PROCESSING AND PRODUCTS Comparison of Enrichment and Plating Media for Isolation of Yersinia N. A. COX and J. S. BAILEY USDAIARS-Richard B. Russell Agricultural Research Center, P.O. Box 5677, Athens, Georgia 30613 F. DEL CORRAL USDA/ARS-Eastern Regional Research Center, 8105 Douglas Road, Philadelphia, Pennsylvania 19118
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602 (Received for publication January 4, 1989) ABSTRACT Yersinia enterocolitica (Serotypes 0:3 or 0:8), Yersinia frederiksenii, Yersinia kristensenii, or Yersinia intermedia along with 108 cells of each of three extraneous organisms (Escherichia coll, Enterobacter aerogenes, Pseudomonas fragi), all commonly found on market poultry, were inoculated into five enrichment media followed by streaking onto 11 plating media to determine the most-efficacious combinations for future surveys or assessment studies. For Yersinia enterocolitica (0:8), infrequent recoveries were made using yeast extract-rosebengal-bile oxalate sorbose broth and phosphate-buffered saline (4 C) followed by plating onto pectin, DNase-Tween 80 (polyoxyethylene sorbitan monooleate)sorbitol, MacConkey-Tween 80, or cefsulodin-irgasan-novobiocin (CIN) agars. With Y. enterocolitica (0: 3), recoveries were most frequently made using phosphate-buffered saline, sorbitol bile (incubated for 17 days) and yeast extract-rosebengal-bile oxalate sorbose broth followed by plating onto pectin, CIN, bismuth sulfite (Difco Laboratories, Detroit, MI), or modified Rimler-Shotts agar. For Y. frederiksenii, Y. kristensenii, and Y. intermedia, incubation in sorbitol bile for 17 days or in yeast extract-rosebengal-bile oxalate sorbose broth, followed by plating onto CIN, pectin, DNase-Tween/80-sorbitol, cellobiosearginine-lysine agar, or MacConkey-Tween 80 agar yielded the most-frequent recoveries. Overall, the CIN and pectin agars performed best for the recovery of the Yersinia bacterium; the modified selenite broth and the bismuth-sulfite plating agars were unsatisfactory in the present study. (Key words: Yersinia bacterium, methodology, recovery, human pathogen, foodborne) 1990 Poultry Science 69:686-693
INTRODUCTION Yersinia enterocolitica is one of the few human pathogens that can grow m properly refrigerated foods at 0 to 5 C (Stern and Oblinger, 1980). The epidemiological knowledge regarding yersiniosis is incomplete; however, clinical data suggest that the majority of infections occur via the digestive tract (Brenner et al., 1977; Hurvell et al., 1980). The psychrotrophic nature of the yersiniosis organism thus presents a unique problem for the food industry. Presently, there is no epidemiological link between poultry and the Yersinia bacterium. However, new pathogens of concern continue to develop as research in poultry microbiology expands.
Increased interest in the detection of Yersinia spp. has led to the development of s e v e r a l e n r i c h m e n t p roC edures and selective p l a t i n g m e d i a N e w l y deve loped and improved for the recovery of Yersinia, p r o c e dures particularly for a specific food type such as p o u ltry, have not been extensively evaluated. T h e objective of the current study was to compare the relative efficacy of the enrichment and plating media most commonly used for the isolation of Yersinia in the presence of a few, selected, extraneous organisms that would ordinarily be found on market broilers. The best method indicated by the current study was used to conduct a limited survey in order to determine the presence of Yersinia, particularly Y. enterocolitica, on market poultry.
686
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 31, 2015
E. B. SHOTTS
687
YERSINIA BACTERIUM METHODOLOGY 1
TABLE 1. Microorganisms employed in media comparisons
Organism
Identification Number
Yersinia enterocolitica, serotype 0:3 Yersinia enterocolitica, serotype 0:8 Yersinia intermedia Yersinia frederiksenii
CDC CDC CDC CDC
Yersinia kristensenii
CDC 2284-79
A1076 A3042 881-82 353-82
Origin, source of the original isolation in a human population, with known details In Spain, clinical source unknown An outbreak in New York From the urine of a 19-yr-old, female patient in Canada From a 68-yr-old, male patient with pharyngitis in North Carolina From the blood of a 68-yr-old patient in Illinois
MATERIALS AND METHODS
Cultures Cultures of Yersinia enterocolitica, Yersinia frederiksenii. Yersinia kristensenii, and Yersinia intermedia were obtained from Dr. George Morris (the Centers for Disease Control, Atlanta, GA). The non-Yersinia cultures (Enterobacter aerogenes, Escherichia coli, and Pseudomonas fragi) were from the culture collection at the Richard B. Russell Agricultural Research Center, Athens, GA. Table 1 lists the cultures used and their origin. Yersinia cultures were maintained on tryptic soy agar (Difco Laboratories, Detroit, MI) at 25 C, without subculturing; other cultures were maintained on nutrient agar at 25 C. Inoculation Inocula for the media comparisons and enrichment sensitivity were made by growing Yersinia on brain-heart-infusion (BHI) agar (Difco Laboratories) plates for 24 h at 30 C, then aseptically removing the visible growth from the plate and suspending it in 3 mL of sterile physiological saline. The suspension was then diluted with sterile saline to an optical density of .206 at 540 nm on a spectrophotometer (Spectronic 710, Bausch and Lomb, Rochester, NY). Serial dilutions were made of the Yersinia per milliliter, and the actual number of Yersinia per milliliter was determined by spreading aliquots of .1 mL from each dilution on six plates of the BHI agar. Approximately 8 cells of Yersinia were inoculated into 10 mL of the enrichment broth. Each culture was introduced separately to each enrichment. In addition to the Yersinia, E. coli, E. aerogenes, and P. fragi were also added to each
enrichment. These cultures were grown in 5 mL of the BHI broth (Difco Laboratories) for 24 h at 37 C and 30 C for P. fragi. One-tenth mL of the broth culture for each organism was introduced per 10 mL of enrichment, which resulted in an initial level of 1.7 x 108 cfu of E. coli per mL, 7.2 x 108 cfu of E. aerogenes per mL, and 8.7 x 107 cfu of P. fragi per mL. These extraneous organisms were selected because of their prevalence in refrigerated broilers and because of the similarity in colonial morphology to Yersinia on some of the selective media used in the current investigation (Harmon et al, 1983). Enrichment Media The enrichment media used to isolate the yersiniae and Y. enterocolitica compared in the present study were: 1. Phosphate-buffered-saline (PBS) M/15 (pH 7.6) with 1.0% sorbitol and .15% bile salts (Mehlman et al., 1978) incubated at 4 C for 11 and 17 days (SBB, 11 days; SBB, 17 days). 2. Modified selenite broth (MSEL) (Lee et al., 1980; Stern and Oblinger, 1980). 3. PBS (Mehlman et al, 1978). Following incubation in the PBS, .5-mL portions of the enrichment culture were treated for both incubation temperatures (4 and 25 C) with 4.5 mL of .25% KOH (potassium hydroxide) in 5% NaCl for 2 min (Aulisio et al., 1980) (PBS, 4 C; PBS, 25 C). 4. Yeast extract rosebengal (YER)-bile oxalate sorbose (BOS) broth (Schiemann, 1982). The YER was incubated at 10 C for 3 days and was transferred at a ratio of .1:10 mL and 1.0:10 mL of BOS, which was incubated for 5 days at 22 C.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 31, 2015
'isolated previously from broiler carcasses at the USDA/ARS-Russell Research Center, Athens, GA: Enterobacter aerogenes, Escherichia coli, and Pseudomonas fragi.
688
COX ET AL.
Differential Selective Plating Media
Identification Procedure Following incubation under the appropriate conditions, one loopful (a 3-mm loop) of each liquid-enrichment medium was streaked for isolation onto each plating medium. After incubation for 48 h at 25 C, two typical Yersinia colonies (maximum) per plate were selected. The isolates were screened by inoculating a triple sugar iron (TSI) agar (Difco Laboratories) and a lysine iron agar (LIA) slant (Difco Laboratories). Cultures exhibiting an acid slant and acid butt on TSI with an acid (yellow) butt on LIA were further tested for motility in a sulfite-indole-motility (Difco Laboratories) tube. The cultures that were motile at 25 C, but not at 37 C, and were oxidase negative were assumed to be Yersinia spp. Further confirmation was obtained with the API-20E system. The entire experiment was replicated three times. Statistical Analysis For each observation of a treatment combination of enrichment and plating media, a positive detection of Yersinia was assigned a numerical value of one; no detection was assigned a value of zero. The resulting data were subjected to ANOVA (Cox, 1977), testing the main effects of the trial (replication), enrichment media, and
RESULTS AND DISCUSSION
The results concerning the performance of enrichment-plating media combinations for the recovery of Y. enterocolitica serotype 0:8 are shown in Table 2. Among the various liquid enrichment media tested in the present study, MSEL performed very poorly, as did PBSKOH, at 25 C with all of the plating media. Those two enrichment media combined were only able to recover serotype 0:8 less than 20% of the time. Perhaps the poor performance of PBS-KOH at 25 C was related to the particular KOH treatment. A 2-min exposure time was probably too harsh (Doyle and Hugdahl, 1983), resulting in the destruction of the Yersinia. At 4 C, the effect of the PBSKOH treatment was not as great: with the longer incubation time, the number of organisms had multiplied to a level such that some cells survived the treatment. Significantly better (P
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602 (Received for publication January 4, 1989) ABSTRACT Yersinia enterocolitica (Serotypes 0:3 or 0:8), Yersinia frederiksenii, Yersinia kristensenii, or Yersinia intermedia along with 108 cells of each of three extraneous organisms (Escherichia coll, Enterobacter aerogenes, Pseudomonas fragi), all commonly found on market poultry, were inoculated into five enrichment media followed by streaking onto 11 plating media to determine the most-efficacious combinations for future surveys or assessment studies. For Yersinia enterocolitica (0:8), infrequent recoveries were made using yeast extract-rosebengal-bile oxalate sorbose broth and phosphate-buffered saline (4 C) followed by plating onto pectin, DNase-Tween 80 (polyoxyethylene sorbitan monooleate)sorbitol, MacConkey-Tween 80, or cefsulodin-irgasan-novobiocin (CIN) agars. With Y. enterocolitica (0: 3), recoveries were most frequently made using phosphate-buffered saline, sorbitol bile (incubated for 17 days) and yeast extract-rosebengal-bile oxalate sorbose broth followed by plating onto pectin, CIN, bismuth sulfite (Difco Laboratories, Detroit, MI), or modified Rimler-Shotts agar. For Y. frederiksenii, Y. kristensenii, and Y. intermedia, incubation in sorbitol bile for 17 days or in yeast extract-rosebengal-bile oxalate sorbose broth, followed by plating onto CIN, pectin, DNase-Tween/80-sorbitol, cellobiosearginine-lysine agar, or MacConkey-Tween 80 agar yielded the most-frequent recoveries. Overall, the CIN and pectin agars performed best for the recovery of the Yersinia bacterium; the modified selenite broth and the bismuth-sulfite plating agars were unsatisfactory in the present study. (Key words: Yersinia bacterium, methodology, recovery, human pathogen, foodborne) 1990 Poultry Science 69:686-693
INTRODUCTION Yersinia enterocolitica is one of the few human pathogens that can grow m properly refrigerated foods at 0 to 5 C (Stern and Oblinger, 1980). The epidemiological knowledge regarding yersiniosis is incomplete; however, clinical data suggest that the majority of infections occur via the digestive tract (Brenner et al., 1977; Hurvell et al., 1980). The psychrotrophic nature of the yersiniosis organism thus presents a unique problem for the food industry. Presently, there is no epidemiological link between poultry and the Yersinia bacterium. However, new pathogens of concern continue to develop as research in poultry microbiology expands.
Increased interest in the detection of Yersinia spp. has led to the development of s e v e r a l e n r i c h m e n t p roC edures and selective p l a t i n g m e d i a N e w l y deve loped and improved for the recovery of Yersinia, p r o c e dures particularly for a specific food type such as p o u ltry, have not been extensively evaluated. T h e objective of the current study was to compare the relative efficacy of the enrichment and plating media most commonly used for the isolation of Yersinia in the presence of a few, selected, extraneous organisms that would ordinarily be found on market broilers. The best method indicated by the current study was used to conduct a limited survey in order to determine the presence of Yersinia, particularly Y. enterocolitica, on market poultry.
686
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 31, 2015
E. B. SHOTTS
687
YERSINIA BACTERIUM METHODOLOGY 1
TABLE 1. Microorganisms employed in media comparisons
Organism
Identification Number
Yersinia enterocolitica, serotype 0:3 Yersinia enterocolitica, serotype 0:8 Yersinia intermedia Yersinia frederiksenii
CDC CDC CDC CDC
Yersinia kristensenii
CDC 2284-79
A1076 A3042 881-82 353-82
Origin, source of the original isolation in a human population, with known details In Spain, clinical source unknown An outbreak in New York From the urine of a 19-yr-old, female patient in Canada From a 68-yr-old, male patient with pharyngitis in North Carolina From the blood of a 68-yr-old patient in Illinois
MATERIALS AND METHODS
Cultures Cultures of Yersinia enterocolitica, Yersinia frederiksenii. Yersinia kristensenii, and Yersinia intermedia were obtained from Dr. George Morris (the Centers for Disease Control, Atlanta, GA). The non-Yersinia cultures (Enterobacter aerogenes, Escherichia coli, and Pseudomonas fragi) were from the culture collection at the Richard B. Russell Agricultural Research Center, Athens, GA. Table 1 lists the cultures used and their origin. Yersinia cultures were maintained on tryptic soy agar (Difco Laboratories, Detroit, MI) at 25 C, without subculturing; other cultures were maintained on nutrient agar at 25 C. Inoculation Inocula for the media comparisons and enrichment sensitivity were made by growing Yersinia on brain-heart-infusion (BHI) agar (Difco Laboratories) plates for 24 h at 30 C, then aseptically removing the visible growth from the plate and suspending it in 3 mL of sterile physiological saline. The suspension was then diluted with sterile saline to an optical density of .206 at 540 nm on a spectrophotometer (Spectronic 710, Bausch and Lomb, Rochester, NY). Serial dilutions were made of the Yersinia per milliliter, and the actual number of Yersinia per milliliter was determined by spreading aliquots of .1 mL from each dilution on six plates of the BHI agar. Approximately 8 cells of Yersinia were inoculated into 10 mL of the enrichment broth. Each culture was introduced separately to each enrichment. In addition to the Yersinia, E. coli, E. aerogenes, and P. fragi were also added to each
enrichment. These cultures were grown in 5 mL of the BHI broth (Difco Laboratories) for 24 h at 37 C and 30 C for P. fragi. One-tenth mL of the broth culture for each organism was introduced per 10 mL of enrichment, which resulted in an initial level of 1.7 x 108 cfu of E. coli per mL, 7.2 x 108 cfu of E. aerogenes per mL, and 8.7 x 107 cfu of P. fragi per mL. These extraneous organisms were selected because of their prevalence in refrigerated broilers and because of the similarity in colonial morphology to Yersinia on some of the selective media used in the current investigation (Harmon et al, 1983). Enrichment Media The enrichment media used to isolate the yersiniae and Y. enterocolitica compared in the present study were: 1. Phosphate-buffered-saline (PBS) M/15 (pH 7.6) with 1.0% sorbitol and .15% bile salts (Mehlman et al., 1978) incubated at 4 C for 11 and 17 days (SBB, 11 days; SBB, 17 days). 2. Modified selenite broth (MSEL) (Lee et al., 1980; Stern and Oblinger, 1980). 3. PBS (Mehlman et al, 1978). Following incubation in the PBS, .5-mL portions of the enrichment culture were treated for both incubation temperatures (4 and 25 C) with 4.5 mL of .25% KOH (potassium hydroxide) in 5% NaCl for 2 min (Aulisio et al., 1980) (PBS, 4 C; PBS, 25 C). 4. Yeast extract rosebengal (YER)-bile oxalate sorbose (BOS) broth (Schiemann, 1982). The YER was incubated at 10 C for 3 days and was transferred at a ratio of .1:10 mL and 1.0:10 mL of BOS, which was incubated for 5 days at 22 C.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 31, 2015
'isolated previously from broiler carcasses at the USDA/ARS-Russell Research Center, Athens, GA: Enterobacter aerogenes, Escherichia coli, and Pseudomonas fragi.
688
COX ET AL.
Differential Selective Plating Media
Identification Procedure Following incubation under the appropriate conditions, one loopful (a 3-mm loop) of each liquid-enrichment medium was streaked for isolation onto each plating medium. After incubation for 48 h at 25 C, two typical Yersinia colonies (maximum) per plate were selected. The isolates were screened by inoculating a triple sugar iron (TSI) agar (Difco Laboratories) and a lysine iron agar (LIA) slant (Difco Laboratories). Cultures exhibiting an acid slant and acid butt on TSI with an acid (yellow) butt on LIA were further tested for motility in a sulfite-indole-motility (Difco Laboratories) tube. The cultures that were motile at 25 C, but not at 37 C, and were oxidase negative were assumed to be Yersinia spp. Further confirmation was obtained with the API-20E system. The entire experiment was replicated three times. Statistical Analysis For each observation of a treatment combination of enrichment and plating media, a positive detection of Yersinia was assigned a numerical value of one; no detection was assigned a value of zero. The resulting data were subjected to ANOVA (Cox, 1977), testing the main effects of the trial (replication), enrichment media, and
RESULTS AND DISCUSSION
The results concerning the performance of enrichment-plating media combinations for the recovery of Y. enterocolitica serotype 0:8 are shown in Table 2. Among the various liquid enrichment media tested in the present study, MSEL performed very poorly, as did PBSKOH, at 25 C with all of the plating media. Those two enrichment media combined were only able to recover serotype 0:8 less than 20% of the time. Perhaps the poor performance of PBS-KOH at 25 C was related to the particular KOH treatment. A 2-min exposure time was probably too harsh (Doyle and Hugdahl, 1983), resulting in the destruction of the Yersinia. At 4 C, the effect of the PBSKOH treatment was not as great: with the longer incubation time, the number of organisms had multiplied to a level such that some cells survived the treatment. Significantly better (P
