Timothy J. Barrett

Establishment of a Universal Size Standard Strain for Use with the PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocols: Converting the National Databases to the New Size Standard

ABSTRACT The PulseNet National Database, established by the Centers for Disease Control and Prevention in 1996, consists of pulsed-field gel electrophoresis (PFGE) patterns obtained from isolates of food-borne pathogens (currently Escherichia coli O157:H7, Salmonella, Shigella, and Listeria) and textual information about the isolates. Electronic images and accompanying text are submitted from over 60 U.S. public health and food regulatory agency laboratories. The PFGE patterns are generated according to highly standardized PFGE protocols. Normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. Originally, a well-characterized strain of each organism was chosen as the reference standard for that particular database. The increasing number of databases, difficulty in identifying an organism-specific standard for each database, the increased range of band sizes generated by the use of additional restriction endonucleases, and the maintenance of many different organism-specific strains encouraged us to search for a more versatile and universal DNA size marker. A Salmonella serotype Braenderup strain (H9812) was chosen as the universal size standard. This strain was subjected to rigorous testing in our laboratories to ensure that it met the desired criteria, including coverage of a wide range of DNA fragment sizes, even distribution of bands, and stability of the PFGE pattern. The strategy used to convert and compare data generated by the new and old reference standards is described.

An Outbreak of Escherichia coli O157:H7 Infections Associated with Leaf Lettuce Consumption

In July 1995, 40 Montana residents were identified with laboratory-confirmed Escherichia coli O157:H7 infection; 52 residents had bloody diarrhea without laboratory confirmation. The median age of those with laboratory-confirmed cases was 42 years (range, 4- 86); 58% were female. Thirteen patients were hospitalized, and 1 developed hemolytic-uremic syndrome. A case-control study showed that 19 (70%) of 27 patients but only 8 (17%) of 46 controls reported eating purchased (not home-grown) leaf lettuce before illness (matched odds ratio, 25.3; 95% confidence interval, 3.9-1065.6). Pulsed-field gel electrophoresis identified a common strain among 22 of 23 isolates tested. Implicated lettuce was traced to two sources: a local Montana farm and six farms in Washington State that shipped under the same label. This outbreak highlights the increasing importance of fresh produce as a vehicle in foodborne illness. Sanitary growing and handling procedures are necessary to prevent these infections.

Rapid Pulsed-Field Gel Electrophoresis Protocol for Subtyping of Campylobacter jejuni

ABSTRACT We developed a rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping Campylobacter isolates based on the standardized protocols used by PulseNet laboratories for the subtyping of other food-borne bacterial pathogens. Various combinations of buffers, reagents, reaction conditions (e.g., cell suspension concentration, lysis time, lysis temperature, and restriction enzyme concentration), and electrophoretic parameters were evaluated in an effort to devise a protocol that is simple, rapid, and robust. PFGE analysis of Campylobacter isolates can be completed in 24 to 30 h using this protocol, whereas the most widely used current protocols require 3 to 4 days to complete. Comparison of PFGE patterns obtained in six laboratories showed that subtyping results obtained using this protocol are highly reproducible.

A Swimming-Associated Outbreak of Hemorrhagic Colitis Caused by Escherichia coli O157:H7 and Shigella Sonnei

BACKGROUND In the summer of 1991, simultaneous outbreaks of bloody diarrhea and hemolytic-uremic syndrome caused by Escherichia coli O157:H7 and of bloody diarrhea caused by Shigella sonnei were traced to a lakeside park near Portland, Oregon. METHODS We identified cases primarily from routine surveillance reports. In case-control studies, the activities of persons with park-associated E. coli O157:H7 or S. sonnei infections were compared independently with those of three sets of controls. We also evaluated environmental conditions at the park and subtyped the bacterial isolates. RESULTS We identified 21 persons with park-associated E. coli O157:H7 infections (all of them children; median age, six years) and 38 persons with S. sonnei infections (most of them children). These 59 people had visited the park over a 24-day period. Their illnesses were not associated with food or beverage consumption. All the case patients reported swimming, however, and in case-control studies swimming was strongly associated with both types of infection (P = 0.015 or less). The case patients were more likely than the controls to report having swallowed lake water, and they had spent more time in the lake. Numbers of enterococci indicative of substantial fecal contamination (geometric mean, > 50 per deciliter) were detected in the swimming area during some but not all of the outbreak period. Park-associated E. coli O157:H7 isolates were identical by pulsed-field gel electrophoresis and were distinguishable from other isolates in the Portland area. CONCLUSIONS Lake water that was fecally contaminated by bathers was the most likely vehicle for the transmission of both the E. coli O157:H7 and the S. sonnei infections. The unusually prolonged outbreak suggests both the survival of these enteric organisms in lake water and a low infectious dose.

The United States National Prospective Hemolytic Uremic Syndrome Study: Microbiologic, Serologic, Clinical, and Epidemiologic Findings

The frequency of Shiga toxin-producing Escherichia coli (STEC) serotypes associated with postdiarrheal hemolytic uremic syndrome (HUS) cases among children and adults in the United States and the proportion with IgM or IgG lipopolysaccharide antibodies to E. coli O157 were determined by use of a nationwide sample from January 1987 through December 1991. Among 83 patients, STEC were isolated from 30 (43%) of 70 whose stool cultures yielded bacterial growth (25 E. coli O157 isolates and 5 non-O157 STEC isolates). Fifty-three (80%) of 66 patients with serum samples had positive O157 lipopolysaccharide antibody titers. Of the 83 patients, 60 (72%) had evidence of STEC infection, including 6 of 8 adults whose illnesses also met criteria for thrombotic thrombocytopenic purpura. Data from a subset of patients suggest that E. coli O157 was the cause of > or = 80% of the STEC infections. All 3 women who were postpartum had evidence of E. coli O157 infection. STEC infection should be considered the likely cause for all persons with postdiarrheal HUS.

Antimicrobial-Resistant Nontyphoidal Salmonella Is Associated with Excess Bloodstream Infections and Hospitalizations

BACKGROUND Nontyphoidal Salmonella is a leading cause of foodborne illness. Few studies have explored the health consequences of antimicrobial-resistant Salmonella. METHODS The National Antimicrobial Resistance Monitoring System (NARMS) performs susceptibility testing on nontyphoidal Salmonella isolates. The Foodborne Diseases Active Surveillance Network (FoodNet) ascertains outcomes for patients with culture-confirmed Salmonella infection, in 9 states, each of which participates in NARMS. We analyzed the frequency of bloodstream infection and hospitalization among patients with resistant infections. Isolates defined as resistant to a clinically important agent were resistant to 1 or more of the following agents: ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and/or trimethoprim-sulfamethoxazole. RESULTS During 1996-2001, NARMS received 7370 serotyped, nontyphoidal Salmonella isolates from blood or stool. Bloodstream infection occurred more frequently among patients infected with an isolate resistant to > or =1 clinically important agent (adjusted odds ratio [OR], 1.6; 95% confidence interval [CI], 1.2-2.1), compared with patients with pansusceptible infection. During 1996-2001, FoodNet staff ascertained outcomes for 1415 patients who had isolates tested in NARMS. Hospitalization with bloodstream infection occurred more frequently among patients infected with an isolate resistant to > or =1 clinically important agent (adjusted OR, 3.1; 95% CI, 1.4-6.6), compared with patients with pansusceptible infection. CONCLUSIONS Patients with antimicrobial-resistant nontyphoidal Salmonella infection were more likely to have bloodstream infection and to be hospitalized than were patients with pansusceptible infection. Mitigation of antimicrobial resistance in Salmonella will likely benefit human health.

Emergence of Multidrug-Resistant Salmonella enterica Serotype Newport Infections Resistant to Expanded-Spectrum Cephalosporins in the United States

We describe a field investigation in New England that identified the emergence and epidemiology of new strains of multidrug-resistant Salmonella, Newport-MDRAmpC, and summarize the Center for Disease Control and Preventions surveillance data for these infections. In Massachusetts, the prevalence of Newport-MDRAmpC among Salmonella serotype Newport isolates obtained from humans increased from 0% (0/14) in 1998 to 53% (32/60) in 2001 (P<.001). In a retrospective case-control study, infection with Newport-MDRAmpC was domestically acquired and was associated with exposure to a dairy farm. Isolates from both humans and cattle had indistinguishable or closely related antibiograms and pulsed-field gel electrophoresis patterns. Nationally, the prevalence of ceftriaxone-resistant Salmonella increased from 0.5% in 1998 to 2.4% in 2001; 85% of the isolates in 2001 were Newport-MDRAmpC, and at least 27 states have isolated these strains from humans, cattle, or ground beef. These data document the widespread emergence of Newport-MDRAmpC strains in the United States and show that the 5-fold increase in the prevalence of Salmonella resistant to expanded-spectrum cephalosporins, between 1998 and 2001, is primarily due to the emergence of Newport-MDRAmpC strains.

Cholera--a possible endemic focus in the United States.

In September and October 1978, after a case of cholera had been discovered in southwestern Louisiana, 10 more Vibrio cholerae O-Group 1 infections were detected in four additional clusters. All 11 infected persons had recently eaten cooked crabs from five widely separated sites in the coastal marsh, and a matched-triplet case-control study showed a significant relation between cholera and eating such crabs (P = 0.007). V. cholerae O1 was isolated from estuarine water, from fresh shrimp, from a leftover cooked crab from a patients refrigerator, and from sewage in six towns, including three without identified cases. All isolates in Louisiana and an isolate from a single unexplained case in Texas in 1973 were biotype El Tor and serotype inaba; they were hemolytic and of a phage type unique to the United States--suggesting that the organism persisted undetected along the Gulf Coast for at least five years.

Antimicrobial resistance among Campylobacter strains, United States, 1997-2001.

We summarize antimicrobial resistance surveillance data in human and chicken isolates of Campylobacter. Isolates were from a sentinel county study from 1989 through 1990 and from nine state health departments participating in National Antimicrobial Resistance Monitoring System for enteric bacteria (NARMS) from 1997 through 2001. None of the 297 C. jejuni or C. coli isolates tested from 1989 through 1990 was ciprofloxacin-resistant. From 1997 through 2001, a total of 1,553 human Campylobacter isolates were characterized: 1,471 (95%) were C. jejuni, 63 (4%) were C. coli, and 19 (1%) were other Campylobacter species. The prevalence of ciprofloxacin-resistant Campylobacter was 13% (28 of 217) in 1997 and 19% (75 of 384) in 2001; erythromycin resistance was 2% (4 of 217) in 1997 and 2% (8 of 384) in 2001. Ciprofloxacin-resistant Campylobacter was isolated from 10% of 180 chicken products purchased from grocery stores in three states in 1999. Ciprofloxacin resistance has emerged among Campylobacter since 1990 and has increased in prevalence since 1997.

Chicken Consumption Is a Newly Identified Risk Factor for Sporadic Salmonella enterica Serotype Enteritidis Infections in the United States: A Case-Control Study in FoodNet Sites

The sources of sporadic Salmonella enterica serotype Enteritidis (SE) infections in the United States are unclear. To determine risk factors for sporadic SE infection, we conducted a population-based case-control study in 5 Foodborne Disease Active Surveillance Network surveillance areas. During the 12-month study, 396 cases of SE infection were ascertained. Among the 182 case patients and 345 controls, SE infection was univariately associated with international travel (matched odds ratio [MOR], 61; 95% confidence interval [CI], 8-447), eating undercooked eggs (MOR, 2.2; 95%CI, 1-5), and eating chicken prepared outside of the home (MOR, 2.2; 95% CI, 1.3-3.4). Multivariate analysis revealed that eating chicken outside of the home remained the only significant risk factor for illness (MOR, 2.0; 95% CI, 1.1-3.6). Chicken consumption has not previously been identified in the United States as a risk factor for SE infection. Measures to prevent SE infections include educating consumers and food handlers about food safety and interventions to decrease contamination of eggs and poultry.