03 - AEROBIC GRAM-NEGATIVE BACTERIA (Exam # 3) Flashcards
1
Q
Explain the principle of the Indole test.
A
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.
2
Q
Explain the procedure and interpretation of the spot indole test.
A
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.
3
Q
Explain the procedure and interpretation of Ehrlich’s indole test.
A
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.
4
Q
Explain the procedure and interpretation of Kovacs’ indole test.
A
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.
5
Q
Explain the principle of the MUG Test (4-Methylumbelliferyl-β-D-Glucuronide)
A
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.
6
Q
Explain the principle of the oxidase test.
A
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.
7
Q
Explain the principle of the Triple Sugar Iron (TSI) Test.
A
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.
8
Q
Explain the procedure and interpretation of the MUG Test (4-Methylumbelliferyl-β-D-Glucuronide)
A
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.
9
Q
Which oxidase reagent is more sensitive?
A
Tetramethyl-p-phenylenediamine dihydrochloride (Kovacs’ oxidase reagent)
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
10
Q
Explain the procedure and interpretation of the oxidase test.
A
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.
11
Q
Explain the principle of the urease test.
A
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.
12
Q
Explain how to innoculate a Triple Sugar Iron (TSI) slant.
A
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.
13
Q
Explain what A, K, H2S, and G mean in relation to triple sugar iron agar slants.
A
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.
14
Q
Explain what is happening in each test tube including, what sugar(s) is (are) being fermented as well as the type of gas production.
A
- Summarize the principle of differential tests and reagent(s) needed to identify various gram negative bacilli.
15
Q
Explain the procedure and interpretation of the urease test.
A
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.
16
Q
Enterobacterales are oxidase _______
A
- 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.
17
Q
What sugar do all Enterobacterales ferment?
A
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.
18
Q
What makes MacConkey agar selective?
A
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
19
Q
What makes MacConkey agar differential?
A
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
20
Q
Enterobacterales have the ability to reduce nitrate (NO3) to
A
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
21
Q
What color are lactose fermenters on MacConkey agar?
A
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.
22
Q
What color are lactose nonfermenters on MacConkey agar?
A
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
23
Q
What two reagents are used in the nitrate reduction test?
A
- N,N-dimethyl – a -naphthylamine
- Sulfanilic acid
1.Summarize the principle of differential tests and reagent(s) needed to identify various gramnegative bacilli.
24
Q
Most members of Enterobacterales are motile by what type of flagella?
A
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.
25
*Which 2 organisms are* **Atrichous (lacking flagella)** making them *nonmotile?*
*Klebsiella* and *Shigella*
Exception: Klebsiella aerogenes
## Footnote
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 reactionsfor identification.
2.3. Shigella spp.
2.5. Klebsiella spp.
26
Which organisms are nonmotile at 35 degrees C but motile at room temp 22 degrees C?
Yersinia enterocolitica and Yersinia pseudotuberculosis
## Footnote
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 reactionsfor identification.
2.16. Yersinia spp.
2.16.1. Yersinia enterocolitica
27
What are the 3 antigens that can be used to serologically group species of *Enterobacterales*?
1. O antigen
2. H antigen
3. K antigen
## Footnote
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
28
The O antigen is located on the \_\_\_\_\_\_\_
and is heat\_\_\_\_\_\_\_\_
* Cell wall (somatic antigen)
* Heat Stable
## Footnote
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
29
The H antigen is located\_\_\_\_\_
and is heat\_\_\_\_\_\_\_\_\_
* On the suface of the flagella (flagellar antigen)
* Heat-labile
## Footnote
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
30
The K antigen is a ______ antigen
and is heat\_\_\_\_\_\_
* Capsular Antigen (found only in certain encapsulated species)
* Heat-labile
## Footnote
4.Describe the antigenic composition, name, location, and heat stability of the three antigens found in many Enterobacterales.
31
Enterobacterales, with a few exceptions, reside normally in what part of the body?
Gastrointestinal (GI) tract
## Footnote
5.Contrast the natural habitat of the Enterobacterales species.
32
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.
## Footnote
6.Contrast the terms “opportunistic pathogens” and “primary pathogen” as they relate to clinical
infections.
33
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.,
## Footnote
6.Contrast the terms “opportunistic pathogens” and “primary pathogen” as they relate to clinical
infections.
34
***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
## Footnote
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
35
E. coli is widely recognized as the most common cause of
UTIs
36
***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.
## Footnote
7. Describe the mode of infection, pathogenesis and virulence factors for Escherichia coli.
7.1. Discuss the pathogenesis of urinary tract infections (UTIs).
37
**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.
## Footnote
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.
38
What 2 cytotoxins does E. coli O157: H7 produce?
Shiga (verotoxin) Toxin I & II
## Footnote
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.
39
Describe the laboratory detection of EHEC.
* Stool culture on highly **differential medium (SMAC agar**), with subsequent serotyping
## Footnote
Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.2. Describe the laboratory detection of EHEC
40
What is SMAC Agar?
A macconkey plate that utilizes sorbitol instead of lactose
## Footnote
Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.2. Describe the laboratory detection of EHEC
41
Does E. coli 0157:H7 ferment sorbitol?
No, it is sorbitol negative. Colonies will appear clear on a SMAC plate
## Footnote
Describe the mode of transmission and pathogenesis of Enterohemorrhagic Escherichia
coli (EHEC).
7.2.2. Describe the laboratory detection of EHEC
42
***Recall which genus is closely linked genetically to E. coli***
**Shigella sp.** (unable to differentiate using MALDI-TOF or Verigene)
## Footnote
7.4. Recall which genus is closely linked genetically to E. coli.
43
What are the five types of diarrheagenic E.coli
1. Enterotoxigenic E.coli (ETEC)
2. Enteroinvasive E.coli (EIEC)
3. Shiga toxin-producing E.coli (STEC)
4. Enteropathogenic E.coli (EPEC)
5. Enteroaggregative E.coli (EAEC)
## Footnote
7.3. List the diarrheagenic E. coli.
44
True or False: Humans are the only reservoir for Shigella.
True
## Footnote
8.Describe the mode of infection, pathogenesis, virulence factors and identification of Shigella spp.
45
Symptoms of shigellosis
* Abdominal cramps, diarrhea, fever
* Presence of blood, mucus, and pus in the stool.
* Referred to as **“bacillary dysentery”**
## Footnote
8.4. Describe clinical manifestations of Shigella infection.
46
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*
## Footnote
8.1. Correlate the four species with their serogroups.
8.1.1. List the four species and associated serogroups.
47
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.
## Footnote
8.Describe the mode of infection, pathogenesis, virulence factors and identification of Shigella spp.
48
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.
## Footnote
8. 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
49
In the U.S., shigellosis is most commonly caused by
S. sonnei
## Footnote
8.3. Describe the epidemiology of each species.
50
In underdeveloped countries, shigellosis is most commonly caused by
S. flexneri
## Footnote
8.3. Describe the epidemiology of each species.
51
Which Shigella spp. produces a Shiga toxin?
S. dysenteriae
## Footnote
8.4.1. List the major virulence factor of S. dysenteriae
52
Shigella culture characteristics
* Lactose negative
* H2S Negative
* Non-motile
* Lysine-decarboxylase negative
53
Salmonella spp. characteristics
* Lactose negative
* Urease negative
* H2S positive (1 exception)
54
H2S– producing colonies of salmonellae growing on xylose-lysine-desoxycholate (XLD) agar appearance:
Black Center
55
All Salmonella spp produce H2S except
Salmonella Paratyphi A
56
What are the 2 species of Salmonella?
1. S. enterica
2. S. bongori (rarely isolated)
57
***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.
58
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.
59
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
60
Enteric fever caused by Salmonella Typhi is known as
typhoid fever
61
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
62
What serotype is associated with Typhoid fever?
The Vi antigen
63
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.
64
How do you treat food poisoning caused by Salmonella?
**Antibiotics are usually not prescribed** as the disease is self-limiting
65
Yersinia pestis causes……
**the plague**
66
Yersinia pseudotuberculosis produces what?
Necrotizing granulomata resembling tuberculosis
67
Yersinia enterocolitica causes what?
**Enteritis, appendicitis-like illness** (most common isolate)
68
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.
69
Recall the three clinical forms of plague.
1. 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.
2. The **septicemic** form occurs when the bacteria spread to the bloodstream.
3. **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.
70
***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.
71
Which species of Yersinia is considered a class A bioterrorism agent?
Y. pestis
72
Is Y. pestis motile at room temp?
No
73
Which species of Yersinia are motile at room temp and not at 37 degrees C?
Y. pseudotuberculosis and Y. enterocolitica
74
The causative agent of plague is most often transmitted to humans by:
Fleas
75
What enteric organism can be acquired by eating improperly prepared and cooked or preserved food contaminated with human feces and produces dysentery?
Shigella spp.
76
# Define Carbapenem-resistant Enterobacteriaceae (CRE):
Resistance to imipenem, meropenem, doripenem, or ertapenem, or documentation that the isolate possesses a carbapenemase.
77
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).
78
Explain the ESBL confirmation test.
* ESBL are inhibited by clavulanic acid, a b-lactamase 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.
79
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.
80
Which organisms are screened for ESBL production?
*Escherichia coli, Klebsiella spp., and Proteus mirabilis*
81
Isolates confirmed to be ESBL producers should ALWAYS be reported as resistant to what antibiotics?
Penicillins, cephalosporins and aztreonam.
82
What gene is most commonly associated with ESBL production?
CTX-M
83
What gene is most commonly associated with Carbapenemase (CPE)-producing Enterobacteriaceae species?
KPC
84
Klebsiella spp is intrinsically resistant to what antibiotic?
Ampicillin
85
Enterobacter spp. is intrinsically resistant to what antibiotic?
Many B-lactam antibiotics including 1st generation cephalosporins
86
Vibrio spp. are commonly found in where?
* In a wide variety of aquatic environments, including fresh water, brackish or estuarine water, and marine or salt water.
* They are temperature sensitive in that in temperate climates in which water temperatures exceed 20 ° C, as in the summer months, vibrios can easily be isolated from water,
87
Pandemics (worldwide epidemics) of **cholera**, a devastating diarrheal disease is caused by what organism?
Vibrio cholerae
88
What are some recognized risk factors that may indicate an infection with Vibrio spp.?
* Recent consumption of raw seafood (especially oysters)
* Recent immigration or foreign travel
* Gastroenteritis with cholera-like or rice-water stools
* Accidental trauma incurred during contact with fresh, estuarine, or marine water or associated products (e.g., shellfish, oyster or clam shells, fishhooks)
89
All species of vibrios, except for V. cholerae and V. mimicus, are halophilic, which means what?
Salt-loving, and require the addition of Na + for growth.
90
Describe the disease Cholera?
* Cholera is an acute diarrheal disease spread mainly through contaminated water.
* The stools produced by patients with cholera are described as “rice water stools”
* Is the result of a powerful enterotoxin known as cholera toxin, or choleragen.
* Hypersecretion of electrolytes resulting in the massive outpouring of watery stools.
91
Vibrio spp are oxidase?
Positive
92
Vibrio cholera is not a strict halophile and therefore
does not require salt to grow. It can be found in fresh water environments.
93
Vibrio parahaemolyticus characteristics:
* Vibrio parahaemolyticus is the second most common Vibrio species implicated in gastroenteritis after V. cholerae. In the United States however, it is the most frequently encountered species in clinical samples.
* Seafood is usually the source
* Halophilic
* Gastrointestinal disease caused by V. parahaemolyticus is generally self-limited. Patients have watery diarrhea, moderate cramps or vomiting, and little if any fever.
94
Vibrio vulnificus characteristics:
* Found in coastal waters and estuaries: causes septicemia and wound infections
* Associated with eating raw oysters
* Persons with liver problems are more susceptible to organism
* The gastroenteritis features both vomiting and diarrhea
* Skin infections may follow trauma, or it may be caused by exposure of already open skin to infected water
95
*Vibrio alginolyticus characteristics*
* Least pathogenic of the vibrios
* Strict halophile
* Almost all isolates originate from extraintestinal sources, such as eye and ear infections or wound and burn infections.
* The organism can be an occupational hazard for people in constant contact with seawater, such as fishermen or sailors.
96
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
97
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
98
Plesiomonas spp
* Now part of Enterobacterales group
* **Oxidase positive**
* Causes gastroenteritis
* No growth on TCBS
99
The most common cause of bacterial gastroenteritis **worldwide** is due to what organism?
Campylobacter jejuni
100
Campylobacter jejuni is associated with undercooked \_\_\_\_\_\_\_\_
Beef (hamburgers) and poultry; causes diarrhea
101
Describe the gram stain of Campylobacter.
* Tiny curved GNB
* Seagull shaped
102
Campylobacter has what type of motility?
Darting motility
103
What test distinguishes C. jejuni from the other Campylobacters?
C. jejuni is hippurate hydrolysis positive
104
Isolating Campylobacter includes:
* Growth on selective media (CAMPY BAP)
* Microaerophilic conditions
* C. jejuni grows at 42 degree C
* C. fetus grows at 37 degree C and not at 42 degree C
105
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.
106
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
107
Helicobacter pylori (H. pylori) is a cause of what disease?
* Peptic ulcers and gastric disease
108
Name 2 ways to presumptively identify H. pylori infections without culture:
1. Collecting a gastric biopsy specimen (tissue) and testing for the presence of a rapid urease reaction (CLOtest)
2. Urease activity can also be detected by the **urea breath test**.
109
# Define fermenters:
Organisms that ferment carbohydrates
110
# 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.
111
Define oxidizers:
Organisms that can oxidize carbohydrates to derive energy for their metabolism
112
Define nonoxidizers:
AKA **asaccharolytic**. Organisms that do not break down carbohydrates at all
113
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**
114
Understanding the Reactions of a TSI slant
115
Signs you might be a nonfermenter
* Oxidase-positive reaction, although reaction can be weak and variable
* Nonreactivity in 24 hours in commercial multitest kit systems used primarily for the identification of Enterobacteriaceae
* No acid production in the slant or butt of TSI or KIA
* Resistance to a variety of classes of antimicrobial agents, such as aminoglycosides, third-generation cephalosporins, penicillins, and fluoroquinolones
116
Members of the Pseudomonas fluorescent group, which includes P. aeruginosa, P. fluorescens, P. putida, produce what type of fluorescent?
**Pyoverdin** (a yellow-green or yellow-brown pigment)
117
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.
118
Pseudomonas aeruginosa infections:
* Associated with nosocomial infections, UTIs, wounds, bacteremia
* **Burn infections**
* **Respiratory infections especially in patients with cystic fibrosis**
119
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
120
Pseudomonas flourescens/putida
* Mostly environmental organism found in soil & water
* Produces pyoverdin
* No growth at 42 degrees C
* Oxidase positive
121
Non-fluorescent pseudomonad group consists of (2)
*Pseudomonas stutzeri & Pseudomonas mendocina*
122
P. aeruginosa is innately resistant to which antimicrobial agents?
Penicillin, ampicillin, first- and second-generation cephalosporins, trimethoprim-sulfamethoxazole (SXT), chloramphenicol, and tetracyclines, along with other agents to which most gram-negative bacilli are resistant.
123
Recall the colonial morphology of P. aeruginosa strains isolated from patients with cystic fibrosis.
Mucoid
124
Burkholderia cepacia is a primary concern of what type of patient?
Cystic fibrosis patient
125
Burkholderia cepacia is resistant to which class of antimicrobials?
Aminoglycosides
126
Describe Burkholderia cepacia on BCSA?
Burkholderia Cepacia Selective Agar produces pink to yellow zone around each colony
127
Which 2 species of Burkholderia are considered agents of bioterrorism?
* Burkholderia mallei
* Burkholderia pseudomallei
128
Burkholderia mallei causes what disease?
**Glanders**, a respiratory tract zoonosis primarily affecting livestock such as horses, mules, and donkeys. It is rare in humans but can produce severe local suppurative or acute pulmonary infections.
129
Burkholderia pseudomallei causes what disease?
**Melioidosis**, an aggressive, granulomatous, pulmonary disease caused by ingestion, inhalation, or inoculation of the organisms, with metastatic abscess formation in lungs and other viscera.
130
St*enotrophomonas maltophilia* characteristics
* Nosocomial pathogen usually in respiratory tract and in immunocompromised pts
* Oxidase Negative
* Inherently resistant to most antipseudomonal drugs
131
The drug of choice for St*enotrophomonas maltophilia is what?*
SXT
132
What are the two most commonly isolated species with of Acinetobacter?
1. A. baumannii
2. A. lwoffii
133
Most Acinetobacter infections are associated with what?
Ventilators, humidifiers, catheters
134
Which species of Acinetobacter is associated with ventilator-associated pneumonia and sepsis and known to have a high mortality rate?
A. baumannii
135
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.
136
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.
137
Describe A. lwoffii and it's susceptibility pattern.
A. lwoffi is susceptible to almost all antimicrobials.
138
Describe A. baumannii and it's susceptibility pattern
* Isolates of A. baumannii are often resistant to many antimicrobials.
* Many strains exhibit resistance to carbapenems; carbapenemases have been reported throughout the United States.
* These isolates have been referred to as CRAB, or **c**arbapenem-**r**esistant **A**. **b**aumannii.
* CRAB isolates are usually only susceptible to colistin and tigecycline.
* Isolation of patients in mobile isolation chamber units is often enforced when a CRAB is isolated.
139
Acinetobacter spp is oxidase what?
Oxidase negative
140
What are the HACEK organisms?
* **H**aemophilus
* **A**ggregatibacter
* **C**ardiobacterium
* **E**ikenella
* **K**ingella
141
Clinical significance of HACEK organisms?
They **reside in the human oral cavity and nasopharynx**, and some species have an **enhanced capacity to cause endocarditis**.
142
Describe Haemophilus' gram stain.
Gram-negative, pleomorphic coccobacilli or rods
143
Haemophilus spp will not grow on what agar?
* BAP
* It will grow on CHOC agar
144
Haemophilus spp may require 2 growth factors present in blood. What are they?
1. X- factor (Hemin)
2. V-factor (NAD)
## Footnote
1. X- factor (Hemin) is found on the BAP
2. V-factor (NAD) not found on BAP unless the cells are lysed, as in choc agar
145
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.
146
Which Haemophilus species requires both X and V factor?
Haemophilus influenzae
147
Which Haemophilus species only requires V factor?
Haemophilus parainfluenzae
148
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.
149
*Haemophilus influenzae* is Porphyrin Synthesis (ALA) what?
Negative
150
*Haemophilus parainfluenzae* is Porphyrin Synthesis (ALA) what?
Positive
151
If an isolate REQUIRES the X factor, what will the porphyrin (ALA) reaction be?
Negative
152
Which of the virulence factors for H. influenza, if present, plays the most significant role?
* The capsule.
* Before widespread use of a vaccine, most invasive infections were caused by the **encapsulated strain H. influenzae serotype b (Hib)** and occurred primarily in young children.
* In unvaccinated children, type b is a leading cause of meningitis.
153
Nontypable strains of H. influenzae are referred as what?
NTHi strains
154
What are the two patterns of disease that are attributed to H. influenzae?
1. **Invasive disease** caused by encapsulated strains, in which bacteremia plays a significant role. Examples of invasive disease include septicemia, meningitis, arthritis, epiglottitis, tracheitis, and pneumonia.
2. The second and the more common pattern of disease, as a result of Hib vaccination, is a more **localized infection** caused by the contiguous spread of NTHi strains, and it occurs within or in close proximity to the respiratory tract. Examples of localized infection include conjunctivitis, sinusitis, and otitis media with effusion (middle ear infections).
155
Haemophilus aegyptius is associated with what type of infection?
Contagious conjunctivitis, commonly referred to as “pinkeye”.
156
Haemophilus ducreyi is associated with what type of infection?
H. ducreyi, a strictly human pathogen, is the causative agent of **chancroid**, a highly communicable sexually transmitted genital ulcer disease (GUD).
157
Which species of Haemophilus, when gram staining the lesion, shows abundant organisms referred to as a “school of fish”?
H. ducreyi
158
Describe H. parainfluenzae and it's pathogenicity.
Usually considered nonpathogenic and normal flora of the upper respiratory tract. Rarely causes endocarditis.
159
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
160
Which HACEK organism is associated with human bite wounds, the colony can “pit” the agar and has a bleach like odor?
Eikenella corodens
161
Which organism infects the aortic valve more frequently compared with the other HACEK organisms?
Cardiobacterium hominis
Gram stain shows “rosettes”
162
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.
163
Which HACEK organism is associated with dental procedures and periodontal disease?
Aggregatibacter
164
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
165
Which organism produces a distinct star shape in the center of its colony and is associated with Acute Juvenile Periodontitis?
Actinobacillus (Aggregatibacter) actinomycetemcomitans
166
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 +
167
Which organism causes **undulating fevers** (characterized by body temperatures that rise in the afternoon and evening and fall in the morning) and is also considered a potential agent of bioterrorism?
Brucella spp.
168
Which organism causes Tularemia (rabbit or deer fever), is considered a select agent, and requires cysteine to grow?
* Francisella spp.
* F. tularensis is a highly infectious agent with as few as 50 organisms causing an infection through the cutaneous (ulceroglandular form) or inhalational (pneumonia) routes and has been the cause of many laboratory-acquired infections.
169
What should you do if you encounter a poorly growing organism with a gram stain of GNB/GNCB after 24hrs of incubation?
Work up of the culture should take place in a biological safety cabinet (BSC) until all select agents of bioterrorism can be ruled out. No MALDI-TOF or automated Identification. Follow CDC rule out procedures.
170
How does the lab detect Legionella?
* **Isolation using special media (BCYE)**
* **Urine antigen detection**
* DFA (no longer recommended)
* Serology
171
Infections caused by Legionella spp. produce a spectrum of symptoms ranging from asymptomatic or mild upper respiratory tract infections to life-threatening pneumonia. What are the names of the 2 diseases linked to Legionella?
1. Legionnaire's disease (pneumonia)
2. Pontiac fever (influenza-like)
172
What is the natural habitat for Legionella?
Legionella spp. are naturally present in warm water, and humans become infected when inhaling contaminated aerosols and droplets, often while traveling.
173
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.
174
Which organism is the etiologic of whooping cough?
* Bordetella pertussis
* Pertussis is one of the most highly communicable diseases of childhood.
* Acquired through the respiratory tract via respiratory droplets or direct contact with infectious secretions.
* Pertussis is a human disease; no animal reservoir or vector has been found.
* Five dose childhood vaccine administered from 6 weeks to 6 years old
175
Describe the 3 phases of pertussis (whooping cough).
B. pertussis infection occurs after exposure to the organism through the respiratory tract and a 1- to 3-week incubation period, usually 7 to 10 days.
* Initial **catarrhal phase** are insidious and nonspecific and include sneezing, mild cough, runny nose. At this stage, the infection is highly communicable because of the large number of organisms in the upper respiratory tract. However, cultures are not often performed at this stage because the symptoms are nonspecific.
* The **paroxysmal phase** follows the catarrhal phase. The hallmark of this phase is the sudden onset of severe, repetitive coughing followed by the **characteristic “whoop” at the end of the coughing spell**. The whooping sound is caused by the rapid gasp for air following the prolonged bout of coughing.
* The **convalescent phase** of disease generally begins within 4 weeks of onset with a decrease in frequency and severity of the coughing spells. Complete recovery may require weeks or months. Adults may or may not experience respiratory symptoms, which can range in severity from persistent cough to acute exacerbation of chronic bronchitis.
176
Describe the general characteristics of most Neisseria spp.
* Fastidious
* Nonmotile
* Gram-negative diplococci
* Oxidase +
* Catalase +
* Capnophilic (requires CO2)
177
Which 2 species of Neisseria are the primary pathogens of the genus?
* **Neisseria gonorrhoeae (often called gonococci) and Neisseria meningitidis (meningococci)**.
* N. gonorrhoeae is not considered part of the normal biota and is always pathogenic.
* N. meningitidis may be found as a commensal inhabitant of the upper respiratory tract of carriers, but it can also become an invasive pathogen.
* All other Neisseria spp. are considered opportunistic pathogens.
178
What are the 2 most common sexually transmitted bacterial infections in the United States?
1. Chlamydia trachomatis
2. Neisseria gonorrhoeae
179
Describe Gonorrhea clinical infections for men.
* Gonorrhea has a short incubation period of approximately 2 to 7 days.
* In men, acute urethritis, usually resulting in **purulent discharge and dysuria (painful urination)**, are the most common manifestations.
* Asymptomatic gonococcal infection in men is uncommon; up to 10% of cases are asymptomatic.
* Complications in male patients include ascending infections, such as **prostatitis and epididymitis.**
180
Describe Gonorrhea clinical infections for women.
* The endocervix is the most common site of infection in women.
* Symptoms of infection, when present, include **dysuria, cervical discharge, and lower abdominal pain**.
* 50% of cases in women may be asymptomatic leading to complications, such as **pelvic inflammatory disease**, which may cause sterility, ectopic pregnancy, or perihepatitis (Fitz-Hugh– Curtis syndrome).
181
What is ophthalmia neonatorum?
* A gonococcal eye infection, during vaginal delivery through an infected birth canal.
* This condition, which can result in blindness if not treated immediately, is rare in the United States because application of antimicrobial eye drops, generally erythromycin, at birth to every infant is legally required.
182
When left untreated, gonococcal infections can result in\_\_\_\_\_\_
Septic arthritis
183
What is specimen of choice when culturing for N. gonorrhea?
* **Genital infections in men is the urethra and in women is the endocervix.**
* In men, purulent discharge can be collected directly onto a swab for culture. When no apparent discharge is present, the swab is inserted up to 2 cm into the anterior urethra and slowly rotated to collect material.
* Swabs for rectal culture should be inserted 4 to 5 cm into the anal canal.
184
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.
185
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
186
What is the preferred assay for the detection of N. gonorrhea?
* **Nucleic acid amplification tests (NAATs)** **are the preferred assays for the detection of N. gonorrhoeae in clinical specimens because of increased sensitivity, specificity, and ability to test with a noninvasive urine specimen.**
* NAATs amplify a specific nucleic acid sequence (e.g., polymerase chain reaction) before detecting the target sequence with a probe.
* NAATs are extremely sensitive and do not require viable organisms.
* Because urine can be used in these procedures, self-collection of the specimen is possible, and pelvic examination or intraurethral swabs are not required.
* NAATs can also detect both N. gonorrhoeae and C. trachomatis in the same specimen.
187
Describe the epidemiology associated with N. meningitiis?
* N. meningitidis can be found on the mucosal surfaces of the nasopharynx and oropharynx in 30% of the population.
* The organism is transmitted by close contact with respiratory droplet secretions from a carrier to a new host.
* Only a few newly colonized hosts develop meningococcal disease, with the highest incidence being found in infants and adolescents.
* Crowded living conditions can facilitate the spread of meningococcus because individuals from different areas can be colonized by different strains. For example military recruits or resident college students.
188
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.
189
What media does N. meningitidis grow on?
* BAP
* CHOC
* ML
190
Why should manipulation of suspected sterile body site (such as CSF) isolates of N. meningitidis be performed in a biosafety cabinet (BSC)?
* **Exposure to N. meningitidis aerosols increases risk of infection.**
* In 2000, two cases of fatal laboratory-acquired meningococcal disease were reported to the CDC. Both clinical microbiologists had examined plates, performed Gram stain, subcultured, or performed slide agglutination serogrouping on patient isolates on the open bench. Isolates recovered from the laboratory scientists were identical to patient organisms.
191
Which Neisseria spp can you be vaccinated against?
N. meningitidis
192
Describe clinical infections of Moraxella catarrhalis.
* M. catarrhalis is an **opportunistic pathogen** and is recognized as a cause of upper respiratory tract infection in otherwise healthy children and in older adults.
* It can also cause lower respiratory infections, especially in adults with chronic obstructive pulmonary disease.
* M. catarrhalis has been reported as the third most common cause of acute otitis media and sinusitis in children
193
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**
194
The nonpathogenic species of Neisseria are typically what color?
Yellow
195
Which Neisseria spp. only utilizes the carbohydrate Glucose?
N. gonorrhea
196
Which Neisseria spp. only utilizes the carbohydrates Glucose and Maltose?
N. meningitidis
197
Which Neisseria spp. utilizes the carbohydrates Glucose, Maltose and Lactose?
N. lactamica
198
M. catarrhalis utilizes what carbohydrates?
None of them.
199
# 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.
## Footnote
1. Summarize the principle of differential tests and reagent(s) needed to identify various gramnegative bacilli.
200
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
## Footnote
1. Summarize the principle of differential tests and reagent(s) needed to identify various gramnegative bacilli.
