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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, , 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 method Acridine Orange Stain

Reagents: Absolute methanol Acridine orange

Purpose: Enhances ability to visualize 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 agar MacConkey agar (MAC) 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 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) difficile Cystine-tellurite blood agar (CTBA) or Tinsdale diphtheriae Löwenstein-Jensen (LJ) 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 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 , (up to 10 % strains are negative) A disc negative (no zone of inhibition) : 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 monocytogenes. Differentiation of and 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 ( 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 – GPC in clusters – 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. 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 (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 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

Hektoen Enteric agar Motility MacConkey agar LIA ONPG # 4 (SIM)

Urea Hektoen Enteric agar STEC Laboratory Identification

Morphology differentiation on BAP, MAC, HE or XLD is not possible SMAC, CHROM agar, MUG STEC toxin testing should be performed on all stools (not all STEC positive stools contain blood) Can be done directly from stool samples, plates or from broth culture (MacConkey broth) Immunoassay method detect Stx1 and Stx 2 Detects both O157 STEC and other non-O157 STEC strains Case Summary #5 A 21-year-old female college student volunteering in Ghana, Africa noticed she had received a bug bite during the night on her right forearm (4 days before she was scheduled to fly home to Michigan). She developed a red, raised inflamed area around the bite that worsened and became extremely painful. She was seen by a physician the day after arriving home in the States (5 days post bite). A culture was ordered and sent to microbiology and a punch biopsy was sent for pathology analysis. Case Summary #5 #5 Blood agar

Catalase

Coagulase

Direct Gram stain Case #5 Key Reactions

Gram stain: GPC in clusters BAP: Cream colored colonies, large zone of beta hemolysis Catalase: positive : positive Latex agglutination Testing: positive (detection of cell wall Protein A and clumping factor) Case #5 Considerations

What other testing needs to be done with Staphylococcus aureus infections? Determination of MRSA, along with AST What other organisms can be isolated as possible cause of skin infection? (BHS Group A) – GPC, chains Pseudomonas aeruginosa – pale and thin GNR’s Pasteurella multocida – GNCB BAP/A disc #5 Catalase

Gram stain

JMS #5 BAP MAC

O/F Glucose

Oxidase

Pseudo F and P agar #5 Catalase

Spot Indole BAP, Choc & MAC

Oxidase Case Summary #6

A 15-year-old male was brought into the ED by his sister. He complained of 24 hours of dysuria and a white substance draining from his penis. He admitted to having several sexual partners in the last 6 months. Observe Gram stain and expected lab results for the organism responsible for this infection. #6 Direct Gram stain from penile drainage

JMS #6 Oxidase

Choc or TM agar NET Rapid NH Case #6 Key Reactions Gram stain: GNDC Choc or Thayer Martin agar: small, gray to tan and translucent colonies Oxidase: positive Chromogenic substrate (NET or Bacticard Neisseria): positive for the enzyme hydroxyprolylaminopeptidase (PRO) Acid production from CHO’s: positive for Glucose only Molecular Methods of Detection: Many nucleic acid amplification tests are available and is most often used for diagnosis of GC in urogenital specimens Case #6 Considerations

Is the Gram stain diagnostic for the patient in the Case study? Yes, the presence of intracellular GNDC in males is diagnostic, and is positive in 95-100% of male infections. Female cervical specimen Gram stains are not diagnostic due to lower positive rates and interference with reading by other similar commensal NF organisms. What other organisms can be isolated as possible cause of urogenital infections? Gardnerella vaginalis – GVR vaginalis – parasite (flagellate) – budding yeast and pseudohyphae #6 Direct Gram stain (BV)/Gardnerella vaginalis

JMS #6 Direct Gram stain (clue cell) Bacterial Vaginosis (BV)/Gardnerella vaginalis

JMS #6 Direct Gram stain Trichomoniasis/

JMS #6 Direct Gram stain Vulvovaginal candidiasis/Candida albicans

JMS