DISCUSSION

A. ENTEROBACTERIACEAE: THE FERMENTATIVE, GRAM-NEGATIVE, ENTERIC BACILLI

Bacteria belonging to the family Enterobacteriaceae are the most commonly encountered organisms isolated from clinical specimens. The Enterobacteriaceae is a large diverse family of bacteria belonging to the order Enterobacteriales in the class Gammaproteobacter of the phylum Proteobacter. Medically important members of this family are commonly referred to as fermentative, gram-negative, enteric bacilli, because they are gram-negative rods that can ferment sugars. Many are normal flora of the intestinal tract of humans and animals while others infect the intestinal tract. Members of this family have the following five characteristics in common:

1. They are gram-negative rods (see Fig. 1)
2. If motile, they possess a peritrichous arrangement of flagella (see Fig. 2)
3. They are facultative anaerobes
4. With the exception of the genus genus Plesiomonas, they are oxidase negative
5. All species ferment the sugar glucose but otherwise vary widely in their biochemical characteristics.

For further information on the gram-negative cell wall, see the following Learning Object in your Lecture Guide:

• The Gram-negative cell wall, Unit 1, Section IIB2b

Forty-four genera and over 130 species of Enterobacteriaceae have been recognized. Some of the more common clinically important genera of the family Enterobacteriaceae include:

Several genera of Enterobacteriaceae are associated with gastroenteritis and food-borne disease. These include:

• Salmonella,
• Shigella,
• certain strains of Escherichia coli, and
• certain species of Yersinia.

All intestinal tract infections are transmitted by the fecal-oral route.

Any infection caused by Salmonella is called a salmonellosis. An estimated 2,000,000 - 3,000,000 people a year in the U.S. become infected with Salmonella and at least 500 die. Since many different animals carry Salmonella in their intestinal tract, people usually become infected from ingesting improperly refrigerated, uncooked or undercooked poultry, eggs, meat, or dairy products contaminated with animal feces.

Enteritis is the most common form of salmonellosis. Symptoms generally appear 6-48 hours after ingestion of the bacteria and include vomiting, nausea, non-bloody diarrhea, fever, abdominal cramps, myalgias, and headache. Symptoms generally last from 2 days to 1 week followed by spontaneous recovery. All species of Salmonella can cause bacteremia but S. typhi, S. paratyphi, and S. choleraesuis are the most common species to cause bacteremia. S. typhi, frequently disseminates into the blood causing a severe form of salmonellosis called typhoid fever. In 1998, 375 cases of typhoid fever were reported in the U.S. but most of these cases were acquired during foreign travel.

Highlighted Bacterium: Salmonella

Any Shigella infection is called a shigellosis. Unlike Salmonella, which can infect many different animals, Shigella only infects humans and other higher primates. There are 20,000 - 30,000 cases of shigellosis a year reported in the US with an estimated 450,000 total cases.

Symptoms of shigellosis include diarrhea, bloody stool, abdominal cramps, and fever. The incubation period is 1-3 days. Initial profuse watery diarrhea typically appears first as a result of enterotoxin. Within 1-2 days this progresses to abdominal cramps, with or without bloody stool. Classic shigellosis presents itself as lower abdominal cramps and stool abundant with blood and pus develops as the Shigella invade the mucosa of the colon.

Highlighted Bacterium: Shigella

While Escherichia coli is one of the dominant normal flora in the intestinal tract of humans and animals, some strains can cause infections of the intestines.

• Enterotoxigenc E. coli (ETEC) produce enterotoxins that cause the loss of sodium ions and water from the small intestines resulting in a watery diarrhea. Over half of all travelers' diarrhea is due to ETEC; almost 80,000 cases a year in the U.S.
• Enteropathogenic E. coli (EPEC) causes an endemic diarrhea in areas of the developing world, especially in infants younger than 6 months. The bacterium disrupts the normal microvilli on the epithelial cells of the small intestines resulting in maladsorbtion and diarrhea.
• Enteroaggregative E. coli (EAEC) is a cause of persistant diarrhea in developing countries. It probably causes diarrhea by adhering to mucosal epithelial cells of the small intestines and interfering with their function.
• Enteroinvasive E. coli (EIEC) invade and kill epithelial cells of the large intestines causing a dysentery-type syndrome similar to Shigella common in underdeveloped countries.
• Enterohemorrhagic E. coli (EHEC), such as E. coli 0157:H7, produce a shiga-like toxin that kills epithelial cells of the large intestines causing hemorrhagic colitis, a bloody diarrhea. In rare cases, the shiga-toxin enters the blood and is carried to the kidneys where, usually in children, it damages vascular cells and causes hemolytic uremic syndrome. E. coli 0157:H7 is thought to cause more than 20,000 infections and up to 250 deaths per year in the U.S.
• Diffuse aggregative E. coli (DAEC) causes watery diarrhea in infants 1-5 years of age. They stimulate elongation of the microvilli on the epithelial cells lining the small intestines.
Scanning electron micrograph E. coli with pili; courtesy of Dennis Kunkel's Microscopy.

Several species of Yersinia, such as Y. enterocolitica and Y. pseudotuberculosis are also causes of diarrheal disease.

Many other genera of the family Enterobacteriaceae are normal flora of the intestinal tract and are considered opportunistic pathogens. The most common genera of Enterobacteriaceae causing opportunistic infections in humans are:

• Escherichia coli,
• Proteus,
• Enterobacter,
• Klebsiella,
• Citrobacter, and
• Serratia.

They act as opportunistic pathogens when they are introduced into body locations where they are not normally found, especially if the host is debilitated or immunosuppressed. They all cause the same types of opportunistic infections, namely:

• urinary tract infections,
• wound infections,
• pneumonia, and
• septicemia.

These normal flora gram-negative bacilli, along with gram-positive bacteria such as Enterococcus species (see Lab 14) and Staphylococcus species (see Lab 15), are among the most common causes of nosocomial infections. The four most common gram-negative bacteria causing nosocomial infections are Escherichia coli, Pseudomonas aeruginosa (discussed below), Enterobacter species, and Klebsiella pneumoniae. Collectively, these four bacteria accounted for 32 percent of all nosocomial infections in the U.S. between 1990 and 1996. There are over two million nosocomial infections per year in the U.S.

The most common infection caused by these opportunistic Enterobacteriaceae is a urinary tract infection (UTI). UTIs account for more than 7, 000,000 physician office visits per year in the U.S. Among the nonhospitalized and nondebilitated population, UTIs are more common in females because of their shorter urethra and the closer proximity between their anus and the urethral opening. (Over 20 percent of women have recurrent UTIs.) However, anyone can become susceptible to urinary infections in the presence of predisposing factors that cause functional and structural abnormalities of the urinary tract. These abnormalities increase the volume of residual urine and interfere with the normal clearance of bacteria by urination. Such factors include prostate enlargement, sagging uterus, expansion of the uterus during pregnancy, paraplegia, spina bifida, scar tissue formation, and catheterization. Between 35 and 40 percent of all nosocomial infections, about 900,000 per year in the U.S., are UTIs and are usually associated with catheterization.

E. coli causes around 90 percent of all uncomplicated urinary tract infections (UTIs) and more than 50 percent of nosocomial UTIs. Staphylococcus saprophyticus (see Lab 15) causes 10 - 20 percent of uncomplicated UTIs and approximately 5 percent of UTIs are caused by other gram-negative enterics such as species of Proteus and Klebsiella or by Enterococcus species (see Lab 14).

Highlighted Bacterium: Escherichia coli

The traditional laboratory culture standard for a UTI has been the presence of more than 100,000 CFUs (colony-forming units; see Lab 4) per milliliter (ml) of midstream urine, or any CFUs from a catheter-obtained urine sample. More recently, this has been modified and counts of as few as 1000 colonies of a single type per ml or as little as 100 coliforms per ml are now considered as indicating a UTI.

Highlighted Infection: Urinary Tract Infections (UTIs)

Wound infections are due to fecal contamination of external wounds or a result of wounds that cause trauma to the intestinal tract, such as surgical wounds, gunshot wounds, knife wounds, etc.

Although they sometimes cause pneumonia, the Enterobacteriaceae account for less than 5% of the bacterial pneumonias requiring hospitalization.

Gram-negative septicemia is a result of these opportunistic gram-negative bacteria getting into the blood. They are usually introduced into the blood from some other infection site, such as an infected kidney, wound, or lung. There are approximately 750,000 cases of septicemia per year in the U.S. and the mortality rate is between 20 and 50 percent. Over 210,000 people a year in the U.S. die from septic shock. Approximately 45 percent of the cases of septicemia are due to gram-negative bacteria. Klebsiella, Proteus, Enterobacter, Serratia, and E. coli, are all common Enterobacteriaceae causing septicemia. (Another 45 percent are a result of gram-positive bacteria (see Labs 14 and 15) and 10 percent are due to fungi, mainly the yeast Candida.)

In the outer membrane of the gram-negative cell wall, the lipid A moiety of the lipopolysaccharide functions as an endotoxin (see Fig 3). Endotoxin indirectly harms the body when massive amounts are released during severe gram-negative infections. This, in turn, causes an excessive cytokine response.

1. The LPS released from the outer membrane of the gram-negative cell wall first binds to a LPS-binding protein circulating in the blood and this complex, in turn, binds to a receptor molecule (CD14) found on the surface of body defense cells called macrophages (see Fig. 4) located in most tissues and organs of the body.

2. This is thought to promote the ability of the toll-like receptor TLR-4 to respond to the LPS, triggering the macrophages to release various defense regulatory chemicals called cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8), and platelet-activating factor (PAF) (see Fig. 4). The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway (see Fig. 4).

3. The complex of LPS and LPS binding protein can also attach to molecules called CD14 on the surfaces of phagocytic white blood cells called neutrophils causing them to release proteases and toxic oxygen radicals for extracellular killing. Chemokines such as interleukin-8 (IL-8) also stimulate extracellular killing. In addition, LPS and cytokines stimulate the synthesis of a vasodilator called nitric oxide.

During minor local infections with few bacteria present, low levels of LPS are released leading to moderate cytokine production by the monocytes and macrophages and in general, promoting body defense by stimulating inflammation and moderate fever, breaking down energy reserves to supply energy for defense, activating the complement pathway and the coagulation pathway, and generally stimulating immune responses (see Fig. 4). Also as a result of these cytokines, circulating phagocytic white blood cells such as neutrophils and monocytes stick to the walls of capillaries, squeeze out and enter the tissue, a process termed diapedesis. The phagocytic white blood cells such as neutrophils then kill the invading microbes with their proteases and toxic oxygen radicals.

However, during severe systemic infections with large numbers of bacteria present, high levels of LPS are released resulting in excessive cytokine production by the monocytes and macrophages and this can harm the body (see Fig. 5). In addition, neutrophils start releasing their proteases and toxic oxygen radicals that kill not only the bacteria, but the surrounding tissue as well. Harmful effects include high fever, hypotension, tissue destruction, wasting, acute respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC), and damage to the vascular endothelium resulting in shock, multiple system organ failure (MOSF), and often death.

This excessive inflammatory response is referred to as Systemic Inflammatory Response Syndrome or SIRS. Death is a result of what is called the shock cascade. The sequence of events is as follows:

During a severe systemic infection, an excessive inflammatory response triggered by overproduction of inflammatory cytokines such as TNF-alpha, IL-1, IL-6, IL-8, and PAF in response to PAMPs often occurs.

The release of inflammatory cytokines eventually leads to vasodilation of blood vessels. Vasodilation (def) is a reversible opening of the junctional zones between endothelial cells (def) of the blood vessels and results in increased blood vessel permeability. Normally, this fights the infection by enabling plasma, the liquid portion of the blood, to enter the surrounding tissue.The plasma (def) contains defense chemicals such as antibody molecules (def), complement proteins (def), lysozyme (def), and beta defensins (def). Increased capillary permeability also enables white blood cells to adhere to the inner capillary wall, squeeze out of the blood vessels, and enter the tissue to fight infection, a process called diapedesis (def).

Excessive productions of cytokines during a systemic infection results in the following events:

1.During diapedesis (def), phagocytic WBCs called neutrophils (def) adhere to capillary walls in massive amounts.

• Chemokines such as IL-8 activate extracellular killing by neutrophils, causing them to release proteases (def) and toxic oxygen radicals (def) while still in the capillaries. These are the same toxic chemicals neutrophils use to kill microbes, but now they are dumped onto the vascular endothelial cells to which the neutrophils have adhered.
• These events result in damage to the capillary walls and leakage of blood into surrounding tissue (see Fig. 3). This contributes to a decreased volume of circulating blood (hypovolemia) (def).
• Hypovolemia then contributes to hypoperfusion (def).

Flash animation summarizing early inflammation and diapedesis.

Flash animation summarizing late inflammation and diapedesis.

Flash animation of extracellular killing by neutrophils

2. Prolonged vasodilation (def) and the resulting increased capillary permeability causes plasma (def) to leave the bloodstream and enter the tissue.

• This contributes to a decreased volume of circulating blood (hypovolemia) (def).
• Hypovolemia then contributes to hypoperfusion (def).

Prolonged vasodilation also leads to decreased vascular resistance within blood vessels.

• This, in turn, contributes to a drop in blood pressure (hypotension).
• Hypotension then contributes to hypoperfusion (def).

Animation of vasodilation.

3. At high levels of TNF, vascular smooth muscle tone and myocardial contractility are inhibited.

• This results in a marked hypotension (def).
• Hypotension then contributes to hypoperfusion (def).

Cytokine-induced overproduction of nitric oxide (NO) by cardiac muscle cells and vascular smooth muscle cells can also lead to heart failure.

4. Activation of the blood coagulation pathway can cause clots called microthrombi to form within the blood vessels throughout the body. This is called disseminated intravascular coagulation (DIC) (def).

• These microthrombi block the capillaries and interfere with perfusion (def).
• Activation of neutrophils also leads to their accumulation and plugging of the vasculature.

5. In the lungs, the increased capillary permeability as a result of vasodilation in the lungs, as well as neutrophil-induced injury to capillaries in the alveoli (def) leads to acute inflammation, pulmonary edema (def), and loss of gas exchange in the lungs. Thiscondition is called acute respiratory distress syndrome (ARDS) (def).

• As a result, the blood does not become oxygenated.
• Lack of oxygenation of the blood via the lungs then causes hypoperfusion (def).

6. In the liver, hypoperfusion (def) and capillary damage results in impaired liver function and a failure to maintain normal blood glucose levels.

• Overuse of glucose by muscles and a failure of the liver to replace glucose can lead to a drop in blood glucose level below what is needed to sustain life. (Glucose is needed to make ATP via aerobic respiration.)

7. Hypoperfusion can also leads to kidney and bowel injury.

8. The combination of hypotension (def), hypovolemia (def), DIC (def), ARDS (def), and the resulting hypoperfusion (def) leads to acidosis (def).

• Without oxygen, cells switch to fermentation and produce lactic acid that lowers the pH of the blood. A blood pH range between 6.8 and 7.8 is needed for normal cellular metabolic activities in humans.
• Changes in the pH of arterial blood extracellular fluid outside this range lead to irreversible cell damage.

In summary:

• Neutrophil-induced damage to the capillaries, as well as prolonged vasodilation, results in blood and plasma leaving the bloodstream and entering the surrounding tissue. This can lead to a decreased volume of circulating blood (hypovolemia).
• Prolonged vasodilation also leads to decreased vascular resistance within blood vessels while high levels of TNF, inhibit vascular smooth muscle tone and myocardial contractility. This results in a marked hypotension.
• Activation of the blood coagulation pathway can cause clots called microthrombi to form within the blood vessels throughout the body causing disseminated intravascular coagulation (DIC).
• Increased capillary permeability as a result of vasodilation in the lungs, as well as neutrophil-induced injury to capillaries in the alveolileads to acute inflammation, pulmonary edema, and loss of gas exchange in the lungs (acute respiratory distress syndrome or ARDS). As a result, the blood does not become oxygenated.
• Hypotension, hypovolemia, ARDS, and DIC result in marked hypoperfusion.
• Hypoperfusion in the liver can result in a drop in blood glucose level from liver dysfunction.
• Hypoperfusion leads to acidosis and the wrong pH for enzymes involved in cellular metabolism resulting in cell death.
• Hypoperfusion also can lead to cardiac failure.

Collectively, this can result in :

• end-organ ischemia (def) Ischemia is a restriction in blood supply that results in damage or dysfunction of tissues or organs.
•  multiple system organ failure (MSOF) (def).
• death.

This is known as Systemic Inflammatory Response Syndrome (SIRS).

Highlighted Infection: Septicemia and Septic Shock

 

The gram-negative cell wall also contains surface proteins that function as adhesins, allowing the bacterium to adhere intimately to host cells and other surfaces in order to colonize and resist flushing. Some gram-negative bacteria also produce invasins, allowing some bacteria to penetrate host cells. Pili, flagella, capsules, and exotoxins also play a role in the virulence of some Enterobacteriaceae.

For further information on virulence factors associated with various Enterobacteriaceae, see the following Learning Objects in your Lecture Guide:

• Endotoxin; Unit 2, Section IC1b
• The Ability to Adhere to Host Cells: Pili and Adhesins; Unit 2, Section IB2
• The Ability to Invade Host Cells; Unit 2, Section IB3
• The Ability to Contact Host Cells; Unit 2, Section IB1
• The Ability to Resist Phagocytic Engulfment; Unit 2, Section IB5b
• The Ability to Resist Phagocytic Destruction and Serum Lysis; Unit 2, Section IB5c
• A-B Toxins; Unit 2, Section IC2b
• Toxins that Damage Cell Membranes; Unit 2, Section IC2c

Many of the Enterobacteriaceae also possess R (resistance) plasmids (see Lab 21). These plasmids are small pieces of circular non-chromosomal DNA that may code for multiple antibiotic resistance In addition, the plasmid may code for a sex pilus, enabling the bacterium to pass R plasmids to other bacteria by conjugation. As mentioned earlier, there are over 2,000,000 nosocomial infections per year in the U.S. Between 50 and 60 percent of the bacteria causing these infections are antibiotic resistant.

For further information on bacterial resistance to antibiotics, see the following Learning Object in your Lecture Guide:

• How Bacteria Resist Our Control Agents; Unit 2, Section IIC

 

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B. PSEUDOMONAS AND OTHER NON-FERMENTATIVE GRAM-NEGATIVE BACILLI

Non-fermentative gram-negative bacilli refer to gram-negative rods or coccobacilli that cannot ferment sugars. The non-fermentative gram-negative bacilli are often normal inhabitants of soil and water. They may cause human infections when they colonize immunosuppressed individuals or gain access to the body through trauma. However, less than one-fifth of the gram-negative bacilli isolated from clinical specimens are non-fermentative bacilli. By far, the most common gram-negative, non-fermentative rod that causes human infections is Pseudomonas aeruginosa. Pseudomonas belongs to the family Pseudomonadaceae in the order Pseudomonadales in the class Gammaproteobacter of the phylum Proteobacter.

Pseudomonas aeruginosa is also an opportunistic pathogen. It is a common cause of nosocomial infections and can be found growing in a large variety of environmental locations. In the hospital environment, for example, it has been isolated from drains, sinks, faucets, water from cut flowers, cleaning solutions, medicines, and even disinfectant soap solutions. It is especially dangerous to the debilitated or immunocompromised patient.

Like the opportunistic Enterobacteriaceae, Pseudomonas is a gram-negative rod, it is frequently found in small amounts in the feces, and it causes similar opportunistic infections: urinary tract infections, wound infections, pneumonia, and septicemia. P aeruginosa is the fourth most commonly isolated nosocomial pathogen, accounting for 10% of all hospital-acquired infections. P. aeruginosa is responsible for 12 percent of hospital-acquired urinary tract infections, 16 percent of nosocomial pneumonia cases, and 10 percent of the cases of septicemia. In addition, P. aeruginosa is a significant cause of burn infections with a 60 percent mortality rate. It also colonizes and chronically infects the lungs of people with cystic fibrosis. Like other opportunistic gram-negative bacilli, Pseudomonas aeruginosa also releases endotoxin and frequently possesses R-plasmids. A number of other species of Pseudomonas have also been found to cause human infections.

• Electron micrograph of Pseudomonas aeruginosa colonizing a vascular catheter.

For further information on virulence factors associated with Pseudomonas, see the following Learning Objects in your Lecture Guide:

• Endotoxin; Unit 2, Section IC1b
• The Ability to Adhere to Host Cells: Pili and Adhesins; Unit 2, Section IB2
• The Ability to Invade Host Cells; Unit 2, Section IB3
• The Ability to Contact Host Cells; Unit 2, Section IB1
• The Ability to Resist Phagocytic Engulfment; Unit 2, Section IB5b
• The Ability to Resist Phagocytic Destruction and Serum Lysis; Unit 2, Section IB5c
• A-B Toxins; Unit 2, Section IC2b
• Toxins that Damage Cell Membranes; Unit 2, Section IC2c

Other non-fermentative gram-negative bacilli that are sometimes opportunistic pathogens in humans include Acinetobacter, Aeromonas, Alcaligenes, Eikenella, Flavobacterium, and Moraxella.

Acinetobacter has become a frequent cause of nosocomial wound infections, pneumonia, and septicemia. The bacterium has become well known as a cause of infections among veterans of the wars in Iraq and Afghanistan and is becoming an growing cause of nosocomial infections in the U.S. Acinetobacter is thought to have been contracted in field hospitals in Iraq and Afghanistan and subsequently carried to veteran's hospitals in the U.S. Because most species are multiple drug resistant, it is often difficult to treat.

Acinetobacter is commonly found in soil and water, as well as on the skin of healthy people, especially healthcare personnel. Although there are numerousr species of Acinetobacter that can cause human disease, Acinetobacter baumannii accounts for about 80% of reported infections.

Concept map for Lab 12

E-Medicine articles on infections associated with organisms mentioned in this lab exercise. Registration to access this website is free. Salmonellosis Typhoid fever Shigellosis Escherichia coli Proteus species Klebsiella species Enterobacter species Serratia species Yersinia enterocolitica Yersinia pseudotuberculosis Acinetobacter baumannii Pseudomonas aeruginosa Urinary tract infections Wound infections Community-acquired pneumonia Nosocomial pneumonia Sepsis Septic shock

C. ISOLATION OF ENTEROBACTERIACEAE AND PSEUDOMONAS

To isolate Enterobacteriaceae and Pseudomonas, specimens from the infected site are plated out on any one of a large number of selective and differential media such as EMB agar (used in Lab 3), Endo agar, Deoxycholate agar, MacConkey agar, Hektoen Enteric agar, and XLD agar.

XLD agar is selective for gram-negative bacteria. In addition, different gram-negative bacilli, due to their biochemical reactions, produce different appearing colonies. Typical reactions for some of the Enterobacteriaceae and Pseudomonas are shown below:

1. Escherichia coli: flat yellow colonies; some strains may be inhibited.
2. Enterobacter and Klebsiella: mucoid yellow colonies.
3. Proteus: red to yellow colonies; may have black centers.
4. Salmonella: usually red colonies with black centers.
5. Shigella and Pseudomonas: red colonies without black centers.

The biochemical reasons for these color reactions will be discussed in Lab 13. Some species and subspecies, however, may not show typical reactions.

Pseudosel agar is selective for Pseudomonas aeruginosa and also stimulates P. aeruginosa to produce its characteristic pigment as well as fluorescent products. Pseudomonas aeruginosa will typically produce a green to blue water-soluble pigment on this agar (see Fig. 7) and will also fluoresce when the plate is placed under a short wavelength ultraviolet light (see Fig. 8).

D. DIFFERENTIATING BETWEEN THE ENTEROBACTERIACEAE AND PSEUDOMONAS

Once the gram-negative rod is isolated, a number of tests can be performed to determine if it is one of the Enterobacteriaceae or if it is Pseudomonas. Several of these tests are listed below:

1. Production of the enzyme oxidase. The oxidase test is based on the bacterial production of an oxidase enzyme. Cytochrome oxidase, in the presence of oxygen, oxidizes the para-amino dimetheylanaline oxidase test reagent in a Taxo-N® disc to form a rose-colored compound indophenol. The Enterobacteriaceae are oxidase-negative (see Fig. 9); Pseudomonas aeruginosa and most other non-fermentative gram-negative rods are oxidase-positive (see Fig. 10). The procedure for the oxidase test is described later in this lab.

2. Fermentation of glucose. All of the Enterobacteriaceae ferment the sugar glucose; Pseudomonas aeruginosa and other non-fermentative gram-negative rods will not.

3. Pigment production. None of the Enterobacteriaceae produces pigment at 37°C; Pseudomonas aeruginosa produces a green to blue, water-soluble pigment called pyocyanin (see Fig. 7). It also produces a product called fluorescein that will fluoresce under short wavelength (254nm) ultraviolet light (see Fig. 8). Pseudosel agar can be used to stimulate the production of pigment and fluorescent products.

4. Odor. Most of the Enterobacteriaceae have a rather foul smell; Pseudomonas aeruginosa produces a characteristic fruity or grape juice-like aroma due to production of an aromatic compound called aminoacetophenone.

Some common biotypes of Pseudomonas as well as all members of the Enterobacteriaceae can also be identified by means of biochemical tests found in commercially produced systems such as the API-20E® System or the Enterotube® II (discussed below).

E. IDENTIFYING THE ENTEROBACTERIACEAE USING THE ENTEROTUBE® II SYSTEM

A number of techniques can be used for the identification of specific species and subspecies of Enterobacteriaceae. Speciation is important because it provides data regarding patterns of susceptibility to antimicrobial agents and changes that occur over a period of time. It is also essential for epidemiological studies such as determination of nosocomial infections and their spread.

In an effort to simplify the speciation of the Enterobacteriaceae and reduce the amount of prepared media and incubation space needed by the clinical lab, a number of self-contained multi-test systems have been commercially marketed. Some of these multi-test systems have been combined with a computer-prepared manual to provide identification based on the overall probability of occurrence for each of the biochemical reactions. In this way, a large number of biochemical tests can economically be performed in a short period of time, and the results can be accurately interpreted with relative ease and assurance.

The Enterotube® II (see Fig. 13) is a self-contained, compartmented plastic tube containing 12 different agars (enabling the performance of a total of 15 standard biochemical tests) and an enclosed inoculating wire. After inoculation and incubation, the resulting combination of reactions, together with a Computer Coding and Identification System (CCIS), allows for easy identification. The various biochemical reactions of the Enterotube® II and their correct interpretation arediscussed in Lab 13. Although it is designed to identify members of the bacterial family Enterobacteriaceae, it will sometimes also identify common biotypes of Pseudomonas and other non-fermentative gram-negative bacilli.

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ORGANISMS (Trypticase Soy agar plate cultures)

Possible unknowns include:

Escherichia coli
Enterobacter aerogenes
Enterobacter cloacae
Proteus mirabilis
Proteus vulgaris
Salmonella enteritidis
Klebsiella pneumoniae
Pseudomonas aeruginosa

CAUTION: TREAT EACH UNKNOWN AS A PATHOGEN!. Inform your instructor of any spills or accidents. WASH AND SANATIZE YOUR HANDS WELL before you leave the lab.

MATERIALS

Taxo N® disk, alcohol, dropper bottle of distilled water, platinum inoculating loop, and either a plate of XLD agar and an Enterotube®II or a plate of Pseudosel agar and an Enterotube®II

PROCEDURE (to be done in pairs)

Each pair will be given one of the above unknowns. You will determine its identity doing the tests below.

1. Using the Trypticase Soy agar culture of your unknown, first perform an oxidase test as follows:

a. Using alcohol-flamed forceps, remove a Taxo-N® disc and moisten it with a drop of sterile distilled water.

b. Place the moistened disc on the colonies of the Trypticase Soy agar plate culture of your unknown.

c. Using a sterile swab, scrape off some of the colonies and spread them on the Taxo-N® disc.

In the immediate test, oxidase-positive reactions will turn a rose color within 30 seconds (see Fig. 10). Oxidase-negative will not turn a rose color (see Fig. 9). This reaction only lasts a couple of minutes. In the delayed test, oxidase-positive colonies within 10 mm of the Taxo-N® disc will turn black within 20 minutes and will remain black (see Fig 11). If the bacterium is oxidase-negative, the growth around the disc will not turn black (see Fig. 12).

Pseudomonas aeruginosa and most other non-fermentative, gram-negative bacilli are oxidase-positive; all of the Enterobacteriaceae are oxidase-negative.

Record your oxidase test results in the Results section of Lab 13.

2. Perform a gram stain on your unknown. All of the Enterobacteriaceae as well as Pseudomonas are gram-negative bacilli (see Fig. 1). Record the results of your gram stain in the Results section of Lab 13.

Instructions for thje Gram Stain from Lab 6.

Instructions for Focusing a Microscope from Lab 1.

• Escherichia coli
• Enterobacter aerogenes
• Enterobacter cloacae
• Proteus mirabilis
• Proteus vulgaris
• Salmonella enteritidis
• Klebsiella pneumoniae
• Pseudomonas aeruginosa

 

3. If your unknown is oxidase-negative, do the following inoculations:

a. Streak your unknown for isolation on a plate of XLD agar using one of the two streaking patterns illustrated in Lab 2, Fig. 4 and Lab 2, Fig. 5. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section.

Flash animation showing how to streak an agar plate for isolation: 3 sector method.

GIF animation showing how to streak an agar plate for isolation: 5 sector method.

b. Inoculate an Enterotube® II as follows:

1. Remove both caps of the Enterotube® II and with the straight end of the inoculating wire, pick off the equivalent of a colony from your unknown plate. A visible inoculum should be seen on the tip and side of the wire.

2. Inoculate the Enterotube® II by grasping the bent-end of the inoculating wire, twisting it, and withdrawing the wire through all 12 compartments using a turning motion.

3. Reinsert the wire into the tube (use a turning motion) through all 12 compartments until the notch on the wire is aligned with the opening of the tube. (The tip of the wire should be seen in the citrate compartment.) Break the wire at the notch by bending. Do not discard the wire yet.

4. Using the broken off part of the wire, punch holes through the cellophane which covers the air inlets located on the rounded side of the last 8 compartments. Your instructor will show you their correct location. Discard the broken off wire in the disinfectant container.

5. Replace both caps and incubate the Enterotube® II on its flat surface at 37°C.

4. If your unknown is oxidase-positive, do the following inoculations:

a. Streak your unknown for isolation on a plate of Pseudosel agar using one of the two streaking patterns illustrated in Lab 2, Fig. 4 and Lab 2, Fig. 5. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section.

Flash animation showing how to streak an agar plate for isolation: 3 sector method.

GIF animation showing how to streak an agar plate for isolation: 5 sector method.

b. Inoculate an Enterotube® II as described above in step 3b.

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After completing this lab, the student will be able to perform the following objectives:

A. ENTEROBACTERIACEAE: FERMENTATIVE, GRAM-NEGATIVE, ENTERIC BACILLI

1. Name the bacterial family to which the most commonly encountered organisms isolated from clinical specimens belong.

2. List five characteristics used to place bacteria into the family Enterobacteriaceae.

3. State what infections are caused by Salmonella and by Shigella and how they are transmitted to humans.

4. Name four strains of Escherichia coli that may infect the gastrointestinal tract.

5. Name five genera of Enterobacteriaceae considered as common opportunistic pathogens, state their normal habitat, and list four common types opportunistic infections that they all may cause.

6. Name several predisposing factors that make one more susceptible to urinary tract infections.

7. In terms of CFUs, state the laboratory culture standards for a urinary tract infection.

8. Define nosocomial infection.

9. State the significance of endotoxins in infections caused by many of the Enterobacteriaceae.

10. Discuss the significance of R-plasmids in our attempts to treat infections caused by the Enterobacteriaceae.

B. PSEUDOMONAS AND OTHER NON-FERMENTATIVE, GRAM-NEGATIVE BACILLI

1. Name the most common non-fermentative gram-negative rod that infect humans and list five types of opportunistic infections it may cause.

2, State 3 infecions being caused with increased frequency by the the gram-negative, non-fermemtative bacillus Acinetobacter. .

C. ISOLATION OF ENTEROBACTERIACEAE AND PSEUDOMONAS

1. State the usefulness of XLD agar and Pseudosel agar for the isolation of Enterobacteriaceae and Pseudomonas.

D. DIFFERENTIATING BETWEEN THE ENTEROBACTERIACEAE AND PSEUDOMONAS

1. State how to differentiate Pseudomonas aeruginosa from the Enterobacteriaceae using the following tests:

a. oxidase test

b. fermentation of glucose

c. production of pigment and fluorescent products

d. odor

E. IDENTIFYING THE ENTEROBACTERIACEAE USING THE ENTEROTUBE®II

1. Briefly describe the Enterotube® II.

SELF-QUIZ

Self-quiz

Answers

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