Secondary selective enrichment of salmonellae from naturally contaminated specimens by using a selective motility system.

A selective motility medium was used as a secondary selective enrichment medium to examine specimens naturally contaminated with salmonellae. The medium, incubated at 37 degrees C, was inoculated from either selenite brilliant green sulfa enrichment broth or Müller-Kauffman tetrathionate broth, both of which had been incubated at 42 degrees C. The use of the selective motility medium resulted in an increase in the number of positive specimens from 65 and 74% to 80 and 82%, when inoculated at 24 and 48 h, respectively, from tetrathionate broth. Tetrathionate broth, when used singly, was significantly better than selenite brilliant green sulfa broth, which detected 55% of positive specimens at both 24 and 48 h. The use of the selective motility medium of Harper and Shortridge (J. Hyg. 67: 181--186, 1969) for the further examination of specimens culturally negative on primary selective enrichment is advocated.     

Secondary selective enrichment of salmonellae from naturally contaminated specimens by using a selective motility system.

A selective motility medium was used as a secondary selective enrichment medium to examine specimens naturally contaminated with salmonellae. The medium, incubated at 37 degrees C, was inoculated from either selenite brilliant green sulfa enrichment broth or Müller-Kauffman tetrathionate broth, both of which had been incubated at 42 degrees C. The use of the selective motility medium resulted in an increase in the number of positive specimens from 65 and 74% to 80 and 82%, when inoculated at 24 and 48 h, respectively, from tetrathionate broth. Tetrathionate broth, when used singly, was significantly better than selenite brilliant green sulfa broth, which detected 55% of positive specimens at both 24 and 48 h. The use of the selective motility medium of Harper and Shortridge (J. Hyg. 67: 181--186, 1969) for the further examination of specimens culturally negative on primary selective enrichment is advocated.     

Improved Isolation of Salmonellae from Naturally Contaminated Meat Products by Using Rappaport-Vassiliadis Enrichment Broth

A total of 454 specimens of meat products were examined for salmonellae by using five procedures of enrichment. The use of a selective motility medium, inoculated from enrichment in Muller-Kauffmann broth, resulted in an increase in the number of positive specimens. However, simple enrichment in Rappaport-Vassiliadis broth, after preenrichment, was more sensitive and specific for recovering salmonellae than the selective motility medium-Muller-Kauffmann broth method.     

Enrichment procedures for isolating salmonellae from raw meat and poultry.

The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and preenrichment in buffered peptone-water followed by enrichment in tetrathionate broth yielded the maximal recovery of salmonellae from raw meat and poultry samples.     

Development of a two-step enrichment procedure for recovery of Yersinia enterocolitica from food.

Two new enrichment media were formulated for the recovery of Yersinia enterocolitica from foods: (i) yeast extract-rose bengal broth for preenrichment at 4 or 10 degrees C; and (ii) bile-oxalate-sorbose broth, a selective enrichment incubated at 22 degrees C. Comparison of these media in a two-step enrichment procedure against cold enrichment and modified Rappaport broth showed improved and more rapid recovery of human strains of Y. enterocolitica from inoculated foods. The use of bile-oxalate-sorbose broth as a selective enrichment also improved the performance of cold enrichment with phosphate-buffered saline. Determination of the best enrichment system for recovery of Y. enterocolitica from samples of retail pork and fresh pork tongues depended on whether the criterion was the number of positive samples, the variety of different serotypes recovered, or the ability to recover the important human serotype O:3. A single enrichment system with the widest selectivity would include preenrichment at 4 degrees C with either phosphate-buffered saline for 14 days or yeast extract-rose bengal broth for 9 days followed by selective enrichment with bile-oxalate-sorbose broth at 22 degrees C for 5 days.     

Enrichment in Muller-Kauffmann's Broth and Rappaport's Broth from Buffered Peptone Water in the Isolation of Salmonellae from Minced Meat

Four hundred and ninetysix samples of minced meat were examined for the presence of salmonellae. The methods of isolation used in the study were: (a) the standardized direct technique of enrichment in Muller-Kauffmann's broth incubated at 43· for 24 and 48 h, described by Edel & Kampelmacher (1969), (b) secondary enrichment in Rappaport's broth from the former medium after 24 h incubation, and (c) pre-enrichments in buffered peptone water incubated at 37·, followed by enrichments in Muller-Kauffmann's and in Rappaport's broth. These last procedures resulted in the higher rates of isolation of salmonellae with a slight, but not statistically significant, superiority of the enrichment in Rappaport's medium.     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Salmonellae as an Index of Pollution of Surface Waters

Screening enrichments of surface water specimens by means of a polyvalent fluorescent antibody reagent for the salmonellae yielded approximately 60% more positive specimens than was obtained by cultural procedures. It is not known what fraction of the excess of fluorescent antibody-positive over culturally positive specimens represents staining of non-salmonellae or non-arizonae as opposed to the staining of non-cultivatable organisms of these two genera. Cotton gauze and rayon-polypropylene fiber swabs were equally sensitive for collecting salmonellae from the streams examined. Tetrathionate enrichment incubated at 41.5 C appeared to be superior to selenite-cystine for isolation of salmonellae from surface waters. Twenty-eight serotypes of Salmonella and two serotypes of Arizona were identified in the 121 positive specimens. In water rated moderately polluted, 65% of all specimens tested were positive; in minimally polluted waters, 38% were positive; and in unpolluted streams, 44% were positive.     

Prevalence of Escherichia coli O157 in Cattle Feeds in Midwestern Feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Isolation of Shigellae: VIII. Comparison of Xylose Lysine Deoxycholate Agar, Hektoen Enteric Agar, Salmonella-Shigella Agar, and Eosin Methylene Blue Agar with Stool Specimens

Two enrichment broths and four plating media were compared for efficiency of detection of enteric pathogens from 1,597 stool specimens. Of 170 salmonellae isolated from the composite of all methods, direct streaking yielded but 54%, whereas enrichment in gram-negative broth found 87% and Selenite-F broth 97%. By contrast, gram-negative broth produced 100% of the 17 shigellae, Selenite-F broth but 77%, and direct streaking only 59%. Thus, enrichment methods produced almost twice the number of both pathogens as direct streaking. Comparison of the plating media revealed xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar to be equal in their abilities to find both pathogens. Both were moderately better than Salmonella-Shigella agar and markedly superior to eosin methylene blue agar. XLD fround 83% of salmonellae produced by the composite of four media and 90% of the shigellae. Hektoen enteric agar found 80% of both. Salmonella-Shigella agar detected 74 and 68%, respectively, and eosin methylene blue agar only 42 and 63%. The numbers of false positives accruing to each medium, however, showed Hektoen enteric and Salmonella-Shigella agars to produce more than twice as many false-positive plates as XLD. Similarly, Selenite-F broth resulted in many more false-positives for all plating media than did gram-negative broth. Consequently, the index of validity, which equates successful isolation of pathogens with total pickings, favored XLD and gram-negative broth as the media of choice, with direct streaking the poorest method by all counts.     

Evaluation of the Growth of Salmonellae in Rappaport's Broth and in Muller-Kauffmann's Tetrathionate Broth

S ummary . The behaviour of 70 strains of salmonellae belonging to 44 serotypes in Rappaport's broth and in Muller-Kauffmann's tetrathionate broth was examined. With an inoculum of 5–25 cells, 5 strains did not grow in Rappaport's medium, 2 multiplied slowly and 63 grew strongly in 24 h. With an inoculum of 100–500 organisms all but one strain grew readily in 24 h. In Muller–Kauffmann's tetrathionate broth inoculated with pure cultures of salmonellae, growth of many strains was markedly inhibited, in the absence of added faeces, at 37° and 43°. This inhibition was more severe with light inocula at 43°. The addition of 0.05% (w/v) of salmonella-free human faeces to Muller–Kauffmann's tetrathionate broth, did not stimulate growth of salmonellae. In contrast, the addition of 5% (w/v) of human stools to this medium resulted in a heavy growth of the added salmonellae, especially at 43°.     

Influence of Incubation Temperature and Sodium Heptadecyl Sulfate (Tergitol No. 7) on the Isolation of Salmonellae from Pork Sausage

Cultures of 68 samples of fresh pork sausage purchased locally were incubated at 37 and 43 C, with and without Tergitol No. 7 (sodium heptadecyl sulfate) added to the tetrathionate-Brilliant Green enrichment broth. The results indicated an advantage in incubating the tetrathionate broth at 43 C rather than 37 C in attempting to isolate salmonellae from pork sausage. Without Tergitol, more samples were positive at 43 C than at 37 C, but with Tergitol there was no difference. The higher temperature suppressed the competing gram-negative bacteria and permitted Salmonella to grow in relatively pure culture, thus providing an advantage for isolating and identifying the organisms. Tergitol dispersed and emulsified the fat which improved the isolation of Salmonella when the cultures were incubated at 37 C but not at 43 C. Brilliant Green-sulfadiazine agar was superior to bismuth sulfite agar for isolating salmonellae from tetrathionate broth cultures of fresh pork sausage.     

Evaluation of different malachite green brands in the performance of rappaport's medium

Summary Three malachite green dyes, Merck's concentrated for microscopy and bacteriology (old), Merck's malachite green oxalate (oxal), and Difco's malachite green, were used for preparation of 3 batches of Rappaport's medium. These media were tested for growth of 40 Salmonella serotypes and for inhibition of competing organisms. All Rappaport's media behaved similarly and supported excellent growth of 38 serotypes. When Salmonella cultures were diluted in fecal-saline suspensions and inoculated in all Rappaport media the competing organisms in these suspensions were efficiently inhibited. However, the inhibition of competing organisms was less efficient when Rappaport's media were inoculated with preenrichment cultures of 20 samples of minced meat in buffered peptone water. In this respect, Difco's malachite green was clearly inferior to the other two dyes. It is concluded that Merck's malachite green oxalate is as efficient as the old dye of the same brand which is no longer produced, and can replace it in the preparation of Rappaport's medium.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport''s Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport's Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport's Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport''s Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

Limitations of lysine-iron-cystine-neutral red broth in the presumptive identification of Salmonella.

Lysine-iron-cystine-neutral red broth performed satisfactorily in the presumptive identification of Salmonella in preenriched food and animal feed samples enriched in tetrathionate-brilliant green broth. Homologous results from selenite-cystine enrichment broths yielded unacceptably high numbers of false negative reactions.     

A System for Detecting Salmonellae in Meat and Meat Products

Leifson's selenite F broth was more selective for salmonellae when incubated at 43° instead of the traditional 37°. Different selective agar media produced different numbers of colonies from similar inocula of salmonella cells, but Difco brilliant green agar consistently gave the highest recoveries when tested in this way. Combined with 43° selenite broth enrichment it provided a useful system for isolating salmonellae from foods. In a short comparative test this system compared favourably with more classical techniques employing enrichment of each sample at 37° in two different enrichment broths, followed by streaking on two selective agars.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

The Use of Enrichment Serology for Salmonella Detection in Human Foods and Animal Feeds

S ummary . Samples (2208) of food raw materials and products were examined for the presence of salmonellae by use of conventional salmonella detection procedures and the enrichment serology (ES) techniques described by Sperber & Deibel (1969); 348 samples were positive for salmonellae by the conventional procedures. Using the ES technique with a 24 h elective enrichment step, 93–98% of samples positive by the conventional procedures were also positive by the ES technique. Selective enrichment of food samples using tetrathionate broth containing novobiocin, incubated at 41°, led to the best recovery of salmonellae by both the conventional and ES techniques.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens.

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.