PulseNet USA standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio parahaemolyticus.

PulseNet is a national molecular subtyping network for foodborne disease surveillance composed of public health and food regulatory agencies. Participants employ molecular subtyping of foodborne pathogens using a standardized method of pulsed-field gel electrophoresis (PFGE) for conducting laboratory-based surveillance of foodborne pathogens. The PulseNet standardized PFGE protocols are developed through a comprehensive testing process. The reproducibility of the protocol undergoes an internal evaluation at the Centers for Disease Control and Prevention and an external evaluation in multiple PulseNet laboratories. Here we describe the development and evaluation of a rapid PFGE protocol for subtyping Vibrio parahaemolyticus for use in PulseNet activities. The protocol was derived from the existing standardized PulseNet protocols for Escherichia coli O157:H7 and Vibrio cholerae. An external evaluation of this protocol was undertaken in collaboration with three PulseNet USA participating public health laboratories. Comparative analysis of the PFGE fingerprints generated by each of these laboratories demonstrated that the protocol is both reliable and reproducible in the hands of multiple users.

Emergence of a new Vibrio parahaemolyticus serotype in raw oysters: A prevention quandary.

CONTEXT: In May and June 1998, reported Vibrio parahaemolyticus infections increased sharply in Texas. OBJECTIVE: To determine factors that contributed to the increase in V parahaemolyticus infections. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional survey of persons reporting gastroenteritis after eating seafood in Texas; survey of environmental conditions in Galveston Bay. MAIN OUTCOME MEASURES: Traceback of oysters, water quality measures in harvest areas, presence of V parahaemolyticus in stool cultures; comparison of median values for environmental conditions before and during the outbreak compared with during the previous 5 years. RESULTS: Between May 31 and July 10, 1998, 416 persons in 13 states reported having gastroenteritis after eating oysters harvested from Galveston Bay. All 28 available stool specimens from affected persons yielded V parahaemolyticus serotype O3:K6 isolates. Oyster beds met current bacteriologic standards during harvest and fecal coliform counts in water samples were within acceptable limits. Median water temperature and salinity during May and June 1998 were 30.0 degrees C and 29.6 parts per thousand (ppt) compared with 28.9 degrees C and 15.6 ppt for the previous 5 years (P<.001). CONCLUSIONS: This is the first reported outbreak of V parahaemolyticus serotype O3:K6 infection in the United States. The emergence of a virulent serotype and elevated seawater temperatures and salinity levels may have contributed to this large multistate outbreak of V parahaemolyticus. Bacteriologic monitoring at harvest sites did not prevent this outbreak, suggesting that current policy and regulations regarding the safety of raw oysters require reevaluation. Consumers and physicians should understand that raw or undercooked oysters can cause illness even if harvested from monitored beds. In patients who develop acute gastroenteritis within 4 days of consuming raw or undercooked oysters, a stool specimen should be tested for Vibrio species using specific media. JAMA. 2000;284:1541-1545.

Laboratory-based surveillance of Salmonella serotype Typhi infections in the United States: antimicrobial resistance on the rise.

CONTEXT: Multidrug-resistant Salmonella serotype Typhi infections have been reported worldwide, but data on the incidence of resistant strains in the United States are lacking. OBJECTIVES: To determine the incidence of antimicrobial-resistant Salmonella Typhi infections and to identify risk factors for infection. DESIGN: Cross-sectional laboratory-based surveillance study. SETTING AND PARTICIPANTS: A total of 293 persons with symptomatic typhoid fever who had Salmonella Typhi isolates and epidemiological information submitted to US public health departments and laboratories from June 1, 1996, to May 31, 1997. MAIN OUTCOME MEASURES: Proportion of Salmonella Typhi isolates demonstrating resistance to 12 antimicrobial agents; patient epidemiological factors associated with drug-resistant infections. RESULTS: Median age was 21 years (range, 3 months to 84 years); 56% were male. Two hundred twenty-eight (80%) were hospitalized; 2 died. In the 6 weeks before illness onset, 81% of patients had traveled abroad. Seventy-four Salmonella Typhi isolates (25%) were resistant to 1 or more antimicrobial agent, and 51 (17%) were resistant to 5 or more agents, including ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole (multidrug-resistant Salmonella Typhi [MDRST]). Although no resistance to ciprofloxacin or ceftriaxone was observed, 20 isolates (7%) were nalidixic acid-resistant (NARST). Patients with MDRST and NARST infections were more likely to report travel outside the United States, particularly to the Indian subcontinent (Bangladesh, India, and Pakistan) (odds ratio [OR], 29.3; 95% confidence interval [CI], 6.8-126.7; P<.001 and OR, 35.9; 95% CI, 3.4-377.3; P<.001, respectively). CONCLUSIONS: Our data suggest that ciprofloxacin and ceftriaxone are appropriate empirical therapy for suspected typhoid fever; however, resistance may be anticipated. Continued monitoring of antimicrobial resistance among Salmonella Typhi strains will help determine vaccination and treatment policies. JAMA. 2000;283:2668-2673.

Outbreak of Salmonella serotype Hartford infections associated with unpasteurized orange juice.

CONTEXT: Acidic foods such as orange juice have been thought to be unlikely vehicles of foodborne illness. OBJECTIVE: To investigate an outbreak of Salmonella enterica serotype Hartford (Salmonella Hartford) infections among persons visiting a theme park in Orlando, Fla, in 1995. DESIGN: Review of surveillance data, matched case-control study, laboratory investigation, and environmental studies. SETTING: General community. PARTICIPANTS: The surveillance case definition was Salmonella Hartford or Salmonella serogroup C1 infection in a resident of or a visitor to Orlando in May or June 1995. In the case-control study, case patients were limited to theme park hotel visitors and controls were matched to case patients by age group and hotel check-in date. MAIN OUTCOME MEASURES: Risk factors for infection and source of implicated food. RESULTS: Sixty-two case patients from 21 states were identified. Both Salmonella Hartford and Salmonella enterica serotype Gaminara (Salmonella Gaminara) were isolated from stool samples of 1 ill person. Thirty-two case patients and 83 controls were enrolled in the case-control study. Ninety-seven percent of case patients had drunk orange juice in the theme park vs 54% of controls (matched odds ratio, undefined; 95% confidence interval, 5.2 to undefined). The orange juice was unpasteurized and locally produced. Salmonella Gaminara was isolated from 10 of 12 containers of orange juice produced during May and July, indicating ongoing contamination of juice probably because of inadequately sanitized processing equipment. CONCLUSIONS: Unpasteurized orange juice caused an outbreak of salmonellosis in a large Florida theme park. All orange juice was recalled and the processing plant closed. Pasteurization or other equally effective risk-management strategies should be used in the production of all juices.

An outbreak of Brainerd diarrhea among travelers to the Galapagos Islands.

In 1992, an outbreak of chronic diarrhea occurred among passengers on a cruise ship visiting the Galapagos Islands, Ecuador. Passengers (548) were surveyed, and stool and biopsy specimens from a sample who reported chronic diarrhea were examined. On completed questionnaires, returned by 394 passengers (72%), 58 (15%) reported having chronic diarrhea associated with urgency (84%), weight loss (77%), fatigue (71%), and fecal incontinence (62%). Illness began 11 days (median) after boarding the ship and lasted 7 to >42 months. Macroscopic and histologic abnormalities of the colon were common, but extensive laboratory examination revealed no etiologic agent. No one responded to antimicrobial therapy. Patients were more likely than well passengers to have drunk the ship's unbottled water or ice before onset of illness and to have eaten raw sliced fruits and vegetables washed in unbottled water. Water handling and chlorination on the ship were deficient. Outbreaks of a similar illness, Brainerd diarrhea, have been reported in the United States. Although its etiology remains unknown, Brainerd diarrhea may also occur among travelers.

Colonic epithelial lymphocytosis associated with an epidemic of chronic diarrhea.

The term Brainerd diarrhea has been applied to outbreaks of chronic watery diarrhea of unknown etiology characterized by acute onset and prolonged duration. Our aim was to describe the histologic changes in gastrointestinal biopsy specimens from patients with Brainerd diarrhea. We examined 52 colonic and 12 small bowel biopsy specimens from 22 patients who were involved in an outbreak of Brainerd diarrhea that was linked to the water supply of a cruise ship visiting the Galapagos Islands. Small bowel biopsy specimens from seven patients were histologically normal. One patient had a duodenal biopsy specimen that resembled celiac sprue. Colonic biopsy specimens from 20 patients revealed surface epithelial lymphocytosis without distortion of mucosal architecture, surface degenerative changes, or thickened subepithelial collagen plates. The degree of surface epithelial lymphocytosis was greater than that seen in control groups of persons with normal colons, acute colitis, and ulcerative colitis (p < 0.001), similar to that seen with collagenous colitis, and less than that seen with lymphocytic colitis (p < 0.001). Three patients showed focal active colitis similar to that described in acute infectious-type colitis in addition to the epithelial lymphocytosis. Two patients had colonic biopsy specimens that were histologically normal. In summary, histologic abnormalities in the small bowel are generally absent in Brainerd diarrhea. Colonic biopsy specimens in Brainerd diarrhea frequently show epithelial lymphocytosis similar to that seen in collagenous and lymphocytic colitis. Although currently Brainerd diarrhea can be diagnosed only with epidemiologic data indicating an epidemic and a point source, the lack of surface degenerative changes and the relatively lower lymphocyte counts seen in our cases of Brainerd diarrhea may serve to distinguish it from lymphocytic colitis, and the lack of a thickened subepithelial collagen plate distinguishes it from collagenous colitis.

Increase in antimicrobial-resistant Salmonella infections in the United States, 1989-1990.

To assess factors associated with antimicrobial-resistant Salmonella infections and trends in resistance, a prospective study of patients with culture-confirmed salmonellosis was done in 1989-1990. Patients with resistant infections were more likely than those with susceptible infections to be hospitalized (P = .006), to be < 1 year old (P = .003), to be black (P = .013), and to have recently been treated with an antimicrobial agent (P = .085). Compared with data from a similar study in 1979-1980, increases were seen in the percentage of patients with resistant infections (from 17% to 31%), in the resistance to ampicillin (10% to 14%), and in the frequency of isolates found in blood (1% to 11%). These data show that treatment of Salmonella infections may be complicated by growing resistance to clinically important antimicrobial agents and by an increasing frequency of extraintestinal complications. Antimicrobial agents with little demonstrated resistance should be considered for patients with complicated illness and at high risk of having a resistant infection.

Epidemic cholera in Ecuador: multidrug-resistance and transmission by water and seafood.

To determine risk factors for cholera in an epidemic-disease area in South America, a case-control investigation was performed in Guayaquil, Ecuador, in July 1991. Residents > 5 years old who were hospitalized for treatment of acute, watery diarrhoea and two matched controls for each were interviewed regarding sources of water and food, and eating, drinking, and hygienic habits. Interviewers inspected homes of case-patients and controls to document water treatment, food-handling, and hygienic practices. Faecal specimens and shellfish were cultured for Vibrio cholerae O 1. Isolates were tested for susceptibility to a variety of antimicrobial agents. Drinking unboiled water (odds ratio [OR] = 4.0, confidence interval [CI] = 1.8-7.5), drinking a beverage from a street vendor (OR = 2.8, CI = 1.3-5.9), eating raw seafood (OR = 3.4, CI = 1.4-11.5), and eating cooked crab (OR = 5.1, CI = 1.4-19.2) were associated with illness. Always boiling drinking water at home (OR = 0.5, CI = 0.2-0.9) was protective against illness. The presence of soap in either the kitchen (OR = 0.3, CI = 0.2-0.8) or bathroom (OR = 0.4, CI = 0.2-0.9) at home was also protective. V. cholerae O 1 was recovered from a pooled sample of a bivalve mollusc and from 68% of stool samples from case-patients. Thirty-six percent of the isolates from stool specimens were resistant to multiple antimicrobial agents. Specific prevention measures may prevent transmission through these vehicles in the future. The appearance of antimicrobial resistance suggests the need for changes in current methods of prevention and treatment.

Antimicrobial-resistant Salmonella spp isolated from healthy broiler chickens after slaughter.

Of 105 Salmonella organisms of any serotype selected from a sample of 1,824 serotyped salmonellae isolated during a nationwide bacteriologic survey of healthy broiler chickens after slaughter, 60 (57%) were resistant to 1 or more antimicrobial agents and 47 (45%) were resistant to 2 or more agents. Highest resistance was to tetracycline (45%), streptomycin (41%), sulfisoxazole (19%), gentamicin (10%), and trimethoprim/sulfamethoxazole (8%). Additional isolates of S typhimurium, heidelberg, agona, and enteritidis were selected from the sample of 1,824 isolates for testing because of the high frequency with which these 4 serotypes are isolated from human patients. The highest frequency of resistance among 104 isolates of S heidelberg, 92 isolates of S typhimurium, and 30 isolates of S agona was to streptomycin (33 to 57%), sulfisoxazole (33 to 50%), tetracycline (26 to 50%), and gentamicin (13 to 40%); 51 to 63% of these isolates were resistant to 1 or more agents and 37 to 59% were resistant to 2 or more agents. Resistance to ampicillin among these 3 serotypes was uncommon (0 to 4%). In contrast, 15 of 19 tested isolates (79%) of S enteritidis were resistant to ampicillin and 13 of the 19 isolates (68%) were resistant only to ampicillin. This pattern of resistance was associated with a specific bacteriophage type and indicated the potential role of bacterial clones in determining the frequency and patterns of antimicrobial resistance in populations of broiler chickens. Resistance to gentamicin and trimethoprim/sulfamethoxazole was higher than that previously reported and is of public health concern because of the frequency with which these drugs are used to treat bacterial infections in human patients.

Diarrhea among African children born to human immunodeficiency virus 1-infected mothers: clinical, microbiologic and epidemiologic features.

Diarrhea and weight loss are common features of pediatric and adult human immunodeficiency type 1 (HIV-1) infection, particularly in developing countries. We studied prospectively episodes of diarrhea in 559 children, ages 10 to 15 months, participating in a longitudinal study of perinatal HIV-1 infection in Kinshasa, Zaire. Children with HIV-1 infection had more frequent episodes of diarrhea and were more likely to present with fever or moderate or severe dehydration and to have persistent or fatal diarrhea. Of 9 HIV-1-positive infants with diarrhea, 3 had enteroadherence factor-positive Escherichia coli, compared with 5 of 74 HIV-1-negative children with diarrhea (P = 0.04); no other pathogen was associated with HIV-1 infection. In a logistic regression model diarrhea was significantly associated with HIV-1 infection in the child, moderate or severe malnutrition and symptoms of acquired immunodeficiency syndrome in the mother. Diarrhea among children with perinatal HIV infection in Zaire is more severe than among uninfected children and is associated with malnutrition and advanced disease in the mother.

Comparison of plasmid profiles, phage types, and antimicrobial resistance patterns of Salmonella enteritidis isolates in the United States.

To evaluate the laboratory techniques for subtyping isolates of Salmonella enteritidis, we compared the plasmid profiles (PP), phage types (PT), and antimicrobial susceptibility patterns (AS) of two nationally representative samples of sporadic human S. enteritidis isolates from 1979 (n = 28) and 1984 (n = 37), 43 isolates from 20 outbreaks of S. enteritidis infections between 1983 and 1987, and 46 animal isolates selected from the U.S. Department of Agriculture Veterinary Services Laboratory in 1986 and 1987. Sporadic and outbreak isolates from humans showed similar rates of resistance to at least one of a panel of antimicrobial drugs (23 and 14%, respectively), PT (91 and 98%, respectively), and PP (97 and 100%, respectively). Sixteen different PP were identified in sporadic, outbreak, and animal isolates; two PP accounted for 76% of sporadic and outbreak isolates. Sporadic human isolates were of PT 8 (42%), of PT 13a (37%), nontypeable (9%), of PT 14b (8%), of PT 9a (3%), and of PT 13 (2%). Outbreak human isolates had similar distributions of PT. PT 8 was associated with poultry: 58% (7 of 12) of the poultry isolates but only 24% (8 of 34) of the isolates from other animals were of PT 8 (P less than 0.04). Although antimicrobial susceptibility patterns do not appear as useful as an epidemiologic marker, PP and PT effectively subtyped S. enteritidis.

Antimicrobial resistance of Shigella isolates in the USA: the importance of international travelers.

A nationwide sample of Shigella isolates was collected and tested for resistance to 12 antimicrobial agents to assess the prevalence and epidemiologic correlates of antimicrobial resistance in Shigella. Of the isolates, 32% were resistant to ampicillin, 7% to trimethoprim-sulfamethoxazole, and 0.4% to nalidixic acid. Fifty (20%) of 252 isolates were associated with foreign travel. The best predictor of clinically important resistance was a history of foreign travel: 20% of isolates from foreign travelers showed trimethoprim-sulfamethoxazole resistance, compared with only 4% of isolates from those without such a history. Quinolone resistance was not identified in travel-related isolates, and quinolones may be more appropriate for initial therapy of travel-related shigellosis than is trimethoprim-sulfamethoxazole.

Epidemiologic evidence that prior antimicrobial exposure decreases resistance to infection by antimicrobial-sensitive Salmonella.

Exposure to antimicrobials is known to increase the risk of infection with antimicrobial-resistant strains of Salmonella, but this effect has not been previously demonstrated with antimicrobial-sensitive strains. The effect of prior antimicrobial exposure was studied during a multistate outbreak of salmonellosis caused by an antimicrobial-sensitive strain of Salmonella havana: 19 (26%) of 72 persons infected had taken an antimicrobial within 30 days before onset of symptoms. Antimicrobial therapy was begun a mean of 15.9 days before and stopped a mean of 7.9 days before onset. In a case-control study, 11 (31%) of 35 case-patients had taken antimicrobials within 30 days of onset compared with 9 (13%) of 70 age- and neighborhood-matched controls (matched odds ratio, 4.3; 95% confidence interval, 1.3, 13.5). The association remained when controlled for the presence of underlying illness or immunosuppression. Prior antimicrobial exposure appears to increase the risk of infection with antimicrobial-sensitive and resistant strains of Salmonella. The delay between antimicrobial use and onset of symptoms suggests that the effect may be due to prolonged alteration of the colonic bacterial flora, resulting in decreased resistance to colonization.

Comparison of a latex agglutination assay and an enzyme-linked immunosorbent assay for detecting cholera toxin.

To determine the pandemic potential of Vibrio cholerae, one must demonstrate both the presence of O1 antigen and the production of enterotoxin (CT). Tissue culture or enzyme-linked immunosorbent assays (ELISAs) for CT have been limited to research and reference laboratories. A kit for detecting CT by reversed passive latex agglutination is now commercially available and was used to test 168 strains of V. cholerae O1 and non-O1. When compared with the routine ELISA, the latex test was 98% accurate (86 of 88) for serogroup O1 strains and 100% accurate (80 of 80) for non-O1 strains. For both O1 and non-O1 study strains, the sensitivity of the latex agglutination test was 0.97 and the specificity was 1.00 when results were compared with ELISA results. The latex test is commercially available and has the advantages of being less complicated and less time-consuming than the ELISA.

Emergence of highly trimethoprim-sulfamethoxazole-resistant Shigella in a native American population: an epidemiologic study.

Resistance to trimethoprim-sulfamethoxazole (TMP-SMX) emerged among Shigella isolates from the Navajo Reservation in the southwestern United States in 1985, years after this antimicrobial agent came into common use. In the study area, TMP-SMX resistance increased dramatically from 3 per cent in 1983 to 21 per cent in 1985. Resistance was polyclonal and occurred in both S. sonnei and S. flexneri. No single plasmid was common to all resistant strains. However, all 28 TMP-SMX resistant isolates examined were resistant to ampicillin and streptomycin and had minimum inhibitory concentrations to sulfamethoxazole of greater than or equal to 4,096 micrograms/ml and to trimethoprim of greater than or equal to 1,024 micrograms/ml. The authors found that 28 of 101 Navajo children with gastrointestinal symptoms who were not taking antimicrobials had TMP-SMX-resistant aerobic fecal flora. To determine risk factors for acquiring resistant strains, they compared 40 case-patients with TMP-SMX-resistant Shigella to 66 controls with TMP-SMX-sensitive Shigella. Case-patients were more likely than controls to have used antimicrobials recently (p = 0.004) and to be hospitalized for shigellosis (p = 0.05). These findings suggest that polyclonal highly TMP-SMX-resistant Shigella emerged by transfer of trimethoprim resistance genes from aerobic bowel flora to endemic Shigella strains, that use of antimicrobials can lead to symptomatic shigellosis and thus the persistence of trimethoprim-resistant Shigella, and that appropriate therapy of shigellosis on the reservation is now a major challenge.

Changes in antimicrobial resistance of Salmonella isolated from humans in the United States.

Changes were examined that occurred in the antimicrobial susceptibility between 1979-1980 and 1984-1985 of Salmonella organisms isolated from humans who resided in a random sample of counties in the United States. Resistance to one or more antimicrobials rose significantly from 16% (82 of 511 isolates) in 1979-1980 to 24% (117 of 485 isolates) in 1984-1985. In addition, statistically significant increases occurred in the proportion of organisms resistant to only one antimicrobial (from 4% to 9%), the rate of resistance of Salmonella typhimurium (from 14% to 26%), and the rate of resistance to tetracycline. For one commonly occurring serotype, Salmonella heidelberg, the proportion of resistant strains fell significantly from 67% in 1979-1980 to 35% in 1984-1985. When epidemiologic characteristics of patients were examined, previous use of antimicrobials for treatment of other illnesses was a significant risk factor for acquiring clinically important infections with antimicrobial-resistant Salmonella. Changes detected in the antimicrobial resistance of Salmonella during this time will ultimately affect the efficacy of antimicrobial therapy in treating human disease.

A multistate outbreak of gastrointestinal illness caused by enterotoxigenic Escherichia coli in imported semisoft cheese.

In September 1983, three clusters of gastrointestinal illness with similar symptoms affected 45 persons in Washington, D.C., after office parties. The illness lasted a mean of 4.4 days and was characterized by watery diarrhea (91%), abdominal cramps (80%), headache (38%), nausea (38%), and subjective fever (20%). Illness was strongly associated with having eaten imported French Brie cheese one to six days before onset of illness (P less than .0001 by Fisher's two-tailed exact test). After publicity about these outbreaks, additional cheese-associated cases were identified over an eight-week period in Illinois, Wisconsin, Georgia, and Colorado. Stool specimens from ill persons in four states yielded Escherichia coli serotype O27:H20. These organisms produced heat-stable enterotoxin and had similar plasmid profiles. When commercially distributed foods are contaminated, enterotoxigenic E. coli can cause widespread disease even in a developed country, and the disease can easily escape correct diagnosis.

Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7.

After two outbreaks of hemorrhagic colitis associated with a previously unrecognized pathogen, Escherichia coli O157:H7, a surveillance system was established to identify and study sporadic cases of this distinct clinical illness in the United States. Between August 1982 and April 1984, we identified 28 persons from 11 states who met our case definition and whose stool specimens yielded E. coli O157:H7. Patients ranged in age from 1 to 80 years. Seventeen patients required hospitalization. All patients recovered, although one developed hemolytic-uremic syndrome 7 days after the onset of bloody diarrhea. Detection of E. coli O157:H7 in stools from persons with hemorrhagic colitis was highly associated with collection of stool specimens within the first 6 days after onset of illness. All E. coli O157:H7 isolates produced a Vero cytotoxin. Hemorrhagic colitis caused by E. coli O157:H7 is widely distributed in the United States as a sporadic illness; clinicians should be aware of its distinctive clinical presentation, and should collect specimens promptly when the diagnosis is suspected.

Outbreak of invasive Escherichia coli gastroenteritis on a cruise ship.

An invasive strain of Escherichia coli (ONT:NM) was isolated from stool specimens from 7 of 10 ill passengers who developed diarrhea during a 5-day ocean cruise. The ill passengers had shared no common exposures off the ship before or during the cruise. Three of the persons whose stools were cultured were part of a tour group of 219 persons, and a food consumption and health history questionnaire was completed by 190 members (87%) of this tour group. Forty-seven (25%) had had diarrhea during the cruise; other symptoms among those with diarrhea included nausea (72%), abdominal cramps (68%), headache (68%), chills (60%), dizziness (53%), myalgias (43%), subjective fever (36%), and vomiting (26%). The median duration of symptoms was 3 days. Eating at cold buffets on ship and eating potato salad, a buffet food item, were significantly associated with illness. No evidence of secondary spread of illness in household contacts of the ill person was found.