Bac Exam 3

What does a pink colony on MacConkey (MAC) mean?

It ferments lactose (lactose fermenter, LF). Acid lowers the pH and the neutral red indicator turns the colony pink.

What does a colorless colony on MAC mean?

It does not ferment lactose (non-lactose fermenter, NLF).

Why is lactose use so important for sorting gram-negative rods?

It is the first split of the enteric flowchart. LF: E. coli, Klebsiella, Enterobacter, Citrobacter. NLF: Salmonella, Shigella, Proteus group, Serratia, Yersinia.

Oxidation versus fermentation: which needs oxygen?

Oxidation needs oxygen and makes less acid. Fermentation does not need oxygen and makes acid (and sometimes gas) from the carbohydrate.

What can carbohydrate fermentation produce, and why add a pH indicator?

Acids, gas and other end products. The pH indicator shows the acid by changing color.

Which medium tells a fermenter from an oxidizer?

OF (oxidative-fermentative) medium: an open tube and a tube sealed with oil. Acid in both = fermenter. Acid in the open tube only = oxidizer.

What is TSI used for?

Triple Sugar Iron agar shows glucose, lactose and sucrose fermentation, H2S production and gas production in one tube.

What ingredients does TSI use to detect each result?

Glucose (1x), lactose and sucrose (10x). Phenol red shows pH change. Ferrous sulfate plus thiosulfate detects H2S (black).

How do the TSI slant and butt differ?

The slant is exposed to oxygen (aerobic). The butt has little oxygen (anaerobic).

How is a TSI tube inoculated and incubated?

Stab the butt, then streak the slant. Cap loosely. Incubate at 35-37 C for 18-24 hours.

How is a TSI reaction written, and what do A and K mean?

Slant/butt. A = acid (yellow). K = alkaline (red).

TSI K/K (red/red): what does it mean?

No fermentation of glucose, lactose or sucrose. Think nonfermenter.

TSI K/A (red/yellow): what does it mean?

Glucose fermented only. Lactose and sucrose were not fermented.

TSI A/A (yellow/yellow): what does it mean?

Lactose and/or sucrose fermented (plus glucose).

Why can a glucose-only fermenter give a yellow butt but a red slant?

Glucose is only 0.1%, so it runs out. On the aerobic slant the organism then uses peptones, which makes alkaline products that cancel the small amount of acid. The anaerobic butt stays acidic.

How is H2S seen in TSI and what do you assume about a black butt?

Black precipitate (ferrous sulfide). H2S forms only in acid, so assume the butt is acidic even if the black hides the yellow.

How is gas recognized in TSI? What are aerogenic and non-aerogenic?

Cracks, bubbles or lifting of the agar. Aerogenic = makes gas. Non-aerogenic = no gas.

Compare delayed lactose fermenters (dLF) with NLF on MAC and TSI.

Both can look like NLF at 24 hours (colorless on MAC, K/A on TSI). A dLF turns pink later because it ferments lactose slowly.

What is the ONPG test for and how does it separate a dLF from an NLF?

ONPG detects beta-galactosidase. dLF is ONPG positive (yellow). True NLF is ONPG negative (no color).

How do permease and beta-galactosidase explain delayed lactose fermentation?

Permease moves lactose into the cell and beta-galactosidase splits it. A dLF has beta-galactosidase but little or slow permease, so fermentation is delayed.

What does a yellow ONPG tube mean? No color change?

Yellow = beta-galactosidase present (o-nitrophenol released). No color change = no beta-galactosidase.

Methyl red (MR): what does it detect?

Mixed acid fermentation of glucose (stable acids, pH below 4.4). Positive = red.

Voges-Proskauer (VP): what does it detect?

The butanediol (acetoin) pathway of glucose fermentation. Positive = red.

How are MR and VP related?

Usually opposite. An organism is typically MR+/VP- or MR-/VP+.

What does a decarboxylase test determine and which amino acids are emphasized?

Whether the organism removes the carboxyl group of an amino acid. Lysine (LDC) and ornithine (ODC) are emphasized.

What products form when lysine and ornithine are decarboxylated?

Lysine gives cadaverine. Ornithine gives putrescine. Both are alkaline amines.

What is in Moeller decarboxylase medium and why must it be acidic and anaerobic?

Peptone, glucose, a pH indicator (bromcresol purple), cofactor and the amino acid. Decarboxylase works at acidic pH and without oxygen.

Why is a Moeller tube overlaid with mineral oil, and what happens first?

Oil keeps it anaerobic. The organism first ferments glucose, which makes the medium acidic (yellow) and turns the enzyme on.

How does a positive decarboxylase create an alkaline shift? Purple versus yellow?

Amine products raise the pH. Purple = positive. Yellow = negative.

Why inoculate a control tube along with the test tube?

The control has no amino acid. It must stay yellow. If it turns purple, the result is invalid.

What does phenylalanine deaminase (PAD) detect? Reagent and positive color?

Conversion of phenylalanine to phenylpyruvic acid. Add ferric chloride. Positive = green. PAD+ means Proteus, Morganella, Providencia.

What does the citrate test determine? What color change is positive?

Whether citrate is the sole carbon source. Simmons citrate: green to blue.

What are the sole carbon and nitrogen sources in citrate medium?

Carbon = citrate. Nitrogen = ammonium salts. Using them makes the pH alkaline.

What does the indole test detect, and which enzyme?

Indole from tryptophan, made by tryptophanase.

Compare Kovac's, Ehrlich's and spot indole.

Kovac's and Ehrlich's: reagent added to a tryptophan broth culture, red/pink ring positive. Spot indole: reagent on filter paper with a colony, blue-green positive.

How is the culture prepared before Ehrlich's or Kovac's indole?

Grown in a tryptophan-containing broth, then reagent is added.

What does malonate test for and what is positive?

Use of malonate as the sole carbon source. Positive = green to blue.

How is motility tested and what is positive?

Stab semisolid medium. Motile = growth spreads away from the stab line (cloudy).

What does nitrate reduction test, and what reagents?

Reduction of nitrate to nitrite (and possibly to N2). Add reagent A (sulfanilic acid) and B (alpha-naphthylamine). Red = nitrite present = positive.

Nitrate: no color after A and B. Why is it ambiguous and what next?

Either nitrate was not reduced or it was reduced all the way to N2. Add zinc dust.

After zinc is added, how do you read nitrate?

Red = nitrate still present, so a true negative. No color = nitrate was already reduced past nitrite, a true positive.

What does the oxidase test detect and what does it separate?

Cytochrome c oxidase. Enterobacteriaceae are negative. Pseudomonas, Vibrio, Aeromonas, Campylobacter are positive.

How are positive oxidase and urease recognized?

Oxidase: purple color quickly on the reagent. Urease: pink/magenta (urea to ammonia raises the pH).

What does the urease test detect?

Urease splits urea into ammonia and CO2, making the medium alkaline (pink).

What is the API 20E used to identify?

Enterobacteriaceae and other gram-negative rods, using 20 miniaturized biochemical tests.

What do ONPG, ADH, LDC, ODC, CIT, H2S, URE, TDA, IND, VP, GEL stand for in API 20E?

ONPG test, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate, H2S, urease, tryptophan deaminase, indole, Voges-Proskauer, gelatinase.

Which API 20E tests are fermentation of sugars?

GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, ARA (glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, arabinose).

How did identification change from before the 1950s to the late 1970s?

From individual conventional tubes to miniaturized multi-test systems (like the API) that are faster and standardized.

What advantages do semi-automated identification and susceptibility instruments give?

Speed, less labor, standardization, and identification with susceptibility in one system.

A gram-negative rod gives no reactions in 24 h on an Enterobacteriaceae kit. What does that suggest?

A nonfermenting gram-negative rod.

What defines Enterobacteriaceae as a group?

Gram-negative rods, facultative anaerobes, oxidase negative, ferment glucose, reduce nitrate to nitrite. Most are motile (Klebsiella and Shigella are nonmotile).

Which media are used for enterics and what do MAC versus HE/XLD show?

MAC separates LF (pink) from NLF. HE and XLD are more selective and help pick out Salmonella and Shigella from normal flora.

How do O, H and K antigens differ?

O = somatic (cell wall LPS), heat stable. H = flagellar, heat labile. K = capsular, heat labile.

Opportunistic versus primary enteric pathogens? Which three genera are primary?

Opportunists cause disease mostly in compromised hosts or outside the gut. Primary pathogens cause disease in healthy people: Salmonella, Shigella, Yersinia.

What is CRE and what does the Xpert Carba-R detect?

Carbapenem-resistant Enterobacterales (resistant to imipenem, meropenem, doripenem, ertapenem). The Xpert Carba-R is a real-time PCR for carbapenemase genes (KPC, NDM, VIM, OXA-48, IMP).

How does the flowchart first split enterics?

By lactose fermentation on MAC. LF: E. coli, Klebsiella, Enterobacter, Citrobacter. NLF: Salmonella, Shigella, Proteus group, Serratia, Yersinia, Edwardsiella.

LF flowchart: how do E. coli, Citrobacter, Enterobacter, Klebsiella separate?

MR, citrate, motility/ODC, lysine and indole. E. coli: MR+, citrate-, indole+. Citrobacter: citrate+. Enterobacter: motile, ODC+. Klebsiella: nonmotile, ODC-.

E. coli biochemical pattern?

Pink LF on MAC, indole +, MR +, VP -, citrate -.

How does citrate separate E. coli from Citrobacter?

E. coli is citrate negative. Citrobacter is citrate positive.

Klebsiella: biochemical and colony characteristics?

LF, mucoid (large polysaccharide capsule), nonmotile, ODC negative, VP positive, citrate positive.

How does the Klebsiella capsule affect colonies and protect it?

Makes colonies large and mucoid. Protects against phagocytosis.

K. pneumoniae: major infections and patient groups?

Pneumonia (alcoholics, diabetics), UTI, bacteremia, nosocomial infections in debilitated patients.

How does indole separate K. oxytoca from K. pneumoniae?

K. oxytoca is indole positive. K. pneumoniae is indole negative.

Enterobacter versus Klebsiella: motility and ODC?

Enterobacter is motile and ODC positive. Klebsiella is nonmotile and ODC negative. Both can look similar on MAC.

Which Enterobacter species are emphasized and how does lysine separate them?

E. cloacae and E. aerogenes. E. aerogenes is lysine decarboxylase positive. E. cloacae is lysine negative. Both are ODC positive.

Serratia: how is it identified among NLFs?

MR negative, DNase positive, may ferment lactose late. Can produce red pigment.

Which Serratia produces brick-red pigment and why might clinical isolates lack it?

S. marcescens. Pigment is often not made at 35 C (more at room temperature) and many clinical isolates are nonpigmented.

Infections and outbreaks linked to S. marcescens?

Hospital outbreaks: UTI, respiratory, wound and bloodstream infections.

S. odorifera: odor?

A musty, rotten-potato odor (biogroups 1 and 2). This detail is standard microbiology, not from your slides.

Hafnia alvei: what is distinctive?

Found in the gut of animals and humans. Rarely a human pathogen. NLF with delayed citrate positivity.

Which genera make up Proteeae and what unique trait?

Proteus, Morganella, Providencia. All NLF and PAD positive.

Infections caused by P. mirabilis and P. vulgaris?

UTIs (including stones from urease and alkaline urine), wound infections, bacteremia.

What does Proteus swarming look like?

Concentric waves spreading over nonselective agar (blood agar) and a burned-chocolate odor.

What do P. mirabilis and P. vulgaris share and how does indole separate them?

Both: swarming, PAD+, H2S+, urease+. P. mirabilis is indole negative. P. vulgaris is indole positive.

Morganella morganii: how does it differ from Proteus?

PAD positive but does not swarm and does not make H2S.

Providencia versus Morganella: citrate? Which Providencia are important?

Providencia is citrate positive, Morganella is negative. P. stuartii (UTI, burn units), P. alcalifaciens (diarrhea), P. rettgeri (UTI).

Edwardsiella tarda: biochemical pattern, and what separates it from Salmonella?

NLF, MR+, H2S+, PAD-. Indole positive separates it from Salmonella (indole negative).

Which Edwardsiella is the human pathogen and what does it cause?

E. tarda: gastroenteritis, wound infections (often after aquatic exposure), bacteremia.

Citrobacter: infections linked to C. freundii and C. koseri?

C. freundii: UTIs and opportunistic infections. C. koseri: neonatal meningitis and brain abscess.

Citrobacter versus E. coli: lactose, MR, citrate?

Citrobacter is a (delayed) LF, MR+, citrate positive. E. coli is LF, MR+, citrate negative.

Why can a dLF Citrobacter be mistaken for Salmonella?

In stool cultures it can look NLF at 24 h and is H2S positive and citrate positive like Salmonella.

Salmonella: biochemical profile?

NLF, MR+, motile, PAD-, H2S+, indole -.

Salmonella on MAC, HE and XLD?

Colorless on MAC. On HE/XLD colonies have black centers (H2S).

Salmonella species and the typhoid serotype?

S. enterica and S. bongori. Serotype Typhi causes typhoid fever.

How is Salmonella acquired from animals? Virulence factors?

Eating contaminated animal products (poultry, eggs, dairy) or contact with animals (reptiles). Virulence: invasion of intestinal cells and survival inside macrophages.

Four clinical outcomes of Salmonella infection?

Gastroenteritis, typhoid fever, nontyphoidal bacteremia, carrier state.

Salmonella gastroenteritis: incubation, course, antibiotics?

Hours to about 2 days. Fever, cramps, diarrhea. Usually self-limited. Antibiotics are usually not given.

How does typhoid differ from Salmonella gastroenteritis?

Typhoid: S. Typhi, human source only, longer incubation (1-3 weeks), invasive systemic enteric fever, serious.

Typhoid diagnostic timing?

Blood culture is positive first (week 1). Stool and urine cultures later. Serology later still.

Shigella versus normal flora?

Close relative of E. coli. NLF (or late), MR+, nonmotile, H2S negative. Causes dysentery.

Four Shigella species and how do they separate?

S. dysenteriae, S. flexneri, S. boydii, S. sonnei, separated by mannitol, ONPG, ODC and serogroup. S. sonnei is ODC positive and the most common in the US.

Why plate Shigella specimens immediately?

Shigella is fragile and dies quickly in stool.

Why is shigellosis highly communicable? Reservoir? Routes?

Very low infectious dose (10-100 organisms). Humans are the reservoir. Fecal-oral, contaminated food, water, flies, person to person.

Shigella symptom progression?

First 24-48 h: fever, cramps, watery diarrhea. Later: bloody, mucus-containing stools (dysentery).

Three Yersinia species that infect humans?

Y. pestis (plague), Y. enterocolitica (gastroenteritis, appendicitis-like illness), Y. pseudotuberculosis (mesenteric adenitis).

Y. pestis: transmission and three forms?

Flea bite from rodents. Bubonic (buboes), septicemic (bloodstream), pneumonic (lungs, person to person).

Y. pestis microscopy and growth?

Bipolar safety-pin staining. Grows best at 25-30 C. Category A bioterror agent.

How is Y. enterocolitica acquired and why does refrigeration not stop it?

Contaminated food (pork, milk). It grows at refrigerator temperature (cold tolerant).

Y. enterocolitica gastroenteritis versus appendicitis-like syndrome?

Gastroenteritis in young children (diarrhea, fever, sometimes bloody). Older children and adults: right lower quadrant pain mimicking appendicitis (mesenteric adenitis).

What is CIN agar used to detect?

Yersinia enterocolitica (cefsulodin-irgasan-novobiocin).