Enterobacteriaceae in ground meats1
Can. J. Microbiol. 1978.24:1574-1582. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/14/15. For personal use only.
AND
MICHAELE. S T I L E S ~ Depot.tt,retrt c prewere streaked onto MacConkey agar (Difco) (MA) and the three sumptive coliform count (LST) > confirmed colselective metlia (VRBA. VKBG, and EMB), incubated at 37°C iform count (BGB) > VRBA-bile-precipitating for 24 h, and :in isolated colony picked from MA and stl.eaked onto nutrient agar (NA). The culture on NA was screened for count > completed coliform count (EMB) > preG~ini-negative.glucose-positive, and oxitlase-negative (Steel sumptive E. coli count (EC). 1961) reactions. Appropriate isolates on NA were i n o c ~ ~ l a t e d Based on the scatterdiagl-ams of the data, and the into 2-1111 ste~.ileinoculuni broth (after BBL Minitek: tryptone 7 . j g . L-tr-yptophcin0.25g, HEPES buffer 0.59g. in l000ml dis- lowel- sank order (Kendall's) correlations, Peartilled water) and shaken vigorously. The same isol;~tewas in- son's lineal- corl-elation coefficients were used to oculated into motility ;ig;lr. (Difco) and a triple sugar- iron (TSI) describe the I-elationship between the counts. The agar slant. A 0.05-ml ;tliquot of suspended culture was inocu- data are shown in Table 3. All correlations were lated into I ~ L I ~ Ytryptose I broth (LST), phenol red lactose broth highly significant ( p = 0.001). For ease of presenta(lactose), duplic;ite t ~ r b e sof EC medium for incubation at 44.5 tion. the I-esultsmay be considered as three groups. and 45.5 + 0.02"C, and onto BBL Minitek discs (BBL. Division of Becton-Dickinson ;inti C o . . Mississa~~ga, Ont., Canada) in a ( t i ) Direct Pl~titlgM e ~ l i ~ i 12-well plastic Minitek plate, including: arabinose, dulcitol, inThe col-relations between counts ranged from ositol. raffinose. I-hamnose. ONPG. lysine, ornithine, phenyl;,lanine, H?S-indole, citfiite, and mnlon;~te.In addition, MR-VI' 0.77 to0.97. Lowest correlations were between the (Difco) and urea (Edwards and Ewing 1972) broths were inocu- bile-precipitating count on VRBA and counts on lated. All tests were incubated at 3 P C for 24 h. except MR-VP bluth at 30°C for 48 h fol. the Voges-Proskn~~elreaction (Barritt other media. The total VRBA count and the bileprecipitating (Enterobacteriaceae) and total VRBG 1936) and 5 days for the methyl red test. TSI was read at 24 h and checked at 48 h for delayed H2S production. Urease production counts had gl-eater than 80% of variation in the was read at 24 and 48h. 4 mid 7 days. Tests inclutled the counts that could be accounted for by the linear biochemical tests used in 'Ente~.otube' s o isol:ites co~iltl be relationship between the counts. identified ~rsing the Encise I I ch;ir:~cte~.istics(Enterobactericiceae Numeric;tI Cotling and Identification System for En( b ) Col$or.rn at~tlE. coli MPN Coirtlts terotube. Roche Diagnostic, 340, Kingsland St., Nutley. N J , Correlations between counts for the different U.S.A.). lMViC tests were included because IMViC results are tests r-anged from 0.44 to 0.92. The propol-tion of ~ ~ s to e ddefine E. c.oli in meats (HPB 1974).
Results Non-fl-ozen ground beef samples, including thl-ee different qualities (based on fat content and method of preparation) and frozen pot-k sausage samples were analyzed. The range of presumptive E. coli counts for each pl.oduct type is shown in Table 1. Enterobacteriaceae and coliform bacteria were also enumerated on these samples. An analysis of variance on the log,,-transformed data on 188 s a m ~ l e was s carried out. A statisticallv significant diffeience between product type (p 0.002) and between media (p = 0.001) was observed. with no interaction effects. Duncan's multiple-range test based on Steel and Tol-I-ie's publication (1960) was carried out at the 5% confidence level. The results are summarized in Table 2. The presumptive E. coli count (EC medium) was significantly less than all othercounts, but the completed coliform (EMB) and coliform (bile-PI-ecipitating)VRBA counts were not statistically different. The presumptive coliform count on LST, bile-precipitating count on VRBG (Enterobacteriaceae), and total count on VRBA were not significantly different, but they were
-
total variation of the confirmed coliform count (BGB) that could be accounted for by linear relationship with the presumptive coliform test (LST) was 85%, similarly the I-elationshipwith the EMB count was only 52%, and 64% with BGB. Correlation coefficients between the other MPN and EC counts were I-elatively low, resulting in a poor relationship with the presumptive E. coli counts. ( c )Direct Pltiting trtlci MPN Techtliclires The strongest relationship existed between LST and BGB and the direct plating counts, but only 40 to 50% of variation could be accounted for by the linear relationship between the counts. The relationship between the direct plating countsand the EMB and EC counts was much less. Since variations in the presumptive E. coli count could not be reliably accounted for by the linear relationship with other Enterobacteriaceae counts included in this study, the possibility of using a direct EC test was studied. For 110 samples, dilutions were inoculated directly and indirectly into EC and incubated at 45.S°C. The log,,-transformed data were analyzed by the t-test, which indicated a highly significant difference ( p < 0.001) betweer
N G AN11 STILES
TABLEI. Counts of presuniptive E. coli (determined in EC medium at 45.5-C) on dilferent ground meats
Can. J. Microbiol. 1978.24:1574-1582. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/14/15. For personal use only.
Count of presumptive E. coli per gram
100 to < 500
< 100
Product
500 to < 1000
1000 or greater
Mean count per gram
Nuniber (percentage) of samples 70 49 12
Ground beef Regular Ground becf Low fat Groi~ndbeef froni store trim Pork sausage Frozen All products
TABLE2. Results of Duncan's multiple-range test for significant differences between counts from ground meats on selective media for Enterobacteriaceae, coliforni bacteria, and E. coli Log,, mean of counts, in increasing order EC
EMB
VRBA-P
BGB
LST
VRBG-P
VRBA-T
VRBG-T
AT 5% level of confidence1 EC
EMB
VRBA-I'
BGB
LST
VRBG-P
VRBA-T
VRBG-T
'The counts for (he media under the common linc arc n o t signifcantly ditTerent. N o r r : V R B A , violet red bile agar: V R B G , Mosscl's Enlerobacteriaceae mcdium ( V R B A , : ; ;1 glucose); -T, total ng at 24 11; count o f bile-prccipilnling and non-bile-prccipitaling colonics at 24 11; -P, c o u n l o f b l l e - p r c c ~ p ~ t n t ~colonics LST, lauryl trvptose b r o l h ; BGB, brilliant green bile 2,7, b r o o i ; E M B , Levine E M B agar; EC, E C b r o l h at 45.5'C.
TABLE3. Pearson's correlation coefficients between counts from ground meats on selective media for Enterobacteriaceae, coliform bacteria, and E. coli VRBA-T
VRBA-P
VRBG-T
VRBG-P
-
VRBA-P VRBG-T VRBG-P LST BGB EMB EC
0.82 0.93 0.90 0.71 0.67 0.51 0.27
-
0.77 0.80 0.70 0.70 0.63 0.46
0.97 0.73 0.69 0.48 0.31
0.72 0.72 0.51 0.36
LST
BGB
EMB
0.80 0.52
0.68
-
0.92 0.72 0.44
N o r r : V R B A , violct rcd bilc agar; V R B G , Mossel's Enterob;~cterinceoe r n e d i ~ l m( V R B A -!- IZ g!iucosc): -T, tot;~lcount o f bilc-precipitating and non-bile-precipitating colonies a t 24 11; -P, count o f bile-prec~pitaling colonies at 24 h : LST, Iauryl tryptosc broth; BGB, brilliant grcen bile 2Z, broth; E M B . Levine E M B agar; EC, E C b r o t h a t 45.5"C.
the methods. Pearson's correlation coefficient for the data was 0.57. The mean for the direct EC test was 3.7 x IOt/g compared with 1.4 x 10Vg for the indirect test. Although the means differed by a factor of 4, the cross-tabulation of the data indicated that differences between these methods could be 10- to 100-fold, which accounted for the poor correlation between the methods. Cross-tabulation of indirect EC counts with the bile-precipitating VRBA counts is shown in Table
4. Of 106 samples with presumptive E. coli > 100, 1000/g and 14 (13.2%) were > lO000/g. Of 30 samples with presumptive E. coli > 1000/g, 12 (40.0%) had bile-precipitating VRBA counts < 1000/g. Results were similar whether pork sausage samples were included in the cross-tabulation or not; o r if cross-tabulations were between indirect EC and total VRBA counts, or between direct EC and bile-precipitating VRBA counts. The
CAN. J. MICROBIOId. VOI..
24. 1978
Can. J. Microbiol. 1978.24:1574-1582. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/14/15. For personal use only.
TABLE 4. Cross-tabulation of indirect EC counts at 45.5"C with bile-precipitating counts on VRBA Indirect EC count (Presumptive E. coli per gram)
Bile-precipitating VRBA count per gram
< 100
i 100
83 (45.9)
100< 500
29 (32.2)
500< 1000
7 (43.8)
> 1000
5 (16.7)
Total
124
100 to
Can. J. Microbiol. 1978.24:1574-1582. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/14/15. For personal use only.
AND
MICHAELE. S T I L E S ~ Depot.tt,retrt c prewere streaked onto MacConkey agar (Difco) (MA) and the three sumptive coliform count (LST) > confirmed colselective metlia (VRBA. VKBG, and EMB), incubated at 37°C iform count (BGB) > VRBA-bile-precipitating for 24 h, and :in isolated colony picked from MA and stl.eaked onto nutrient agar (NA). The culture on NA was screened for count > completed coliform count (EMB) > preG~ini-negative.glucose-positive, and oxitlase-negative (Steel sumptive E. coli count (EC). 1961) reactions. Appropriate isolates on NA were i n o c ~ ~ l a t e d Based on the scatterdiagl-ams of the data, and the into 2-1111 ste~.ileinoculuni broth (after BBL Minitek: tryptone 7 . j g . L-tr-yptophcin0.25g, HEPES buffer 0.59g. in l000ml dis- lowel- sank order (Kendall's) correlations, Peartilled water) and shaken vigorously. The same isol;~tewas in- son's lineal- corl-elation coefficients were used to oculated into motility ;ig;lr. (Difco) and a triple sugar- iron (TSI) describe the I-elationship between the counts. The agar slant. A 0.05-ml ;tliquot of suspended culture was inocu- data are shown in Table 3. All correlations were lated into I ~ L I ~ Ytryptose I broth (LST), phenol red lactose broth highly significant ( p = 0.001). For ease of presenta(lactose), duplic;ite t ~ r b e sof EC medium for incubation at 44.5 tion. the I-esultsmay be considered as three groups. and 45.5 + 0.02"C, and onto BBL Minitek discs (BBL. Division of Becton-Dickinson ;inti C o . . Mississa~~ga, Ont., Canada) in a ( t i ) Direct Pl~titlgM e ~ l i ~ i 12-well plastic Minitek plate, including: arabinose, dulcitol, inThe col-relations between counts ranged from ositol. raffinose. I-hamnose. ONPG. lysine, ornithine, phenyl;,lanine, H?S-indole, citfiite, and mnlon;~te.In addition, MR-VI' 0.77 to0.97. Lowest correlations were between the (Difco) and urea (Edwards and Ewing 1972) broths were inocu- bile-precipitating count on VRBA and counts on lated. All tests were incubated at 3 P C for 24 h. except MR-VP bluth at 30°C for 48 h fol. the Voges-Proskn~~elreaction (Barritt other media. The total VRBA count and the bileprecipitating (Enterobacteriaceae) and total VRBG 1936) and 5 days for the methyl red test. TSI was read at 24 h and checked at 48 h for delayed H2S production. Urease production counts had gl-eater than 80% of variation in the was read at 24 and 48h. 4 mid 7 days. Tests inclutled the counts that could be accounted for by the linear biochemical tests used in 'Ente~.otube' s o isol:ites co~iltl be relationship between the counts. identified ~rsing the Encise I I ch;ir:~cte~.istics(Enterobactericiceae Numeric;tI Cotling and Identification System for En( b ) Col$or.rn at~tlE. coli MPN Coirtlts terotube. Roche Diagnostic, 340, Kingsland St., Nutley. N J , Correlations between counts for the different U.S.A.). lMViC tests were included because IMViC results are tests r-anged from 0.44 to 0.92. The propol-tion of ~ ~ s to e ddefine E. c.oli in meats (HPB 1974).
Results Non-fl-ozen ground beef samples, including thl-ee different qualities (based on fat content and method of preparation) and frozen pot-k sausage samples were analyzed. The range of presumptive E. coli counts for each pl.oduct type is shown in Table 1. Enterobacteriaceae and coliform bacteria were also enumerated on these samples. An analysis of variance on the log,,-transformed data on 188 s a m ~ l e was s carried out. A statisticallv significant diffeience between product type (p 0.002) and between media (p = 0.001) was observed. with no interaction effects. Duncan's multiple-range test based on Steel and Tol-I-ie's publication (1960) was carried out at the 5% confidence level. The results are summarized in Table 2. The presumptive E. coli count (EC medium) was significantly less than all othercounts, but the completed coliform (EMB) and coliform (bile-PI-ecipitating)VRBA counts were not statistically different. The presumptive coliform count on LST, bile-precipitating count on VRBG (Enterobacteriaceae), and total count on VRBA were not significantly different, but they were
-
total variation of the confirmed coliform count (BGB) that could be accounted for by linear relationship with the presumptive coliform test (LST) was 85%, similarly the I-elationshipwith the EMB count was only 52%, and 64% with BGB. Correlation coefficients between the other MPN and EC counts were I-elatively low, resulting in a poor relationship with the presumptive E. coli counts. ( c )Direct Pltiting trtlci MPN Techtliclires The strongest relationship existed between LST and BGB and the direct plating counts, but only 40 to 50% of variation could be accounted for by the linear relationship between the counts. The relationship between the direct plating countsand the EMB and EC counts was much less. Since variations in the presumptive E. coli count could not be reliably accounted for by the linear relationship with other Enterobacteriaceae counts included in this study, the possibility of using a direct EC test was studied. For 110 samples, dilutions were inoculated directly and indirectly into EC and incubated at 45.S°C. The log,,-transformed data were analyzed by the t-test, which indicated a highly significant difference ( p < 0.001) betweer
N G AN11 STILES
TABLEI. Counts of presuniptive E. coli (determined in EC medium at 45.5-C) on dilferent ground meats
Can. J. Microbiol. 1978.24:1574-1582. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/14/15. For personal use only.
Count of presumptive E. coli per gram
100 to < 500
< 100
Product
500 to < 1000
1000 or greater
Mean count per gram
Nuniber (percentage) of samples 70 49 12
Ground beef Regular Ground becf Low fat Groi~ndbeef froni store trim Pork sausage Frozen All products
TABLE2. Results of Duncan's multiple-range test for significant differences between counts from ground meats on selective media for Enterobacteriaceae, coliforni bacteria, and E. coli Log,, mean of counts, in increasing order EC
EMB
VRBA-P
BGB
LST
VRBG-P
VRBA-T
VRBG-T
AT 5% level of confidence1 EC
EMB
VRBA-I'
BGB
LST
VRBG-P
VRBA-T
VRBG-T
'The counts for (he media under the common linc arc n o t signifcantly ditTerent. N o r r : V R B A , violet red bile agar: V R B G , Mosscl's Enlerobacteriaceae mcdium ( V R B A , : ; ;1 glucose); -T, total ng at 24 11; count o f bile-prccipilnling and non-bile-prccipitaling colonics at 24 11; -P, c o u n l o f b l l e - p r c c ~ p ~ t n t ~colonics LST, lauryl trvptose b r o l h ; BGB, brilliant green bile 2,7, b r o o i ; E M B , Levine E M B agar; EC, E C b r o l h at 45.5'C.
TABLE3. Pearson's correlation coefficients between counts from ground meats on selective media for Enterobacteriaceae, coliform bacteria, and E. coli VRBA-T
VRBA-P
VRBG-T
VRBG-P
-
VRBA-P VRBG-T VRBG-P LST BGB EMB EC
0.82 0.93 0.90 0.71 0.67 0.51 0.27
-
0.77 0.80 0.70 0.70 0.63 0.46
0.97 0.73 0.69 0.48 0.31
0.72 0.72 0.51 0.36
LST
BGB
EMB
0.80 0.52
0.68
-
0.92 0.72 0.44
N o r r : V R B A , violct rcd bilc agar; V R B G , Mossel's Enterob;~cterinceoe r n e d i ~ l m( V R B A -!- IZ g!iucosc): -T, tot;~lcount o f bilc-precipitating and non-bile-precipitating colonies a t 24 11; -P, count o f bile-prec~pitaling colonies at 24 h : LST, Iauryl tryptosc broth; BGB, brilliant grcen bile 2Z, broth; E M B . Levine E M B agar; EC, E C b r o t h a t 45.5"C.
the methods. Pearson's correlation coefficient for the data was 0.57. The mean for the direct EC test was 3.7 x IOt/g compared with 1.4 x 10Vg for the indirect test. Although the means differed by a factor of 4, the cross-tabulation of the data indicated that differences between these methods could be 10- to 100-fold, which accounted for the poor correlation between the methods. Cross-tabulation of indirect EC counts with the bile-precipitating VRBA counts is shown in Table
4. Of 106 samples with presumptive E. coli > 100, 1000/g and 14 (13.2%) were > lO000/g. Of 30 samples with presumptive E. coli > 1000/g, 12 (40.0%) had bile-precipitating VRBA counts < 1000/g. Results were similar whether pork sausage samples were included in the cross-tabulation or not; o r if cross-tabulations were between indirect EC and total VRBA counts, or between direct EC and bile-precipitating VRBA counts. The
CAN. J. MICROBIOId. VOI..
24. 1978
Can. J. Microbiol. 1978.24:1574-1582. Downloaded from www.nrcresearchpress.com by University of Waterloo on 01/14/15. For personal use only.
TABLE 4. Cross-tabulation of indirect EC counts at 45.5"C with bile-precipitating counts on VRBA Indirect EC count (Presumptive E. coli per gram)
Bile-precipitating VRBA count per gram
< 100
i 100
83 (45.9)
100< 500
29 (32.2)
500< 1000
7 (43.8)
> 1000
5 (16.7)
Total
124
100 to
