03 - AEROBIC GRAM-NEGATIVE BACTERIA (Exam # 3) Flashcards
Explain the principle of the Indole test.
Detects bacteria who produce tryptophanase, an enzyme needed to breakdown tryptophan into indole, pyruvate, and ammonia.
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the procedure and interpretation of the spot indole test.
Procedure:
1. Saturate a piece of filter paper with DMACA indole reagent (1% p dimethylaminocinnamaldehyde in 10% HCl).
2. Using a sterile wooden stick or loop, pick up several isolated 18-24 old colonies growing on a 5% sheep blood agar plate and smear the isolate onto the saturated filter paper.
Interpretation:
Positive: Blue color change within 20 seconds
Negative: No color development or a pinkish color
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the procedure and interpretation of Ehrlich’s indole test.
Procedure:
1. Aliquot 2 mL of indole broth, incubated at 35-37 degrees C for 24-72 hours, to a separate tube.
2. Add 1 mL of xylene and shake tube.
3. Allow tube to stand so that solvent rises to the surface
4. Dispense 5 drops of Ehrlich’s reagent (p-Dimethylaminobenzaldehyde) down the side of the tube. Do not shake.
Interpretation:
Positive test= Pink/red ring between broth and solvent within 15 minutes
Negative Test= No color development in the form of aring within 15 minutes.
- Summarize the principle of differential tests and reagent(s) needed to identify various gramnegative bacilli.
Explain the procedure and interpretation of Kovacs’ indole test.
Procedure:
1. Aliquot 2mL of indole broth, which has been incubated overnight at 35-37 degrees C to a separate tube.
2. Dispense 5 drops of Kovacs’ reagent (p-dimethylaminobenzaldehyde) down the side of the tube. Shake gently.
Interpretation:
Positive test= A pink/red ring between broth and solvent
Negative test= No color development
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the principle of the MUG Test (4-Methylumbelliferyl-β-D-Glucuronide)
Detects the enzyme beta-glucuronidase, which is used to cleave the compound 4-Methylumbelliferyl-β-D-Glucuronide(MUG) resulting in methlymbelliferone, a fluorescen compound. Most strains of E.coli produce beta-glucuronidase.
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the principle of the oxidase test.
Detects bacteria that make cyctochrome oxidase, an enzyme that reduces molecular oxygen in the electron transport chain. The test utilizes an artifical electron donor that turns indophenol blue when oxidzed by cytochrome C.
The artifical electron donor can be either be tetramethyl-p-phenylenediamine dihydrochloride ( Kovacs’ oxidase reagent) or dimethyl-p-phenylenediamine dihydrochloride (Gordon and McLeod reagent).
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the principle of the Triple Sugar Iron (TSI) Test.
Differential medium used for gram-negative enteric organisms. Detects the ability of bacteria to ferment glucose, lactose, sucrose, and produce hydrogen sulfide gas.
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the procedure and interpretation of the MUG Test (4-Methylumbelliferyl-β-D-Glucuronide)
Procedure:
Disk Method
1. Place a MUG Disk in an empty sterile petri dish and add
one drop of demineralized water. Alternatively, the
disk can be placed directly on the agar surface, in which
case, it will not require the addition of water because
moisture from the medium will rehydrate the disk.
2. Smear 2-3 isolated colonies on the disk.
3. Place a piece of filter paper saturated with water in the
lid of the petri dish to provide a humid environment.
4. Incubate aerobically for up to 30 minutes at 35-37°C.
5. Following incubation, examine the disk for fluorescence
using a longwave ultraviolet light (360 nm) in a darkened
room.
Tube Method
1. Add 0.25 ml of demineralized water to a clean, plastic
or glass tube.
2. Make a heavy suspension of the test isolate (3-5
colonies from an 18-24 hour blood agar plate) in the
tube.
3. Using forceps place a MUG Disk in the tube and shake
vigorously to ensure adequate elution of the substrate
in the surrounding liquid.
4. Incubate aerobically for 1 hour at 35-37°C.
5. Following incubation, examine the tube for
fluorescence using a longwave ultraviolet light (360
nm) in a darkened room.
Interpretation:
* Positive =blue fluorescence
* Negative = no fluorescence
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Which oxidase reagent is more sensitive?
Tetramethyl-p-phenylenediamine dihydrochloride (Kovacs’ oxidase reagent)
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the procedure and interpretation of the oxidase test.
Procedure:
1. Soak a small piece of filter paper in 1% Kovács oxidase reagent (or Gordon and McLeod reagent) and let dry.
2. Use a loop and pick a well-isolated colony from a fresh (18- to 24-
hour culture) bacterial plate and rub onto treated filter paper.
3. Observe for color changes.
Interpretation:
Positive reaction = purple or deep blue color change within 30 seconds
Weak positive reaction = purple or blue color within 30 to 60 seconds
Negative reaction = no color change in 60 seconds
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the principle of the urease test.
Bacteria who produce the enzyme urease are able to break down urea into ammonia. The medium contains urea and phenol red.
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain how to innoculate a Triple Sugar Iron (TSI) slant.
Stab the butt and streak the slant. Incubate with caps loosened at 35 °C in an ambient incubator and examine after 18–24 h for carbohydrate fermentation, gas production and hydrogen sulfide production. Any combination of these reactions may be observed. Do not incubate longer than 24 h because the acid reaction in the slant of lactose and sucrose fermenters may revert to an alkaline reaction.
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain what A, K, H2S, and G mean in relation to triple sugar iron agar slants.
Reactions include:
- Acid reaction (A) = yellow color
- Alkaline reaction (K) = red color
- Hydrogen sulfide production (H2S) = black color or precipitate
- Gas production (G) = bubbles, cracks, or media displacement
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain what is happening in each test tube including, what sugar(s) is (are) being fermented as well as the type of gas production.
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Explain the procedure and interpretation of the urease test.
Procedure:
1. Incoulate urea agar with a heavy inoculum from a pure,18-24 hours culture, streaking back and forth over the slant. Do not stab the butt.
2. Incubate in ambient air with cap loosened at 35-37 degrees C.
3. Examine for color development after 2,6, 24 hours, and daily for up to 6 days.
Interpretation:
Positive reaction = intense pink-red color development
Negative reaction = No color change
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
Enterobacterales are oxidase _______
- Oxidase negative (except Plesiomonas),
- Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales
order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions
for identification.
What sugar do all Enterobacterales ferment?
glucose
- Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales
order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions
for identification.
What makes MacConkey agar selective?
Crystal violet
Bile salts
2.Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions for identification
What makes MacConkey agar differential?
Lactose
2.Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions for identification
Enterobacterales have the ability to reduce nitrate (NO3) to
Nitrites (NO2)
Red color indicates presence of nitrites after reagents are added
2.Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions for identification
What color are lactose fermenters on MacConkey agar?
Pink
2.Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions for identification.
What color are lactose nonfermenters on MacConkey agar?
Clear
2.Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions for identification
What two reagents are used in the nitrate reduction test?
- N,N-dimethyl – a -naphthylamine
- Sulfanilic acid
1.Summarize the principle of differential tests and reagent(s) needed to identify various gramnegative bacilli.
Most members of Enterobacterales are motile by what type of flagella?
Peritrichous
- Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactionsfor identification.
Which 2 organisms are Atrichous (lacking flagella) making them nonmotile?
Klebsiella and Shigella
Exception: Klebsiella aerogenes
- Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactionsfor identification.
2.3. Shigella spp.
2.5. Klebsiella spp.
Which organisms are nonmotile at 35 degrees C but motile at room temp 22 degrees C?
Yersinia enterocolitica and Yersinia pseudotuberculosis
- Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactionsfor identification.
2.16. Yersinia spp.
2.16.1. Yersinia enterocolitica
What are the 3 antigens that can be used to serologically group species of Enterobacterales?
- O antigen
- H antigen
- K antigen
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
The O antigen is located on the _______
and is heat________
- Cell wall (somatic antigen)
- Heat Stable
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
The H antigen is located_____
and is heat_________
- On the suface of the flagella (flagellar antigen)
- Heat-labile
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
The K antigen is a ______ antigen
and is heat______
- Capsular Antigen (found only in certain encapsulated species)
- Heat-labile
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
Enterobacterales, with a few exceptions, reside normally in what part of the body?
Gastrointestinal (GI) tract
5.Contrast the natural habitat of the Enterobacterales species.
Opportunistic pathogens
Are often a part of the usual intestinal microbiota of both humans and animals. However, outside their normal body sites, these organisms can produce serious extraintestinal, opportunistic infections.
6.Contrast the terms “opportunistic pathogens” and “primary pathogen” as they relate to clinical
infections.
What are the Primary pathogens or “true pathogens” not present as commensal biota in the GI tract of humans?
Salmonella enterica, Shigella spp., and Yersinia spp.,
6.Contrast the terms “opportunistic pathogens” and “primary pathogen” as they relate to clinical
infections.
Escherichia coli (E.coli)
- 90% are lactose positive
- Indole positive
- Wild type beta hemolytic on BAP and lactose positive
- can be NH & LP, NH & LN, H & LP, and H & LN
2.Categorize the genera and species of the gram-negative bacilli belonging to the Enterobacterales order utilizing their macroscopic appearance, lactose fermentation and key biochemical reactions
for identification.
2.1. Escherichia coli
E. coli is widely recognized as the most common cause of
UTIs
Discuss the pathogenesis of urinary tract infections (UTIs).
The E. coli strains that cause UTIs usually originate in the large intestine as resident biota and can exist either as the predominant E. coli population or as a small part of the E. coli strains in the large intestine.
Strains that cause UTIs produce factors that allow them to attach to the urinary epithelial mucosa. The primary virulence factor associated with the ability of E. coli to cause UTIs is the production of pili, which allow uropathogenic strains to adhere to epithelial cells and not be washed out with urine flow.
- Describe the mode of infection, pathogenesis and virulence factors for Escherichia coli.
7.1. Discuss the pathogenesis of urinary tract infections (UTIs).
Enterohemorrhagic Escherichia coli (EHEC) is commonly associated with what strain of E.coli and what clinical symptoms?
- E. coli O157: H7
- associated with hemorrhagic diarrhea, colitis, and hemolytic uremic syndrome (HUS). HUS is characterized by low platelet count, hemolytic anemia, and kidney failure.
7.2. Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.1. Recall the two cytotoxins and the common serotype responsible for EHEC illness.
7.2.3. Correlate hemolytic uremic syndrome with EHEC infection.
What 2 cytotoxins does E. coli O157: H7 produce?
Shiga (verotoxin) Toxin I & II
7.2. Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.1. Recall the two cytotoxins and the common serotype responsible for EHEC illness.
Describe the laboratory detection of EHEC.
- Stool culture on highly differential medium (SMAC agar), with subsequent serotyping
Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.2. Describe the laboratory detection of EHEC
What is SMAC Agar?
A macconkey plate that utilizes sorbitol instead of lactose
Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.2. Describe the laboratory detection of EHEC
Does E. coli 0157:H7 ferment sorbitol?
No, it is sorbitol negative. Colonies will appear clear on a SMAC plate
Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.2. Describe the laboratory detection of EHEC
Recall which genus is closely linked genetically to E. coli
Shigella sp. (unable to differentiate using MALDI-TOF or Verigene)
7.4. Recall which genus is closely linked genetically to E. coli.
What are the five types of diarrheagenic E.coli
- Enterotoxigenic E.coli (ETEC)
- Enteroinvasive E.coli (EIEC)
- Shiga toxin-producing E.coli (STEC)
- Enteropathogenic E.coli (EPEC)
- Enteroaggregative E.coli (EAEC)
7.3. List the diarrheagenic E. coli.
True or False: Humans are the only reservoir for Shigella.
True
8.Describe the mode of infection, pathogenesis, virulence factors and identification of Shigella spp.
Symptoms of shigellosis
- Abdominal cramps, diarrhea, fever
- Presence of blood, mucus, and pus in the stool.
- Referred to as “bacillary dysentery”
8.4. Describe clinical manifestations of Shigella infection.
Shigella spp. are divided into four major antigen groups and must be identified by serologic grouping. This is based on what type of antigen?
- The O antigen. (Dirty Fingers Bring Shigella)
A S. dysenteriae
B S. flexneri
C S. boydii
D S. sonnei
8.1. Correlate the four species with their serogroups.
8.1.1. List the four species and associated serogroups.
Transmission of Shigella occurs by
Direct person-to-person contact, and spread can take place via the fecal-oral route, with carriers as the source. Shigellae may also be transmitted by flies, fingers, and food or water contaminated by infected persons.
8.Describe the mode of infection, pathogenesis, virulence factors and identification of Shigella spp.
The “O” antigen can be masked by what other antigen, making it unable to type?
The K antigen. It is heat labile and the capsule can be removed (or unmasked) by boiling a cell suspension.
- Describe the mode of infection, pathogenesis, virulence factors and identification of Shigella spp.
8.1.2. Resolve the problem of strains that fail to serotype
In the U.S., shigellosis is most commonly caused by
S. sonnei
8.3. Describe the epidemiology of each species.
In underdeveloped countries, shigellosis is most commonly caused by
S. flexneri
8.3. Describe the epidemiology of each species.
Which Shigella spp. produces a Shiga toxin?
S. dysenteriae
8.4.1. List the major virulence factor of S. dysenteriae
Shigella culture characteristics
- Lactose negative
- H2S Negative
- Non-motile
- Lysine-decarboxylase negative
Salmonella spp. characteristics
- Lactose negative
- Urease negative
- H2S positive (1 exception)
H2S– producing colonies of salmonellae growing on xylose-lysine-desoxycholate (XLD) agar appearance:
Black Center
All Salmonella spp produce H2S except
Salmonella Paratyphi A
What are the 2 species of Salmonella?
- S. enterica
- S. bongori (rarely isolated)
Describe the mode of infection for Salmonella spp.
- Humans acquire the infection by ingesting the organisms in food, milk, and water contaminated with human or animal excreta.
- With the exception of Salmonella Typhi and Salmonella Paratyphi, salmonellae organisms infect various animals that serve as reservoirs and sources of human infections.
- Salmonella serotypes Typhi and Paratyphi have no known animal reservoirs, and infections seem to occur only in humans. Carriers are often the source of infection.
What factors are responsible for the virulence of salmonellae?
- Fimbriae used in adherence in initiating intestinal infection.
- The ability to traverse intestinal mucosa. (can lead to septicemia)
- Enterotoxin produced by certain Salmonella strains that cause gastroenteritis has been implicated as a significant virulence factor.
Describe, in humans, how salmonellosis may present clinically:
- Acute gastroenteritis or food poisoning characterized by vomiting and diarrhea
- Typhoid fever, the most severe form of enteric fever, caused by Salmonella serotype Typhi, and enteric fevers caused by other Salmonella serotypes (e.g., Salmonella Paratyphi and Choleraesuis)
- Nontyphoidal bacteremia
- Carrier state following Salmonella infection
Enteric fever caused by Salmonella Typhi is known as
typhoid fever
Describe the carrier state of Salmonella:
Individuals who recover from infection may harbor the organisms in the gallbladder, which becomes the site of chronic carriage. Such individuals excrete the organisms in their feces either continuously or intermittently; nevertheless, they become an important source of infection for susceptible persons. The carrier state may be terminated by antimicrobial therapy if gallbladder infection is not evident. Otherwise, cholecystectomy has been the only solution to the chronic state of enteric carriers.
I.E. Typhoid Mary
What serotype is associated with Typhoid fever?
The Vi antigen
Resolve the problem of strains that fail to serotype for salmonella
- A few strains may possess capsular (K) antigens, designated Vi antigen. The Vi antigen often blocks the O antigen during serologic typing but may be removed by heating.
- The capsular antigen plays a significant role in preventing phagocytosis of the organism.
How do you treat food poisoning caused by Salmonella?
Antibiotics are usually not prescribed as the disease is self-limiting
Yersinia pestis causes……
the plague
Yersinia pseudotuberculosis produces what?
Necrotizing granulomata resembling tuberculosis
Yersinia enterocolitica causes what?
Enteritis, appendicitis-like illness (most common isolate)
Describe the specific morphologic feature of Yersinia pestis on Methylene blue or the Wayson stains.
It shows intense staining at each end of the bacillus, referred to as bipolar staining, which gives it a “safety-pin” appearance.
Recall the three clinical forms of plague.
- The bubonic form, the most common, usually results from the bite of an infected flea. Characteristic symptoms appear 2 to 5 days after infection. The symptoms include high fever with painful regional lymph nodes known as buboes (swollen lymph nodes) begin to appear.
- The septicemic form occurs when the bacteria spread to the bloodstream.
- Pneumonic plague occurs secondary to bubonic plague or the septicemic form when organisms proliferate in the bloodstream and respiratory tract. Pneumonic plague can be a primary infection if the bacteria are inhaled. Subsequent epidemic outbreaks can arise from the respiratory transmission of the organisms. The fatality rate in pneumonic plague is high— essentially 100%— in untreated patients.
Recall the optimal culture conditions for Yersinia spp.
- Optimal growth temperature of 25 ° to 30 ° C.
- Y. enterocolitica is clearly motile at 25 ° C but not at 35 ° C.
- Culture on a specific Yersinia medium at 25 ° C
- Cold enrichment can be used to increase the recovery in fecal samples suspected of containing this organism. Fecal material is inoculated into isotonic saline and kept at 4 ° C for 1 to 3 weeks, with weekly subculturing to selective agar for Yersinia.
Which species of Yersinia is considered a class A bioterrorism agent?
Y. pestis
Is Y. pestis motile at room temp?
No
Which species of Yersinia are motile at room temp and not at 37 degrees C?
Y. pseudotuberculosis and Y. enterocolitica
The causative agent of plague is most often transmitted to humans by:
Fleas
What enteric organism can be acquired by eating improperly prepared and cooked or preserved food contaminated with human feces and produces dysentery?
Shigella spp.
Define Carbapenem-resistant Enterobacteriaceae (CRE):
Resistance to imipenem, meropenem, doripenem, or ertapenem, or documentation that the isolate possesses a carbapenemase.
Explain the resistance associated with ESBL
Most isolates of K. pneumoniae, Klebsiella oxytoca, and many E. coli are susceptible to later-generation cephalosporins and aztreonam; however, spontaneous mutations occur that may result in novel β-lactamases that can inactivate extended-spectrum cephalosporins, penicillins, and aztreonam. These β-lactamases are known as extended-spectrum β-lactamases (ESBLs).
Explain the ESBL confirmation test.
- ESBL are inhibited by clavulanic acid, a b-lactam inhibitor. This property is used in the lab to identify ESBL.
- A beta-lactam antibiotic such as ceftazidime or cefotaxime is tested with and without clavulanic acid and compared.
Looking at the picture. The diameter of the zone around cefotaxime– clavulanic acid (4-o’clock position) is more than 5 mm larger than the zone around cefotaxime (5-o’clock position), which indicates a positive reaction as clavulanic acid restores the activity of cefotaxime. Although the zones for ceftazidime and ceftazidime– clavulanic acid are comparable, only one set of drugs needs to be positive to confirm an isolate as an ESBL producer.
List the indicator drugs used to determine the need for the ESBL test.
If ceftazidime, ceftriaxone, cefotaxime (or other screening antibiotic) has MIC >2 ug/ml, an ESBL may be present.
Which organisms are screened for ESBL production?
Escherichia coli, Klebsiella spp., and Proteus mirabilis
The media used to Isolate Vibrio spp.
Thiosulfate Citrate Bile Salts Sucrose (TCBS)
(Stool Culture Media)
Selective & Differential
• Isolation of Vibrio spp. from stool specimen
• Salt content inhibits most other gram-positive & gram-negative organisms
• Yellow colonies (sucrose positive) = V. cholerae or V. alginolyticus
• Green colonies (sucrose negative) = V. parahaemolyticus
Ingredients: Yeast extract & peptone base; sucrose is fermentable carbohydrate; Oxgall; sodium cholate, sodium chloride, bile salt; sodium thiosulfate; sodium citrate; bromthymol & thymol blue pH indicator
Aeromonas spp. characteristics
- Found in fresh & brackish water
- Cause diarrhea & wound infections (not all strains pathogenic; presence in stool culture does not necessarily indicate infection
- Most are lactose fermenters on MAC, No growth on TCBS
- Oxidase postive.
- Spot indole positive
Describe the gram stain of Campylobacter.
- Tiny curved GNB
- Seagull shaped
Campy Blood Agar (CAMPY)
Stool Culture Media
Enriched & Selective
• Isolation of Campylobacter species in stool cultures
• Antibiotics inhibit gram-positive and gram-negative flora organisms in stool
• Cultures are incubated at 42°C in microaerophilic conditions to help inhibit normal enteric (stool) flora & allow Campylobacter to grow
Ingredients: Brucella agar base with sheep blood provide heme and other growth factors; five antimicrobial agents: trimethoprim, vancomycin, amphotericin B, polymyxin, cephalothin.
Campy Filter Agar Plate
(Stool Culture Media)
Physical selection on enriched media
• Organisms are very narrow and motile, allowing passage through pores in the filter; other enteric organisms found in stool are retained on top of filter
• Cultures are incubated at 42°C in microaerophilic conditions to help inhibit normal enteric (stool) flora & allow Campylobacter to grow
Ingredients: Sheep blood agar (BAP) with 0.45 micron filter
Define nonfermenters
Organisms that do not ferment carbohydrates.
Nonfermenters fail to acidify an oxidative-fermentative (OF) medium when it is overlaid with mineral oil or fail to acidify triple sugar iron agar (TSIA) butts.
OF Media
- Detects acid production from oxidation of glucose in open tube
- Low peptone / high carbohydrate ratio helps detect small amounts of acid from oxidation; you must have a pH indicator that turns color near higher acidic range pH 7.0 The smaller amounts of acid won’t be masked by the alkaline reaction from peptone utilization.
- Semi-solid to “suspend” the smaller amounts of acidity so it doesn’t get diluted out from jiggling the tube
- Best for oxidizers and asaccharolytic organisms
- NOTE: Acid from fermenters > acid from oxidizers
Understanding the Reactions of a TSI slant
Most strains of P. aeruginosa will also produce the blue, water-soluble pigment called what?
Pyocyanin. Pyocyanin combining with pyoverdin produces the green color characteristic of P. aeruginosa colonies.
No other nonfermentative, gram-negative bacillus produces pyocyanin, so its presence can be used to specifically identify P. aeruginosa.
Pseudomonas aeruginosa Identifying Characteristics:
- Recognizable colony morphology on BAP (metallic or pearlescent, flat with rough edges or very mucoid; produce pigment)
- Grape or tortilla like odor
- Ability to grow at 42 degrees C in culture and in the environment (hot tubs)
- Oxidase positive
- Oxidizer
- Motile by means of polar monotrichous flagella
Recall the colonial morphology of P. aeruginosa strains isolated from patients with cystic fibrosis.
Mucoid
Describe Burkholderia cepacia on BCSA?
Burkholderia Cepacia Selective Agar produces pink to yellow zone around each colony
Stenotrophomonas maltophilia characteristics
- Nosocomial pathogen usually in respiratory tract and in immunocompromised pts
- Oxidase Negative
- Inherently resistant to most antipseudomonal drugs
Describe Acinetobacter and it’s reaction to gram stain.
They appear as gram-negative coccobacilli or even gram-negative cocci on Gram stain (Fig. 21.8). Acinetobacter organisms can resist decolonization and retain the crystal violet stain, leading to misidentification. They can appear as gram-positive cocci in smears made from blood culture bottles.
Describe Acinetobacter and how it looks on MacConkey agar.
They are lactose negative. However, the purplish hue produced by some species on this medium may resemble that of a lactose-fermenting bacterium.
Describe Haemophilus’ gram stain.
Gram-negative, pleomorphic coccobacilli or rods
Haemophilus spp will not grow on what agar?
- BAP
- It will grow on CHOC agar
What is satellitism and how does it help detect Haemophilus?
- Satellitism occurs when an organism, such as Staphylococcus aureus, produces V factor as a by-product of metabolism. The Haemophilus isolate obtains X factor from the BAP agar and the V factor from one of these organisms after hemolyzing the BAP.
Which Haemophilus species requires both X and V factor?
Haemophilus influenzae
Which Haemophilus species only requires V factor?
Haemophilus parainfluenzae
Which test is an alternative method for differentiating the heme-producing species of Haemophilus?
- The porphyrin test or ALA Differentiation Disk
- The test detects the presence of porphyrin compounds, which are intermediates in the hemin biosynthetic pathway. Porphyrins are detected by the emission of a red-orange fluorescence under UV light (366nm) and indicate that the organism is capable of synthesizing hemin and is not dependent on hemin (X-Factor) for growth. Conversely, hemin requiring strains lack the enzymes to synthesize hemin and do not produce intermediate porphyrin compounds.
Which species of Haemophilus, when gram staining the lesion, shows abundant organisms referred to as a “school of fish”?
H. ducreyi
Isolates of H. influenza should be tested for beta lactamase production VIA cefinase disc. If the cefinase disc were positive, which antibiotic should be considered to be resistant?
Ampicillin
Which HACEK organism is associated with human bite wounds, the colony can “pit” the agar and has a bleach like odor?
Eikenella corodens
Which organism infects the aortic valve more frequently compared with the other HACEK organisms?
Cardiobacterium hominis
Gram stain shows “rosettes”
Which HACEK organism causes infections in pediatric patients in bones and joints?
Kingella.
Gram stain is plump bacilli in chains and tends to resist decoloization.
Which HACEK organism is associated with dental procedures and periodontal disease?
Aggregatibacter
Which organism causes soft tissue infections after dog or cat bites, requires increased CO2 for growth, has a fusiform GNB gram stain and displays “gliding motility” on an agar plate?
Capnocytophaga
Which organism produces a distinct star shape in the center of its colony and is associated with Acute Juvenile Periodontitis?
Actinobacillus (Aggregatibacter) actinomycetemcomitans
Which organism is related to Zoonosis (a disease humans acquire from animals), primarily wound infections from cat and other animal bites?
Pasteurella spp.
Grows well on BAP & CHOC. Not on MAC
CAT +
OXIDASE +
INDOLE +
Describe the method for culturing Legionella from a respiratory specimen?
Legionella spp. are fastidious, aerobic bacteria that do not grow on SBA and require L-cysteine for growth. Tiny colonies may appear on CHOC agar that contains L-cysteine; however, BCYE agar with L-cysteine is best for Legionella isolation. BCYE agar is available commercially as nonselective and selective media, and both should be used for optimal isolation. Selective BCYE agar contains polymyxin B, anisomysin, and either vancomycin or cefamandole. Although selective medium improves recovery of Legionella spp. from highly contaminated specimens, it can inhibit growth of some Legionella spp., and so it should not be used alone. Inoculated media are incubated at 35 ° C in air; increased CO2 can enhance the growth of some of the more fastidious species. Legionella spp. colonies are generally visible within 3 to 5 days.
How is the direct gram stain used to diagnose gonococcal infection?
- The demonstration of gram-negative intracellular diplococci from the urethral discharge of a symptomatic male correlates at a rate of 89% with culture and is evidence of gonococcal infection. The gonococci are in pairs with adjacent sides flattened, giving them a kidney shape.
- Because women have vaginal commensal microbiota that resembles gonococci, direct Gram stain correlates in only 50% to 70% of cases with culture. Direct Gram stain may be helpful in a symptomatic woman with discharge, but culture is necessary for confirmation. Gram stain with more than five polymorphonuclear neutrophils per field but no bacteria (see Fig. 17.4B) suggests nongonococcal urethritis with other organisms, such as C. trachomatis or Ureaplasma urealyticum.
What is culture media used to isolate N. gonorrhea and inhibit the growth of indigenous flora?
- A chocolate agar with antibiotics. (Thayer-Martin agar, Martin-Lewis, or New York City Agar)
- Does not grow well on BAP
Describe the clinical infections associated with N. meningitiis?
- After exposure to meningococcus, the incubation period ranges from 1 to 10 days.
- The bacteria adhere to the nasopharyngeal mucosa, leading to colonization. In most individuals, colonization of the mucosa results in a subclinical infection or mild symptoms.
- In a few hosts, the bacteria gain access to the bloodstream and potentially the central nervous system, resulting in meningitis, sepsis, or both.
- When N. meningitidis enters the bloodstream, two main diseases can occur— fulminant meningococcemia or meningitis.
- Meningococcemia, or sepsis, may occur with or without meningitis and carries a 25% mortality rate, even if treated. Purpura (hemorrhaging of blood into skin and mucous membranes, producing bruises) with petechial skin rash (pinpoint red spots caused by hemorrhage) (Fig. 17.8A), tachycardia, and hypotension can develop during bacteremia, and thrombosis is common. Death may occur in 12 to 48 hours from onset.
- Meningitis is characterized by an abrupt onset of frontal headache, stiff neck (nuchal rigidity), confusion, and photophobia. The fatality rate is 10% to 15%; an additional 10% to 20% have serious sequelae, such as neurologic complications or seizures.
What media does N. meningitidis grow on?
- BAP
- CHOC
- ML
Describe the characteristics of M. catarrhalis in the lab?
- GNDC
- CAT +, OX +
- Grows well on BAP, CHOC. Not ML
- The term hockey puck has been used to describe the colony because it remains intact when pushed across the plate with a loop.
- Butyrate esterase positive
The nonpathogenic species of Neisseria are typically what color?
Yellow
Which Neisseria spp. only utilizes the carbohydrate Glucose?
N. gonorrhea
Which Neisseria spp. only utilizes the carbohydrates Glucose and Maltose?
N. meningitidis
Which Neisseria spp. utilizes the carbohydrates Glucose, Maltose and Lactose?
N. lactamica
M. catarrhalis utilizes what carbohydrates?
None of them.
Organism
Explain the principle, procedure, and interpretation of the O/F glucose test.
Princple: Semisolid medium containing glucose and bromthymol blue indicator. In the presence of acid, bromthymol blue indicator changes from green to yellow. The test is used to categorized bacteria as oxidizers, fermenters, or nonsacchrolytic.
Procedure:
For each test isolate (18-24 hours old and in pure culture), two tubes should be incoulated and incubated in parallel with the carbohydrate tubes. Touch the colony using an inoculating needle and stab once down the center of the medium to within approximately 1/4th an inch from the bottom. overlay one tube with sterile mineral oil. Incubate the open tube with cap loosened and the closed tube with cap tight at 335-37 degrees C for up to 7 days. Examine daily for a color change from green to yellow.
Interpretation:
See image.
- Summarize the principle of differential tests and reagent(s) needed to identify various gramnegative bacilli.
Explain the principle, procedure, and purpose of the citrate test.
Principle: Use to detect bacteria capable of using citrate as a sole carbon source and ammonium dihydrogen phosphate as a sole nitrogen source. The medium (Simmons Citrate Agar) contains citrate, ammonium dihydrogen phosphate, and bromthymol blue. Capable bugs will grow on this medium and breakdown ammonium dihydrogen phosphate to ammonia.
Procedure:
1. Streak the surface of the slant with a single colony from a 18-24 hour culture.
2. Incubate in ambient air with caps loosened at 35-37 degrees C for up to 4 days
3. Examine daly for growth and an intesnse blue-green color development.
Interpretation:
Positive test: Growth with an intense blue-green color on the slant
Negative test: No growth to trace growth with the slant remaining dark green
- Summarize the principle of differential tests and reagent(s) needed to identify various gramnegative bacilli.
