Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet.

Standardized rapid pulsed-field gel electrophoresis (PFGE) protocols for the subtyping of Escherichia coli O157:H7, Salmonella serotypes, and Shigella species are described. These protocols are used by laboratories in PulseNet, a network of state and local health departments, and other public health laboratories that perform real-time PFGE subtyping of these bacterial foodborne pathogens for surveillance and outbreak investigations. Development and standardization of these protocols consisted of a thorough optimization of reagents and reaction conditions to ensure that the protocols yielded consistent results and high-quality PFGE pattern data in all the PulseNet participating laboratories. These rapid PFGE protocols are based on the original 3-4-day standardized procedure developed at Centers for Disease Control and Prevention that was validated in 1996 and 1997 by eight independent laboratories. By using these rapid standardized PFGE protocols, PulseNet laboratories are able to subtype foodborne pathogens in approximately 24 h, allowing for the early detection of foodborne disease case clusters and often aiding in the identification of the source responsible for the infections.

Cholera in the United States, 1995-2000: trends at the end of the twentieth century.

To evaluate recent trends in cholera in the United States, surveillance data from all cases of laboratory-confirmed toxigenic Vibrio cholerae O1 and O139 infection reported to the Centers for Disease Control and Prevention between 1995 and 2000 were reviewed. Sixty-one cases of cholera, all caused by V. cholerae O1, were reported. There was 1 death, and 35 (57%) of the patients were hospitalized. Thirty-seven (61%) infections were acquired outside the United States; 14 (23%) were acquired through undercooked seafood consumed in the United States, 2 (3%) were acquired through sliced cantaloupe contaminated by an asymptomatically infected food handler, and no source was identified for 8 (13%) infections. The proportion of travel-associated infections resistant to trimethoprim-sulfamethoxazole, sulfisoxazole, streptomycin, and furazolidone increased from 7 (8%) of 88 in 1990-1994 to 11 (31%) of 35 in 1995-2000. Foreign travel and undercooked seafood continue to account for most US cholera cases. Antimicrobial resistance has increased among V. cholerae O1 strains isolated from ill travelers.

An outbreak of Yersinia enterocolitica O:8 infections associated with pasteurized milk.

In October 1995, an outbreak of Yersinia enterocolitica O:8 infections occurred in the Upper Valley of Vermont and New Hampshire. Ten patients were identified, median age 9 years (range, 6 months-44 years). Three patients were hospitalized; 1 underwent an appendectomy. Consumption of bottled pasteurized milk from a local dairy was associated with illness (matched odds ratio undefined; lower 95% confidence interval, 1.9). No deficiencies in pasteurization procedures or equipment were detected. Y. enterocolitica O:8 was isolated from 1 raw-milk sample and from a fecal sample from 1 dairy pig. The route of contamination was not determined; this outbreak likely resulted from postpasteurization contamination of milk. Dairy pigs were the most likely source of contamination. Milk bottles were likely contaminated by rinsing with untreated well water prior to filling or by other environmental routes. Educating dairy owners about Y. enterocolitica and postpasteurization contamination is necessary to prevent further outbreaks.

Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Israel Vibrio Study Group.

BACKGROUND: Vibrio vulnificus is a gram-negative bacterium that causes septicaemia and wound infection. Cases occur sporadically, and no previous outbreaks due to a common source or a clonal strain have been reported. In the summer and autumn of 1996 and 1997, an outbreak of invasive V. vulnificus infection occurred in Israel in people who had recently handled fresh, whole fish purchased from artificial fish-ponds. METHODS: We reviewed clinical and epidemiological information, and undertook an environmental investigation to assess disease characteristics, modes of transmission, phenotypic characteristics of the bacterium, and fish-marketing policy. The clonal nature of 19 isolates was studied by biotyping, pulsed-field gel electrophoresis, and restriction-fragment length polymorphism (RFLP) analysis of a PCR fragment. FINDINGS: During 1996-97, 62 cases of wound infection and bacteraemia occurred. 57 patients developed cellulitis, four had necrotising fasciitis, and one developed osteomyelitis. In all cases, the fish were cultivated in inland fish-ponds. In the summer of 1996, fish-pond managers initiated a new marketing policy, in which fish were sold alive instead of being packed in ice. Phenotypically, the isolates had five atypical biochemical test results. The isolates were non-typeable by pulsed-field gel electrophoresis, and all had the same PCR-RFLP pattern which had not been seen previously. INTERPRETATION: The cause of the outbreak was a new strain of V. vulnificus, classified as biogroup 3. A new fish-marketing policy that began in 1996 may have exposed susceptible people to the organism.

A prolonged outbreak of Shigella sonnei infections in traditionally observant Jewish communities in North America caused by a molecularly distinct bacterial subtype.

During 1994-1996, Shigella sonnei outbreaks occurred in 8 North American traditionally observant Jewish communities. These communities remain relatively separate from neighboring populations while maintaining close contact by travel with coreligionists in other cities. Epidemiologic investigations suggested community-to-community transmission via travel. Outbreak-related and control isolates of S. sonnei from each city were subtyped by pulsed-field gel electrophoresis (PFGE) to confirm an epidemiologic linkage between outbreaks. Forty-three (94%) of 46 outbreak-related isolates had closely related PFGE patterns, constituting a single subtype; 33 (94%) of 35 control isolates demonstrated unrelated PFGE patterns. Several patterns differing by < or = 3 bands were identified within the outbreak subtype; one of these accounted for 65% of outbreak isolates. Hence, a single subtype of S. sonnei caused an international outbreak involving 8 traditionally observant Jewish communities, but not neighboring populations, over a 2-year period, suggesting sustained propagation of the epidemic strain between communities.

Cholera.

Few diseases invoke public fear as readily as cholera. In its most severe state, cholera can cause death from hypotensive shock within 12 h of the first symptom. Cholera typically occurs in epidemics, spreading rapidly within the community, especially if hygeinic conditions are poor. Fortunately, effective water treatment has limited the spread of cholera in most of the developed world, and the treatment of cholera by oral rehydration has dramatically reduced the mortality rate.

An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E. coli.

An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991. Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days. Median incubation periods ranged from 50 to 56 h. A nonmotile strain of E. coli (E. coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons. This organism produced enteroaggregative E. coli heat-stable enterotoxin 1 and contained the enteropathogenic E. coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells. E. coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.

Epidemic cholera in Guatemala, 1993: transmission of a newly introduced epidemic strain by street vendors.

Epidemic cholera reached Guatemala in July 1991. By mid-1993, Guatemala ranked third in the hemisphere in reported cases of cholera. We conducted a case-control study with two age-, sex-, and neighbourhood-matched controls per patient in periurban Guatemala City. Twenty-six patients hospitalized for cholera and 52 controls were enrolled. Seven (47%) of 15 stool cultures obtained after admission yielded toxigenic Vibrio cholerae O1. All seven were resistant to furazolidone, sulfisoxazole, and streptomycin, and differed substantially by pulsed-field gel electrophoresis from the Latin American epidemic strain dominant in the hemisphere since 1991. In univariate analysis, illness was associated with consumption of left-over rice (odds ratio [OR] = 7.0, 95% confidence interval [CI] = 1.4-36), flavored ices (-helados') (OR = 3.6, CI = 1.1 - 12), and street-vended non-carbonated beverages (OR = 3.8, CI = 1.2-12) and food items (OR = 11.0, CI = 2.3-54). Street-vended food items remained significantly associated with illness in multivariate analysis (OR = 6.5, CI = 1.4-31). Illness was not associated with drinking municipal tap water. Maintaining water safety is important, but slowing the epidemic in Guatemala City and elsewhere may also require improvement in street vendor food handling and hygiene.

Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.

By DNA-DNA hybridization, we classified 26 human strains, 4 dog and cat strains, and 4 hamster strains putatively identified as Helicobacter cinaedi as well as 2 human strains and 2 animal strains of Helicobacter fennelliae. All but one human strain belonged to the same hybridization group as the type strain of H. cinaedi. The animal strains also appeared to belong to this hybridization group. Both human strains of H. fennelliae were shown to be H. fennelliae by DNA-DNA hybridization, but both animal strains were less than 15% related to the type strain. All strains were also characterized by plasmid profiles and ribotyping. Plasmids were found in 23% of the human strains, 100% of the hamster strains, and 33% of the dog and cat strains. Human strains were essentially identical by ribotyping, but were clearly differentiated from the hamster and dog and cat strains. Some strains may be difficult to culture on primary isolation; we found that our strains grew well on anaerobic CDC agar, brucella agar, and tryptic soy agar II. Our H. cinaedi and H. fennelliae strains differed from those previously described because some were resistant to cephalothin: some H. cinaedi strains were also resistant to nalidixic acid. All isolates were also characterized by antimicrobial susceptibility testing. We found that human strains of H. cinaedi were more resistant to clindamycin and erythromycin than were animal isolates; 19% of the human strains were resistant to ciprofloxacin. Therefore, we recommend that antimicrobial susceptibility results be obtained before initiating therapy for H. cinaedi and H. fennelliae infections.

The emerging diversity of the electrophoretic types of Vibrio cholerae in the Western Hemisphere.

Since the Latin American cholera epidemic began in 1991, 447 isolates of Vibrio cholerae O1 from the Western Hemisphere have been assayed by multilocus enzyme electrophoresis (MEE) to determine allelic variation among 16 enzyme-encoding genes. Two electrophoretic types (ETs) were identified among toxigenic isolates from Latin America: 323 were ET 4, the ET associated with the Latin American epidemic, and 29 were ET 3. Twenty-three of these ET 3 isolates had a distinctive antimicrobial resistance pattern also seen in isolates imported into the United States from Latin America and Southeast Asia. These resistant isolates had an identical ribotype and nearly identical pulsed-field gel electrophoresis (PFGE) patterns. Most nontoxigenic isolates analyzed were not precursors or descendants of toxigenic epidemic strains. MEE provided a population genetic frame-work for the interpretation of PFGE and ribotype data from the isolates in this study. All three methods identified 2 distinct strains of toxigenic V. cholerae O1 currently epidemic in Latin America.

Molecular subtyping of toxigenic Vibrio cholerae O139 causing epidemic cholera in India and Bangladesh, 1992-1993.

Since October 1992, > 150,000 cases of cholera have been reported from India and Bangladesh; the great majority of Vibrio cholerae isolates belong to the newly established serogroup O139. To better understand the interaction of genetic and epidemiologic factors responsible for their sudden appearance and rapid spread, representative toxigenic V. cholerae O139 isolates were molecularly characterized and compared with a set of toxigenic V. cholerae O1 and non-O1/non-O139 strains. DNA sequences of the cholera toxin B subunit gene and multilocus enzyme electrophoresis markers of V. cholerae O139 strains were identical to those of V. cholerae O1 isolates of the seventh pandemic. Two distinct ribotypes and four pulsed-field gel electrophoretic patterns were observed for O139 strains. V. cholerae O139 strains were very similar to V. cholerae O1 strains of the seventh pandemic but clearly different from the toxigenic V. cholerae strains of serogroups other than O1 and O139.

Molecular characterization of Vibrio cholerae O1 strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was performed on 180 isolates of Vibrio cholerae serogroup O1 representing 6 different multilocus enzyme electrophoresis (MEE) types and 27 rRNA restriction fragment length polymorphism types (ribotypes). Isolates were digested with the restriction enzyme NotI and were separated into 63 patterns on the basis of differences in band arrangements. In general, strains which were different by MEE or ribotyping also had different PGFE patterns. PFGE identified individual strains within a single MEE type or ribotype; isolates with one PFGE pattern were less frequently distinguished by ribotyping. All V. cholerae O1 isolates tested from the Latin American epidemic were indistinguishable by their MEE, ribotype, or PFGE patterns. PFGE could further distinguish strains of this same ribotype isolated in Africa, Europe, the South Pacific, or Southeast Asia. Although both MEE and PFGE could identify the strain from the Latin American epidemic, PFGE was more rapid and less labor intensive. PFGE also distinguished nontoxigenic isolates endemic to the U.S. Gulf Coast from unrelated nontoxigenic isolates. In the present study PFGE was more discriminating than other previously described subtyping assays for V. cholerae O1 and appears to be a useful epidemiologic tool.

Evaluation of ribotyping techniques as applied to Arcobacter, Campylobacter and Helicobacter.

Restriction fragment length polymorphisms of ribosomal DNA (ribotyping) of many bacterial species has been useful for both epidemiologic subtyping and species identification. However, the use of ribotyping has been confined to major research and reference laboratories due to two factors: (a) the procedure must be carefully optimized for each organism one wishes to investigate and (b) most currently available protocols use hazardous chemicals or radioisotopes. The purpose of this study is to suggest an overall scheme that a clinical or research microbiologist could apply to ribotyping of any organism. In general, we recommend using a guanidium extraction method for DNA extraction, careful optimization of restriction conditions, and hybridization with non-radioactive digoxigenin-labelled probes; these procedures do not use hazardous chemicals or radioisotopes.

Molecular subtyping of Vibrio cholerae O1 strains recently isolated from patient, food and environmental samples in Spain.

Nineteen Vibrio cholerae O1 strains isolated in Spain from patient, food and environmental samples in the period 1990-1992 were characterized by detection of cholera toxin by enzyme immunoassay, detection of cholera toxin gene by polymerase chain reaction, and by biotyping, ribotyping and pulsed-field gel electrophoresis. Ten isolates were toxigenic and were further characterized by multilocus enzyme electrophoresis. Molecular subtyping methods allowed precise differentiation between isolates, indicating their geographic origin. Isolates associated with the ongoing seventh pandemic were distinguishable from those associated with the present Latin American epidemic. All isolates from the environment and seafood were nontoxigenic, and were genetically different and more diverse than toxigenic isolates. The data suggest that a focus of endemic cholera does not exist in Spain, and that the analyzed nontoxigenic Vibrio cholerae O1 isolates from imported seafood were not a threat to public health.

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.

Differentiation of Salmonella enteritidis phage type 8 strains: evaluation of three additional phage typing systems, plasmid profiles, antibiotic susceptibility patterns, and biotyping.

Three additional phage typing systems for Salmonella enteritidis, plasmid analysis, biochemical tests, and antimicrobial susceptibility tests, were used in an attempt to subdivide 30 phage type 8 (phage typing system used by the WHO International Center for Enteric Phage Typing, London, England) isolates. These isolates represented 18 different egg-related outbreaks (21 strains) and 9 reference strains or strains that were not egg-associated. Only 7 of the 30 strains (28%) were subdivided by one or more of the methods used; this included 3 of the 21 strains from egg-related outbreaks. Twenty-seven strains contained a 55-kb plasmid that is associated with S. enteritidis. Of 65 additional phages tested, 2 from the phage typing system obtained from the Pasteur Institute, Paris, France, were useful in differentiating the three strains that lacked the 55-kb plasmid. Although the results obtained for the 21 strains from egg-related outbreaks showed that the strains had minor phenotypic differences, the overall results suggested that the strains may represent a single clone. Studies are planned to test additional phages and other typing methods to see whether strains of phage type 8 can be further differentiated.

A multistate outbreak of Salmonella javiana and Salmonella oranienburg infections due to consumption of contaminated cheese.

OBJECTIVE: To determine the source of an outbreak of Salmonella javiana and Salmonella oranienburg infections. DESIGN: Laboratory-based statewide surveillance for Salmonella infections and two separate case-control studies. SETTING: Community- and industry-based studies conducted from May through October 1989. PARTICIPANTS: Thirty-one culture-confirmed outbreak-associated cases of S javiana infection and 60 community controls matched for telephone prefix, gender, and age in case-control study I; 50 cases, 100 community controls, and 64 family member controls in case-control study II. RESULTS: One hundred thirty-six culture-confirmed cases of S javiana infection and 11 cases of S oranienburg infection were associated with the outbreak in Minnesota. Outbreak-associated cases were also identified in Wisconsin (15 cases), and in Michigan and New York (one case each). Cases were more likely than controls to have consumed mozzarella cheese manufactured at a single cheese plant (plant X) or cheese that had been shredded at processing plants that also shredded cheese manufactured at plant X (odds ratio [OR], 7.2; 95% confidence interval [CI], 1.7 to 23.2; P < .01). The outbreak-associated strains of both serovars were isolated from two unopened 16-oz (0.45-kg) blocks of mozzarella cheese produced at plant X. The most probable numbers of Salmonella organisms in these samples were 0.36/100 g and 4.3/100 g. CONCLUSIONS: The potential for bacterial pathogen contamination of cheese during manufacture and processing has important epidemiologic implications, particularly because cheese consumption has recently increased in the United States. Low-level contamination of a nationally distributed food product can cause geographically dispersed foodborne outbreaks that may be difficult to detect.

An international foodborne outbreak of shigellosis associated with a commercial airline.

OBJECTIVE: To determine the source of an international outbreak of shigellosis associated with consumption of food served by a Minnesota-based airline. DESIGN: Cohort studies of players and staff of a Minnesota-based professional football team and passengers on flights with a confirmed case of outbreak-associated Shigella sonnei infection. SETTING: Community- and industry-based studies conducted from October through November 1988. PARTICIPANTS: Sixty-five football team players and staff, and 725 airline passengers in the cohort studies. RESULTS: Twenty-one (32%) of 65 football players and staff developed shigellosis that was associated with consumption of cold sandwiches prepared at the airline flight kitchen (relative risk [RR], 17.1; 95% confidence interval [Cl], 2.4 to 120; P < .001). Confirmed or probable shigellosis was identified among 240 passengers on 219 flights to 24 states, the District of Columbia, and four countries between September 14 and October 13. An outbreak-associated strain of S sonnei was isolated from football players and staff, airline passengers, and flight attendants. Thirty (4.1%) of 725 passengers on 13 flights with confirmed cases had confirmed or probable shigellosis. Illness was associated with consumption of cold food items served on the flights and prepared by hand at the airline flight kitchen (RR, 5.7; 95% Cl, 1.4 to 23.5; P < .01). CONCLUSIONS: This international outbreak of shigellosis was identified only because of the occurrence of an index outbreak involving a professional football team. Prevention of Shigella transmission in mass catering establishments may require reduction of hand contact in the preparation of cold food items or elimination of these items from menus.

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.

Vibriophage VcA-3 as an epidemic strain marker for the U.S. Gulf Coast Vibrio cholerae O1 clone.

Toxigenic and nontoxigenic Vibrio cholerae O1, El Tor biotype strains, which are endemic to the U.S. Gulf Coast, can be lysogenic for bacteriophage VcA-3. To evaluate the presence of VcA-3 as an indicator of toxigenicity and as an epidemic strain marker, phage production and the presence of phage and cholera toxin genes were assayed in 98 strains of V. cholerae O1 (35 U.S. and 63 foreign strains). By using a HindIII chromosomal digest for Southern blot analysis, 39 of the study strains hybridized with the VcA-3 probe in 10 banding patterns. The 15 toxigenic and 6 of the 20 nontoxigenic U.S. isolates gave four VcA-3-related patterns. Among the foreign isolates, 12 of 12 toxigenic classical biotype strains, 1 of 43 toxigenic El Tor biotype strains, and 3 of 8 nontoxigenic atypical strains gave six patterns that were clearly distinct from that of VcA-3. Compared with Southern blot analysis, the phage production assay had a sensitivity of 1.0 and a specificity of 0.48, while the colony hybridization assay had a sensitivity of 1.0 and a specificity of 0.77 for identification of VcA-3. Neither assay reliably identified the toxigenic Gulf Coast clone. The presence of VcA-3, as defined by Southern blot analysis, always separated toxigenic U.S. from foreign isolates and often from nontoxigenic U.S. isolates of V. cholerae O1.