Identification of \u3ci\u3eVibrio\u3c/i\u3e Isolates by a Multiplex PCR Assay and \u3ci\u3erpoB\u3c/i\u3e Sequence Determination

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

Non-O157 Shiga Toxin–Producing \u3ci\u3eEscherichia coli\u3c/i\u3e 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% stx1 but not stx2; 22% stx2 but not stx1; 17% both stx1 and stx2; 84% intimin (eae); and 86% enterohemolysin (E-hly). stx2 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

Evaluation of Commercial Latex Reagents for Identification of O157 and H7 Antigens of \u3ci\u3eEscherichia coli\u3c/i\u3e

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

Accuracy of Six Commercially Available Systems for Identification of Members of the Family \u3ci\u3eVibrionaceae\u3c/i\u3e

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

Identification of \u3ci\u3eVibrio\u3c/i\u3e Isolates by a Multiplex PCR Assay and \u3ci\u3erpoB\u3c/i\u3e Sequence Determination

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

Serological Cross-Reactions between \u3ci\u3eEscherichia coli\u3c/i\u3e 0157 and Other Species of the Genus \u3ci\u3eEscherichia\u3c/i\u3e

The antigenic relatedness of Escherichia coli 0157 and four sorbitol-negative species of the genus Escherichia was examined. Isolates of Escherichia hermannii, E. fergusonii, E. vulneris, and E. blattae were tested in the tube agglutination assay by using polyclonal antisera and in the slide agglutination assay by using latex reagents. Only four isolates (17%) of E. hermannii exhibited serological cross-reactivity

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

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

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation

Fil: Brengi, Silvina P. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Sun, Qiangzheng. Chinese Center for Disease Control and Prevention. National Institute for Communicable Diseases Control and Prevention. State Key Laboratory for Infectious Disease Prevention and Control, Changping, Beijing; China.Fil: Bolaños, Hilda. Centro Nacional de Referencia de Bacteriología, Inciensa, Cartago; Costa Rica.Fil: Duarte, Francisco. Centro Nacional de Referencia de Bacteriología, Inciensa, Cartago; Costa Rica.Fil: Jenkins, Claire. Public Health England. Gastrointestinal Bacteria Reference Unit, Londres, Inglaterra.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Shahnaij, Mohammad. International Center for Diarrheal Disease Research. Laboratory Sciences and Services Division. Enteric and Food Microbiology Laboratory Dhaka; Bangladés.Fil: Sowers, Evangeline G. Centers for Disease Control and Prevention. National Institute for Escherichia and Shigella Reference Unit, Atlanta, Georgia, Estados Unidos.Fil: Strockbine, Nancy. Centers for Disease Control and Prevention. National Institute for Escherichia and Shigella Reference Unit, Atlanta, Georgia, Estados Unidos.Fil: Talukder, Kaisar A. International Center for Diarrheal Disease Research. Laboratory Sciences and Services Division. Enteric and Food Microbiology Laboratory Dhaka; Bangladés.Fil: Derado, Gordana. Centers for Disease Control and Prevention. Biostatistics and Information Management Office. Waterborne and Environmental Diseases. Division of Foodborne, Atlanta, Georgia, Estados Unidos.Fil: Viñas, María R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Enterobacterias; Argentina.Fil: Kam, Kai Man. The Chinese University of Hong Kong. Faculty of Medicine. School of Public Health and Primary Care. Stanley Ho Centre for Emerging Infectious Diseases, Hong Kong; China.Fil: Xu, Jianguo. Chinese Center for Disease Control and Prevention. National Institute for Communicable Diseases Control and Prevention. State Key Laboratory for Infectious Disease Prevention and Control, Changping, Beijing; China.Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance