Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes.

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

Automated ribotyping differentiates vibrio parahaemolyticus O3:K6 strains associated with a Texas outbreak from other clinical strains.

Automated ribotyping with a Qualicon Riboprinter was used to determine whether clinical isolates of Vibrio parahaemolyticus O3:K6 recovered during two U.S. outbreaks of oyster-associated gastroenteritis in 1998 were related to each other and to a previously identified highly virulent Asian clone of this serotype. The patterns produced using the restriction enzymes Eco RI and Pst I suggest that the outbreak in the Northeastern United States was caused by a single strain closely related to the Asian clone. In contrast, it appears that multiple strains were involved in the Texas outbreak and that the predominant type was genetically distinct from the Northeastern and Asian clone.

Growth of Escherichia coli O157:H7 during sprouting of alfalfa seeds.

AIMS: Escherichia coli O157:H7 was monitored daily during sprouting of alfalfa seeds inoculated at high (3.92 log10 cfu g(-1)) and low (1.86 log10 cfu g(-1)) levels to assess the extent of pathogen growth during production. METHODS AND RESULTS: Sprouts and rinse water were tested by direct and membrane filter plating on modified sorbitol MacConkey agar and BCM O157:H7(+) agar; the antibody-direct epifluorescent filter technique; and rapid immunoassays. The pathogen reached maximum populations after one and two days of sprouting seeds inoculated at high and low levels, respectively; in either case, populations of 5-6 log10 cfu g(-1) were reached. Detection limits of two rapid immunoassays, Reveal and VIP, without enrichment were determined to be 5-7 log10 cfu ml(-1). CONCLUSION: These results show the ability of E. coli O157:H7 to grow to high levels during sprouting; however, because these levels may be below detection limits, it is necessary to include enrichment when monitoring sprout production for E. coli O157:H7 by the rapid test kits. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that sprouts may harbor high levels of pathogens. The appropriate use of rapid test methods for pathogen monitoring during sprouting is indicated.

Use of spent irrigation water for microbiological analysis of alfalfa sprouts.

Numerous outbreaks of foodborne illness have been linked to the consumption of raw sprouts. Sprout producers have been advised by the Food and Drug Administration to include microbiological testing of spent irrigation water during production as part of an overall strategy to enhance the safety of sprouts. Alfalfa sprouts and irrigation water were analyzed to show the feasibility of using irrigation water for monitoring the microbiological safety of sprouts. Sprouts and water were produced and harvested from both commercial-scale (rotary drum) and consumer-scale (glass jars) equipment. Rapid increases of aerobic mesophiles occurred during the first 24 h of sprouting, with maximum levels achieved after 48 to 72 h. The counts in irrigation water were on average within approximately 1 log of their respective counts in the sprouts. Similar results were obtained for analysis of Escherichia coli O157:H7 in irrigation water and sprouts grown from artificially inoculated seeds. Testing of spent irrigation water indicated the contamination status of alfalfa sprouts grown from seeds associated with outbreaks of Salmonella infection.

Growth of Salmonella during sprouting of alfalfa seeds associated with salmonellosis outbreaks.

Growth of Salmonella was assessed during sprouting of naturally contaminated alfalfa seeds associated with two outbreaks of salmonellosis. Salmonella was determined daily in sprouts and sprout rinse water samples by a three-tube most probable number (MPN) procedure and a commercial enzyme immunoassay (EIA). Growth of Salmonella in the sprouts was reflected in the rinse water, and the MPNs of the two samples were generally in agreement within approximately 1 log. The results from EIA testing of sprouts and water samples were also in agreement. The pathogen was present in the seed at less than 1 MPN/g, and it increased in number to maximum population levels of 102 to 10(3) MPN/g in one seed lot and 10(2) to 10(4) MPN/ g in the other seed lot. Maximum populations of the pathogen were apparent by day 2 of sprouting. These results show the ability of the pathogen to grow to detectable levels during the sprouting process, and they provide support for the recommendation to test the sprout water for the presence of pathogens 48 h after starting seed sprouting. The effectiveness of a 10-min, 20,0000-microg/ml (ppm) calcium hypochlorite treatment of the outbreak-associated seeds was studied. For both seed lots, the hypochlorite treatment caused a reduction, but not elimination, of Salmonella contamination in the finished sprouts. These results confirm the need to test each production batch for the presence of pathogens, even after 20,000 microg/ml (ppm) hypochlorite treatment of seeds, so that contaminated product is not distributed.

Differentiation between types and strains of Clostridium botulinum by riboprinting.

The ability of automated ribotyping to differentiate between major types and individual strains of Clostridium botulinum was tested using the Qualicon Riboprinter Microbial Characterization System. Pure spores of C. botulinum type A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35 degrees C. Plates were rinsed with buffer (2 mM Tris + 20 mM EDTA) to remove vegetative cells that were heated for 10 min at 80 degrees C, treated with a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the enzyme EcoRI. Riboprint patterns were obtained for 30 strains of the four major types of C. botulinum most commonly involved in human foodborne botulism. Proteolytic strains yielded the best and most consistent results. Fifteen ribogroups were identified among the 31 strains tested. Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging to evolutionarily distinct Clostridium lineages. This degree of differentiation between strains of C. botulinum may be useful in hazard analysis and identification, hazard analysis and critical control point monitoring and validation, environmental monitoring, and in inoculation studies.

Ribotype analysis of strain distribution in Listeria monocytogenes.

Changes in the temporal and spatial patterns of strain distribution for the foodborne pathogen Listeria monocytogenes were studied by ribotyping using the Qualicon Riboprinter system. Ribotype patterns were obtained by using the restriction enzymes EcoRI and PvuII for 72 isolates of L. monocytogenes recovered from smoked salmon samples over a period of 3 years. Each pattern was classified both by comparison to a pattern library and by comparison among the 72 isolate patterns. Eleven EcoRI-based ribogroups and 16 PvuII groups were identified. Eight of the 11 EcoRI ribogroups were found in isolates obtained over a period of >12 months, and 75% of the EcoRI ribogroups that were found in more than one food sample were distributed nationally. Within the set of isolates, there were 26 instances where more than one isolate was obtained from a single food sample. In 35% of these instances, the co-isolates produced different ribotype patterns, indicating that multiple strains of L. monocytogenes commonly coexist in the same environment. Overall, these data indicate that the population of L. monocytogenes consists of a number of widely dispersed strains with little geographic or temporal stratification.