The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.Brucellosis is a zoonotic disease that causes abortion, fetal death, and genital infections in animals. In humans, this highly diverse illness initially presents as fever, malaise, and myalgia and may later develop into a chronic illness affecting various organs and tissues. The causative agents of brucellosis are facultative intracellular gram-negative bacteria belonging to the genus Brucella. This genus has been further classified into six species according to host preference and pathogenicity. Genetic studies, however, indicate that the six classical species of Brucella represent a single genomospecies based upon DNA-DNA reassociation (14). In spite of this evidence, the conventional classification scheme has been maintained. Human brucellosis has been attributed to B. abortus, B. melitensis, B. suis, and B. canis and more recently to strains resembling Brucella isolated from marine mammals (13). Laboratory detection of Brucella and species identification is based largely on culture isolation and phenotypic characterization. This process is lengthy and labor-intensive and has been associated with a heightened risk of laboratory-acquired infections (7). To surmount these problems, nucleic acid amplification has been explored for the rapid detection and confirmation of Brucella.A number of nucleic acid sequences have been targeted for the development of Brucella genus-specific PCR assays, including 16S rRNA, the 16S-23S intergenic spacer region, omp2, and bcsp31 (1, 9, 11, 12). The most frequently described PCR target for the diagnosis of human brucellosis is the bcsp31 gene encoding a 31-kDa antigen conserved among Brucella spp. (8,9,15). PCR identification of Brucella strains at the species or biovar level has been more challenging. Recently, Redkar et al.(10) described real-time PCR assays for the detection of B. abortus, B. melitensis, and B. suis biovar 1. These PCR assays target the specific integration of IS711 elements within the genome of the respective Brucella species or biovar. The assays, however, were designed to be tested in separate PCRs. Using similar PCR targets, but in a multiplex format, we have developed a real-time triplex assay that permits rapid confirmation of Brucella spp., B. abortus, and B. melitensis isolates in a single test.The primers and TaqMan probes (Qiagen, Alameda, Calif

We describe here the isolation and identification of a Shiga toxin 1 (Stx1)-producing Enterobacter cloacae strain, M12X01451, from a human clinical specimen. The bacterial isolate was identified as E. cloacae using a polyphasic approach that included phenotypic, genetic, and proteomic analyses. The M12X01451 stx 1 was sequenced, and the holotoxin was found to share only 87% amino acid sequence identity with the nearest Stx1 subtype reference sequence. Sequence analysis of the regions immediately flanking stx 1 displayed similarities with bacteriophage-related sequences, suggesting a prophage origin. The

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A comparative analysis of the Bordetella pertussis, B. bronchiseptica, and B. parapertussis genome assemblies permitted the identification of regions with significant sequence divergence and the design of two new real-time PCR assays, BP283 and BP485, for the specific detection of B. pertussis. The performance characteristics of these two assays were evaluated and compared to those of culture and an existing real-time PCR assay targeting the repetitive element IS481. The testing of 324 nasopharyngeal specimens indicated that, compared to culture, the BP283 assay had a sensitivity and specificity of 100 and 96.8% and the BP485 assay had a sensitivity and specificity of 92.3 and 97.1%. Notably, B. holmesii was isolated from two specimens that were positive by the IS481 assay but negative by the BP283 and BP485 assays. These two assays represent an improvement in specificity over those of PCR assays targeting only IS481 and may be duplexed or used in conjunction with existing PCR assays to improve the molecular detection of B. pertussis.Bordetella pertussis, the causative agent of pertussis, or whooping cough, is reemerging as a significant respiratory pathogen in many parts of the world (2, 14, 26). In the United States, the number of pertussis cases has increased rapidly over the last several years (2). Explanations for this reemergence include waning herd immunity, the antigenic drift of B. pertussis strains away from the antigenic composition of vaccines, better pertussis awareness and reporting, and the increasing use of nucleic acid amplification tests for the laboratory detection of B. pertussis (3,16). Nucleic acid amplification tests, such as the PCR test, offer an improvement in sensitivity over that of culture for the diagnosis of pertussis (23). Unfortunately, there is no standardized PCR test available for B. pertussis detection by clinical microbiology laboratories. Among the previously described PCR assays for pertussis diagnosis, the most frequently utilized target sequence is that of IS481. Over 200 copies of this insertion element are found in the B. pertussis genome (18). This high copy number offers a significant advantage in analytical sensitivity for PCR assays utilizing this target compared to assays with single-copy target sequences (23). However, the enhanced sensitivities of assays targeting IS481 also make them more prone to give false-positive results through laboratory contamination. Moreover, assays detecting the IS481 target sequence also lack specificity, as IS481 elements are also found in B. holmesii and some B. bronchiseptica strains (21,22). Reports of respiratory disease outbreaks attributed to B. pertussis based on errant results of PCR tests targeting IS481 have prompted recommendations for the use of additional PCR targets for the confirmation of B. pertussis detection by PCR (2,5,7,11,19,20).In this study, we describe the identification of two new PCR target sequences through comparative genomics. Real-time PCR assays targeting these sequences were developed in simplex and d...

We compared a set of commercial Salmonella somatic and flagellar serotyping antisera to in-house-prepared antisera from the Microbial Diseases Laboratory, California Department of Public Health, using 327 Salmonella enterica strains belonging to subgroups I, II, IIIa, IIIb, and IV. The sensitivities of Denka Seiken (Tokyo, Japan) somatic and flagellar antisera (using a tube agglutination assay) were 94.0% and 99.2%, respectively, and the specificity was 100% for both sets of sera. Polyvalent O and O1 antiserum sensitivity and specificity were >90%, with the exception of polyvalent O1 antiserum, for which sensitivity was 88.9%. When Denka Seiken flagellar antisera were used in a slide agglutination assay, the sensitivity and accuracy dropped to 88.9% and the specificity fell to 91%. Overall, Denka Seiken commercial antisera performed very well and, together with the comprehensive range of factors available, offer laboratories quality reagents suitable for serotyping strains of salmonellae.Infections caused by nontyphoidal Salmonella strains generally vary from asymptomatic conditions to a self-limiting gastroenteritis characterized by mildly watery stools, mucus, and occasionally occult or visible blood. Bacteremia occurs in ϳ1% to 4% of immunocompetent patients, and 5% to 10% of these persons will develop other extraintestinal complications, including reactive arthritis, central nervous system infections, endocarditis, osteomyelitis, and urinary tract infections (2, 5). These infections have a significant societal impact in terms of both personal suffering and economic consequences. Between 1996 and 1999, the Food-Borne Diseases Active Surveillance Network estimated that nontyphoidal Salmonella strains were responsible for ϳ1.4 million human cases of gastroenteritis per year in the United States, resulting in 168,000 physician office visits, 15,000 hospitalizations, and 400 deaths annually (6). The annual cost of these infections is estimated to range between $0.5 billion and $2.3 billion (1). These figures are based on medical expense and loss of productivity due to salmonellosis and do not include the substantial additional costs incurred by the food industry (due to product recall, litigation, etc). Although most cases of salmonellosis are sporadic, in 2004 over 120 food-borne outbreaks of salmonellosis in the United States were reported to the Centers for Disease Control and Prevention (CDC) (4). Many salmonella infections and subsequent deaths can be prevented if outbreaks are identified rapidly and epidemiologically linked food products are removed from the market. Since the strain of salmonella involved in an outbreak is typically tracked by its serotype and the molecular subtype of that serotype, it is essential that serotyping be performed with precision to ensure that all strains involved in an outbreak are recognized. Most clinical and public health laboratories rely on commercially prepared antisera to serotype salmonellae. However, in our laboratory, where we serotype between 3,600 and 5,400 salmone...