1 PATH 417 Case 4: One Too Many HamburgersThe Microbiology Laboratory Summary By: Sunny Chen

2 Topic Overview Potential bacterial candidates for this infectious scenario Possible samples taken Possible tests performed Expected results for the potential bacterial candidates

3 Topic Overview Potential bacterial candidates for this infectious scenario Possible samples taken Possible tests performed Expected results for the potential bacterial candidates

4 Potential Bacterial CandidatesPossible disease: gastroenteritis Possible bacterial causes include: Salmonella typhimurium(S. typhimurium) Campylobacter jejuni (C. jejuni) Escherichia coli O157:H7 Shigella

5 Potential Bacterial CandidatesPossible contaminated food: ground beef (in hamburger) Most common bacterial pathogens associated with this food: Salmonella Shiga-toxin producing Escherichia coli (STECs)-especially E. coli O157:H7 Campylobacter jejuni Listeria monocytogenes Staphylococcus aureus

6 Possible Infectious Mechanismgrinding of meat  bacteria present on the surface get distributed throughout the meat  in the “Danger Zone” (temperatures between 4.4°C and 60°C)bacteria multiply why necessary to cook the meat to a safe internal temperature of 71.1 °C and to keep ground beef stored below 4.4 °C

7 Potential Bacterial Candidatesbacterial candidates associated with bloody diarrhea: Escherichia coli (particularly E. coli O157:H7) Campylobacter jejuni (C. jejuni) Salmonella Typhimurium (S. Typhimurium) Shigella sonnei (S. sonnei) Thus, potential bacterial candidates for this case can include the above bacteria

8 Potential Bacterial Candidate-C. jejunigram-negative bacterium (figure on the right) microaeroplic, non-fermentative, and coccoid or round in shape have flagellum to navigate in the host

9 Potential Bacterial Candidate-C. jejunicell wall structure: inner lipid membrane, thin peptidoglycan layer and an outer membrane on the surface of the cell wall, lipopolysaccharides embedded to the outer membrane LPS: endotoxic b/c can trigger Toll-Like-Receptor 4s

10 Potential Bacterial Candidate-C. jejuniprimary reservoir locale: gastrointestinal tract of animals transmitted to humans via: food sources unclean water direct physical contact with an infected animal primary symptoms: bloody stool abdominal pain fever nausea diarrhea vomiting other complications involving the digestive system

11 Potential Bacterial Candidate- E. coli O157:H7facultative anaerobic bacteria, gram-negative, rod-shaped able to produce Shiga-toxin, can cause extensive damage to the walls of the large intestinehemorrhagic colitis (diarrhea and fever followed by bloody diarrhea)/ hemolytic uremic syndrome (HUS), particularly in children and the elderly

12 Potential Bacterial Candidate- E. coli O157:H7found in the normal flora of the gastrointestinal tract that doesn’t normally cause infections can naturally be found in the intestinal contents of some cattle, goats, and even sheep present in ground beef transmission: usually spread through the improper preparation of beef products

13 Potential Bacterial Candidate- E. coli O157:H7Primary symptoms: abdominal pains blood and watery diarrhea vomiting mild fevers

14 E. coli O157:H7 & HUS Hemolytic uremic syndrome (HUS):A condition that is caused by the abnormal destruction of red blood cells red blood cells overwhelm and clog up the filtration system of the kidneyskidney failure usually develops 5-10 days after the start of bloody diarrhea caused by bacterial infections avoid the use of antibiotics or antimotility agents during diarrheal illness antimotility shown to increase the incidence of HUS motility slows, the gut is exposed to the toxins for a longer period of time antibiotic-induced injury to the bacterial membrane favors the acute release of large amounts of toxins shown to increase the risk of full-blown HUS by 17-fold recommendation: avoid usage except in cases of sepsis

15 Potential Bacterial Candidate- S. typhimuriuma flagellated gram-negative facultative anaerobic bacilli bacteria (right figure) toxicity due to an outer membrane consists largely of lipopolysaccharides (LPS)protect the bacteria from the environment

16 Potential Bacterial Candidate- S. typhimuriumfound in the intestinal tract of animals causes two different diseases enteric fever (typhoid) occurs as the bacterium enters the bloodstream acute gastroenteritis foodborne infections of the gastrointestinal tract most common transmission method: ground meat and the improper preparation of the meat before consumption primary symptoms: abdominal pain cramps vomiting diarrhea

17 Potential Bacterial Candidate- Shigella (most likely SPotential Bacterial Candidate- Shigella (most likely S. sonnei in North America) Gram-negative facultative anaerobic, nonspore-forming, nonmotile, rod-shaped bacteria species classified into four serogroups: S.dysenterae S.flexneri S.boydii S.sonnei (shown here)

18 Potential Bacterial Candidate- Shigella (most likely SPotential Bacterial Candidate- Shigella (most likely S. sonnei in North America) Disease caused: shigellosis affects the gastrointestinal tract associated with the symptoms: bloody diarrhea fever stomach cramps abdominal pains Molecular cause: Shiga toxin produced causes inflammatory response to the enteric cell wall and necrotic cell death of the colonic epithelium Infection due to consumption of infected food or liquids

19 Topic Overview Potential bacterial candidates for this infectious scenario Possible samples taken Possible tests performed Expected results for the potential bacterial candidates

20 Possible Samples TakenStool Blood Urine Details summarized in the table coming next

21 Possible Samples TakenWhen to Collect How much to Collect Method of Collection Storage Transportation Stool As soon as the onset of diarrhea or bloody stool Whole stools or several rectal swabs Whole stool collected in a sterile plastic container with a tight, re-sealable lid Rectal swabs can be done rectally or on whole stool The swabs are inserted 1 to 1.5 inches into the rectum and rotated gently, then placed into the transport medium Whole stool that is not processed within 2 hours of collection should be refrigerated at 4°C A portion of the stool can be frozen at -15°C for antigen testing and PCR Specimens are refrigerated for transport using ice by overnight mail Frozen stool samples are transported using dry ice

22 Possible Samples TakenWhen to Collect How much to Collect Method of Collection Storage Transportation Blood During the onset of gastroenteritis symptoms 3mL for children; 15mL for adults A tourniquet is applied and a sterile needle is used for the puncture site Withdrawn blood is collected in labelled tubes The tubes are inverted several times to ensure proper mixing Collection tubes may contain anticoagulants, gels or other additives in preparation for laboratory testing Samples are stored in temperatures between 4 to 25°C Unspun serum should be refrigerated for transport using ice Spun serum can be frozen for shipping by using dry ice

23 Possible Samples TakenWhen to Collect How much to Collect Method of Collection Storage Transportation Urine First passed urine in the morning or midstream catch any time of day, during the onset of symptoms 10 to 50mL Collected in sterile plastic container with a lid Specimens refrigerated at 4-6°C Samples are centrifuged for 5 to 10 minutes, then the supernatant is discard and the sediment is re-suspended in a transport medium Samples should be refrigerated during transported and processed within 48 hours

24 Importance of Microbiology Laboratorycritical in deducing the pathogen responsible for the patient’s symptoms and diagnosis of the disease essential that the correct bacterial pathogen is isolated from stool cultures for the management of and development of a treatment plan for patients important in determining what antibiotics to use some strains of bacteria are resistant to specific types of antibiotics some types of antibiotics may be more effective against certain bacteria urine and blood samples: have important role in the diagnosis of the disease blood test: allows physicians to understand the patient’s overall health status by determining their renal function, electrolytes, overall blood cell values Help to determine if the bacteria have spread to the systemic circulation urine test: provides information of whether the bacteria have spread to other sites

25 Topic Overview Potential bacterial candidates for this infectious scenario Possible samples taken Possible tests performed Expected results for the potential bacterial candidates

26 Possible Tests PerformedStool culture laboratory will ‘grow’ (culture) the pathogens present in the stool sample to determine whether a pathogen is present if so, identify (at least narrow down) the possible pathogen based on the media conditions and the characteristics demonstrated sample processed selected for portions that may contain blood or mucus ensures the highest number surviving of pathogens for culture

27 Possible Tests PerformedStool culture (Conti) plated on different media for identification purposes: MacConkey (MAC) agar for general recovery of gram negative rod shaped bacteria Selective and differential medium used to isolate gram negative bacteria and determine if they are fermenting or non-fermenting A selective/differential medium designed for the recovery of Salmonella E.g. Bismuth sulfite agar A medium designed for the recovery of Campylobacter E.g. Campy blood agar A medium designed for the recovery of E. coli O157:H7 and/or enrichment broth for testing for the presence of Shiga toxin E.g. MacConkey agar with sorbitol (SMAC) or cefixime-tellurite SMAC (CT-SMAC) A medium designed for the recovery of Shigella E.g. HardyChrom Salmonella Shigella (SS) Note: Any blood samples/urine samples would undergo similar culturing protocol as the stool sample

28 Possible Tests Performed— Different MediaMedium Composition Principle MacConkey (MAC) agar • Pancreatic digest of gelatin and peptones provide the essential nutrients, vitamins and nitrogenous factors required by bacteria • NaCl- maintains osmotic balance • Agar solidifies medium • Lactose monohydrate a fermentable source of C,H,and Os • Crystal violet and bile salts inhibit the growth of most gram positive bacteria • Neutral red a pH indicator A selective and differential medium Used to isolate gram-negative bacteria by: inhibit growth of gram-positive bacteria differentiate between fermenting (pink colonies) and non-fermenting (colourless colonies) gram-negative bacteria Fermentation causes a local pH decreasecauses the indicator to turn pink

29 Possible Tests Performed-MAC Agar

30 Possible Tests Performed— Different MediaMedium Composition Principle Bismuth sulfite agar • Beef extract and peptone provide nitrogen • Dextrose energy source • Disodium phosphate buffering agent • Ferrous sulfate detects H2S production • Bismuth sulfite indicator and brilliant green inhibit the growth of gram-positive bacteria and members of the coliform group (e.g. E. coli) • Agar solidifies the medium A highly selective/differential medium for Salmonella sp. including typhoid fever causing serotypes Bismuth sulfite indicator and brilliant green inhibit growth of gram-positive and coliform bacteria (e.g. E. coli) to allow Salmonella to grow freely Most Salmonella enterica subspecies are capable of producing H2S (detected by ferrous sulfate in the medium) - iron precipitated formation of colonies that have a brown/black colour with a metallic sheen - Other bacteria would have inhibited growth/ different colored colonies (likely greenish)

31 Possible Tests Performed-Bismuth Sulfite Agar

32 Possible Tests Performed— Different MediaMedium Composition Principle Campy blood agar • Casein and meat peptone provide nutrients for Campylobacter growth • Sodium provides electrolytes and maintains osmotic balance • Dextrose provides an energy source Yeast extract provides essential vitamin B • Sheep’s blood provides growth factors • Cephalothin, amphotericin B, trimethoprim, vancomycin, and polymyxin B inhibit the growth of Enterobacteriaceae, staphylococcus, and yeast A highly selective medium for Campylobacter sp. Growth is optimized for Campylobacter Growth of yeast, Enterobacteriaceae (e.g. E. coli, Salmonella, and staphylococcus) are inhibited Colonies should be yellowish/greyish and non-hemolytic

33 Possible Tests Performed-Campy Blood Agar

34 Possible Tests Performed— Different MediaMedium Composition Principle MacConkey agar with sorbitol (SMAC) or cefixime-tellurite SMAC (CT-SMAC) • Same composition as MacConkey agar except replace lactose with sorbitol E. coli O157:H7 is unable to ferment • Allows differentiation of pathogenic O157:H7 E. coli from other serotypes (as it is unable to ferment sorbitol) • Would be likely done as a subculture of E. coli (of unknown serotype) cultured first upon regular MAC agar • O157:H7 E. coli colonies would be colourless they can’t ferment the sorbitol (which would lower the pH around the colony and change the colour of the colony to red) colourless colonies

35 Possible Tests Performed— SMAC or CT-SMACSMAC with E. Coli O157

36 Possible Tests Performed— Different MediaMedium Composition Principle HardyChrom Salmonella Shigella (SS) • Agar solidifies medium • Bile salts and sodium deoxycholate inhibit growth of gram-positive bacteria Peptones provide nutrients for bacterial growth • Selective antimicrobial agents inhibit other types of enteric bacteria • Fermentable C,H,Os allows distinguish between enteric pathogens and delayed lactose fermenters • A pH indicator • Ferric ammonium citrate and sodium thiosulfate allow visualization of H2S producing colonies • Patented chromogenic mixture produces different colours depending on which bacterial enzymes degrade them • A selective medium allows differentiation between Salmonella and Shigella. Color differentiation: • Most Salmonella subtypes produce H2S would appear as colonies with black centers • Non-H2Sproducing Shigella would appear as teal-blue colonies • Other bacterial species would be completely/partially inhibited if present, would be a different colour (due to chromogenic mixture)

37 Possible Tests Performed— SS

38 Side note: A table of more media and their intended use (Pt 1)Medium Intended use and notes All-purpose broths Gram-negative (GN) broth Selective enrichment for Gram-negative rods, specifically Salmonella and Shigella (subculture after 6–8 h of incubation, not part of routine setup unless for STEC EIA), can be used for STEC EIA Selenite F broth Selective enrichment for Gram-negative rods, specifically Salmonella and Shigella (subculture after 18–24 h of incubation, may inhibit growth of some Shigella species) (not part of routine setup) Organism-specific broths Alkaline peptone water Selective enrichment broth for Vibrio, when requested (subculture to TCBS after 24 h of incubation) MAC broth Can be used for STEC EIA, enrichment for Y. enterocolitica if incubated at 25°C (not part of routine setup) All-purpose agars Hektoen enteric (HE) Selective medium for Gram-negative rods, differentiates lactose fermenters (yellow-orange) from nonfermenters (blue or green), H2S production can be detected (black precipitate) MacConkey (MAC) Selective medium for Gram-negative rods, differentiates lactose fermenters (pink) from nonfermenters (colorless) Salmonella-shigella (SS) Selective medium for Gram-negative rods, differentiates lactose fermenters (pink/red) from nonfermenters (colorless), H2S production can be detected (black precipitate) Xylose-lysine-deoxycholate (XLD) Selective medium for Gram-negative rods, differentiates lactose fermenters (yellow) from nonfermenters (red), H2S production can be detected (black precipitate)

39 Side note: A table of more media and their intended use (Pt 2)Medium Intended use and notes Highly selective/differential agars Bismuth sulfite Isolation of Salmonella, including Salmonella Typhi (black on this medium) Brilliant green Isolation of Salmonella (red, pink, or white with red halo on this medium), inhibits Salmonella Typhi and Salmonella Paratyphi Blood agar with ampicillin Isolation of Aeromonas (not part of routine setup unless specifically requested) Campy Blood Isolation of Campylobacter Campy CVA Campylosel Cefsulodin-Irgasan-novobiocin (CIN) Isolation of Yersinia enterocolitica or Aeromonas (deep red center and transparent margin [bull's eye appearance] on this medium) (not part of routine setup) Charcoal selective Charcoal-cefoperazone-deoxycholate agar (CCDA) CHROMagar Salmonella Isolation of Salmonella (mauve-rose on this medium)

40 Side note: A table of more media and their intended use (Pt 3)Medium Intended use and notes CHROMagar O157 Isolation of O157 STEC (mauve on this medium) CHROMagar STEC Isolation of 6 most common STEC serogroups (mauve on this medium) Cycloserine-cefoxitin-egg yolk/cycloserine-cefositin-fructose Isolation of Clostridium difficile (not part of routine setup unless requested) HardyChrom SS Isolation of Salmonella (black on this medium) and Shigella (teal on this medium) nositol-brilliant green-bile salt Isolation of P. shigelloides (white to pink on this medium) (not part of routine setup unless requested) MacConkey agar with sorbitol (SMAC) or cefixime-tellurite SMAC (CT-SMAC) Isolation of E. coli O157 (colorless on this medium) Thiosulfate-citrat e-bile salts-sucrose (TCBS) Isolation of Vibrio (not part of routine setup unless requested), V. cholerae is yellow on this medium, V. parahaemolyticus is green on this medium, some vibrios are inhibited

41 Possible Tests Performed— Secondary Biochemical TestsSlanted media test used to subculture suspicious colonies for pathogen identity confirmation involved the classic “3-tube” biochemical test triple sugar iron agar (TSI) lysine iron agar (LIA) Christensen urea

42 Possible Tests Performed— Triple Sugar Iron Agar (TSI)Involves 3 sugars: lactose, sucrose, and glucose determine the fermentation preferences of the bacteria contains “butt” (bottom) and “slant” (top) regions Represent poorly or richly oxygenated regions Contain pH indicator for detection of fermentation Phenol red indicator turns yellow for acidic environment

43 Possible Tests Performed— Triple Sugar Iron Agar (TSI)Reaction Principles lactose or sucrose is fermentedboth butt and slant turns yellow, gas may be generated, bubbles/crackles observed only small amount of glucose is fermentedbutt (more media, more glucose)turns yellow , slant (less media, less glucose, acid oxidized by the organisms) remains red none of the sugars are fermentedboth the butt and slant would be red H2S producedindicator turns black for presence of ferrous sulfide

44 Possible Tests Performed— Triple Sugar Iron Agar (TSI)Expected Results: Slant/Butt Description Color Presentation (Slant/Butt) Interpretation Alkaline slant/no change in butt (K/NC) Red/Red glucose, lactose and sucrose non-fermenter Alkaline slant/Alkaline butt (K/K) Alkaline slant/acidic butt (K/A) Red/Yellow glucose fermentation only, gas (+ or -), H2S (+ or -) Acidic slant/acidic butt (A/A) Yellow/Yellow glucose, lactose and/or sucrose fermenter gas (+ or -), H2S (+ or -)

45 Possible Tests Performed— Lysine Iron Agar (LIA)also contains slant and butt regions tests the ability of bacteria to deaminate lysine or decarboxylate lysine occur in the slant or butt of the media respectively

46 Possible Tests Performed— Lysine Iron Agar (LIA)Reaction Principles production of decarboxylate lysine= creation of a product reacts with the bromcresol purple indicatorpurple butt, slant remains purple negative decarboxylation=yellow butt, purple slant Deamination of lysine=production of ammonia reacts with the ferric ammonium citrate  dark red slant, yellow butt Negative deamination=purple slant Presence of H2Sblack precipitate in the butt

47 Possible Tests Performed— Lysine Iron Agar (LIA)Expected Results: Slant/Butt Description Color Presentation (Slant/Butt) Interpretation Alkaline slant/Alkaline butt (K/K) Purple/Purple negative lysine deamination (slant),positive lysine decarboxylation (butt) Acidic slant/Alkaline butt (A/K)* Yellow/ Purple positive lysine deamination (slant), positive lysine decarboxylation (butt) Red slant/Acidic butt (R/A) Red/ Yellow positive lysine deamination (slant), negative lysine decarboxylation (butt) Acidic slant/Acidic butt (A/A) Yellow/Yellow Tube 1:  K/K Tube 2:  R/A * There might be a mistake on the wiki generated

48 Possible Tests Performed— Additional TestsGram staining A common microbiology lab technique used to differentiate between gram-positive (blue/purple) and gram-negative (red/pink) cells involves staining bacterial cells with crystal violet (a water soluble dye)+ add Gram’s iodine solution iodine forms insoluble complex with crystal violet followed by decolorization-decolorizer dehydrates the peptidoglycan layer Gram-Positive: crystal violet-iodine complex is trapped within the bacteria, dye is retained, can’t be counter-stained Gram-Negative: crystal violet-iodine complex is lost (dye is lost), counter stained to be red/pink

49 Possible Tests Performed— Additional TestsGram staining

50 Possible Tests Performed— Additional TestsOxidase Test determines if the bacteria has the cytochrome C oxidase enzyme responsible for catalyzing electron transport to electron acceptors as a part of the bacteria’s respiratory chain test involves catalyzing a redox reaction turns the reagent (tertramethyl-p-phenylene-diamine dihydrochloride) from colorless to a deep purple Different methods: dry filter paper method direct plate method swab method impregnated oxidase strip method test tube method

51 Possible Tests Performed— Additional TestsOxidase Test General Principle: introduce the colony to be tested to the reagent observe for color change Expected Results Positive=color change to purple Negative=no/delayed color change (remain colorless) Wet filter paper method protocol Soak a strip of filter paper in 1% solution of reagent Rub a speck of the culture onto the filter paper Observe for colour change, where positive is deep purple in 5-10s, delayed positive is purple in 10-60s, and negative is colourless or colour change after 60s

52 Possible Tests Performed— Additional TestsOxidase Test

53 Possible Tests Performed— Additional TestsCatalase Test tests bacteria for the presence of enzyme catalase catalyzes the breakdown of H2O2 into H2O and O2 introduce the bacteria to H2O2 Positive=rapid bubbling observed (O2 produced) Negative=no bubbling observed Many different methods Test Tube method protocol Ensure the colonies tested are 18-24hrs old, scrape several colonies up with a sterile tool (wooden stick, glass rod) Immerse colonies in solution Look for immediate bubbling (positive test result)

54 Possible Tests Performed— Additional TestsCatalase Test

55 Possible Tests Performed— Additional TestsPCR a method of amplifying the gene of interest general protocol: Denaturation – heating the mixture to 94ºC, the two strands of the DNA molecule separate into single strands Annealing – the mixture is cooled down to 50-70ºC, allowing primers to bind to complementary sites on the single strands Extension – the mixture is heated to 72ºC, allowing DNA polymerase to extend a new, complementary strand from the primers Result –doubling of DNA at the end of every cycle

56 Possible Tests Performed— Additional TestsPCR Application in this case: E. coli O157:H7 possesses the genes Stx1 and Stx2 for Shiga toxin production The other suspected pathogens of this case lack this gene Target these genes using PCR would allow for identification of E. coli O157:H7 as the causative agent in this case

57 Possible Tests Performed— Additional TestsELISA(Enzyme-linked Immunosorbent Assay) used to detect the presence of a specific antigen An example protocol: fix unknown antigen to an immobile surface, add antibodies specific to the antigen of interest if the unknown antigen is the antigen of interest, binding would occur wash the plate to remove any unbound antibodies any antibody bound to the plate after washing can be detected (they’re usually conjugated with an enzyme or some other molecules for detection) indicative of the presence of the antigen of interest

58 Possible Tests Performed— Additional TestsELISA(Enzyme-linked Immunosorbent Assay) Application in this case (E. coli O157:H7): Antigen of interest: Shiga toxin only antigen from E. coli O157:H7 would have the specific antibodies bound to it detection of E. coli O157:H7 Direct ELISA shown on the right as an example of ELISA

59 Possible Tests Performed— Additional TestsAntibiotic Sensitivity Test Aka susceptibility testing, a technique used: to test for antibiotic resistant pathogens to determine which antibiotic treatments would be most effective for the treatment of a particular patient a variety of procedural methods available E.g. the disc diffusion test Inoculate a Mueller-Hinton Agar (MHA) plate with the bacteria of interest Apply (typically up to 12) disks containing antimicrobial agents Incubate the plate for hours (depending on specific tests) Observe zones of inhibition around disks, measure the diameter of these zones Zones are dependent on: diffusion rate of the antimicrobial agent susceptibility of the pathogen to the antimicrobial agent

60 Possible Tests Performed— Additional TestsAntibiotic Sensitivity Test

61 Possible Tests Performed— Additional TestsAgglutination Test final test for pathogen identity confirmation general protocol: Place a homogenous suspension of the colony of interest in a test tube or a slide Test against antiserum (of known antibodies) specific to pathogen of interest Visible clumping seen within 1 min=positivepathogen of interest detected

62 Possible Tests Performed— Additional TestsAgglutination Test

63 Topic Overview Potential bacterial candidates for this infectious scenario Possible samples taken Possible tests performed Expected results for the potential bacterial candidates

64 Expected Results for E.coli 0157:H7Test Expected Result MacConkey plate pink colonies (lactose-fermenting) EMB plate blue colonies (lactose-fermenting) SM colourless colonies (does not ferment sorbitol) Oxidase Test positive Catalase Gram staining Red(Gram negative) PCR Positive (bacteria possess Stx1 and Stx2 genes) ELISA positive (produces Shiga toxin) TSI K/A or A/A, gas LIA K/A or A/A Antibiotic Sensitivity Test Susceptible to nitrofurantoin, ciprofloxacin, and norflaxocin Not susceptible tetracycline, erythromycin, and amoxicillin

65 Expected Results for CampylobacterTest Expected Result Campylobacter Skirrow agar small, mucoid, flat or slightly raised, non-hemolytic translucent and creamy-grey colonies (selective media for the bacteria) Oxidase Positive Catalase Gram staining Red (gram-negative) PCR negative (bacteria does not possess Stx1 and Stx2 genes) ELISA negative (does not produces Shiga toxin) Antibiotic Sensitivity Test Susceptible to erythromycin Not susceptible to ciprofloxacin

66 Expected Results for SalmonellaTest Expected Result MacConkey media colorless colonies (non-lactose fermenting) EMB colourless colonies (non-lactose fermenting) XLD media red colonies with black centers (decarboxylates lysine-red, H2S production under alkaline conditions-black) HE media  bluish-green colonies (H2S production) Oxidase Negative Catalase Positive Gram staining Red (Gram negative) PCR negative (bacteria does not possess Stx1 and Stx2 genes) ELISA negative (does not produces Shiga toxin) TSI K/A, H2S, ± gas LIA K/K or A/A, H2S Antibiotic Sensitivity Test Susceptible to ciprofloxacin Not susceptible to ampicillin

67 Microbiologic CharacteristicsSide Note: Some additional characteristics of the potential bacterial candidates Bacterial Species Detection Method Microbiologic Characteristics Campylobacter Campylobacter Skirrow agar Curved gram-negative rod Rapidly motile E.coli MacConkey, EMB, or SM agar Gram-negative rod Lactose-producing Salmonella MacConkey, EMB, XLD, or HE agar Non-lactose H2S-producing Lysine decarboxylator

68 Image source Slide 7-http://enfo.agt.bme.hu/drupal/sites/default/files/Campylobacter%20jejuni.JPG Slide 10-https://foodlawlatest.files.wordpress.com/2014/07/e-coli-o157-h7.jpg Slide 14-http://www.dzif.de/fileadmin/user_upload/news/zeitlos/bacteria/Salmonella_typhimurium_490.jpg Slide 16-http://atlas.microumftgm.ro/bacteriologie/bactsp/shigella/frotiu/pic/sh_me.jpg Slide 29-http://apchute.com/wellmeyer/media/McConUnin.jpg Slide 31- https://www.dlsweb.rmit.edu.au/set/LearningObjects/FoodMicroTutes/images/S4Dscn1249.jpg Slide 33- https://lh4.googleusercontent.com/-UCdKPz8K9hU/VL-dXLvc4gI/AAAAAAAAAP8/yQCjv1KKYY8/w318-h324/1021.png Slide 35-http://www.oxoid.com/omd/library/fullsize/CM0813.jpg Slide 37-http://files.constantcontact.com/fbc /84a3781c-f3c1-4c5f-af1c-906fe7fbd227.jpg Slide 44- Slide 47-http://iws2.collin.edu/dcain/CCCCD%20Micro/LIA.jpg Slide 49-http://laboratoryinfo.com/wp-content/uploads/2016/01/gram-positive-vs-gram-negative.png Slide 52-https://www.cdc.gov/meningitis/lab-manual/images/chapt7-figure04.gif

69 Image source Slide 54-http://www.telmeds.org/wp-content/uploads/2009/10/CatalaseResults1.jpg Slide 56-http://www.scielo.br/img/revistas/bjm/v43n2/13f03.jpg Slide 58-http://www.studyread.com/wp-content/uploads/2016/09/Elisa-test-direct.jpg Slide 60-https://www.cdc.gov/meningitis/lab-manual/images/chapt11-figure01.gif; Slide 62-https://www.cdc.gov/groupbstrep/images/lab-agglutinationtests-lg.jpg Text source from the Wiki page generated

70 Thank you!