Outbreaks of non-O157 Shiga toxin-producing Escherichia coli infection: USA.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections are increasingly detected, but sources are not well established. We summarize outbreaks to 2010 in the USA. Single-aetiology outbreaks were defined as ⩾2 epidemiologically linked culture-confirmed non-O157 STEC infections; multiple-aetiology outbreaks also had laboratory evidence of ⩾2 infections caused by another enteric pathogen. Twenty-six states reported 46 outbreaks with 1727 illnesses and 144 hospitalizations. Of 38 single-aetiology outbreaks, 66% were caused by STEC O111 (n = 14) or O26 (n = 11), and 84% were transmitted through food (n = 17) or person-to-person spread (n = 15); food vehicles included dairy products, produce, and meats; childcare centres were the most common setting for person-to-person spread. Of single-aetiology outbreaks, a greater percentage of persons infected by Shiga toxin 2-positive strains had haemolytic uraemic syndrome compared with persons infected by Shiga toxin 1-only positive strains (7% vs. 0·8%). Compared with single-aetiology outbreaks, multiple-aetiology outbreaks were more frequently transmitted through water or animal contact.

Cloning of Shiga-like toxin structural genes from a toxin converting phage of Escherichia coli.

The genes controlling high-level production of Shiga-like toxin (SLT) in Escherichia coli were cloned from the SLT converting phage 933J. This phage was isolated from a strain of E. coli that caused a foodborne outbreak of hemorrhagic colitis. The genes that convert normal E. coli to organisms producing high levels of toxin were cloned into the plasmid pBR328 and expressed in E. coli HB101. DNA restriction mapping, subcloning, examination of the cloned gene products by minicell analysis, neutralization, and immunoprecipitation with antibodies to SLT were used to localize the toxin converting genes and identify them as structural genes for SLT. Southern hybridization studies established that the DNA fragment carrying the cloned toxin structural genes had homology with the DNA of Shigella.

Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii.

Escherichia coli, Escherichia albertii, and Escherichia fergusonii are closely related bacteria that can cause illness in humans, such as bacteremia, urinary tract infections and diarrhea. Current identification strategies for these three species vary in complexity and typically rely on the use of multiple phenotypic and genetic tests. To facilitate their rapid identification, we developed a multiplex PCR assay targeting conserved, species-specific genes. We used the Daydreamer™ (Pattern Genomics, USA) software platform to concurrently analyze whole genome sequence assemblies (WGS) from 150 Enterobacteriaceae genomes (107 E. coli, 5 Shigella spp., 21 E. albertii, 12 E. fergusonii and 5 other species) and design primers for the following species-specific regions: a 212bp region of the cyclic di-GMP regulator gene (cdgR, AW869_22935 from genome K-12 MG1655, CP014225) for E. coli/Shigella; a 393bp region of the DNA-binding transcriptional activator of cysteine biosynthesis gene (EAKF1_ch4033 from genome KF1, CP007025) for E. albertii; and a 575bp region of the palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase (EFER_0790 from genome ATCC 35469, CU928158) for E. fergusonii. We incorporated the species-specific primers into a conventional multiplex PCR assay and assessed its performance with a collection of 97 Enterobacteriaceae strains. The assay was 100% sensitive and specific for detecting the expected species and offers a quick and accurate strategy for identifying E. coli, E. albertii, and E. fergusonii in either a single reaction or by in silico PCR with sequence assemblies.

Bacterial, viral and parasitic enteric pathogens associated with acute diarrhea in hospitalized children from northern Jordan.

To determine the etiology of acute diarrhea in Jordanian children under 5 years of age, we examined stool samples from 265 children admitted to the pediatric ward at Princess Rahma Hospital for Children, Irbid, Jordan, for parasites, rotavirus and enteric bacteria. Using both traditional and molecular diagnostic techniques, we detected enteropathogens in 66.4% of patients with diarrhea. A single enteric pathogen was detected in 50.9% of the children, and multiple pathogens were detected in 15.5%. The prevalence of enteropathogens identified was as follows: rotavirus (32.5%), enteropathogenic Escherichia coli (12.8%), enteroaggregative E. coli (10.2), enterotoxigenic E. coli (5.7%), Shigella spp. (4.9%), Entamoeba histolytica (4.9%), Salmonella spp. (4.5%), Campylobacter jejuni/coli (1.5%), Cryptosporidium spp. (1.5%), enteroinvasive E. coli (1.5%), eae-, Ehly-positive E. coli (0.8%), Giardia lamblia (0. 8%) and Yersinia enterocolitica (0.4%). No Vibrio cholerae, Shiga toxin-producing E. coli, microsporidia, adenovirus or small round virus were detected. Findings from this study demonstrate that rotavirus and several types of diarrheagenic E. coli, which are not screened for during routine examinations of stool samples in public health laboratories, were the most frequently detected enteropathogens in these children. Our findings highlight the value of using a combination of traditional and molecular techniques in the diagnosis of diarrheal disease in this population.

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.

Cloning of genes for production of Escherichia coli Shiga-like toxin type II.

Genes controlling production of Shiga-like toxin type II (SLT-II) in Escherichia coli were cloned from the SLT-II-converting bacteriophage 933W and compared with the Shiga-like toxin type I (SLT-I) genes previously isolated and described from phage 933J. Subcloning analysis identified a region within the 4.9-kilobase EcoRI fragment of phage 933W that was associated with SLT-II production. Experiments with E. coli minicells containing these subclones demonstrated that the 4.9-kilobase EcoRI fragment encodes the structural genes for SLT-II. These experiments additionally showed the genetic organization of the SLT-II genes to be the same as that of the SLT-I genes, with the coding sequence for the large A subunit adjacent to that for the smaller B subunit. The mobilities of the SLT-II subunits in sodium dodecyl sulfate-polyacrylamide gels were slightly greater than those determined for the SLT-I subunits. Although apparent processing of the SLT-I subunits was observed with polymyxin B treatment of the labeled minicells, no processing of the SLT-II subunits was detected. Southern blot hybridization studies suggested that the DNA fragment carrying the SLT-II structural genes shares approximately 50 to 60% homology with the DNA of the SLT-I structural genes.

Evaluation of commercial latex reagents for identification of O157 and H7 antigens of Escherichia coli.

Agglutination reactions obtained with three commercial latex reagents for detecting Escherichia coli O157 antigen (Oxoid Diagnostic Reagents, Hampshire, England; Pro-Lab Inc., Richmond Hill, Ontario, Canada; and Remel Microbiology Products, Lenexa, Kans.) and one for detecting H7 antigen (Remel) were compared with those obtained by standard serologic methods by using the Centers for Disease Control and Prevention (CDC) reference antisera for O157 and H7 antigens. For 159 strains of E. coli and related organisms, the Oxoid, Pro-Lab, and Remel O157 latex reagents each had a sensitivity and specificity of 100% compared with the CDC reference antiserum. For 106 strains of E. coli and related organisms that were not enhanced for motility through semisolid medium, the Remel H7 latex reagent had a sensitivity of 96% and a specificity of 100% compared with the standard tube agglutination method with CDC H7 antiserum. Measures to enhance motility were needed for some strains to detect the H7 antigen. Our findings demonstrate that the commercial latex reagents are good alternatives to standard serologic methods for identifying the O157 and H7 antigens of E. coli.

Evidence for participation of the macrophage in Shiga-like toxin II-induced lethality in mice.

Seven strains of inbred mice were compared for their susceptibility to the lethal effects of Shiga-like toxin II (SLT II). A/J mice, which are unable to produce the C5 component of complement, did not differ from C5 normal mice in susceptibility to SLT II. CBA/NJ mice (hemizygous for X-linked immunodeficiency) did not differ from the B-cell sufficient CBA/J strain. C3H/HeJ mice, defective in macrophage response to lipopolysaccharide (Lpsd), showed a consistently and significantly longer mean time to death than did the normally responsive C3H/HeN strain. C57BL/10ScN mice, which also carry the Lpsd allele, showed a similar but smaller difference in mean time to death compared with the C57BL/10SnJ strain. Production of tumor necrosis factor could be induced in vitro by SLT II treatment of C3H/HeN, but not C3H/HeJ macrophages. These results imply that antibody and complement production do not modulate SLT II lethality in mice, but that the macrophage may contribute to SLT II-induced injury.

Production and characterization of three monoclonal antibodies to Candida albicans proteins.

Three monoclonal antibodies, designated A2C7, C2C7, and F19, were produced which recognize proteins from Candida albicans. All are of the immunoglobulin G1 heavy chain and kappa light chain class. A2C7 and C2C7 immunoprecipitated three proteins contained in a partially purified fraction (region A) of a mycelial cytoplasmic extract of C. albicans. The apparent molecular weights of these proteins are 120,000 (120K) to 135K, 44K to 52K, to 38K. Monoclonal antibody F19 was reactive with proteins of 42K, 43K, and 50K in immunoblotting experiments. F19 was also able to form a precipitin band in agarose gel with protein(s) contained in region A. Limited proteolytic digestion of the three proteins immunoprecipitated by A2C7 and C2C7 demonstrated that both monoclonal antibodies recognized the same three Candida proteins and that there exists a significant degree of relatedness in primary structure among the three proteins. Proteins with apparent molecular weights of 120K to 135K, 44K to 52K, and 35K to 38K that were immunoprecipitated by sera from two patients with invasive candidiasis and by the serum from a rabbit immunized against a 48K (44K to 52K) Candida protein were also analyzed by limited proteolysis. Patterns of peptide fragments generated by enzymatic digestion of these proteins showed that the proteins recognized by the monoclonal antibodies are the same proteins recognized by antibodies in the sera of patients during an invasive Candida infection and by antibodies in the serum of the immune rabbit.

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes.

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.

Isolation and characterization of a beta-D-glucuronidase-producing strain of Escherichia coli serotype O157:H7 in the United States.

A phenotypic variant of Escherichia coli serotype O157:H7 (G5101) was isolated from a patient with bloody diarrhea. Strain G5101 does not ferment sorbitol but is beta-D-glucuronidase and urease positive. Serotyping and colony hybridization using a serotype-specific DNA probe confirmed that the isolate was O157:H7. G5101 produces Shiga-like toxins I and II and contains an eae gene that is highly conserved in the O157:H7 serotype. This strain would have been missed by laboratories that screen for the sorbitol-negative, beta-D-glucuronidase-negative phenotype in isolating E. coli O157:H7 from clinical and food specimens.

Molecular subtyping of Salmonella enteritidis phage type 8 strains from the United States.

Salmonella enteritidis is now the most common serotype of the genus Salmonella reported in the United States. Bacteriophage typing has been helpful for subdividing S. enteritidis strains from different sources in the United States. Most S. enteritidis outbreaks reported were egg related, and the majority of them were caused by strains of phage type 8. To determine whether restriction fragment length polymorphism of the rRNA genes (ribotyping) and of the genomic DNAs from two lysogenic phages from S. enteritidis could be used to discriminate between S. enteritidis phage type 8 strains, we conducted Southern hybridization studies on 24 isolates from different outbreaks and six non-outbreak-associated strains using DNA probes for 16S and 23S rRNA genes and S. enteritidis typing phages 1 and 2 from the Ward typing system (L. R. Ward, J. D. H. de Sa, and B. Rowe, Epidemiol. Infect. 99:291-294, 1987). Of seven restriction endonucleases screened with the probe for rRNA genes, AccI provided the best discrimination between strains; six distinct patterns were observed. AccI ribosomal DNA patterns 1 to 6 were detected among 76.7, 3.3, 6.7, 3.3, 3.3, and 6.7% of isolates tested, respectively. Strains of AccI ribosomal DNA pattern 3 could be further subdivided into two additional patterns by using SmaI. Epidemiologically related strains had identical patterns. No discrimination between strains was achieved by probes for phages 1 and 2. No sequences homologous to the phage I probe were detected among phage type 8 strains, and all strains tested with six restriction enzymes had the same hybridization pattern with the phage 2 probe. These findings demonstrate that ribotyping with AccI and SmaI provides an additional means of discriminating between some phage type 8 strains; however, ribotyping and the phage 2 hybridization results from egg-related outbreak strains support previous findings that these strains are closely related.

Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli.

Three monoclonal antibodies, designated MAb 16E6, MAb 13C4, and MAb 19G8, were produced which recognize Shiga-like toxin (SLT) from Escherichia coli. All three monoclonal antibodies neutralized the cytotoxicity of E. coli SLT and were able to immunoprecipitate intact labeled toxin with Staphylococcus aureus protein A. The three antibodies were of the G1 heavy and kappa light chain classes. MAb 16E6 bound to the B subunit of SLT in Western blots and also neutralized the lethality of the toxin for mice and the enterotoxicity of the toxin in ligate rabbit ileal loops. The ability of MAb 16E6 to neutralize the cytotoxicity, lethality, and enterotoxicity of E. coli confirms the hypothesis that all three activities are associated with a single toxin. MAb 16E6 and MAb 13C4 also neutralized the cytotoxicity of purified Shiga toxin from Shigella dysenteriae type 1 and Shiga-like toxic activities in crude cell extracts from Shigella flexneri, Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella typhimurium. Thus, Shiga toxin and the SLTs from E. coli, Shigella flexneri, V. cholerae, V. parahaemolyticus, and Salmonella typhimurium share a common B subunit epitope that is involved in neutralization. MAb 13C4 has been successfully used in a colony blot assay for the detection of bacterial colonies which produce high levels of SLT. Sixty-two different strains of bacteria were tested by both the cytotoxicity and colony blot assays for the presence of SLT. The colony blot assay detected all strains of bacteria which produce greater than or equal to 10(5) 50% cytotoxic doses of SLT per ml of sonic lysate. There were no false-positive results among the 62 samples tested.

A nested PCR followed by magnetic separation of amplified fragments for detection of Escherichia coli Shiga-like toxin genes.

The Shiga-like toxin (SLT) I and II genes in cytotoxic Escherichia coli strains were detected using a polymerase chain reaction (PCR) procedure. Identification and differentiation of SLT I and II was carried out using primers giving PCR-generated DNA fragments of different size for the two cytotoxins. A two-step PCR procedure utilizing three primers in a nested configuration for both SLT I and II was combined with magnetic separation to identify the toxin genes in a rapid, specific and sensitive test system designated DIANA (Detection of Immobilized Amplified Nucleic Acid). The first PCR was carried out using standard methods, and the product generated was used as primer in the second PCR. In this procedure one of the primers from the first PCR was used with biotin label, and the second (inner) primer was 32P-labelled. The double-stranded DNA fragments generated containing the two primers, were biotinylated on one 5' end and 32P-labelled on the other 5' end. These fragments were separated from the solution using streptavidin-coated super-paramagnetic microscopic beads. The test could detect and differentiate between SLT I and II in a positive/negative ratio of more than 20. The assay could detect five SLT-positive E. coli organisms in the 5 microliters test sample. The presence of 100-fold more SLT-negative strains in a sample did not adversely affect the test signal.

Molecular epidemiologic techniques in analysis of epidemic and endemic Shigella dysenteriae type 1 strains.

During 1988 the number of Shigella dysenteriae type 1 infections reported in the United States increased fivefold. To determine if recent isolates from Mexico were related to those that caused epidemics of dysentery worldwide, Southern hybridization analysis was done with Shiga toxin and ribosomal RNA gene probes. Western hemisphere and Eastern Hemisphere strains differed by the size of a single EcoRI fragment carrying the Shiga toxin genes. Three ribosomal DNA (rDNA) patterns were observed, which correlated with the strain's continental origin for 81 of 83 isolates tested. Together the Shiga toxin and rDNA probe results indicated that recent Mexican isolates were chromosomally similar to earlier Central American isolates and distinct from Asian and African strains. This suggests there has been no significant exchange of organisms between continents in recent decades and that the 1988 outbreak in Mexico was caused by strains present in Central America since at least 1962.

Genetic polymorphism of the ipaH multicopy antigen gene in Shigella spps. and enteroinvasive Escherichia coli.

The ipaH loci comprise a multicopy antigen gene family unique to Shigella species and enteroinvasive Escherichia coli (EIEC). DNA probes derived from the Shigella flexneri serotype 5 ipaH7.8 gene were used to compare the molecular arrangement of ipaH alleles in a variety of Shigella and EIEC strains. Multiple copies of ipaH-homologous sequences were detected in all invasion plasmids examined. Oligonucleotide probes covering discrete 24 bp segments of the ipaH7.8 gene and sequences flanking the ipaH4.5 (probe H25) and ipaH2.5 (probe H24) loci were used to define the extent of homology among invasion plasmid copies of ipaH in S. flexneri serotypes 1, 2 and 5 and in S. sonnei. IpaH alleles carried by these invasion plasmids were not structurally equivalent and showed sequence divergence at their amino- and carboxy-terminal ends. The H25 probe was shown to correspond to an IS629 sequence genetically linked to the ipaH alleles, while the H24 probe defined a DNA sequence found only in Shigella invasion plasmids. Chromosomal DNA from invasion plasmid-cured S. flexneri and S. sonnei strains hybridized a core ipaH7.8 gene segment, indicating that portions of the ipaH7.8 structural gene were reiterated and contained within the shigellae chromosomes. Based on the specificity of the ipaH7.8 core probe and the detection of ipaH sequences on the invasion plasmids and chromosomes of Shigella strains, three polymorphic groups within a collection of forty S. dysenteriae 1 isolates received by the United States Centers for Disease Control in 1988 were identified using this probe. These results suggest that ipaH restriction fragment length polymorphisms may be useful in genetic lineage and epidemiologic studies of virulent shigellae.

Characterization of Shigella dysenteriae serotypes 11, 12, and 13.

We conducted serologic and biochemical studies on strains of three provisional Shigella serotypes. Included were 19 strains of serotype 3873-50 from three countries (1 from Mexico, 1 from Netherlands Antilles, and 17 from the United States), 13 strains of serotype 3341-55 from three countries (1 from Canada, 1 from Bulgaria, and 11 from the United States), and 19 strains of serotype 19809-73 from two countries (16 from Israel and 3 from the United States). Reactions of these strains with homologous and heterologous Shigella antisera showed that serotypes 3873-50 and 19809-73 were unique. Strains of serotype 3341-55 shared an identical antigen with Shigella boydii 18; however, they were distinguished from S. boydii 18 biochemically and with absorbed sera. Strains of the three serotypes were biochemically most similar to Shigella dysenteriae. Like other shigellae, strains from each serotype were invasive in the Serény assay. Although biochemically similar to S. dysenteriae, none of the provisional serotypes produced Shiga toxin or the biologically related toxin, Shiga-like toxin II, produced by some strains of Escherichia coli. On the basis of these results and other published findings, we support earlier proposals to add the three provisional serotypes 3873-50, 3341-55, and 19809-73 to the Shigella schema and will include them as S. dysenteriae 11, 12, and 13, respectively.

Isolation of a Citrobacter freundii strain which carries the Escherichia coli O157 antigen.

A biochemically typical strain of Citrobacter freundii which carries the Escherichia coli O157 antigen is described. The significance of differentiating such strains from typical E. coli O157 strains is stressed.

Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera.

Antigenic components in the cytoplasmic extract of Candida albicans were examined after fractionation by concanavalin A-Sepharose and DEAE-Sephacel ion-exchange chromatography. Fractions from the DEAE column were tested by fused rocket immunoelectrophoresis for their reactivity with antibodies in the sera of 20 patients with disseminated candidiasis. Three groups of fractions (regions A, B, and C) from the DEAE column were defined by their reactivity with these sera. Immunoblot analysis with 20 human sera identified 18 antigenic components in regions A, B, and C. Region A contained nine antigens, region B contained four antigens, and region C contained five antigens. Region A contained an antigen with an apparent molecular weight of 48,000 that was recognized by 7 of 10 sera from patients with disseminated candidiasis. Immunoprecipitation experiments with labeled proteins from region A and 51 human sera also demonstrated the presence of a major antigen whose apparent molecular weight is 48,000 to 52,000. The 48- to 52-kilodalton protein is an abundant protein in region A and is the most frequently recognized protein by antibodies in the sera of patients with disseminated candidiasis. Patients with disseminated candidiasis had significantly higher levels of antibody (immunoglobulin G) (P less than 0.001) directed against the 48- to 52-kilodalton protein than did patients with noninvasive forms of candidiasis, patients with other fungal infections, or normal, healthy persons.