RESUMEN

During September 1999, a multistate outbreak of Salmonella serovar Muenchen infection associated with eating raw alfalfa sprouts was identified in Wisconsin. Despite use of a calcium hypochlorite sanitizing procedure to pretreat seeds before sprouting, at least 157 outbreak-related illnesses were identified in seven states having sprouters who received alfalfa seed from a specific lot. The continued occurrence of sprout-related outbreaks despite presprouting disinfection supports the concern that no available treatment will eliminate pathogens from seeds before sprouting and reinforces the need for additional safeguards to protect the public. A lack of consumer knowledge regarding exposure to sprouts documented in this investigation suggests that more-targeted outreach to high-risk individuals may be needed to reduce their risk.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Six methods were compared for detection of three strains of Escherichia coli O157:H7 in enrichments of inoculated apple juice. Juice was inoculated at levels varying from 0.1 to 100 CFU/ml and centrifuged after overnight storage at 4 degrees C, and pellets were incubated at 37 degrees C in nonselective enrichment broth. At hourly intervals between 5 and 10 h and at 24 h, the enrichments were tested for E. coli O157:H7 by direct fluorescent antibody (DFA), antibody-direct epifluorescent filter technique (Ab-DEFT), direct selective plating on sorbitol MacConkey agar (SMA), immunomagnetic separation coupled to either selective plating (IMS-SMA) or the polymerase chain reaction (IMS-PCR), and flow cytometry (FC). The most consistent detection of 0.1 CFU/ml of the slowest growing strain of the pathogen was provided by the IMS-SMA and IMS-PCR after 8 h of enrichment. The time required for detection at the level of 0.1 CFU/ml for each assay was Ab-DEFT, 11 h; IMS-PCR, 16 h; FC, 24 h; IMS-SMA, 32 h; and SMA, 48 h. Absolute detection limits (without enrichment) were: IMS-PCR, 10(3) CFU/ml; Ab-DEFT and IMS-SMA, 10(4) CFU/ml; SMA, 10(5) CFU/ml; and DFA, 10(6) CFU/ml. Recovery of the pathogen (10 CFU/ml) in apple juice after 28 days of 4 degrees C storage was possible by means of an 8-h enrichment and Ab-DEFT, IMS-PCR, or IMS-SMA.

Asunto(s)

RESUMEN

The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157: H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g-1 faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157: H7 in less than 8 h, but with a sensitivity of approximately 10(3) cfu g-1 faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers.

Asunto(s)

RESUMEN

The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables. Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 microns pore nylon filter, and passage of filtrate through a 0.4 micron pore black polycarbonate filter to collect and concentrate Listeria cells. After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted. A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating. Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 10(7) colony forming units (CFU)/mL. Microbial backgrounds as high as 10(8) CFU/mL did not affect performance of Ab-DEFT. In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h. Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.

Asunto(s)

RESUMEN

Flow cytometry is a potentially valuable analytical method in microbiology providing the ability to analyze rapidly large numbers of individual microorganisms by several parameters. With a flow cytometer with enhanced light scatter sensitivity and a conventionally configured sorting cytometer, a series of comparative studies to determine the ability of the two flow systems and the antibody-direct epifluorescent filter technique (Ab-DEFT) to detect and enumerate Escherichia coli O157:H7 were made. Initial experiments used culture-derived mixtures of non-pathogenic E. coli and serial dilutions of E. coli O157:H7. Subsequent studies involved analysis of enrichment cultures from ground beef inoculated with E. coli O157:H7. Comparison of flow cytometry with microscopy and plate counts produced similar results at higher concentrations in both culture mixtures and beef enrichments. At the lowest concentrations Ab-DEFT was more sensitive, however, the time required for analysis was much less with flow cytometry. With a cytometer with enhanced light scatter sensitivity designed for bacterial analysis, O157:H7 could be distinguished from E. coli strain HB101 on the basis of light scatter. This instrument also provided direct count data for selected populations. In experiments using cell sorting to isolate target organisms, the purity of fluorescent-labeled E. coli O157:H7 sorted from beef enrichment cultures and plated was not affected by the level of background organisms, as is often the case in conventional plating procedures.

Asunto(s)

RESUMEN

The antibody-direct epifluorescent filter technique (Ab-DEFT) was adapted for direct detection of Escherichia coli O157:H7 in bovine feces. The method involved suspension of bovine feces in buffer, centrifugation for 30 s, treatment of the supernatant with trypsin and Triton X-100 at 50 degrees C for 10 min, pre-filtration through 5 and 1.2 microns pore filters, and filtration through a 0.4 micron pore filter. The final filter was stained with fluorescein-labeled polyclonal antibody specific for the O157 antigen and examined by epifluorescence microscopy. The Ab-DEFT was correlated with viable plate counts for enumeration of the pathogen in artificially inoculated bovine feces (r = 0.96). The limit of detection was approximately 10(4)-10(5) CFU/g feces. The procedure provided a clean background for microscopic visualization of cells; however, cell loss and inaccurate quantitation sometimes resulted. E. coli O157:H7 was detected in feces of an inoculated calf for more than 3 weeks post-inoculation. The Ab-DEFT may be useful for rapid screening of cattle for the presence of the E. coli O157:H7 and as an analytical method in ecological studies of the pathogen.

Asunto(s)

RESUMEN

Artificially inoculated Escherichia coli O157:H7 was directly enumerated in ground beef and beef exudate, without enrichment or selection, by the antibody-direct epifluorescent filter technique (Ab-DEFT). The total assay time of the Ab-DEFT was less than 1 h. The beef was homogenized, treated for 15 min with trypsin and Triton X-100, and passed through a 5-microns-pore-size prefilter and then through a 0.2-microns-pore-size black polycarbonate filter. The final filter was stained directly with fluorescein-labeled anti-O157 polyclonal antibody, rinsed, and examined by epifluorescence microscopy. The sensitivity of the Ab-DEFT was compared with that of a standard enrichment culture technique. Both methods reliably determined the presence of the pathogen in beef at 16 CFU/g. The Ab-DEFT was also useful for quantifying the pathogen and monitoring its growth in beef.

Asunto(s)

RESUMEN

Three easy and rapid microtiter plate assays for determining phage sensitivity of lactococci and enterococci have been developed. In the microlysis assay, the degree of sensitivity was measured on the basis of the ability of the bacterial cells to grow in the presence of various concentrations of phage and to effect a color change of an acid-base indicator as a result of acid production. Two assays that specifically measure phage adsorption to bacterial cells have been developed on the basis of the enzyme-linked immunosorbent assay (ELISA) technique. In the direct phage adsorption ELISA, adsorption of phage particles to cells immobilized onto microtiter plate wells was measured using specific anti-phage antibody. In the competitive phage adsorption ELISA, phage adsorption was assayed by allowing phage to compete with specific antibody binding to the bacterial cell surface. All three assays were quantifiable photometrically.

Asunto(s)

RESUMEN

The Streptococcus faecalis plasmid pCF-10 is representative of a class of plasmids that enables its host cells to respond to sex pheromones produced by other S. faecalis cells. The pheromone response has been previously shown to result in increased conjugal plasmid transfer, cell clumping, and multiple cell-surface antigenic changes. To test for other effects of pheromone induction, cells carrying pCF-10 were used as recipients in matings with an isogenic donor strain carrying a derivative of pCF-10, tagged with a transposon to provide an additional selective marker. Pheromone induction of the "male recipients" decreased their recipient ability by a factor of 10-300 in comparison to uninduced cells or plasmid-free recipients. These results indicate that an entry exclusion (surface exclusion) function, similar to that described in studies of plasmids in Gram-negative bacteria, is induced during the S. faecalis pheromone response process. The exclusion operates only against homologous plasmids. Immunological, biochemical, and genetic experiments using monoclonal antibodies reactive with C130, the predominant protein antigen associated with the pheromone response of cells carrying pCF-10, indicate that this antigen is involved in surface exclusion. The data also support the notion that synthesis of C130 involves a posttranslational modification of a precursor of C130 to a final product of higher molecular weight form.

Asunto(s)

RESUMEN

High-molecular-weight surface antigens, obtained by ammonium sulfate precipitation of culture supernatants and identified in Western blots of sodium dodecyl sulfate-polyacrylamide gels, have been correlated with the sex pheromone response of Streptococcus faecalis donor cells. Pheromone-induced cells carrying the conjugative plasmid pCF10 produced both an antigenic component (C130) composed of at least four bands in the range of 130 kilodaltons and a 73-kilodalton antigen (SA 73). The concentration of the C130 antigen in culture supernatants increased with time after exposure of donor cells to pheromone preparations. Gel filtration studies indicated that this antigen exists in the native state as a very large complex that is more than 180,000 daltons in size. The C130 antigen was susceptible to digestion by proteinase K and was not reactive with either concanavalin A or wheat germ agglutinin. The antigenicity of C130 was not destroyed by treatment of blots with trypsin, chymotrypsin, or papain before development with antibody, whereas the antigenicity of SA73 was susceptible to these treatments.

Asunto(s)

Asunto(s)

RESUMEN

Veillonella parvula, which cannot ferment or incorporate most sugars, incorporated radioactivity from [14C]ribose and [14C]fructose into cellular lipopolysaccharide (LPS) in the presence of lactate as an energy source. It was shown that virtually all of the fructose carbon which was assimilated into LPS material appeared in hydrophilic LPS components, and almost none was assimilated into fatty acid LPS components. The assimilation of lactate carbon into LPS in the presence of fructose was shifted from the hydrophilic toward the fatty acid components.

Asunto(s)

RESUMEN

8-Methoxypsoralen has been shown to act as a radiosensitizer of hypoxic bacterial cells with uvrA, recA and uvrB and/or lexA mutations. No effect of the drug on the radiosensitivity of oxic bacteria with these mutations was observed. This drug differs from O2 and electron-affinic radiosensitizers in that its effect is not purely dose-modifying and can exceed the oxygen effect in certain mutants.

Asunto(s)

Asunto(s)