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The new, more sinister face of a familiar pathogen: increasing antibiotic resistance and association with hemolytic uremic syndrome are causes for concern. (Cover Story).

Escherichia coli (E. coli) is a pathogen very familiar to any laboratory professional. For many of us, it was one of the first gram-negative rods we were taught to identify. We came to know that this organism ferments lactose. The methyl red reaction is positive, and the VogesProskauer test is negative. Both phenylalanine deaminase and urease activity are absent. Citrate cannot be used as the sole carbon source, and 2 is not produced. The organism will not grow in the presence of potassium cyanide (KCN). (1)

We were taught, and rapidly came to see for ourselves in isolates from urine, blood, and sputum, that E. coli is the most frequent cause of some of the most common bacterial infections, including traveler's diarrhea, urinary tract infections, and bacteremia. It can cause pneumonia and is a leading pathogen in neonatal meningitis. (1)

E. coli is indeed one of the most prevalent infecting organisms of Enterobacteriaceae, and its genome, genetic characteristics, and virulence properties have been extensively studied. In the past few years, laboratory professionals have begun to see a more sinister face of this familiar pathogen. Some serotypes, in particular E. coli O157:H7, have been associated with a foodborne, severe gastrointestinal illness presenting with sudden onset of bloody diarrhea. This can lead to the potentially fatal hemolytic uremic syndrome (HUS). More and more E. coli isolates are proving resistant to many antibiotics, causing physicians and other healthcare workers to rethink their testing and treatment strategies.

A case study

The colorectal surgeon was awakened by a telephone call at 2 a.m. one day. It was a staff member from the local emergency room. A 22-year-old patient with a three-day history of crampy abdominal pain and bloody diarrhea was brought in for treatment. The pain had been generalized, but it increased in severity and then localized to the right lower abdominal quadrant, causing her to seek emergency care. The emergency room physician thought it was probably appendicitis. Could the surgeon come and evaluate her?

To the surgeon, this did not sound like the usual case of appendicitis. The patient stated that she had had a rare steak for dinner several nights before, and the abdominal pain started shortly thereafter. She denied any recent travel. The patient had no prior history of Crohn's disease or ulcerative colitis. She was otherwise in good health.

She had mild tenderness in the right lower quadrant and grossly bloody stool on rectal examination. The CBC was normal on admission. An abdominal computerized tomography scan showed inflammatory changes involving the entire right colon. Clostridium difficile toxin and stool cultures were obtained, as well as stool for an ova and parasite examination. The surgeon also added an order asking the laboratory to check for B. coli O157:H7 in the stool.

The patient underwent a colonoscopy that showed severe right-sided colitis. Biopsies of the right colon showed ulceration with marked acute and chronic inflammation and multiple fibrin thrombi in capillaries (Figure 1). Although nonspecific, these findings suggested either ischemic colitis (unlikely in a 22-year-old patient) or possible E. coli 01 57:1-17-associated colitis.

Over the next several days, the patient developed leukocytosis with left-shifted neutrophilia, and her platelet count fell from 122,000 to 37,000. Her BUN and creatinine began to rise, and a urinalysis was positive for ketones and protein. Stool cultures were reported positive for presumptive B. coli O157, and a diagnosis of infectious colitis with associated hemolytic uremic syndrome was confirmed.

The patient was treated with supportive measures and underwent plasmapheresis. Over the next several weeks, she recovered without complications.

Defining antigenic characteristics of E. coli and the 0157:H7 serotype

About 80 percent of significant gram-negative bacteria isolated in the clinical laboratory are Enterobacteriaceae. It has been estimated that about one-third of the septicemia isolates, two-thirds of the bacterial gastroenteritis isolates, and three-fourths of the urinary tract isolates are accounted for by Enterobacteriaceae. (1-3) In addition to E. coli, this family includes such important human intestinal pathogens as Shigella, Salmonella, and Yersinia. Enterobacter and Klebsiella are also members of this family.

E. coli and other Enterobacteriaceae share certain antigenic groups that react with antisera. These three classes of antigens include the O, or somatic antigens; the H, or flagellar antigens; and the K, or capsular antigens. The O antigens consist of the polysaccharide side chains of the envelope lipopolysaccharide of the organism's outer cell membrane.

In E. coli, some O serogroups act primarily as markers for a certain infectious process. For example, some O serogroups possess colonization factors and toxins necessary for gastroenteritis, while others possess adhesive factors important in urinary tract infection. The enterohemorrhagic E. coli usually belong to the serotype O157:H7 and were first associated with a multistate outbreak of hemorrhagic colitis in 1982. (4) In this case, more than 20 people in Oregon suffered from food poisoning manifested by severe, bloody diarrhea after eating hamburgers at a fast-food restaurant.

Meat can become infected with the organism during the slaughter of cattle. Ground beef is particularly vulnerable, as the organism becomes thoroughly distributed during the meat-grinding process. Produce, mayonnaise, and unpasteurized milk have also been suspected sources of this infection. It has been transmitted in an unchlorinated municipal water supply and to people swimming in a lake contaminated with feces. The ease with which E. coli O157:H7 is spread from person to person suggests that the infectious dose is low, similar to Shigella. In the United States alone, E. coli O157:H7 is estimated to cause more than 20,000 infections and as many as 250 deaths each year. (5)

In E. coli, the K1 antigen is associated with neonatal meningitis, bacteremia, and urinary tract infection. The K1 capsule is a virulence factor, acting as a shield to the organism by decreasing the ability of antibodies to bind to the bacteria and inhibiting the ability of white blood cells to phagocytize this bacteria. (1)

Other virulence factors

A number of virulence factors have been identified in Enterobacteriaceae. Fimbriae or pili act as adhesins, promoting the adherence of bacteria to mucosal surfaces. In E. coli, these adhesins are called colonization factor antigens and are an important factor in E. coli-induced gastroenteritis. P fimbriae are associated with E. coli strains that are capable of producing urinary tract infections and pyelonephritis. S fimbriae have been found on E. coli strains associated with neonatal sepsis or meningitis. (1,6,7)

Bacterial toxins are the most obvious example of virulence factors. E. coli produces hemolysins that can destroy red blood cells and inhibit phagocytosis and chemotaxis of white blood cells. (8) Secretion of enterotoxins causes diarrhea. E. coli O157:H7 produces one or both types of toxins known as verocytotoxins or Shiga toxins. These toxins are designated as verocytotoxins because of their toxic effects on cultured Vero cells or Shiga-like toxins because of their similarity to the cytotoxin produced by Shigella dysentery type 1. Shiga toxins are divided into several types. Most cases of human disease are associated with Shiga toxin 1 or Shiga toxin 2. More than 100 other serotypes of E. coli have been found to produce Shiga toxins as well, but not all of these E. coli have been implicated in illness. (59)

Unlike the cytotoxins and enterotoxins, whose distribution is restricted to pathogenic strains of E. coli, all gram-negative bacteria can produce endotoxins. Endotoxins consist of the lipopolysaccharide of the outer membrane. This endotoxin can cause a septic shock response. (1)

E. Coli 0157:H7 and hemolytic uremic syndrome (HUS)

Infection with E. coli O157:H7 typically begins with severe abdominal cramps and nonbloody diarrhea that may become grossly bloody by the second or third day. Unlike most bacterial infections, E. coli O157:H7 is usually characterized by a low-grade fever or no fever. This may lead a clinician to suspect inflammatory bowel disease or ischemic colitis. As in our case, if a colonoscopy and biopsy are performed, the findings may simulate ischemia with patchy distribution and fibrin microthrombi. Symptoms usually subside within a week. However, 6 percent of patients will go on to develop HUS. (5)

The term "hemolytic-uremic syndrome" was coined by Gasser, et al., in 1955 to describe a devastating illness consisting of nonimmune hemolytic anemia, thrombocytopenia, and acute renal failure. (10) As in the above case involving a 22-year-old woman, it often occurs after a prodromal period of bloody diarrhea caused by Shiga toxin from E. coli, in particular serotype O157:H7.

HUS develops in approximately 6 percent of patients infected with E. coli O157:H7. It most frequently occurs in children under the age of 5 years and the elderly, although it can be seen in adolescents and adults. In addition to age, risk factors for the development of HUS include bloody diarrhea, fever, an elevated white blood cell count, and treatment with antimotility agents. (5) Laboratory features of HUS are listed in Table 1.

The disease may end with multiorgan failure involving the heart, pancreas, central nervous system, and other organs. Neurologic complications including seizures, coma, and hemiparesis develop in one-quarter of the patients. About one-half will require dialysis, and three-quarters receive red blood cell transfusions. The mortality rate is 3 percent to 5 percent. It is one of the most common causes of acute renal failure in children. (5)

The differential diagnosis of HUS includes disseminated intravascular coagulation, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, and primary hemolytic anemia. Once recognized, current therapy is primarily supportive, consisting of blood transfusion, nutritional support, and dialysis, if necessary. In children, more than 95 percent will recover from the acute illness, but some will go on to develop chronic renal failure, hypertension, and proteinuria.

No specific therapy has proven effective in patients infected with E. coli O157:H7. Some studies have suggested that HUS is more likely to develop in patients treated with antibiotics, while others have shown the opposite. Antibiotics should probably be avoided until there is a multicenter trial of early antibiotic therapy. Antimotility agents are contraindicated in patients with bloody diarrhea. (5)

Chandler, et al., studied 53 children infected with E. coli O157:H7 to better elucidate the pathophysiology of this illness. Their study showed that the concentration of prothrombin fragments was high in the early days of the illness, before the development of anemia, thrombocytopenia, and renal failure. These findings confirm that thrombogenesis is occurring at an early stage in the disease, and suggest that damage to the kidney tubules may be due to formation of fibrin thrombi rather than direct injury to the tubules by circulating Shiga toxin. This demonstrated in a prospective clinical study that enhanced thrombin generation and impaired fibrinolysis clearly precede and therefore play a role in the development of the clinical manifestations of HUS. (11,12)

As the differential diagnosis is broad, recovery of E. coli O157:H7 from the stool can be key to making the diagnosis. Therefore, it is important that this disease be suspected and appropriate culture techniques used.

Laboratory identification of E. coli O157:H7

There are hundreds of strains of E. coli. How does a lab determine if a particular strain isolated from stool is pathogenic? Nonpathogenic E. coli cannot be distinguished from E. coli 0157:1-17 on routine media. Special culture media must be used. Some authors suggest that all diarrheal stools, and especially bloody stools, be automatically cultured for E. coli O157:H7. (5)

As in the case described above, if a patient has bloody diarrhea or is suspected of having this infection, the clinician should ask that the stool be screened for E. coli O157:H7 by using sorbitol MacConkey (SMAC) agar as one of the culture media. E. coli O157:H7 does not ferment D-sorbitol rapidly, and the colonies are thus colorless on this media. Usually, this organism will also leave a small zone of hemolysis on a blood agar plate. An organism that ferments sorbitol will produce a pink color within 24 hours.

MacConkey agar with Cefixime and tellurite is also used as a selective agar for detection of E. coli serotype O157:H7. Cefixime inhibits Proteus species, and tellurite inhibits non- 0157 E. coli and other organisms, thus improving selectivity. Bile salts and crystal violet that inhibit gram-positive bacteria such as enterococci and staphylococci are also included in the media.

SMAC will not identify all clinically important Shiga toxin-producing E. coli. There are two commercially available enzyme immunosassays that are approved by the U.S. Food and Drug Administration that will detect both Shiga toxin 1 and 2. One drawback is that the level of free toxin may be below the limit of detection.

Following isolation of sorbitol-negative colonies, biochemical tests are performed using an automated system to confirm the organism as E. coli. The next step is determining whether the organism has the 0157 antigen. This is done with a commercial latex agglutination rest. If positive, these organisms can then be reported as presumptive E. coli O157:H7. Testing for the H7 antigen is often done in a reference laboratory. (5,9)

Emerging antibiotic resistance in E coli

Although much attention and literature has focused on E. coli O157:H7 due to its association with outbreaks of hemorrhagic colitis and hemolytic uremic syndrome, another problem is developing with this common organism. The management of urinary tract infections is now complicated by the emergence of antibiotic-resistant strains of E. coli.

Urinary tract infection is extremely common in the United States. It is estimated that 11 percent of women report one physician-diagnosed urinary tract infection per year. (13) The lifetime probability that a woman will have a urinary tract infection is 60 percent. In recent U.S. studies, 15 percent to 18 percent of E. coli isolates from urinary tract infections have been resistant to trimethoprim-sulfamethoxazole. In other areas of the world, such as Israel and southern Europe, the prevalence of resistance is now 30 percent to 50 percent. (14-17)

Multiple factors have led to the emergence and spread of trimethoprim-sulfamethoxazole-resistant E. coli. One postulated cause is the widespread use of antibiotics in animal feeds. Antibiotics have been used in food animals in North America and Europe for nearly half a century. These antibiotics are used for prophylaxis and treatment of infection, as well as for growth promotion and enhanced feed efficiency. It has been estimated that 24.6 million pounds of antimicrobials are given to animals for nontherapeutic purposes and 2 million pounds are given for therapy each year. In contrast, 3 million pounds are given to humans. (18)

Although there is great debate over the impact of antimicrobials in food animals on human health, their use does seem to select for resistant strains and enhances their persistence in the environment. Another concern is the horizontal spread of the resistance genes from bacteria in food animals to commensal strains in human intestinal microflora. (18)

Resistance related to antibiotic prophylaxis

Not all antibiotic resistance in human pathogenic bacteria can be ascribed to use of these drugs in food animals. Another cause for trimethoprim-sulfamethoxazole resistance in E. coli is use of the drug in adults and children to treat urinary tract and respiratory infections. It is also prescribed as prophylaxis against Pneumocystis carinii in patients with human immunodeficiency virus. This widespread use has created selective pressures favoring resistant strains in fecal flora. (14)

In E. coli, resistance to trimethoprim-sulfamethoxazole is often genetically linked to other antimicrobial agents. Therefore, exposure to ampicillin and other commonly used drugs may contribute to this resistance. (14)

There is evidence that the initial event leading to community-acquired urinary tract infection is intestinal colonization with an uropathogenic strain of E. coli. It persists by way of adhesions that also promote attachment to urothelium. (19) Prolonged colonization of the intestine may facilitate the acquisition of antibiotic-resistant genes.

Manges, et al., studied three geographically diverse communities in California, Minnesota, and Michigan. They collected E. coli isolates from women with urinary tract infections in a university setting. These isolates were evaluated for antibiotic susceptibility, O:H serotype, and several other factors. In these three geographically diverse communities, the researchers found that a single clonal group accounted for nearly half of the trimethoprim-sulfamethoxazole resistant E. coli infections. These authors suggest that their findings are consistent with wide dissemination of a clonal strain from a common source, perhaps contaminated food. (15)

In areas of the country with a high prevalence of resistance to trimethoprim-sulfamethoxazole, the Infectious Disease Society of America now recommends alternative agents for treatment. This may cause rethinking of the empiric treatment of urinary tract infections without culture and susceptibility testing, which has become common in many clinical settings. (20)

Conclusions

E. coli is a common and well-known pathogen isolated every day in clinical laboratories across the country. With its various virulence factors and toxins, this common pathogen can cause serious disease with significant morbidity and mortality. As in our patient, certain serotypes such as E. coli O157:H7 can cause severe gastrointestinal illness when ingested in undercooked beef. This can lead to the potentially devastating disorder called hemolytic uremic syndrome.

Increasingly, E. coli is becoming resistant to antibiotics. This may be due to use in animal feed or widespread use of antibiotics for prophylaxis and other illnesses in humans. New studies suggest related clonal groups of E. coli may be contributing to these antibiotic-resistant infections.

Our patient survived her episode of infectious colitis and hemolytic uremic syndrome, and recovered without complications. High clinical suspicion led to appropriate culture techniques for detection. An understanding of the many faces of E. coli is important for laboratorians so that they recognize and accurately report potentially dangerous serotypes and antibiotic-resistant organisms.
Table 1

Laboratory features of : hemolytic uremic syndrome


Anemia -- often severe
Moderate neutrophilia
Fragmented RBCs, burr cells,
and microspherocytes
Reticulocytosis
Thrombocytopenia
Hemoglobinemia
Low haptoglobin
Mildly elevated bilirubin
Elevated urea nitrogen and
creatinine
Hemoglobinuria with RBCs,
WBCs, and casts


Acknowledgement

The author thanks Mary Jacobs, MT (ASCP), section head of microbiology, Cleveland Clinic Hospital, for help with researching this article.

References

(1.) Mandell GL, Bennett JE, Dolin R. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases. 5th ad. Philadelphia, PA: Churchill Livingston; 2000:2294-2310.

(2.) Schaberg DR. Major trends in the microbial etiology of nosocomial infection. Am J Med. 1991;91(suppl.3B):72S-75S.

(3.) Banerjee SN, Emori G, Culver DH, at al. Secular trends in nosocomial primary bloodstream infections in the United States, 1980-1989. Am J Med. 1991;91(suppl. 3B):86S-89S.

(4.) Riley LW, Remis RS, Helgerson SD, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med. 1983;308:881-685.

(5.) Boyce TG, Swerdlow DL, Griffin PM. Escherichia coli 0157:H7 and the hemolytic-uremic syndrome. N Engl J Med. 1995;333:364-368.

(6.) Evans DJ, Silver RP, Evans DJ Jr. Plasmid-controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect Immun. 1975;12:656-667.

(7.) Mooi FR, de Graff FK. Molecular biology of fimbria of enterotoxigenic Escherichia coil. Curr Top Microbiol Immunol. 1985;118:119-138.

(8.) Bohach GA, Snyder IS. Chemical and immunological analysis of the complex structure of Escherichia coli alpha-hemolysin. J Bacterial. 1985;164:1071-1080.

(9.) Hayes C. Detecting a public health risk: Escherichia call 0157:H7. Laboratory Medicine. 1998;29:347-355.

(10.) Gasser VC, Gautier E, Stack A, Siebenmans RE, Oechslin R. Hamolytisch-uramische Syndrome: Bilaterale Nierenrindennekrosen bei akuten erworbenen hamolytischen Anamien. Schweiz Med Wochenschr. 1955;85:905-909.

(11.) Chandler WL, Jelacic S, Boater DR, et al. Prothrombotic coagulation abnormalities preceding the hemolytic-uremic syndrome. N Engl J Med. 2002;346:23-32.

(12.) Grabowski EF. The hemolytic-uremic syndrome -- toxin, thrombin and thrombosis [editorial]. N Engl J Med. 2002;346:58-61.

(13.) Foxman B, Barlow R, Darcy H, Gillespie B, Sobel JD. Urinary tract infection: self-reported incidence and associated costs. Ann Epidemiol. 2000;10:509-515.

(14.) Stamm WE. An epidemic of urinary tract infections? [editorial] N Engl J Med. 2001;345:1055-1057.

(15.) Manges AR, Johnson JR, Foxman B, O'Bryan TT, Fullerton KE, Riley L. Widespread distribution of urinary tract infections caused by a multidrug-resistant Escherichia coli clonal group. N Engl J Med. 2001;345:1007-1013.

(16.) Kahlmeter G. The ECO_SENS Project: a prospective, multinational, multicentre epidemiological survey of the prevalence and antimicrobial susceptibility of urinary tract pathogens -- interim report. J Antimicrob Chemother. 2000;46(suppl. A):15-22.

(17.) Gupta K, Scholes D, Stamm WE. Increasing prevalence of antimicrobial resistance among uropathogens causing acute uncomplicated cystitis in women. JAMA. 1999;281:736-738.

(18.) Gorbach S. Antimicrobial use in animal feed -- time to stop [editorial]. N Engl J Med. 2001;345:1202-1203.

(19.) Wold AE, Caugant DA, Lidin-Janson G. de Man P, Svanborg C. Resident colonic Escherichia coli strains frequently display uropathogenic characteristics. J Infect Dis. 1992;165:46-52.

(20.) Warren JW, Abrutyn E, Hebel JR, Johnson JR, Schaeffer AJ, Stamm WE. Guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women. Clin Infect Dis. 1999;29:745-758.

Sherry Woodhouse. M.D., is chair of the Division of Pathology and Laboratory Medicine, Cleveland Clinic Florida, Weston, FL.
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Date:Dec 1, 2002
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