Nancy A. Strockbine

Non-O157 Shiga Toxin–Producing Escherichia coli Infections in the United States, 1983–2002

BACKGROUND Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a well-recognized cause of bloody diarrhea and hemolytic-uremic syndrome (HUS). Non-O157 STEC contribute to this burden of illness but have been underrecognized as a result of diagnostic limitations and inadequate surveillance. METHODS Between 1983 and 2002, 43 state public health laboratories submitted 940 human non-O157 STEC isolates from persons with sporadic illnesses to the Centers for Diseases Control and Prevention reference laboratory for confirmation and serotyping. RESULTS The most common serogroups were O26 (22%), O111 (16%), O103 (12%), O121 (8%), O45 (7%), and O145 (5%). Non-O157 STEC infections were most frequent during the summer and among young persons (median age, 12 years; interquartile range, 3-37 years). Virulence gene profiles were as follows: 61% stx(1) but not stx(2); 22% stx(2) but not stx(1); 17% both stx(1) and stx(2); 84% intimin (eae); and 86% enterohemolysin (E-hly). stx(2) was strongly associated with an increased risk of HUS, and eae was strongly associated with an increased risk of bloody diarrhea. STEC O111 accounted for most cases of HUS and was also the cause of 3 of 7 non-O157 STEC outbreaks reported in the United States. CONCLUSIONS Non-O157 STEC can cause severe illness that is comparable to the illness caused by STEC O157. Strains that produce Shiga toxin 2 are much more likely to cause HUS than are those that produce Shiga toxin 1 alone. Improving surveillance will more fully elucidate the incidence and pathological spectrum of these emerging agents. These efforts require increased clinical suspicion, improved clinical laboratory isolation, and continued serotyping of isolates in public health laboratories.

Multicenter Evaluation of a Sequence-Based Protocol for Subtyping Shiga Toxins and Standardizing Stx Nomenclature

ABSTRACT When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli

ABSTRACT PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx1 and stx2) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx1, eae, and E-hly genes and 96 and 100%, respectively, for the stx2 gene. No stx2 genes were detected from 10 stx2f-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.

Identification of Vibrio Isolates by a Multiplex PCR Assay and rpoB Sequence Determination

ABSTRACT Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species—Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus—account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.

Evolutionary Genetics of a New Pathogenic Escherichia Species: Escherichia albertii and Related Shigella boydii Strains

A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at approximately 7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor approximately 28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.

Shigella Isolates From the Global Enteric Multicenter Study Inform Vaccine Development

Shigella case isolates from the Global Enteric Multicenter Study were serotyped to guide vaccine development. A quadrivalent vaccine that includes O antigens from S. sonnei, S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 should provide broad protection.

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.

Diagnostic Microbiologic Methods in the GEMS-1 Case/Control Study

To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged <5 years at 7 sites. Each site employed an identical case/control study design and each utilized a uniform comprehensive set of microbiological assays to identify the likely bacterial, viral and protozoal etiologies. The selected assays effected a balanced consideration of cost, robustness and performance, and all assays were performed at the study sites. Identification of bacterial pathogens employed streamlined conventional bacteriologic biochemical and serological algorithms. Diarrheagenic Escherichia coli were identified by application of a multiplex polymerase chain reaction assay for enterotoxigenic, enteroaggregative, and enteropathogenic E. coli. Rotavirus, adenovirus, Entamoeba histolytica, Giardia enterica, and Cryptosporidium species were detected by commercially available enzyme immunoassays on stool samples. Samples positive for adenovirus were further evaluated for adenovirus serotypes 40 and 41. We developed a novel multiplex assay to detect norovirus (types 1 and 2), astrovirus, and sapovirus. The portfolio of diagnostic assays used in the GEMS study can be broadly applied in developing countries seeking robust cost-effective methods for enteric pathogen detection.

Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2.

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

Accuracy of Six Commercially Available Systems for Identification of Members of the Family Vibrionaceae

ABSTRACT Six commercially available bacterial identification products were tested with Vibrio alginolyticus (12 strains), V. cholerae (30 strains), Photobacterium (Vibrio) damselae (10 strains), V. fluvialis (10 strains), V. furnissii (4 strains), V. hollisae (10 strains), V. metschnikovii (9 strains), V. mimicus (10 strains), V. parahaemolyticus (30 strains), and V. vulnificus (10 strains) to determine the accuracy of each system for identification. The products included API 20E, Crystal E/NF, MicroScan Neg ID2 and Rapid Neg ID3, and Vitek GNI+ and ID-GNB. Each product was tested only with those species that were listed in its database. Overall, the systems correctly identified 63.9, 80.9, 63.1, 73.6, 73.5, and 77.7% of the isolates to species level, respectively. Error rates ranged from 0.8% for the API 20E to 10.4% for the Rapid Neg ID3. The API 20E gave “no identification” for 13.1% of the isolates, while the Neg ID2, GNI+, ID-GNB, and Crystal were unable to identify 1.8, 2.9, 5.0, and 6.9%, respectively. For V. cholerae, specifically, accuracy ranged from 50.0 to 96.7%, with the API 20E having the worst performance and Crystal having the best. V. fluvialis presented the biggest challenge for the API 20E and the GNI+, with probabilities averaging 10%, while V. mimicus was a major problem with the Crystal E/NF, which identified none of the strains correctly. With the Neg ID2, correct answers were often obtained only after a modified inoculation of the panel with a bacterial suspension prepared with 0.85% NaCl. Additional tests required for identification often included growth in the absence of NaCl, which is not readily available in most clinical laboratories. The only product to correctly identify at least 90% of V. cholerae strains was the Crystal E/NF, and only three of the six products, the API 20E and both of the Vitek cards, correctly identified more than 90% of the V. parahaemolyticus strains. Thus, extreme care must be taken in the interpretation of answers from these six commercially available systems for the identification of Vibrio species.