Microbiology Review: Bacteriological Cases Jeanne Stoddard MT (ASCP) MHS ASCLS-MI Meeting Kellogg Conference Center East Lansing, MI April 1, 2016 Objectives 1. Review bacteriological media and stains utilized in the clinical laboratory. 2. Recognize clinical picture for commonly isolated bacterial pathogens. 3. Correlate plate morphology, Gram stain, and biochemical results with organism identification. Stain and Media Review Gram positive yeast and hyphae Gram Stain Reagents: Crystal violet Iodine Acetone alcohol Safranin JMS Gram negative organisms stain red Gram positive organisms stain purple/blue Acid Fast Stain-Kinyoun Reagents: Carbolfuchsin Acid/Alcohol Methylene Blue JMS Acid fast positive organisms stain red (magenta) Acid fast negative organisms stain pale blue Fluorescent AFB Detection- Auramine-Rhodamine Reagents: Auramine O & Rhodamine Acid-alcohol Potassium permanganate Purpose: Screening for detection of mycobacteria Mycobacteria stain bright yellow-orange against a dark greenish-black background Confirm positives with Kinyoun staining method Acridine Orange Stain Reagents: Absolute methanol Acridine orange Purpose: Enhances ability to visualize bacteria Useful when not sure if NOS All MO’s stain a fluorescent bright orange KOH and Calcofluor White Stain Reagent: 10% KOH & Calcofluor white Purpose: JMS KOH dissolves keratin and other debris to make fungi more visible CalcoflUor dye is absorbed by chitin in fungal cell wall and fluoresces blue-white Media Media Routine-for set up and general purpose Blood agar MacConkey agar (MAC) Chocolate agar CNA agar Enriched-additional nutrients to support fastidious bacterial growth Blood, Choc, Thayer Martin Selective and Differential Media MAC, EMB, HE , Others MacConkey Agar Selective-Crystal violet and bile, inhibits GPO’s and fastidious GNR’s Differential-Lactose and neutral red, pH shift enables identification of Lactose fermenting GNR’s Staphylococcus aureus Escherichia coli Salmonella typhimurium JMS Selective and Differential Media for GNR’s HE XLD EMB JMS Differential Media Used on Stool Cultures Organism MacConkey (MAC) Hektoen-Enteric Xylose-Lysine- (HE) Deoxycholate (XLD) Escherichia coli and LF Bright orange to Yellow colonies other lactose Dark pink, salmon colored fermenters sometimes mucoid colonies colonies Salmonella species NLF Green (colorless) to Red colonies with Colorless colonies blue green colonies black centers with black centers Shigella species NLF Green (colorless) Colorless colonies Colorless colonies colonies Yersinia NLF Salmon colored Yellow or colorless enterocolitica Colorless colonies colonies colonies More Media Sorbitol MacConkey (SMAC) E. coli O157:H7 Skirrow’s medium Campylobacter spp. V-agar Gardnerella vaginalis Buffered Charcoal Yeast Extract (BCYEα) Legionella Bordet-Gengou or Regan-Lowe medium Bordetella pertussis Thayer Martin agar Neisseria gonorrhoeae More Miscellaneous Media Thiosulfate-citrate-bile salts-sucrose agar (TCBS) Vibrio cholerae Cycloserine cefoxitin fructose agar (CCFA) Clostridium difficile Cystine-tellurite blood agar (CTBA) or Tinsdale Corynebacterium diphtheriae Löwenstein-Jensen (LJ) Mycobacterium tuberculosis Oxidative-Fermentative base- Polymyxin B- Bacitracin-Lactose (OFPBL) Burkholderia cepacia Case Studies for Review Case Summary #1 A 21 year old pregnant woman vaginally delivered a female infant at term, 15 hours after her water broke. She had good prenatal care. The newborn appeared normal at birth, but at 24 hours developed labored breathing and a fever. Blood and CSF cultures were obtained from the infant. Observe test results on the following slides and use to make an organism identification. #1 Blood Culture Gram Stain JMS #1 BAP Catalase CAMP test BE Na Hippurate #1 Antigen Latex Agglutination Case #1 Key Reactions Gram Stain: GPC, pairs and chains BAP: 24o colonies are small, translucent with a small zone of beta hemolysis, (up to 10 % strains are negative) A disc negative (no zone of inhibition) Catalase: negative Bile esculin: negative (no change) Sodium hippurate: positive (purple) CAMP: positive with arrow of hemolysis Strep Antigen: positive (agglutination) Group B Case #1 Considerations How is this infection acquired? Strep Group B is passed vertically from a colonized mother to infant during vaginal birth process, obstetrical complications are a predisposing factor. How could it have been prevented? Diagnosis in the mother at 35-37 weeks gestation with vaginal/rectal culture screen, prophylactic treatment of the mother with penicillin or ampicillin should prevent transmission of Early onset disease in newborn (< 7 days old). What other organisms need to be ruled out when newborn sepsis or meningitis is considered? Most common etiology for newborn sepsis and meningitis is Gp B strep, E. coli, and Listeria monocytogenes. Differentiation of Listeria monocytogenes and Streptococcus agalactiae Organism/ Colony Gram Esculin Sodium CAMP Catalase Test morphology stain hydrolysis hippurate test BAP Listeria Flat, grey GPR positive positive + + monocytogenes with small or rectangle zone of beta GPCB hemolysis Streptococcus Translucent, GPC negative positive + – agalactiae raised with pairs arrow small zone and of beta chains hemolysis BAP CAMP test CSF Gram stain Sodium hippurate Catalase SIM (RT) BE Case Summary #2 An expectorated sputum arrives in the laboratory from a 57-year old male with a diagnosis of pneumonia and a 30 year history of smoking. Observe the culture plates and biochemical results given and give most likely organism identification (Genus and species). #2 Sputum Direct Gram Stain (10X) JMS #2 Sputum Direct Gram Stain (100X) JMS TSIA #2 Urea Citrate SIM VP Case #2 Key Reactions BAP and Choc: Grey, mucoid colonies MAC: Mucoid, LF Indole: negative (NO red ring) Motility: negative (no growth away from stab) Voges-Proskauer: positive (red) Citrate: positive (blue) Urea: positive (magenta pink) TSIA: A/A G Case #2 Considerations Direct smear Gram stain should be helpful with preliminary information to clinician. What other bacteria can be found as common etiology of pneumonia? Haemophilus influenzae –Tiny pleomorphic GNR’s or GNCB Staphylococcus aureus – GPC in clusters Streptococcus pneumoniae – GPC, pairs, lancets and chains #2 Sputum Direct Gram Stain JMS #2 BAP, MAC and Choc X and V factor disks QUAD agar #2 Sputum Direct Gram Stain JMS #2 Sputum Direct Gram Stain JMS #2 Gram stain BAP P disk Catalase JMS Case Summary #3 A 19 year old female with a past history of urinary tract infection presents to the ED with complaints of left flank pain, fever, chills and a noted increase in urinary frequency. Urine from a clean-catch, mid-stream (CCMS) specimen is plated using a 1.0 µl (0.01 ml) calibrated loop which reveals the following results: #3 Urine Culture-Workup? JMS MAC #3 Motility Urea TSIA MR VP Indole Citrate Case #3 Key Reactions BAP: Grey colonies, beta hemolytic MAC: LF colonies Indole: positive (red ring ) or positive spot test Methyl red (MR): positive (red) Voges Proskauer (VP): negative (no change) Citrate: negative (green) Motility: positive Urea: negative TSIA: A/A gas Case #3 Considerations Urine Culture Quantitaion- Plate 1 µl; 1 colony = 1000 CFU/ml Plate 10 µl; 1 colony = 100 CFU/ml Clean catch urine: > 105 CFU/ml Cath urine: >104 CFU/ml Common bacteria causing UTI = Escherichia coli Other Enterics Enterococcus faecalis Staphylococcus saprophyticus #3 MAC and BAP ODC TSIA Urea Spot and Tube Indole PDA #3 MacConkey agar LIA IMViC Motility BAP #3 Catalase Gram stain BE 6.5% NaCl JMS #3 BAP Catalase Novobiocin disc Gram stain Case Summary #4 A 45-year old man hosted a final four basketball tournament party at his house, where he served barbequed chicken. Two days after the party, several of his friends developed abdominal pain, fever and diarrhea. Exam of the stool samples submitted by these patients revealed the presence of RBC’s and WBC’s. Stool culture results are seen in subsequent slides. All patients recovered in 7-10 days. #4 Gram stain Growth at 42oC and in microaerophilic environment #4 Catalase Campy BAP growth Oxidase Sodium hippurate Case #4 Key Reactions Gram stain: GNR’s, “bird’s wings” or “gull wings” BAP: mucoid and grey, colonies coalesce Oxidase: positive Catalase: positive Sodium hippurate: will differentiate species C. jejuni = positive C. coli, C. lari, and C. fetus = negative Latex agglutination tests also available Stool Pathogen Analysis of Blood, WBC’s, Mucus, and Fever Organism or Toxin Blood (RBCs) WBCs Mucus Fever Enteropathogenic E. - - (rare) +++ No coli (EPEC) Enterotoxigenic E. coli - - - No (ETEC) Enterohemorrhagic ++ -/± - No E.coli O157:H7 (EHEC) Enteroinvasive E. coli + ++ + Yes (EIEC) Enteroaggregative - - ++ No E.coli (EAEC) Campylobacter jejuni ± + + Yes Salmonella spp. ± ± - Yes Shigella spp. + + + Yes Aeromonas hydrophila V V V V Yersinia enterocolitica + + - Yes Plesiomonas - V - V shigelloides Vibrio cholera - - - No Vibrio parahemolyticus ± + - Yes Clostridium difficile - V - Yes (toxin) Viruses - - - No Case #4 Considerations What complications need to be considered with Campylocbacter infections? Guillain-Barré syndrome and reactive arthritis Name primary plating media used routinely for stool cultures? Blood agar MacConkey agar Hektoen Enteric agar Campy BAP (or Skirrow’s or Campy CVA) What fecal pathogens have to be considered as possible cause of gastroenteritis when working up a routine stool culture? Salmonella Shigella (4 serogroups) Campylobacter species STEC MacConkey agar XLD agar # 4 TSIA LIA SIM Urea