Gram-Negative Bacilli Flashcards
1
Q
What is the clinical significance of Acinetobacter?
A
It is an opportunistic pathogen with a high prevalence in hospitals. Because it frequently colonizes the skin and respiratory tracts of patients, its clinical significance in patient specimens can be difficult to establish.
2
Q
What are some of the characteristics of Acinetobacter?
A
They are non-fermentative, oxidase-negative, gram-negative rods that grow on MacConkey agar. On Gram stain, they are plump coccobacilli that may be mistaken for Neisseria.
3
Q
What are the characteristics of the family Enterobacteriaceae?
A
Gram-negative non-sporeforming rods Falcutative anaerobes Oxidase-negative Glucose fermenters Most reduce nitrates to nitrites Most are catalase-positive.
4
Q
Which of the following is a characteristic of all Enterobacteriaceae: acid butt in TSI, acid slant in TSI, H2S production, or colorless colonies on MacConkey?
A
Acid butt in TSI. All Enterobacteriaceae ferment glucose. Not all ferment lactose or sucrose or produce H2S.
5
Q
Discuss the antigens used for serological grouping of some of the Enterobacteriaceae,
A
O antigen - Somatic antigen - Cell wall - Lipopolysaccharide, heat stable
H antigen - Flagellar antigen - Flagella - Proteins, heat labile
K antigen - Capsular antigen - Capsule - Polysaccharide, heat labile, may mask O antigen. Removed by heating.
6
Q
Which of the Enterobacteriaceae are routinely serotyped?
A
Salmonella, Shigella, and E. coli O157:H7.
7
Q
What error may result from selecting organisms for serotyping from a sugar-containing medium such as MaConkey or TSI?
A
Auto-agglutination. Colonies should be selected from blood agar.
8
Q
Which antigen would be expected in bacteria that grow away from the stab line in motility agar?
A
O and H. All bacteria have the somatic antigen (O). Bacteria that grow away from the stab line are motile by means of flagella. The H antigen is associated with flagella.
9
Q
Following presumptive identification of Salmonella and Shigella by biochemical testing, what further testing is required?
A
Serotyping. Salmonella is typed by its O and H antigens, Shigella by O antigens only. (It is nonmotile, so does not have H antigens.)
10
Q
Which antigens are associated with Salmonella typhi?
A
D and Vi, a capsular antigen associated with virulence.d
11
Q
A suspected Salmonella is tested with polyvalent O antiserum and no agglutination is observed. What should be done and why?
A
Typing should be repeated on a suspension of the organism that has been heated. Heating destroys the K antigen which may mask the O antigens.
12
Q
Which three enterics are most commonly isolated from clinical specimens?
A
E. coli, Klebsiella, and Proteus.
13
Q
Which species of Proteus is most frequently isolated?
A
P. mirabilis.
14
Q
Which Enterobacteriaceae are pathogens to the intestinal tract?
A
Salmonella, Shigella, diarrheagenic E. coli, and Yersinia.
15
Q
Which Enterobacteriaceae are nonlactose fermenters (NLF)?
A
SAlmonella, Shigella, Proteus, Morganella, Providencia, Yersinia, Serratia, and Edwardsiella. Some Citrobacter and E. coli are lactose-negative also.
16
Q
Describe lactose fermenters (LF) and nonlactose fermenters (NLF) on EMB, MacConkey, Hektoen Enteric, and XLD.
A
Lactose Fermenters: EMB - Purple or green-black MaConkey - Pink Hektoen Enteric - Orange to salmon pink XLD - Yellow
Nonlactose Fermenters: EMB - Colorless MacConkey - Colorless Hektoen Enteric - Green to blue-green XLD - Colorless or red
17
Q
What color are E. coli colonies on XLD?
A
Yellow.
18
Q
What color are Shigella colonies on XLD?
A
Colorless.
19
Q
What color are Salmonella colonies on XLD?
A
Red with black center.
20
Q
Which enteric is characterized by swarming growth on blood agar?
A
Proteus.
21
Q
Which lactose-positive enterics are often “stringy” when touched with a loop (mucoid)?
A
Klebsiella and Enterobacter. This is due to their polysaccharide capsules.
22
Q
Which enteric produces a characteristic green metallic sheen on EMB?
A
E. coli.
23
Q
Which facultative gram-negative rod sometimes produces a red pigment?
A
Serratia marcescens.
24
Q
Which member of the family Enterobacteriaceae is thought to be a potential agent of bioterrorism?
A
Yersinia pestis, the causative agent of plague. Although usually contracted by the bite of a flea, person-to-person transmission by droplets is possible. The key characteristic for rapid diagnosis of plague is the closed safety pin appearance of Y. pestis when stained Wayson stain. It grows best at 28C.
25
WHich sugars are contained in TSI tubes?
Glucose (0.1%), lactose (1%), and sucrose (1%).
26
Explain how to interpret TSI tubes.
Yellow butt = glucose-positive
Pink butt = glucose-negative
Yellow slant = lactose- or sucrose-positive
Pink slant = lactose- and sucrose-negative
Black precipitate = hydrogen sulfide production
Bubbles = gas production
27
How should the TSI reaction be recorded when the butt is totally black and the slant is yellow?
A/A, H2S. H2S is only produced in an acid environment.
28
Describe TSI reactions for E. coli.
A/A, Gas.
29
Describe TSI reactions for Proteus vulgaris.
A/A with H2S. The yellow slant is due to fermentation of sucrose, not lactose.
30
Describe TSI reactions for Salmonella.
K/A, Gas, H2S.
31
Describe TSI reactions for Shigella.
K/A.
32
Which test detects acetoin, an intermediary of glucose metabolism, by the development of a red complex when KOH and alpha-napthol are added?
Voges-Proskauer.
33
Which Enterobacteriaceae are Voges-Proskauer-positive?
Klebsiella, Enterobacter, and Serratia.
34
Name four enterics that are H2S-positive.
Salmonella, Proteus, Citrobacter, and Edwardsiella.
35
What does the ONPG test detect?
Slow lactose-fermenters.
36
What might cause a false-positive oxidase test?
Use of an iron-containing wire fro transferring growth. A wooden stick or a platinum wire should be used.
37
From which medium should organisms be selected fro oxidase testing?
One without fermentable sugars, for example, blood or chocolate agar. Fermentation of carbohydrates may result in false-negative oxidase reactions due to acidification of the medium.
38
A bacterial colony turns dark blue-black when a 1% solution of tetramethyl-para-phenylenediamene dihydrichloride is applied. What can be said about the organism?
It is oxidase-positive.
39
Describe a positive citrate test.
Growth on the medium, with or without a color change from green to blue. The citrate test determines the ability of an organism to utilize soidum citrate as its only carbon source and inorganic ammonium salts as its only nitrogen source.
40
What might cause a false-negative citrate test?
Over-tightening the cap on the tube. The cap should be applied loosely to allow oxygen to enter the tube.
41
Name three members of the family Enterobacteriaceae that are urease-positive.
Proteus, Morganella, Providencia rettgeri, and Klebsiella (slowly).
42
Which genera of the family Enterobactriaceae are positive for phenylalanine deaminase?
Proteus, Morganella, and Providencia.
43
Which genera of Enterobacteriaceae are usually nonmotile at 35C?
Shigella, Klebsiella, and Yersinia. Yersinia is motile at 25C.
44
What must be present in the medium from which colonies are selected for spot indole test?
Trytophan, otherwise a false-negative result will occur. Blood and chocolate agars contain tryptophan; MacConkey does not.
45
How is the spot indole test used?
To presumptively identify an organism with characteristic colonial appearance on MacConkey agar as E. coli and to quickly differentiate the swarming Proteae. O. mirablis is indole-negative and P. vulgaris is indole-positive.
46
In the nitrate reduction test, after the addition of solution #1 and solution @2, no red color developed. Zinc dust was added to the reaction and immediately a red color developed. Should the reaction be recorded as positive or negative?
A red color after the addition of zinc dust indicates the presence of residual nitrates, therefore the test is negative.
47
Compare IMViC reactions for E. coli, Klebsiella, and Enterobacter.
E. coli: Indole (+), Methyl Red (+), Voges-Proskauer (-), Citrate (-)
Klebsiella: Indole (-), Methyl Red (-), Voges-Proskauer (+), Citrate (+)
Enterbacter: Indole (-), Methyl Red (-), Voges-Proskauer (+), Citrate (+)
48
How can Klebsiella and Enterobacter be differentiated?
Klebsiella is ornithine decarboxylase-negative; Enterbacter is positive. Klebsiella is nonmotile; Enterobacter is motile.
49
Which species of Klebsiella is indole-positive?
K. oxytoca.
50
What are shiga-toxin producing E. coli (STEC)?
Enterohemorrhagic E. coli (EHEC), also known as verotoxin-producing E. coli. The most common serotype is O157:H7. STEC is characterized by the presence of RBCs and the absence of WBCs in the stool.
51
What diseases does enterohemorrhagic E. coli cause?
Hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.
52
How is enterohemorrhagic E. coli contracted?
From undercooked meat, raw milk, unpasteurized apple cider, and contaminated produce, such as lettuce.
53
How can enterohemorrhagic E. coli be differentiated from nonpathogenic E. coli?
Enterohemorrhagic E. coli does not ferment sorbitol. It produces colorless colonies on MacConkey sorbitol agar (SMAC). Following isolation on SMAC, serotyping is required for definitive identification.
54
What is enteropathogenic E. coli?
A non-invasive, non-toxigenic E.coli that causes diarrhea in infants. No RBCs or WBCs are found in the stool.
55
Klebsiella is inoculated onto a freshly prepared citrate slant as part of the laboratory's protocol for quality control of media prepared in-house. After overnight incubation at 37C, no growth is observed and the color of the slant is green. What is the most likely explanation for these findings?
A false-negative reaction occurred because the lid was screwed on too tightly.
56
Name a rapid test to differentiate Proteus mirabilis from Proteus vulgaris.
P. mirabilis is indole-negative; P. vulgaris is indole-positive.
57
Which biochemical test differentiates Salmonella and Shigella from most normal intestinal gram-negative rods?
Salmonella and Shigella are lactose-negative.
58
Name two tests that differentiate Salmonella and shigella.
Salmonella is motile and hydrogen sulfide-positive; Shigella is nonmotile and hydrogen sulfide-negative.
59
Which species of Salmonella produces only trace amounts of H2S and citrate-negative?
S. typhi.
60
Which Shigella is most commonly isolated in the U.S.?
S. sonnei.
61
Which Shigella ferments lactose slowly?
S. sonnei (rarely).
62
A colorless colony was isolated on MacConkey agar from a stool culture. What would be the single best test to rule out Salmonella or Shigella.
Urease. Salmonella and Shigella are urease-negative. A positive urease would be suggestive of Proteus.
63
Name a medium selective for Yersinia enterocolitica.
Cefsulodin-irgasan-novobiocin agar (CIN). Y. entercolitica colonies are red "bull's eye" colonies surrounded by a colorless halo.
64
Which rapid test, when positive, rules out a member of the family Enterobacteriaceae?
Oxidase. Enterobacteriaceae are oxidase-negative.
65
List several enteric organisms that cause polymorphonuclear leukocytes (PMNs / segmented neutrophils / polys) in the stool.
Salmonella, Shigella, Campylobacter, enteroinvasive E. coli, and Clostridium difficile.
66
An organism is isolated from a stool culture on Campy Blood Agar incubated at 42C in increased CO2. Gram stain reveals curved gram-negative rods, some resembling sea gulls. What two biochemical tests can be used fro presumptive identification of this organism?
Catalase and oxidase. Campylobacter is positive for both.
67
Name the enteric Campylobacters.
C. jejuni and C. coli.
68
Which species of Campylobacter is isolated in blood cultures?
C. fetus.
69
Which organism is thought to be the causative agent of some cases of gastric ulcers?
Helicobacter pylori. Its mode of transmission is not known.
70
How is H. pylori diagnosed?
Presumptive evidence is a positive test when biopsy material is placed into urease broth. The organism can also be cultured from biopsy material. Incubation for one week in a microaerobic atmosphere is required. Presumptive identification is based on cellular morphology (lik Campylobacter) and positive results for oxidase, catalase, and rapid urease tests. Non-invasive tests include the urea breath test, which detects H. pylori urease, EIA H. pylori stool antigen tests, and PCR assays for direct detection in the stool. Another approach is serodiagnosis in which EIA tests detect H. pylori antibodies in the blood.
71
Name a curved gram-negative rod that is found in coastal waters and causes wound infections and food poisoning associated with consumption of raw oysters.
Vibrio vulnificus.
72
Explain how to interpret OF (oxidation/fermentation) tubes.
Open Tube: +, +, -
Closed Tube: -, +, -
Interpretation: Oxidizer, Fermenter, Non-oxidizer, nonfermenter
73
Which non-fermenter is most frequently encountered in the clinical lab?
Pseudomonas aeruginosa.
74
A gram-negative rod is isolated from a patient with second and third degree burns. The isolate produces a bluish green pigment and a characteristics fruity odor. It is oxidase-positive and the TSI reactions are K/K. What reaction would you expect if you inoculated this organism into oxidation/fermentation media containing glucose?
Color change in the open tube; no color change in closed tube. The organism is Pseudomonas aeruginosa which is an oxidizer and a non-fermenter.
75
What pigment is specific for Pseudomonas aeruginosa?
Pyocyanin, a blue-green pigment.
76
How does Stenotophomonas (Xanthomonas) maltophilia differ form Pseudomonas?
It is oxidase-negative.
77
An organism is isolated from the urine of a catheterized in-patient. The colonies on blood agar are large and smooth with uneven and lavender-green to light purple pigment. A greenish discoloration to the agar underneath the growth is noted, along with an ammonia smell. The colonies on MacConkey agar are colorless. The Gram stain shows shot to medium-sized, straight, gram-negative rods. The organism is oxidase-negative and resistant to most antibiotics. What would you suspect?
Stenotrophomonas maltophilia. The species name, "maltose loving", refers to the fact that it oxidizes maltose faster than glucose. An important distinguishing biochemical reaction of S. maltophilia is a negative oxidase reaction. (Note: Don't confuse oxidation of carbohydrates and the oxidase reaction.) Stenotrophomonas is widely distributed in nature and is not usually part of the normal flora. Its resistance to a wide array of antibiotics allows it to survive in the hospital environment, where it is a common cause of nosocomial infections.
78
With what type of infection is Achromobacter (Alcaligenes) xylosoxidans associated?
Nosocomial infections involving compromised patients. Achromobacter survives in the hospital environment because of its resistance to disinfectants and antibiotics. It may be transmitted in contaminated IV fluids, hemodialysis fluids, irrigation fluids, soaps, and disinfectants. The species name refers to its strong oxidation of xylose. This gram-negative rod does not ferment glucose and is oxidase-positive.
79
Which gram-negative rods are frequently associated with pneumonia in cystic fibrosis patients?
Burkholderia cepacia, Pseudomonas aeruginosa, Achromobacter (Alcaligenes) xylosoxidans, and Stenotrophomonas maltophilia.
80
Which species of Burkholderia causes glanders, a contagious disease of horses that is communicable to man?
B. mallei.
81
Which species of Burkholderia causes melioidosis?
B. pseudomallei.
82
Plesiomonas shigelloides can cross react with Shigella grouping antisera. How could misidentification be avoided?
By the oxidase test. Shigella is oxidase-negative; Plesiomonas is oxidase-positive.
83
Describe Haemophilus on Gram stain.
Small, pale staining, pleomorphic gram-negative coccobacilli.
84
Discuss the growth requirements of Haemophilus.
Depending on the species, X and/or V factors may be required for growth. Growth is best with increased carbon dioxide. Haemophilus are susceptible to chilling and drying.
85
What is X factor?
Hemin, a heat stable substance found in hemoglobin.
86
What is V factor?
Nicotinamide adenine dinucleotide (NAD), a coenzyme found in blood yeast, potato extract, and synthesized by Staphylococcus, S. pneumoniae, and Neisseria.
87
What medium is used to grow Haemophilus?
Chocolate agar because it supplies X and V factors.
88
Where are Haemophilus species part of the normal flora?
In the upper respiratory tract.
89
Which species of Haemophilus is most commonly associated with infection?
H. influenzae.
90
Which serotype of Haemophilus influenzae causes the majority of causes of meningitis?
Type b. The incidence has been greatly reduced since a vaccine became available in the mid-80s.
91
What age group is most susceptible to H. influenzae meningitis?
Children 3 months to 6 years of age.
92
Which Haemophilus species causes conjunctivitis or pink eye?
H. influenzae, biotype aegyptius.
93
What is satellitism?
Tiny colonies of Haemophilus growing around colony on blood agar. Normally Haemophilus will not grow on blood agar unless it is near an organism that produces V factor such as Staphylococcus.
94
Explain how the different species of Haemophilus are identified.
```
H. influenzae--
Requirement For X Factor (hemin): +
Requirement For V Factor (NAD): +
Hemolysis on Rabbit or Horse Blood Agar: -
Porphyrin (ALA): -
```
```
H. parainfluenzae--
Requirement For X Factor (hemin): -
Requirement For V Factor (NAD): +
Hemolysis on Rabbit or Horse Blood Agar: -
Porphyrin (ALA): +
```
```
H. haemolytica--
Requirement For X Factor (hemin): +
Requirement For V Factor (NAD): +
Hemolysis on Rabbit or Horse Blood Agar: +
Porphyrin (ALA): -
```
```
H. parahaemolyticus--
Requirement For X Factor (hemin): -
Requirement For V Factor (NAD): +
Hemolysis on Rabbit or Horse Blood Agar: +
Porphyrin (ALA): +
```
```
H. aphrophilus--
Requirement For X Factor (hemin): -
Requirement For V Factor (NAD): -
Hemolysis on Rabbit or Horse Blood Agar: -
Porphyrin (ALA): +
```
```
H. ducreyi--
Requirement For X Factor (hemin): +
Requirement For V Factor (NAD): -
Hemolysis on Rabbit or Horse Blood Agar: -
Porphyrin (ALA): -
```
95
As suspected colony of Haemophilus is inoculated on trypticase soy agar and X and V strips are placed on the surface. After overnight incubation, growth is observed between the X and V strips only. Which species of Haemophilus has bee isolated?
Either H. influenzae or H. haemolyticus. Both require X and V factors. Hemolysis on rabbit or horse blood agar can differentiate the two. H. haemolyticus is hemolytic.
96
Explain the porphyrin or ALA test.
It is a test that can be used in place of X and V strips. The Haemophilus species that do not require X factor (H. parainfluenzae and H. parahemolyticus) can use delta aminolevulinic acid (ALA) as a substrate for the synthesis of porphyrin (hemin). Porphyrins fluoresce under UV light.
97
A porphyrin tube was inoculated with a colony suspected of being Haemophilus. After incubation, the tube was examined under UV light and orange fluorescence was observed. Which species of Haemophilus has been isolated?
Either H. parainfluenzae or H. parahemolyticus.
98
Two colony types are observed on a blood agar plate form a culture of an infant's eye. One type is large, white, convex, and opaque. The other is pinpoint, transparent, and only grows around the larger colony. There is no growth on the MacConkey agar. What tests should be performed?
The pinpoint colonies should be tested to determine if they are Haemophilus. A Gram stain, X and V strips or porphyrin test, and growth on rabbit blood agar should be performed. The large colony must be identified also, beginning with a Gram stain and catalase test.
99
Which species of Haemophilus resembles schools of fish on Gram stain?
H. ducreyi.
100
What does H. ducreyi cause?
Chancroid, a type of sexually transmitted disease that is most comon in teh tropics.
101
How does Haemophlius aphrophilus differ from other Haemophilus species?
It does not require X or V factors.
102
What is the HACEK group?
Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella. These organisms are normal flora of the month and opportunistic pathogens that cause endocarditis. All require increased carbon dioxide for growth.
103
What are some of the unique identifying characteristics of Eikenella corrodens?
A fastidious gram-negative coccobacillus that requires increased carbon dioxide and hemin. It often produces a yellow pigment, pitting of the agar, and a bleach-like odor. It is associated with human bites.
104
What it the name of the infection caused by Brucella?
Brucellosis. it is contracted from unpasteurized milk, direct contact with infected animals, or inhalation of aerosolized organisms. Brucella is a potential bioterrorism agent.
105
In suspected cases of brucellosis, what is the optimal specimen to collect?
Blood or bone marrow.
106
Why should suspected isolates of Burcella be referred to a Public Health laboratory?
Brucella is a BSL-3 pathogen and is the most commonly reported laboratory-associated bacterial infection. Cases have occurred from sniffing cultures, direct skin contact with cultures, and aerosol generating procedures.
107
What tests constitute a presumptive diagnosis of Brucella?
Tiny, faintly staining, gram-negative coccobacilli from blood or bone marrow
Slow growth on sheep blood agar (2-3 days from colony appearance)
Oxidase-positive
Urease-positive
Isolates that meet these criteria should be referred to a Public Health laboratory.
108
Which gram-negative rod causes tularemia and requires cystine or cysteine for growth?
Francisella tularensis.
109
How is Francisella contracted?
From ticks, handling infected animals, inhalation, or ingestion of contaminated food or water. Francisella is a potential bioterrorism agent.
110
Why should suspected isolates of Francisella be referred to a Public Health laboratory?
Francisella is a BSL-3 pathogen. It is one of the most virulent organisms known. As few as 1-10 cells can cause illness. It should not be manipulated at the bench.
111
What tests constitute a presumptive diagnosis of Francisella?
Poorly staining, tiny gram-negative coccobacilli from blood, lymph node aspirate, or respiratory specimens
Blood isolates that will grow slowly on chocolate agar but poorly or not at all on blood agar in 24 hours
Faint growth in thio (requires cysteine in other broths)
Isolates that meet these criteria should be referred to a Public Health laboratory.
112
What causes Legionnaire's disease?
Legionella pneumophilia.
113
How is Legionnaire's disease contracted?
Through inhalation of aerosols from air conditioners, cooling towers, shower heads, whirlpools, and misters.
114
Who is at highest risk for Legionnaire's disease?
The immonosuppressed, elderly, debilitated, and smokers.
115
What are Legionella's special growth requirements?
It requires 1-cysteine and iron for growth. It grows on buffered charcoal yeast extract medium (BCYE).
116
How can Legionella be rapidly diagnosed?
By direct Fluorescent antibody stain (DFA) of tissue or respiratory secretions. DFA is less sensitive than culture.
117
An organism causing respiratory disease with characteristic paroxysms produces colonies on Bordet-Gengou medium that resemble mercury droplets. What is the organism?
Bordetella pertussis.
118
Who is at risk for contracting whooping cough?
Once considered a childhood disease, whooping cough is now recognized as a disease in adults as well.
119
What is the preferred specimen specimen for isolation of Bordetella pertussis?
Although swabs made of calcium alginate are commonly sued to collect nasopharyngeal specimens, aspirated nasopharyngeal secretions are the best specimens.
120
What other media will grow Bordetella in addition to Bordet-Gengou?
Regan Lowe and charcoal-horse blood agar.
121
What rapid test can be used for the diagnosis of Bordetella directly from the clinical specimen?
A direct fluorescent antibody stain.
