Two new Escherichia coli O groups: O172 from "Shiga-like" toxin II-producing strains (EHEC) and O173 from enteroinvasive E. coli (EIEC).

Two Escherichia coli strains were established as antigenic test strains for two new O groups, O172 and O173. The O172 strain (EHEC) which produces "Shiga-like" toxin II (verocytotoxin 2) was isolated from a case of haemorrhagic colitis while the enteroinvasive O173 strain (EIEC) originated from a child with diarrhoea.

The use of plasmid profiles and nucleic acid probes in epidemiologic investigations of foodborne, diarrheal diseases.

The application of nucleic acid analyses to investigations of infectious disease outbreaks has resulted in useful molecular strain markers that distinguish the epidemic clone of a particular pathogen and help identify specific vehicles of infection. We have successfully used plasmid profile analysis, restriction endonuclease digestion of plasmid and whole-cell DNAs, and nucleic acid hybridization to investigate recent outbreaks of foodborne diarrheal illness. Plasmid analysis has been important in identifying epidemic strains of Salmonella enteritidis and Escherichia coli O157:H7. In a culture survey of S. enteritidis isolates from humans and a variety of animals, including chickens and chicken eggs, we identified 16 distinct plasmid profiles and used these to differentiate strains, especially within commonly occurring phage types (Colindale 8 and 13a). HindIII digests of plasmid DNA were useful in distinguishing plasmids of similar mass but dissimilar enzyme target sequences; they clearly distinguished S. enteritidis strains causing systemic infections in children in parts of Africa from U.S. isolates. Investigations of outbreaks of hemorrhagic colitis have also been assisted by plasmid analysis. Restriction endonuclease digests of whole-cell DNA and Southern blot analysis, hybridizing with E. coli 16S and 23S rRNA (ribotyping), have been effective subtyping techniques, especially for plasmidless isolates of Campylobacter jejuni. In five outbreaks of C. jejuni infections, ribotyping of PvuII and ClaI digests distinguished individual epidemic strains within one commonly occurring C. jejuni serotype (Penner 2, Lior 4). Preliminary data show that ribotyping of NcoI digests can also distinguish individual epidemic strains of E. coli O157:H7 and may provide a more stable marker than plasmid profiles. Specific DNA probes derived from cloned virulence genes of E. coli have been invaluable in epidemic investigations and surveys. Using colony hybridization, we found in one survey of stool specimens from 174 dairy cattle that 11% of animals were asymptomatically carrying Shiga-like toxigenic E. coli other than O157:H7. We also found that newly synthesized oligonucleotide probes for the Shiga-like toxins I and II agreed 100% with cloned gene probes in a study of 613 E. coli strains. Future studies of these organisms will include the use of additional synthetic oligonucleotides as primers to amplify the toxin genes directly in patient and animal specimens by the polymerase chain reaction. There is a continuing and expanding role for molecular approaches in epidemiological investigations. The DNA methods described above are not based on the often complex expression of phenotypic characteristics, and, unlike sensitive and specific techniques such as phage typing, a single method can be used to study a variety of Gram-positive and negative bacterial pathogens.

Hazard analysis and critical control point systems in the United States Department of Agriculture regulatory policy.

Recent outbreaks of foodborne illness and studies by expert groups have established the need for fundamental change in the United States meat and poultry inspection programme to reduce the risk of foodborne illness. The Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA) has embarked on a broad effort to bring about such change, with particular emphasis on the reduction of pathogenic micro-organisms in raw meat and poultry products. The publication on 25 July 1996 of the Final Rule on pathogen reduction and hazard analysis and critical control point (HACCP) systems was a major milestone in the FSIS strategy for change. The Final Rule provides a framework for change and clarifies the respective roles of industry and government in ensuring the safety of meat and poultry products. With the implementation of this Final Rule underway, the FSIS has been exploring ways in which slaughter inspection carried out under an HACCP-based system can be changed so that food safety risks are addressed more adequately and the allocation of inspection resources is improved further. In addition, the FSIS is broadening the focus of food safety activities to extend beyond slaughter and processing plants by working with industry, academia and other government agencies. Such co-operation should lead to the development of measures to improve food safety before animals reach the slaughter plant and after products leave the inspected establishment for distribution to the retail level. For the future, the FSIS believes that quantitative risk assessments will be at the core of food safety activities. Risk assessments provide the most effective means of identifying how specific pathogens and other hazards may be encountered throughout the farm-to-table chain and of measuring the potential impact of various interventions. In addition, these assessments will be used in the development and evaluation of HACCP systems. The FSIS is currently conducting a quantitative risk assessment for eggs, and several surveys and studies are being performed to supply data needed to conduct other risk assessments. The FSIS has established a food safety research agenda which will fill data gaps.

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.

Genetic evidence of clonal descent of Escherichia coli O157:H7 associated with hemorrhagic colitis and hemolytic uremic syndrome.

Genetic relatedness of 100 strains of Escherichia coli, isolated mostly from patients with hemorrhagic colitis or hemolytic uremic syndrome, was determined for chromosomal genotypes on the basis of allelic variation at 17 enzyme-encoding loci detected by multilocus enzyme electrophoresis. Fifteen of the 17 loci were polymorphic, with an average of 3.5 alleles per locus. Comparison of the observed combinations of alleles among strains revealed 25 distinct multilocus genotypes, which were used to define naturally occurring cell lineages or clones. Cluster analysis of the genotypic data revealed that isolates of serotype O157:H7 fall into a well-defined group of clonal genotypes that share alleles, on average, at 90% of their enzyme loci. The O157:H7 clonal group is only distantly related to other Verotoxin-producing strains belonging to other serotypes of E. coli. The results strongly support the hypothesis that isolates of E. coli O157:H7 obtained from geographically separate outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome belong to a pathogenic clone that occurs throughout North America.

Bacterial endotoxin both enhances and inhibits the toxicity of Shiga-like toxin II in rabbits and mice.

The ability of bacterial lipopolysaccharide (LPS) to enhance the toxicity of Shiga-like toxin II (SLT-II) was investigated in rabbits and mice. Rabbits were continuously infused with 0.5 50% lethal dose (LD50) of SLT-II per day. Rabbits that received a 30-micrograms/kg dose of LPS (0.02 LD50) on day 3 of infusion were significantly more likely to die than were rabbits receiving SLT-II only. Rabbits receiving SLT-II and a lower dose of LPS (3 micrograms/kg) did not die but lost an average 3.3% +/- 1.0% of initial body weight during the first 5 days of infusion, compared with weight gains of 4.2% +/- 0.6% and 17.1% +/- 0.9% for rabbits receiving only SLT-II or LPS, respectively. Rabbits that were pretreated with LPS 20 h before challenge with a single dose of SLT-II showed highly significant protection from both the diarrheagenic and lethal effects of SLT-II. Pretreatment of endotoxin-responsive C3H/HeN mice protected the animals from challenge with an LD50 but not an LD100 of SLT-II. LPS enhanced the lethal toxicity of SLT-II for C3H/HeN mice when it was given at 8 or 24 h but not 0 or 72 h after SLT-II challenge. LPS did not affect the lethal toxicity of SLT-II for endotoxin-resistant C3H/HeJ mice. These results suggest that LPS enhances the effects of SLT-II in vivo. Since cecal changes that increase mucosal permeability occur in response to SLT in rabbits, this synergy may be directly relevant to disease processes.

Plasmid-mediated properties of a heat-stable enterotoxin-producing Escherichia coli associated with infantile diarrhea.

The plasmid mediation and transmissibility of heat-stable enterotoxin production and multiple antibiotic resistance have been demonstrated for Escherichia coli O78:K80:H12 epidemiologically incriminated in a hospital outbreak of infantile diarrhea. The conjugal transfer of a 67 X 10(6) - and a 30 X 10(6)-dalton plasmid was associated with the transfer of resistances and enterotoxin production, respectively. Using antibiotics to select E. coli K-12 transconjugants from a one-step bacterial cross, all of the monitored resistances were transferred concurrently, and 36% of the resistant transconjugants produced enterotoxin.

Prevalence of toxigenic and invasive strains of Escherichia coli in a hospital population.

The pathogenesis of gastrointestinal infections caused by Escherichia coli has been attributed to the ability of some strains to produce exotoxins (enterotoxins) or to invade the intestinal mucosa directly. To investigate the possible role of invasiveness and of production of toxin in the pathogenesis of nonenteric E. coli infections, we studied E. coli isolated from 152 patients with urinary tract infections, bacteremia, or both. None of the isolates from urine or blood exhibited invasiveness in the guinea pig eye or produced toxin in tissue culture or in infant mice; only one patient was a gastrointestinal carrier of toxin-producing E. coli. We concluded that, in the population of this study, production of toxin and invasiveness were not important in the pathogenesis of most urinary tract infections or bacteremia caused by E. coli, and that gastrointestinal colonization with such organisms was uncommon.

In vitro susceptibilities of aerotolerant Campylobacter isolates to 22 antimicrobial agents.

We evaluated the in vitro activities of 22 antimicrobial agents against 78 human and animal isolates belonging to two aerotolerant Campylobacter species, C. cryaerophila and C. butzleri, using a broth microdilution technique. An additional 10 antimicrobial agents were included at concentrations found in selective Campylobacter media. Strains of C. cryaerophila belonged to two DNA hybridization groups: DNA hybridization group 1A, which includes the type strain of C. cryaerophila, and DNA hybridization group 1B. The aminoglycosides, fluoroquinolones, and one tetracycline (minocycline) demonstrated the most activity against all DNA hybridization groups (C. cryaerophila DNA groups 1A and 1B and C. butzleri). Most isolates were resistant to cephalosporin antibiotics, with the exception of cefotaxime, and were variably susceptible to trimethoprim-sulfamethoxazole. C. cryaerophila DNA hybridization group 1A isolates were generally susceptible to the tetracyclines, chloramphenicol, nalidixic acid, azithromycin, erythromycin, and roxithromycin and moderately susceptible to clindamycin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam. The MICs of tetracyclines were higher for C. butzleri and C. cryaerophila DNA hybridization group 1B isolates than for C. cryaerophila DNA hybridization group 1A isolates, but most strains were still susceptible to doxycycline and tetracycline; all isolates were susceptible to minocycline. C. butzleri and C. cryaerophila DNA hybridization group 1B isolates were generally resistant to the macrolide antibiotics (including erythromycin), chloramphenicol, clindamycin, nalidixic acid, ampicillin, and trimethoprim-sulfamethoxazole. Differences in antimicrobial susceptibility between aerotolerant Campylobacter species and more common Campylobacter species, e.g., C. jejuni, suggest that different treatment strategies may be necessary. Strains of all three DNA hybridization groups of aerotolerant Campylobacter isolates were susceptible to colistin, polymyxin B, and rifampin at concentrations commonly used in selective media. These results suggest that primary isolation methods for Campylobacter species may need to be modified to include aerotolerant Campylobacter strains.

Serotypes of enterotoxigenic Escherichia coli isolated in the United States.

Strains of enterotoxigenic Escherichia coli isolated from humans in the United States were found in 11 of 16 serotypes that previously were documented in the international literature as associated with enterotoxin production. Of 68 strains belonging to these 11 serotypes, 28 (41%) were enterotoxigenic; none of 46 strains belonging to 5 other previously implicated serotypes was enterotoxigenic. Control cultures of various serotypes were selected for comparison and found to contain 0 to 7% enterotoxigenic E. coli. E. coli belonging to documented enterotoxin-associated serotypes, characterized by both O and H antigens, were selected for toxin testing to determine their prevalence and potential pathogenicity in this country. In this study, a strain possessing any combination of an enterotoxin-associated serotype O antigen and H antigen was more likely to be enterotoxigenic than strains possessing only the specific O antigen or H antigen or neither. Five E. coli strains belonging to undocumented enterotoxin-associated serotypes did contain a combination of previously reported enterotoxin-associated serotype O and H antigens and did produce enterotoxin.

Hemolytic activity in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli.

We screened 223 strains of Escherichia coli belonging to serotypes previously associated with the production of enterotoxin for hemolytic activity, using horse erythrocytes in liquid and in agar media. Thirty-eight were hemolytic. They belonged to nine different serotypes; most (65.8%) belonged to one serotype, O6: H-. Additionally, all 38 strains were specifically assayed for a filterable, heat-labile hemolytic activity previously associated with a hemolysin plasmid. A comparison of hemolytic activity and enterotoxicity showed that none of 32 strains hemolytic in both media was enterotoxigenic; 28 of the 32 expressed heat-labile hemolytic activity. Four of the six strains hemolytic in only one of the media were enterotoxigenic; none of these six expressed heat-labile hemolytic activity. Of 223 strains, 176 that were of human origin and isolated in the United States were further assayed for three traditionally plasmid-mediated characteristics: heat-labile enterotoxin, heat-stable enterotoxin, and colonization factors. The interrelationships of these characteristics, including hemolytic activity, may reflect varying degrees of plasmid compatibility.

Antibiotic resistance in Enterotoxigenic and non-enterotoxigenic Escherichia coli.

Antibiotic disk susceptibility tests were done on 220 strains of Escherichia coli belonging to serotypes reported in the literature to be associated with the production of enterotoxin. A total of 128 (58%) were resistant to one or more antibiotics, sulfa drugs, or chemotherapeutic agents. An analysis of these strains revealed primary, secondary, and tertiary drug resistance patterns that indicated a selective pattern in the formation of multiple drug resistance in E. coli. Resistances to certain antibiotics were more likely to occur in pairs and triads (secondary resistance patterns) that were often combined or coexisted in a single strain of E. coli to produce tertiary drug resistance patterns, conferring drug resistance to five or six different antibiotics. Among enterotoxin-associated serotypes, single and multiple drug resistance was less frequently associated with enterotoxin-produced strains than with strains from the same serotype that were not enterotoxigenic. Within the enterotoxigenic E. coli, single and multiple resistance to antibiotics was more frequent in strains producing only heat-stable enterotoxin (ST) than in strains producing only heat-labile enterotoxin (LT) or both. The number of resistances to different antibiotics per resistant strain averaged approximately 1.4 for LT plus ST or LT strains, and 3.9 for ST strains and nonenterotoxigenic strains. Phenotypic characterization of 170 strains for four usually plasmid-mediated characteristics showed that the number of antibiotics to which a strain was directly resistant varied with the type and number of plasmid-mediated characteristics present.

Ureolytic Escherichia coli of human origin: serological, epidemiological, and genetic analysis.

Forty-five strains of ureolytic Escherichia coli of human origin, isolated in the United States between 1956 and 1977, were characterized by geographical distribution, site of infection, serotype, resistance to antibiotics, and biochemical reactions. All strains were studied for the ability to generate clones of nonureolytic E. coli (segregants), and a subset of these were selected for plasmid analysis and a variety of bacterial matings. There did not appear to be a common geographical distribution, serotype, antibiogram, or other aberrant biochemical reactions other than the hydrolysis of urea among these strains. The predominance of urinary tract isolates (46.7% total) may reflect a relationship between urea hydrolysis and pathogenesis at this site. Ten of the strains (22.2%) did segregate nonureolytic E. coli colonies, and all possessed at least one common plasmid species with a molecular weight of about 65 X 10(6). Only strain 1138-77 serotype O16:H6 conjugally transfered the ability to hydrolyze urea, ferment sucrose, and resist inhibition by sulfadiazide simultaneously. The resulting, recombination-deficient E. coli K-12 tranconjugant was found to possess a plasmid with a molecular weight of about 80 X 10(6) to 90 X 10(6).

An outbreak of antibiotic-resistant Salmonella enteritidis in Liberia, West Africa.

Between October 1980 and August 1982, 100 patients in the pediatric population at Curran Lutheran Hospital, Zorzor, Liberia were identified as having multiple drug-resistant Salmonella enteritidis serotype enteritidis. The illness usually presented as an enteric fever but also as meningitis, gastroenteritis, empyema, subcutaneous abscesses, chronic otitis media, or a combination of these conditions. Predisposing factors were young age and debilitation from malnutrition or measles. The mortality of infected patients was 27.8%. The organism was originally misidentified as a Citrobacter species because of a delayed reaction on lysine decarboxylase medium. Incubation of the medium for five days resulted in a positive reaction that identified the organism as a Salmonella species. The isolates were resistant to multiple antibiotics. Genes mediating resistance were located on a 120-megadalton conjugative plasmid. A cryptic nonconjugative 40-megadalton plasmid was also present in several isolates.

Molecular epidemiology of Yersinia enterocolitica O:3 infections: use of chromosomal DNA restriction fragment length polymorphisms of rRNA genes.

Yersinia enterocolitica is a major enteric pathogen associated with a wide variety of clinical and immunologic manifestations, including transfusion-associated disease, from which there is a high mortality. Although previously rare in the United States, in the late 1980s Y. enterocolitica O:3 emerged as the predominant serotype in the United States, as it has been in Canada, Europe, and Japan. Epidemiologic investigation of this serogroup has been hampered by the limited availability of a phage typing system and the fact that Y. enterocolitica harbors few plasmids that are useful as strain markers. We therefore analyzed whole-cell DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to study a group of 61 (50 human, 11 porcine) Y. enterocolitica isolates. Initially, 20 different restriction enzymes were used: NciI appeared to give the best discrimination of hybridization banding patterns (ribotypes) within Y. enterocolitica O:3. Ribotyping distinguished seven clones among all the study isolates and four clones within Y. enterocolitica O:3 (53 isolates studied) and clearly differentiated Y. enterocolitica O:3 from Y. enterocolitica O:9; O:1,2,3; O:20; and O:5,27. Most serogroup O:3 isolates belonged to two clones, ribotypes I and II, including 23 of 24 Y. enterocolitica O:3 (13 human, 11 porcine chitterling) isolates recovered from a recent outbreak of Y. enterocolitica in children in Atlanta associated with chitterling preparation and 3 transfusion-associated O:3 isolates from the United States. Y. enterocolitica O:3 ribotypes I and II were also isolated in Japan, ribotypes II and IV were isolated in Belgium, and ribotype I was isolated in Canada. Ribotype patterns I and II corresponded to phage types 9b and 8, respectively. Ribotyping was able to distinguish individual strains of Y. enterocolitica O:3, but suggests that a limited number of clones have disseminated within the United States and globally. The finding of identical ribotype patterns in chitterling and human specimens from the Atlanta outbreak supports epidemiologic evidence that swine were the source of infection and major reservoir for Y. enterocolitica O:3.

Use of cholera toxoid in an enzyme-linked immunosorbent assay for antitoxin.

A glutaraldehyde-inactivated toxoid was evaluated as a coating antigen in an ELISA for cholera antitoxin. A reference panel of 36 human sera with antitoxin levels determined by several other assay systems and 58 sera from an outbreak of illness due to Vibrio cholerae were studied. Toxoid compared favorably with two purified cholera toxins, and the microtiter assay using all three antigens was effective in detecting antibody in known convalescing cholera patients.

Resistance plasmid transfer by Serratia marcescens in urine.

Resistance plasmids were transferred in urine from a multi-drug-resistant Serratia marcescens to Escherichia coli. Transfer of resistance to kanamycin, tetracycline, chloramphenicol, streptomycin, ampicillin, and carbenicillin occurred readily after 4 h of incubation at room temperature (25 degrees C). The urinary catheter collection bag is postulated as a potential site for extraintestinal resistance plasmid transfer in the Enterobacteriaceae, especially for pathogens such as Serratia, which do not frequently colonize the intestinal tract.

Escherichia coli heat-labile enterotoxin: comparison of antitoxin assays and serum antitoxin levels.

The mouse adrenal tumor cells (Y-1 strain) and the Chinese hamster ovary cells, two routinely used tissue culture assays for Escherichia coli heat-labile enterotoxin (LT), were used to detect serum antitoxin responses in culture-positive patients from several well-defined sources. There was no correlation between a significant antitoxin response and isolation of LT-producing E. coli in two "domestic" diarrheal outbreaks. Serum samples from a third group of individuals in a rural cholera-endemic area consistently demonstrated significant rises in neutralizing antibody to LT.

Utilization of exogenous siderophores by Campylobacter species.

The availability of iron to potentially pathogenic bacterial strains is restricted by the iron-binding proteins of the host. In this study, we examined 40 strains of Campylobacter species grown under iron-limiting conditions. While the strains produced no detectable siderophores, all the isolates freely utilized exogenous siderophores produced by other organisms as iron carriers. These data suggest that the use of an exogenous siderophore (either purified or present in a coinfecting microorganism) may be important in developing a suitable laboratory model for campylobacteriosis.