Gram-Negative Bacilli

What are the general characteristics of the family Enterobacteriaceae?

-Gram-negative non-sporeforming rods

-Facultative anaerobes

-Oxidase negative (Plesiomonas shigelloides is an exception)

-Glucose fermenters

-Most reduce nitrates to nitrites

-Most are catalase positive

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?

Acid butt in TSI. All Enterobacteriaceae ferment glucose. Not all ferment lactose or sucrose or produce H2S.

What are the 3 antigens used for serological grouping of some of the Enterobacteriaceae, and where are they located?

Antigen/Alternate Name/Location/Characteristics

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.

Which of the Enterobacteriaceae are routinely serotyped?

Salmonella, Shigella, and E. coli O157:H7

What error may result from selecting organisms for serotyping from a sugar-containing medium such as MacConkey or TSI?

Auto-agglutination. Colonies should be selected from blood agar.

Which antigens would be expected in bacteria that grow away from the stab line in motility agar?

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.

Following presumptive identification of Salmonella and Shigella by biochemical testing, what further testing is required?

Serotyping. Salmonella is typed by its O and H antigens., Shigella by O antigens only. (It is nonmotile, so does not have H antigens.)

Which antigens are associated with Salmonella typhi?

O and Vi, a capsular antigen associated with virulence.

A suspected Salmonella is tested with polyvalent O antiserum and no agglutination is observed. What should be done and why?

Typing should be repeated on a suspension of the organism that has been heated. Heating destroys the K antigens which may mask the O antigens.

Which 3 enterics are most commonly isolated from clinical specimens?

E. coli, Klebsiella, and Proteus.

Which species of Proteus is most frequently isolated?

P. mirabilis

Which Enterobacteriaceae are pathogens in the intestinal tract?

Salmonella, Shigella, diarrheagenic E. coli, and yersinia.

Which Enterobacteriaceae are nonlactose fermenters (NLF)?

Salmonella, Shigella, Proteus, Morganella, Providencia, Yersinia, Serratia, and Edwardsiella. Some Citrobacter and E. coli are lactose negative also.

Describe lactose fermenters (LF) and nonlactose fermenters (NLF) on EMB, MacConkey, Hektoen Enteric, and XLD

Media/Lactose Fermenters/Nonlactose Fermenters

EMB / Purple or green-black / Colorless

MacConkey / Pink / Colorless

Hektoen Enteric / Orange to salmon pink / Green to blue-green

XLD / Yellow / Colorless or red

What color are E. coli colonies on XLD?

Yellow

What color are Shigella colonies on XLD?

Colorless (the red medium shows through)

What color are Salmonella colonies on XLD?

Red with black center

Which enteric is characterized by swarming growth on blood agar?

Proteus

Which lactose-positive enterics are often "stringy" when touched with a loop (mucoid)?

Klebsiella and Enterobacter. This is due to their polysaccharide capsules.

Which enteric produces a characteristic green metallic sheen on EMB?

E. coli

Which facultative gram-negative rod sometimes produces a red pigment?

Serratia marcescens

Which member of the family Enterobacteriaceae is thought to be a potential agent of bioterrorism?

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 with Wayson stain. Y. pestis grows best at 28°C.

Which sugars are contained in TSI tubes?

Glucose (0.1%), lactose (1%), and sucrose (1%)

Explain how to interpret TSI tubes

Yellow butt: glucose positive

Pink butt: glucose negative

Yellow slant: lactose and/or sucrose positive

Pink slant: lactose and sucrose negative

Black precipitate: hydrogen sulfide production

Bubbles: gas production

How could 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.

Describe TSI reactions for E. coli

A/A, Gas

Describe TSI reactions for Proteus vulgaris

A/A with H2S. The yellow slant is due to fermentation of sucrose, not lactose

Describe TSI reactions for Salmonella

K/A, Gas, H2S

Describe TSI reactions for Shigella

K/A

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

Which Enterobacteriaceae are Voges-Proskauer positive?

Klebsiella, Enterobacter, and Serratia

Name 4 enterics that are H2S positive

Salmonella, Proteus, Citrobacter, and Edwardsiella

What does the ONPG test detect?

B-galactosidase, and enzyme required for organisms to utilize lactose. Both rapid lactose fermenters and late (slow) lactose fermenters have the enzyme.

What might cause a false-positive oxidase test?

The use of stainless steel or Nichrome inoculating loops or wires. A wooden stick or a platinum wire should be used.

From which medium should organisms be selected for oxidase testing?

One without fermentable sugars, for example, blood or chocolate agar. Fermentation of carbohydrates may result in a false-negative oxidase reactions due to acidification of the medium.

A bacterial colony turns dark blue-black when a 1% solution of tetramethyl-para-phenylenediamine dihydrochloride is applied. What can be said about the organism?

It is oxidase positive

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 sodium citrate as its only carbon source and inorganic ammonium salts as its only nitrogen source.

Name 3 members of the family Enterobacteriaceae that are urease positive.

Proteus, Morganella, and Providencia rettgeri. Klebsiella is also positive, but slowly.

Which genera of the family Enterobacteriaceae are positive for phenylalanine deaminase?

Proteus, Morganella, and Providencia

Which genera of Enterobacteriaceae are usually nonmotile at 35°C?

Shigella, Klebsiella, and Yersinia. Yersinia is motile at 25°C.

What must be present in the medium from which colonies are selected for a spot indole test?

Tryptophan, otherwise a false-negative result will occur. Blood and chocolate agars contain tryptophan; MacConkey does not.

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 Proteus P. vulgaris is indole positive.

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.

Compare IMViC reactions for E. coli, Klebsiella, and Enterobacter

Org./Indole/Methyl Red/Voges-Proskauer/Citrate

E. coli / + / + / = / =

Klebsiella / = / = / + / +

Enterobacter / = / = / + / +

How can Klebsiella and Enterobacter be differentiated?

Klebsiella is ornithine decarboxylase negative; Enterobacter is positive. Klebsiella is nonmotile; Enterobacter is motile.

Which species of Klebsiella is indole positive?

K. oxytoca

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.

What diseases does enterohemorrhagic E. coli cause?

Hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.

How is enterohemorrhagic E. coli contracted?

From undercooked meat, raw milk, unpasteurized apple cider, and contaminated produce, such as lettuce.

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.

What is enteropathogenic E. coli?

A noninvasive, nontoxigenic E. coli that causes diarrhea in infants. No RBCs or WBCs are found in the stool.

Which rapid test can differentiate Proteus mirabilis from Proteus vulgaris?

Spot indole. P. mirabilis is indole negative; P. vulgaris is indole positive.

Which biochemical test differentiates Salmonella and Shigella from most normal intestinal gram-negative rods?

Lactose. Salmonella and Shigella are lactose negative.

Name 2 tests that differentiate Salmonella and Shigella.

Salmonella is motile and hydrogen sulfide positive; Shigella is nonmotile and hydrogen sulfide negative.

Which species of Salmonella produces only trace amounts of H2S and is citrate negative?

S. typhi

What infection does Shigella cause?

Bacillary dysentery. Dysentery is a condition in which diarrhea is accompanied by cramping abdominal pain and tenesmus (painful straining when passing a stool). It is caused by microorganisms that penetrate the mucosa of the intestinal tract and cause inflammation. The stools often contain white blood cells, red blood cells, and mucus.

In which patient population are Shigella infections most common?

Children. Outbreaks can occur at any time of the year, but are most common in the summer. Shigella is the most easily communicated of the bacterial diarrheas because so few organisms are required to establish an infection.

Which Shigella is most commonly isolated in the U.S.?

S. sonnei

Which Shigella ferments lactose slowly?

S. sonnei (rarely)

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.

Name a medium that is selective for Yersinia enterocolitica

Cefsulodin-irgasan-novobiocin agar (CIN). Y. enterocolitica colonies are red "bull's eye" colonies surrounded by a colorless halo.

Which rapid test, when positive, rules out most members of the family Enterobacteriaceae?

Oxidase. All of the Enterobacteriaceae, with the exception of the recently added Plesiomonas shigelloides, are oxidase negative.

Why was Plesiomonas shigelloides reclassified as a member of the family Enterobacteriaceae?

Because ribosomal RNA sequencing showed that it is very similar to Proteus. The species name reflects its biochemical and antigenic similarities to Shigella.

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.

What types of infection does Plesiomonas shigelloides cause?

Diarrheal disease, often associated with raw or undercooked seafood, and occasionally extraintestinal infections, such as bacteremia and meningitis in immunocompromised patients.

Which biochemical reactions are most helpful in the identification of Plesiomonas shigelloides?

It is positive for oxidase, inositol fermentation, ornithine, lysine, and arginine.

What are several enteric organisms that cause polymorphonuclear leukocytes (PMNs/segmented neutrophils/polys) in the stool?

Salmonella, Shigella, Campylobacter, enteroinvasive E. coli, and Clostridium difficile

An organism is isolated from a stool culture on Campy blood agar incubated at 42°C in a microaerophilic, capnophilic atmosphere. Gram stain reveals curved gram-negative rods, some resembling sea gulls. What 2 biochemical tests can be used for presumptive identification of this organism?

Catalase and oxidase. Campylobacter is positive for both.

What is the ideal atmosphere for culturing Campylobacter?

5% O2, 10% CO2, and 85% N2

Why is selective Campylobacter medium incubated at 42°C?

C. jejuni can grow from 4°C-42°C. The higher incubation temperature inhibits the growth of normal enteric flora.

Which are the enteric camplyobacters?

C. jejuni and C. coli.

Which species of Campylobacter is isolated in blood cultures?

C. fetus

Which organism is thought to be the causative agent of some cases of gastric ulcers?

Helicobacter pylori. Its mode of transmission is not known.

How is H. pylori diagnosed?

Usually by nonculture methods, such as a rapid urease reaction from a gastric biopsy specimen or by the urea breath test. Another approach is serodiagnosis in which H. pylori antibodies in the blood are detected by ELISA.

Which curved gram-negative rod is found in coastal waters and causes wound infections and food poisoning associated with consumption of raw oysters, especially in patients with liver disease?

Vibrio vulnificus

Which organism causes cholera?

Vibrio cholerae. It is transmitted by contaminated food and water and causes a profuse diarrhea that leads to dehydration. The stool is described as "rice water stool" because it is watery with flecks of mucus.

Which test separates V. cholerae from most members of the family Enterobacteriaceae?

Oxidase. Vibrio is oxidase positive.

How does V. cholerae differ from most other Vibrio species?

It does not grow in 6.0% NaCl. Other species are halophilic (salt loving) and require some NaCl to grow. They grow in concentrations ranging from 1.0-12.0%; V. cholerae grows in 0-1.0% NaCl.

What can be used as a transport medium and enrichment broth for a stool sample from a patient suspected of having cholera?

Alkaline peptone water (APW). The pH of 8.6-9.0 inhibits normal flora.

What is a selective and differential medium for Vibrio?

Thiosulfate-citrate-bile salts-sucrose (TCBS) agar. V. cholera and a few other species ferment sucrose and produce yellow colonies. Species that do not ferment sucrose remain blue-green.

How is the OF (oxidation/fermentation) test interpreted?

Open tube/Closed tube/Interpretation

+ / = / Oxidizer

+ / + / Fermenter

= / = / Nonoxidizer, nonfermenter

Which nonfermenter is most frequently encountered in the clinical lab?

Pseudomonas aeruginosa

Which pigment is specific for Pseudomonas aeruginosa?

Pyocyanin, a blue-green pigment

A gram-negative rod is isolated from a patient with second and third degree burns. The isolate produces a bluish green pigment and a characteristic 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 nonfermenter.

How does Stenotrophomonas maltophilia differ from Pseudomonas?

It is oxidase negative

An organism is isolated from the urine of a catheterized in-patient. The colonies on blood agar are large and smooth with uneven edges 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 short 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. 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.

What are some of the characteristics of Acinetobacter?

It is a nonfermentative, oxidase-negative, gram-negative rod that grows on MacConkey agar. The appearance on Gram stain is that of plump coccobacilli that may be mistaken for Neisseria.

What is the clinical significance of Acinetobacter?

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.

A respiratory specimen from an immunocompromised patient grows feather-edged colonies surrounded by a zone of green discoloration on blood agar. The colonies on MacConkey agar are colorless. A fruity odor of apples or strawberries is detected. The Gram stain shows medium to long, straight gram-negative rods. The organism is oxidase positive and does not ferment glucose. What would you suspect?

Alcaligenes faecalis

With which type of infection is Achromobacter 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.

Which gram-negative rods are frequently associated with pneumonia in cystic fibrosis patients?

Burkholderia cepacia, Pseudomonas aeruginosa, Alcaligenes xylosoxidan, and Stenotrophomonas maltophilia

Which species of Burkholderia causes glanders, a contagious disease of horses that is communicable to man?

B. mallei

Which species of Burkholderia causes melioidosis?

B. pseudomallei

How does Haemophilus appear on Gram stain?

Small, pale staining, pleomorphic gram-negative coccobacilli

What are several considerations in ensuring the recovery of Haemophilus from culture?

Depending on the species, X and/or V factors may be required for growth. Growth is best with increased CO2. Haemophilus is susceptible to chilling and drying.

What is X factor?

Hemin, a heat stable substance found in hemoglobin.

What is V factor?

Nicotinamide adenine dinucleotide (NAD), a coenzyme found in blood, yeast, potato extract, and synthesized by Staphylococcus, S. pneumoniae, and Neisseria.

What medium is used to grow Haemophilus?

Chocolate agar because it supplies X and V factors.

Where are Haemophilus species part of the normal flora?

In the upper respiratory tract.

Which species of Haemophilus is most commonly associated with infection?

H. influenzae