Molecular confirmation of oysters as the vector for hepatitis A in a 2005 multistate outbreak.

Numerous hepatitis A outbreaks were linked to the consumption of raw molluscan shellfish in the United States between 1960 and 1989. However, there had been no major molluscan shellfish-associated hepatitis A outbreaks reported in the United States for more than a decade (1989 to 2004). Beginning in late August 2005, at least 10 clusters of hepatitis A illnesses, totaling 39 persons, occurred in four states among restaurant patrons who ate oysters. Epidemiologic data indicated that oysters were the source of the outbreak. Traceback information showed that the implicated oysters were harvested from specific Gulf Coast areas. A voluntary recall of oysters was initiated in September. Hepatitis A virus (HAV) was detected in multiple 25-g portions in one of two recalled samples, indicating that as many as 1 of every 15 oysters from this source was contaminated. Comparing 315 nucleotides within the HAV VPl-2B region, 100% homology was found among four amplicons recovered from a total of six independent experiments of the implicated oysters, and an identical HAV sequence was detected in sera from all 28 patient serum specimens tested. Ten percent heterogeneity over 315 nucleotides (31 variants) was observed between the outbreak strain (subgenotype 1A) and an HM-175 strain (subgenotype 1B) used in the laboratory where the oysters were processed. To our knowledge, this investigation is the first in the United States to identify an HAV-identical strain in persons with hepatitis A as well as in the food that was implicated as the source of their infections.

Detection and identification of bacteria in a juice matrix with Fourier transform-near infrared spectroscopy and multivariiate analysis.

The use of Fourier transform-near infrared (FT-NIR) spectroscopy combined with multivariate pattern recognition techniques was evaluated to address the need for a fast and senisitive method for the detection of bacterial contamination in liquids. The complex cellular composition of bacteria produces FT-NIR vibrational transitions (overtone and combination bands), forming the basis for identification and subtyping. A database including strains of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus cereus, and Bacillus thuringiensis was built, with special care taken to optimize sample preparation. The bacterial cells were treated with 70% (vol/vol) ethanolto enhance safe handling of pathogenic strains and then concentrated on an aluminum oxide membrane to obtain a thin bacterial film. This simple membrane filtration procedure generated reproducible FT-NIR spectra that allowed for the rapid discrimination among closely related strains. Principal component analysis and soft independent modeling of class analogy of transformed spectra in the region 5,100 to 4,400 cm(-1) were able to discriminate between bacterial species. Spectroscopic analysis of apple juices inoculated with different strains of E. coli at approximately 10(5) CFU/ml showed that FT-NIR spectralfeatures are consistent with bacterial contamination and soft independent modeling of class analogy correctly predicted the identity of the contaminant as strains of E. coli. FT-NIR in conjunction with multivariate techniques can be used for the rapid and accurate evaluation of potential bacterial contamination in liquids with minimal sample manipulation, and hence limited exposure of the laboratory worker to the agents.

Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease.

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.

Rapid detection and identification of bacterial strains by Fourier transform near-infrared spectroscopy.

The use of Fourier transform near-infrared (FT-NIR) spectroscopy and multivariate pattern recognition techniques for the rapid detection and identification of bacterial contamination in liquids was evaluated. The complex biochemical composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that can be used for classification and identification. Bacterial suspensions (Escherichia coli HB101, E. coli ATCC 43888, E. coli 1224, Bacillus amyloliquifaciens, Pseudomonas aeruginosa, Bacillus cereus, and Listeria innocua) were filtered to harvest the cells and eliminate the matrix, which has a strong NIR signal. FT-NIR measurements were done using a diffuse reflection-integrating sphere. Principal component analysis showed tight clustering of the bacterial strains at the information-rich spectral region of 6000-4000 cm(-1). The method reproducibly distinguished between different E. coli isolates and conclusively identified the relationship between a new isolate and one of the test species. This methodology may allow for the rapid assessment of potential bacterial contamination in liquids with minimal sample preparation.

Comparative molecular analysis of erythromycin-resistance determinants in staphylococcal isolates of poultry and human origin.

The ermA, ermB, ermC and msrA/msrB genes were detected in multidrug-resistant Staphylococcus spp. strains by PCR. Among 25 human clinical staphylococcal isolates the ermA, ermB, ermC and the msrA/msrB genes were detected in 88, 72, 4 and 100% of the strains, respectively. Among 24 poultry isolates the ermA, ermB, ermC and the msrA/msrB genes were detected in 100, 16.6, 50 and 12.5% of the strains, respectively. The ermA gene was found exclusively on the chromosome, whereas the ermC gene was found on 2.4-4.2 kb plasmids. Restriction fragment length polymorphism (RFLP) analysis of the ermA gene with Eco RI revealed five patterns (25.0, 21.0, 10.5, 6.2 and 4. 8 kb) for the clinical strains and two (8.0 and 6.2 kb) for the poultry strains. The 6.2 kb RFLP pattern, in both the poultry and human clinical isolates, indicates a common lineage for the ermA gene.

Vibrio cholerae non-O1 serogroup associated with cholera gravis genetically and physiologically resembles O1 E1 Tor cholera strains.

Until recently, only Vibrio cholerae strains of the O1 serogroup have been associated with epidemic cholera. In December 1992, an outbreak of cholera gravis in Vellore, India, was attributed to a new serogroup of V. cholerae recently designated O139. Serogroup O139 cholera has since spread to 13 countries and has reached pandemic proportions. Serogroup O139 cholera evades immunity to O1 cholera and is not detected by the standard O1 antigen test. Understanding the origins of O139 cholera and determining the relatedness of O139 to O1 cholera are necessary to device strategies for detecting, reporting, and controlling this new pandemic. In order to determine the origins of this novel cholera serogroup, O139 was analyzed for virulence genes, for virulence proteins and their regulation, and for its genomic background. We found that O139 and O1 V. cholera strains of the E1 Tor biotype possess highly homologous virulence genes encoding cholera toxin and toxin-coregulated pili and that the regulation of virulence protein expression likewise was indistinguishable between O139 and O1. Pulsed-field gel electrophoresis (PFGE) revealed the restriction digest pattern of O139 strains to be closely related to that of O1 serogroup E1 Tor biotype cholera strains from the Indian subcontinent. However, PFGE showed minor differences among individual O139 cholera isolates, suggesting that O139 V. cholerae is evolving.

Application of pulsed-field gel electrophoresis to the epidemiological characterization of Staphylococcus intermedius implicated in a food-related outbreak.

An outbreak of food intoxication involving over 265 cases in western United States occurred in October 1991. Staphylococcus intermedius was implicated as the aetiologic agent. Representative outbreak isolates (five clinical and ten from foods) produced type A enterotoxin. DNA fragments generated by four restriction endonucleases and analysed by pulsed-field gel electrophoresis (PFGE) provided definitive evidence that all isolates from nine different counties in California and Nevada were derived from a single strain. The PFGE pattern of these outbreak isolates was distinct from those of a heterogeneous collection of seven S. intermedius strains of veterinary origin and five unrelated S. aureus laboratory strains. The data show a significant PFGE pattern heterogeneity not only among members of different Staphylococcus species but also within members of the same species and even the same enterotoxin type. The results indicate that PFGE is a valuable strain-specific discriminator for the epidemiological characterization of S. intermedius. To our knowledge, this represents the first documented foodborne outbreak caused by S. intermedius. These findings suggest that the presence of S. intermedius and other species such as S. hyicus in food should be reason for concern.

International dissemination of epidemic Vibrio cholerae by cargo ship ballast and other nonpotable waters.

In 1991 and 1992, toxigenic Vibrio cholerae O1, serotype Inaba, biotype El Tor, was recovered from nonpotable (ballast, bilge, and sewage) water from five cargo ships docked in ports of the U.S. Gulf of Mexico. Four of these ships had taken on ballast water in cholera-infected countries; the fifth took on ballast in a noninfected country. Isolates examined by pulsed-field gel electrophoresis were indistinguishable from the Latin American epidemic strain, C6707; however, they differed significantly from the endemic Gulf Coast strain (VRL 1984), the sixth-pandemic strain (569-B), and a V. cholerae non-O1 strain isolated from a ship arriving from a foreign port. On the basis of our findings, the Food and Drug Administration recommended that the U.S. Coast Guard issue an advisory to shipping agents and captains requesting that ballast waters be exchanged on the high seas before entry of ships into U.S. ports.

Molecular characterization of Vibrio cholerae O1 strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was performed on 180 isolates of Vibrio cholerae serogroup O1 representing 6 different multilocus enzyme electrophoresis (MEE) types and 27 rRNA restriction fragment length polymorphism types (ribotypes). Isolates were digested with the restriction enzyme NotI and were separated into 63 patterns on the basis of differences in band arrangements. In general, strains which were different by MEE or ribotyping also had different PGFE patterns. PFGE identified individual strains within a single MEE type or ribotype; isolates with one PFGE pattern were less frequently distinguished by ribotyping. All V. cholerae O1 isolates tested from the Latin American epidemic were indistinguishable by their MEE, ribotype, or PFGE patterns. PFGE could further distinguish strains of this same ribotype isolated in Africa, Europe, the South Pacific, or Southeast Asia. Although both MEE and PFGE could identify the strain from the Latin American epidemic, PFGE was more rapid and less labor intensive. PFGE also distinguished nontoxigenic isolates endemic to the U.S. Gulf Coast from unrelated nontoxigenic isolates. In the present study PFGE was more discriminating than other previously described subtyping assays for V. cholerae O1 and appears to be a useful epidemiologic tool.

Molecular genetics of the flgI region and its role in flagellum biosynthesis in Caulobacter crescentus.

The differentiating bacterium Caulobacter crescentus has been studied extensively to understand how a relatively simple life form can govern the timing of expression of genes needed for the production of stage-specific structures. In this study, a clone containing the 5.3-kb flaP region was shown to contain the flgI, cheL, and flbY genes arranged in an operon with transcription proceeding from flgI to flbY. The predicted flgI polypeptide shows remarkable identity (44%) to the flagellar basal body P-ring protein encoded by the flgI gene of Salmonella typhimurium. flgI mutations case a reduction in the levels of flagellin production and the overproduction of the hook proteins. Therefore, the flgI-encoded P-ring protein is required for normal flagellin and hook protein synthesis, suggesting that basal body assembly may play a role in the regulation of flagellar gene expression. The flbY gene probably is a basal body component as well, since flbY mutants have flagellin and hook protein synthesis patterns similar to those exhibited by other basal body mutants. The smaller cheL gene complements a mutant that is unable to respond to chemotactic signals despite possessing a functional flagellum. This is the first example of an operon containing both flagellar and chemotaxis genes in C. crescentus.