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Asubstantial number of bacillary dysentery cases are due to infection with the Shigella species, and result in endemic situations and frequent epidemics in the developing countries. The organisms produce diarrhea by invasion of the colon. This action is mediated by a plasmid (a subcellular component outside the nucleus that has a genetic function) that carries the determinants that code invasion by each of the Shigella species and the related enteroinvasive Escherichia coli strain (EIEC). Identification of isolates of Shigella in the feces is usually accomplished by culture and biochemical test responses. A new procedure, polymerase chain reaction (PCR), when applied to Shigella identification eliminates the need for biochemical testing. Following growth on solid media, colonies were analyzed by DNA hybridization. DNA sequences were identified that were specific for Shigella species and all tested EIEC isolates. Analyses of these sequences resulted in the development of a test process using PCR for identification of the organisms without a requirement for in vitro cultivation. This process can provide results on the same day the specimens are received. The advantage of a quick test turnaround is obvious in the cases of institutional outbreaks and disasters. There are disadvantages, which have yet to be reconciled, including the fact that the procedure does not distinguish between species of the Shigella nor EIEC from Shigella. (Consumer Summary produced by Reliance Medical Information, Inc.)

The U.S. Food and Drug Administration Escherichia coliIdentification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin–producing E. coli(STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. colistrains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxAand eaegenes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx2a, stx2c, and stx2dgenes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxAand eaeand accurately subtyped the eaealleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stxand eaesubtyping, and ehxAdetection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx2a, stx2c, and stx2dsubtypes.

In July–August 2009, eight patients with bloody diarrhea complicated by hemolytic uremic syndrome (HUS) were admitted to hospitals in Tbilisi, Georgia. We started active surveillance in two regions for bloody diarrhea and post-diarrheal HUS. Of 25 case-patients who developed HUS, including the initial 8 cases, half were ⩾15years old, 67% were female and seven (28%) died. No common exposures were identified. Among 20 HUS case-patients tested, Shiga toxin was detected in the stools of 2 patients (one with elevated serum IgG titers to several Escherichia coliserogroups, including O111 and O104). Among 56 persons with only bloody diarrhea, we isolated Shiga toxin-producing E. coli(STEC) O104:H4 from 2 and Shigellafrom 10; 2 had serologic evidence of E. coliO26 infection. These cases may indicate a previously unrecognized burden of HUS in Georgia. We recommend national reporting of HUS and improving STEC detection capacity.

OBJECTIVE To describe pathogens identified through routine clinical practice and factors associated with identifying Shiga toxin–producing Escherichia coli (STEC) infection in patients with postdiarrheal hemolytic uremic syndrome (D+HUS). DESIGN Population-based active surveillance. SETTING Hospitals in the FoodNet surveillance areas from 2000 through 2010. PARTICIPANTS Children younger than 18 years with D+HUS. MAIN EXPOSURES Testing for STEC and demographic and clinical characteristics. MAIN OUTCOME MEASURES Percentage of patients with evidence of infection with likely HUS-causing agents and associations between exposures and evidence of STEC infection. RESULTS Of 617 patients, 436 (70.7%) had evidence of infection with likely HUS-causing agents: STEC O157 (401 patients), non-O157 STEC (21 patients), O157 and non-O157 STEC (1 patient), Streptococcus pneumoniae (11 patients), and other pathogens (2 patients). Among patients without microbiological evidence of STEC, 76.9% of those tested had serologic evidence of STEC infection. Children more likely to have evidence of STEC infections included those patients tested for STEC less than 4 days after diarrhea onset, 12 months or older (71.6% vs 27.8% if <12 months of age), with infections as part of an outbreak (94.3% vs 67.3%), with bloody diarrhea (77.2% vs 40.4%), with onset during June through September (76.9% vs 60.1%), with a leukocyte count greater than 18 000/μL (to convert to ×109/L, multiply by 0.001) (75.7% vs 65.3%), or with only moderate anemia (hemoglobin >7.0 g/dL [to convert to grams per liter, multiply by 10] or hematocrit greater than 20% [to convert to a proportion of 1, multiply by 0.01]) (75.1% vs 66.3%). However, many of these associations were weaker among children with thorough STEC testing. CONCLUSIONS Early stool collection for E coli O157 culture and Shiga toxin testing of all children with possible bacterial enteric infection will increase detection of STEC strains causing HUS. In the absence of microbiological evidence of STEC, serologic testing should be performed.

ABSTRACTWhen 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 dysenteriaetype 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 stxgenes 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.

AbstractClinical laboratory practices affect patient care and disease surveillance. It is recommended that laboratories routinely use both culture for Escherichia coliO157 and a method that detects Shiga toxins (Stx) to identify all Stx-producing E. coli(STEC) and that labs send broths or isolates to a public health laboratory. In 2007, we surveyed laboratories serving Foodborne Diseases Active Surveillance Network sites that performed on-site enteric disease diagnostic testing to determine their culture and nonculture-based testing practices for STEC identification. Our goals were to measure changes over time in laboratory practices and to compare reported practices with published recommendations. Overall, 89% of laboratories used only culture-based methods, 7% used only Stx enzyme immunoassay (EIA), and 4% used both Stx EIA and culture-based methods. Only 2% of laboratories reported simultaneous culture for O157 STEC and use of Stx EIA. The proportion that ever used Stx EIA increased from 6% in 2003 to 11% in 2007. The proportion that routinely tested all specimens with at least one method was 66% in 2003 versus 71% in 2007. Reference laboratories were less likely than others to test all specimens routinely by one or more of these methods (48% vs. 73%, p= 0.03). As of 2007, most laboratories complied with recommendations for O157 STEC testing by culture but not with recommendations for detection of non-O157 STEC. The proportion of laboratories that culture stools for O157 STEC has changed little since 2003, whereas testing for Stx has increased.

ABSTRACTVibrio, 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 Vibrioinfections in humans. Rapid and accurate identification of Vibriospecies 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 Vibrioisolates 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. parahaemolyticusisolates among the other 119 isolates. Sequence analysis based on rpoBwas used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticussequences. The rpoBsequences for 12 of 15 previously unidentified isolates clustered with other Vibriospecies in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibriospecies. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibriopathogens in clinical samples, and rpoBsequencing provides an additional identification tool for other species in the genus Vibrio.

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:

ABSTRACTTo facilitate the diagnosis of enterotoxigenic Escherichia coli(ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coliisolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.

A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.

ABSTRACTA collection of 20 Escherichia colistrains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtBsequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coliCdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdtsequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.

ABSTRACTSix 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. fluvialispresented the biggest challenge for the API 20E and the GNI+, with probabilities averaging 10%, while V. mimicuswas 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. choleraestrains 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. parahaemolyticusstrains. Thus, extreme care must be taken in the interpretation of answers from these six commercially available systems for the identification of Vibriospecies.

ABSTRACTPCR 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 (stx1and 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. colinegative for stxgenes, 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-hlygenes and 96 and 100%, respectively, for the stx2gene. No stx2genes 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.

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%), Shigellaspp. (4.9%), Entamoeba histolytica(4.9%), Salmonellaspp. (4.5%), Campylobacter jejuni/coli(1.5%), Cryptosporidiumspp. (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 ipaHloci comprise a multicopy antigen gene family unique to Shigellaspecies and enteroinvasive Escherichia coli(EIEC). DNA probes derived from the Shigella flexneriserotype 5 ipaH7.8gene were used to compare the molecular arrangement of ipaHalleles in a variety of Shigellaand EIEC strains. Multiple copies of ipaH-homologous sequences were detected in all invasion plasmids examined. Oligonucleotide probes covering discrete 24 bp segments of the ipaH7.8gene 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 ipaHin S. flexneriserotypes 1, 2 and 5 and in S. sonnei. IpaHalleles 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 IS629sequence genetically linked to the ipaHalleles, while the H24 probe defined a DNA sequence found only in Shigellainvasion plasmids. Chromosomal DNA from invasion plasmid-cured S. flexneriand S. sonneistrains hybridized a core ipaH7.8gene segment, indicating that portions of the ipaH7.8structural gene were reiterated and contained within the shigellae chromosomes. Based on the specificity of the ipaH7.8core probe and the detection of ipaHsequences on the invasion plasmids and chromosomes of Shigellastrains, three polymorphic groups within a collection of forty S. dysenteriae1 isolates received by the United States Centers for Disease Control in 1988 were identified using this probe. These results suggest that ipaHrestriction fragment length polymorphisms may be useful in genetic lineage and epidemiologic studies of virulent shigellae.

The Shiga-like toxin (SLT) I and II genes in cytotoxic Escherichia colistrains 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. coliorganisms in the 5 μl test sample. The presence of 100-fold more SLT-negative strains in a sample did not adversely affect the test signal.

Shigellaspp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneribeing the most frequently isolated species in developing countries.

Subinhibitory concentrations of trimethoprim-sulfamethoxazole increased the total yield of Shiga-like toxin (SLT), produced by Shigella dysenteria 1 and by enterophathogenic and enterohemorrhagic strains of Escherichia coli. Stimulation of SLT synthesis by trimethoprim-sulfamethoxazole was demonstrated by an increase in cytotoxic activity for HeLa cells and the diameter of the zone formed around bacterial colonies probed with monoclonal antibodies to SLT. Thus, supplementation of culture media with trimetroprimsulfamethoxazol will facilitate SLT purification and detection of SLT-producing bacteria.

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.

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 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.