn Molecular biology test IMViC DNA-DNA Hybridization ◆ Can form acetate and formate from glucose ➔ Heat-stable even at 100 C Voges-Proskauer Test ➔ Poorly antigenic ➔ NO red ring is formed after the addition of Negative ST V α-naphthol and KOH Infant mouse assay DNA-DNA Hybridization Citrate Determination Test Rabbit Ileal Test ➔ It retains the green color. Negative C 1 Incision is made in the abdominal wall of an anesthetized rabbit. ◆ Lacks enzyme Citritase Rabbit’s ileum is pulled out, and several sections are tied depending on the 2 number of test organisms. (e.g., 4+1 for (-) control). Each section = 4 inches in length. 3 Inject organisms into ligated sections. After inoculation, place ligated loops into the rabbit and stitch/staple the 4 incision.. 5 After 4 hours, rabbit is sacrificed and examined. Chinese Hamster Ovary Cell Assay 1 Tissue culture made of Chinese hamster ovary cell are available in the market. 2 Inoculate organism into cells/tray and incubate. 3 Cell death is observed due to LT cytotoxicity. Infant Mouse Assay 1 Organism is injected intraperitoneally into the mouth of a 1-4 day old mouse. Fig. TSI and IMViC 2 Measure amount of fluid accumulated after 4 hours. TWO GROUPS OF PATHOGENIC E. COLI II. EIEC (Enteroinvasive E. coli) I. Extraintestinal Pathogens ➔ Can invade the enterocytes (intestinal epithelium) (1) Uropathogenic E. coli (UPEC) ◆ Causing bloody diarrhea as a result of direct invasion ➔ Major cause of E. coli-associated community-acquired of enterocytes in the colon Pathogenesis UTI ➔ Causes cystitis and acute pyelonephritis ◆ Ex. E. coli O112 Sereny Test ➔ Haemolysin Inoculate organism into one eye of the guinea pig (test); Virulence ➔ Pili 1 Factors the other eye is used as a negative control. ➔ Cytotoxic necrosing factor Positive Result: Keratoconjunctivitis in 24 hours characterized by combined 2 E. coli O6K2H1 inflammation of the cornea and conjunctiva. ➔ Will agglutinate with O6, K2, and H1 antiserum 3 Eye is treated with antibiotic eye drop. Example ★ Kit for E. coli antisera has different numbers (O, K, and H antisera) III. EHEC (Enterohemmorrhagic E. coli) ○ Used for agglutination or serologic test ➔ AKA VTEC or STEC ◆ Verotoxin-producing E. coli (2) Meningitis / Sepsis-associated E. coli (MNEC) ◆ Shiga-like toxin-producing E. coli Produced by Shigella dysenteriae ➔ Causes neonatal meningitis Most frequently associated EHEC Pathogenesis ◆ Results in high morbidity and mortality ➔ E. coli O157:H7 ➔ E. coli O157:NM (non motile) ➔ K1 antigen ◆ Causing hemorrhagic colitis Virulence Factors ◆ 80% of MNEC has this antigen ◆ Caused outbreaks in… ◆ (1) USA traced to a burger joint 6  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT ◆ (2) Europe from mongo sprouts Lactose ➔ Lactose fermenter ◆ (3) Japan infecting 6,000 school children ➔ Aerogenic (acid over acid gas) TSI rxn. Verotoxin or Shiga-like toxin ➔ (--++) ➔ A cytotoxin which damages vascular endothelial cells IMVIC rxn. ➔ Opposite to E.coli ◆ Causing Hemorrhagic Colitis that can lead to Hemolytic Uremic Syndrome (HUS) ➔ Positive Direct String Test Hemolytic Uremic Syndrome ➔ K1 capsular clonal complex ➔ Characterized by (1) CC23k1 ➔ Associated with pyogenic liver ◆ Hemolytic anemia abscesses ◆ Low platelet count Virulence Factor ◆ Can result in kidney failure and death ➔ K1 capsular complex EHEC is isolated using (2) CC82k1 ➔ Associated with severe pneumonia ➔ Sorbitol instead of Lactose (ordinary MAC) and septicemia ➔ EHEC produces colorless colonies MacConkey ➔ Resistant to bactericidal effects of serum ◆ Does not ferment sorbitol Development of Hypervirulent Sorbitol Agar (hypermucoviscous) phenotype hvKP complement and neutrophils (MAC-SOR or ➔ Other E. coli strains (80%) produce pink colonies ➔ Ampicillin Smac) ◆ Ferment sorbitol ➔ May demonstrate multidrug resistance ◆ Due to plasmids with enzymes.. Cefixime- ➔ More sensitive and specific than Smac Resistances Tellurite- containing ➔ Inhibits other strains ◆ Such as cabapenemase & MacConkey Agar cephalosporinase CHROMagar ➔ Higher sensitivity and specificity and better diagnostic efficiency than SMAC POP UP ASSIGNMENT ★ 🫨 Find tests that demonstrate effects of cabapenamase O157 ➔ Mauve colored colonies KLEBSIELLA OXYTOCA ➔ Has a heat-labile cytotoxin ➔ Isolated from patients with.. ◆ Self-limiting antibiotic-associated haemorrhagic colitis KLEBSIELLA RHINOSCLEROMATIS AND OZAENAE ➔ Agents of chronic infections Rhinoscleroma and Atrophic Rhinitis, Fig. MAC-SOR, CT-Smac, CHROMagar O157 Ozena After colonies are grown into the chosen media… KLEBSIELLA GRANULOMATIS ➔ Antisera O157 and H7 ➔ New member of Klebsiella Serotyping ➔ Formerly known as Calymatobacterium granulomatis ➔ Needs tissue culture made up of Vero Cells Cytotoxin Assay ◆ African green monkey kidney cells ➔ Transferred to genus Klebsiella ◆ As they share the same properties on their capsule Cytotoxin Assay Into the tray containing the Vero cells, add the bacterial suspension of the test ➔ Causative agent for Granuloma Inguinale or Donovanosis 1 ◆ A sexually transmitted disease (STD) organism. 2 Incubate. ◆ Characterized by genital ulceration and inflammation After incubation, cell death is observed 3 ➔ Difficult to cultivate in vitro EHEC is cytotoxic to the Vero Cells ◆ Thus diagnosis is based on the findings of Donovan IV. EPEC (Enteropathogenic E.coli) bodies in scrapings of lesions obtained from the patient and… ➔ Does NOT produce exotoxins ➔ Associated with attachment and effacement of the enterocytes ◆ Stained with Wright’s or Giemsa stain in colon intestinal cell wall ◆ E.g E. coli O111 and O125 I. Donovan Bodies ➔ Serotyping can be done to.. Bacteria stain ◆ Detect or diagnose the presence of these EPEC ➔ Blue rods with polar granules ➔ Common cause of orphanage/nursery outbreaks “Safety-pin” Appearance ➔ Large pink capsules within large macrophages V. EAEC (Enteroaggregative E.coli) ➔ Both ends have metachromatic granules ➔ Associated with the aggregative regulator gene, (AggR) ➔ Rod-shaped bacteria inside the cytoplasm of the macrophage ◆ Responsible for the cellular adherence of the organism, causing Diarrhea Bacterium is surrounded by a large pink capsule ➔ The same doctor discovered Leishmania donovani and the ➔ AggR may be detected by molecular studies, like.. Donovan bodies ◆ DNA-DNA hybridization ◆ Caused by Calymatobacterium granulomatis (K. granulomatis) ➔ Stools typically are NOT bloody and do NOT contain pus cells – Dr. Donovan ➔ Inflammation is accompanied by fever and abdominal pain GENUS ENTEROBACTER Description ➔ Fish-eye colonies GENUS KLEBSIELLA EMB ◆ Dark center, periphery is paler ➔ Inhabitants of Nasopharynx and GIT ➔ Smaller capsules, less mucoid (compared to Cause Capsule Klebsiella) Liver Abscesses Primary Pneumonia UTI Wound Infection ➔ Motile Motility Septicemia Meningitis ➔ Urease (-) Urease Test KLEBSIELLA PNEUMONIAE ◆ Except ss of E. cloacae & agglomerans ➔ AKA Friedlander’s Bacillus Lactose ➔ Lactose fermenter ➔ Most commonly isolated spp. of Klebsiella in the lab ➔ Aerogenic TSI rxn. Parameters Description ➔ (--++) ➔ Large, pink, mucoid colonies EMB or MacConkey IMVIC rxn. ◆ Similar to Klebsiella ◆ Due to being capsulated agar ◆ Has O antigen ➔ Capsulated COMMONLY FOUND ENTEROBACTER IN THE LAB Capsule ➔ Differentiated by two tests: Motility ➔ Nonmotile (lacks H antigen) ◆ (1) Lysine decarboxylase Urease Test ➔ Urease (+) ◆ (2) Ornithine decarboxylase 7  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT L decarboxylase O decarboxylase GENUS EDWARDSIELLA E. cloacae - + E. tarda E. aerogenes + + ➔ Normal flora of GIT of cold-blooded animals E. agglomerans - - ◆ Reptiles, fish, turtles ★ Pantoea agglomerans = new designation of E. agglomerans ○ Yellow pigmented colonies ➔ May cause wound infxn that leads to… ○ Arginine dehydrolase negative ◆ Myonecrosis - necrotic dmg to muscle ➔ Immunocompromised persons are susceptible Cause CRONOBACTER SPP. Gastroenteritis Meningitis C. sakazakii C. malonaticus Septicemia Liver dss. Neonatal infxn Adult infxn Description ➔ Motile Motility ➔ Formerly Enterobacter sakazakii ◆ Peritrichous flagella Lactose ➔ Non-lactose fermenter Bacteremia Associated with Meningitis H2S ➔ (+) ◆ Enzyme: Cysteine desulfhydrase Necrotizing colitis TSI ➔ Alk/Ac with H2S Description Indole ➔ (+) ➔ Best demonstrated at 25°C Yellow pigment ➔ Ampicillin GENUS CITROBACTER ➔ 1st and 2nd generation cephalosporins C. freundii Resistance ◆ Due to AmpC chromosomal beta-lactamase Description ➔ Produce colonies with greenish metallic sheen ➔ -, -, -, + (like E.coli) IMVIC rxn EMB ➔ TSI and IMVIC differentiates E. coli from C. freundii GENUS HAFNIA Motility ➔ Motile ➔ Lactose fermenters H. alvie Lactose ◆ Some are slow lactose fermenters ➔ Formerly Enterobacter hafniae Urease ➔ Variable ➔ Slow lactose fermenter H2S ➔ Most strains are H2S (+) Lactose fermenters Tests Reactions Indole (-) ➔ Ac/Ac gas (aerogenic) with H2S Citrate (-) TSI Slow lactose fermenters Urease (-) Lysine and Ornithine (+) ➔ Alk/Ac with H2S decarboxylase IMVIC rxn ➔ (-, +, -, +) ➔ More biochemical test will produce (+) results when grown at ➔ May harbor inducible AmpC genes that will encode 25°C resistance to… Resistance ◆ Ampicillin and ◆ 1st gen cephalosporins GENUS SERRATIA S. marcescens COLIFORMS ➔ Nosocomial infxns ➔ Proteus ◆ With medical devices (urinary catheters, respirators, ➔ Providencia IV fluids) ➔ Morganella General Characteristics Description ➔ Non-lactose fermenters Motility ➔ Motile ➔ Motile ➔ Variable Urease Test ◆ Some strains are (+), some (-) ➔ Produce rapid & abundant urease ➔ Deaminate lysine & phenylalanine Lactose ➔ Slow lactose fermenter ONPG ➔ (+) ◆ Has beta-galactosidase GENUS PROTEUS ➔ Alkaline/acid ➔ Colonies exhibit foul odor TSI ◆ 24 hours incubation is not enough for fermentation of lactose ◆ “Chocolate cake” or “burnt chocolate” IMVIC rxn ➔ (-, -, +, +) ➔ Associated with UTI, next to E.coli detect enzyme tryptophanase Ornithine decarb —> detect ornithine decarboxylase 8  Lecture 6. MacConkey (+), Oxidase(-), Gram (-) Rods. Ms. Nida C. Gelig, RMT Weil- Felix reaction Differentiation based on given characteristics ➔ Serologic test which uses proteus antigens TSI IMViC LIA OD ◆ (1) P. vulgaris OX2 and OX9 Alk/A -/+/-/- P/P - ◆ (2) P. mirabilis OXK S. typhi w/ H2S no gas ➔ For dx of Rickettsial diseases S. paratyphi-A Alk/A -/+/-/- P/Y R/Y + ◆ Caused by rickettsia bacteria Alk/A w/ H2S P/P S. enteritidis -/+/-/+ + Salmonellosis GENUS PROVIDENCIA ➔ Salmonella infection P. rettgeri P. stuartii ➔ MOT = Fecal-oral route ➔ Former member of Proteus ➔ Major pathogen in burn ➔ Infective dose = ingestion of 100,000 organisms to get ◆ Old name = Proteus rettgeri infection infected ➔ Can cause Urinary tract infection 3 General Categories of Salmonellosis in Man (UTI) ➔ Self limiting ➔ Lasts 2-5 days Both are H2S (-) unlike the proteus ➔ Most common salmonellosis in man Citrate (+) = both can utilize citrate ◆ That infects small and large intestines OD (-) = both lack ornithine decarboxylase ➔ Causative agent remain localized in GIT ◆ Does not invade bloodstream GENUS MORGANELLA M. morganii ➔ Caused by any species, most commonly by ➔ Proteus morganii ◆ S. typhimurium Old Name (1) Gastro ◆ S. enteritidis (Gartner’s Bacillus) -enteritis ➔ Associated with Urinary tract infection Infection (UTI) ➔ Incubation period = 1-2 days ◆ Time between ingestion of contaminated food and appearance of symptoms ➔ H2S (-) H2S Test ➔ Does not produce H2S ➔ Symptoms: ◆ Nausea ➔ Citrate (+) Citrate Test ◆ Vomiting ➔ Cannot utilize citrate ◆ Diarrhea ➔ OD (+) Ornithine ➔ Contains ornithine decarboxylase Decarboxylase Test ➔ From GIT, organism enters bloodstream Morganella and ➔ Citrate utilization test ➔ Infects any tissue Providencia ➔ Ornithine decarboxylation test ◆ Pneumonia, meningitis, osteomyelitis Differentiation ➔ Does not involve GIT ◆ Only pass through (2) Bacteremia & Extra- ➔ Caused by any species, most commonly intestinal ◆ S. choleraesius Infections ◆ S. dublin ➔ Symptoms: Fig. (Left to right) Plate showing swarming growth of proteus; P. vulgaris smear; TSI proteus rxn; ◆ Fever TSI providencia and morganella rxn ◆ Chills ◆ Anorexia ★ Plate shows site of inoculum on upper portion