International Journal of Food Microbiology.,, 15 ( 1992 ) ! 21 - 130
12 I
'i~) 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1605/92/$115.00 FOOD 1111466
Efficacy of prolonged (48 h) selective enrichment for the detection of foodborne Salmonella J.-Y. D ' A o u s t , A . M . Seweil and A. Jean Food Directorate. fh'alth and Welfare Canada, Sir l'¥ederick G. Bant#lg Research Centre, Ottawa, Ontario, Canada (Received 25 September 1991; accepted 26 December 1991)
The effect of prolonged (48 h) incubation on the productivity of five enrichment-temperature conditions (tetralhi~mate brilliant green, 35 and 43°C; Muller-Kauffman tctrathionate brilliant green, 43°C; Rappaport-Vassiliadis, 43°C; selenite cystine, 35°C) was compared to homologous results obtained under standard (24 h) conditions of selective enrichment. Of 797 high moisture and 166 low moisture foods tested, 171 121.5%) and 80 (48.2%), respectively, were found to contain salmonellae by one or more analytical condition. Combined results of the five enrichment conditions after 24 and 48 h of incubation identified 247 (98.4%) and 250 (99.6%) of the 251 contaminated samples identified in this study. Our results are at variance with earlier reports on the greater method sensitivity with extended ( >/48 h) periods of selective enrichment. The productivities of individual enrichment conditions after each period of incubation varied markedly where reco,,ery rates with TBGa3 and MKTBG4s exceeded that obtained with SC35 and TBG35. Our findings also underline the determinant role of enrichment at an elevated temperature (43°C), and use of multiple enrichment and plating media for the optimal recovery of foodborne Salmonella. Key words: Salmonella; Incubation; Selective enrichment; Foodborne pathogen
Introduction The ubiquitous distribution of Salmonella in the natural environment and in the global food chain, and the growing evidence that iow numbers of this pathogen can cause human illness, predicate the need for highly sensitive methods of detection (D'Aoust, 1989; D'Aoust, 1991). Research efforts into the development of abbreviated analytical procedures have demonstrated greater recovery of foodborne Salmonella with overnight 116-24 h) than with short (6 h) periods of pre-enrichment and selective enrichment (Andrews, 1989; D'Aoust et al., 1990). Although standard cultural methods provide for an 18-24 h period of selective enrichment (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984; International Organization for Standardization, 1990), isolated reports have shown Correspondence address: J.-Y. D'Aoust, Food Directorate, Health and Welfare Canada, Sir Frederick G. Banting Research Centre, Tunney's Pasture, Ottawa, Ontario, Canada KIA 0L2.
122
increased method sensitivity with extended ( > 48 h) enrichment (Harvcy and Price, 1981; Yde and Ghysels, 1984; Fricker et al., 1985). The present study compares the productivity of five selective c~richment conditions after 24 and 48 h of incubation.
Materials and Methods
Naturally-contaminated high moisture (797) and low moisture (166) foods were obtained as a result of monitoring or compliance activities of Canadian government agencies, or were purchased at local retail outlets. Most fish and shellfish were imported from Asiatic and from South and Central Americar~ countries, whereas raw meat and poultry samples were generally obtained from local shops. Cultural analysis of each test sample (100 g) consisted of pre-enrichment in 9 volumes of appropriate nonselective broth medium (Health Protection Branch, 1978) for 24 h at 35°C. Poultry carcasses were thoroughly rinsed in 1 liter of nutrient broth, and the entire rinse solution incubated as previously described. Replicate portions (1 ml) of each pre-enrichment culture were inoculated separately into the following enrichment media: tetrathionate brilliant green incubated at 35°C (TBG35) and a separate tube of broth incubated at 43°C (TBG43); selenite cystine at 35°C (SC35); Muller-Kauffman tetrathionate brilliant green at 43°C (MKTBG43); Rappaport-Vassiliadis at 43°C (RV43). Following incubation for 24 h, each of the five enrichment cultures was streaked in quadrants on bismuth sulfite (BSA) and brilliant green sulfa (BGS) agar media; plates were incubated for 16-18 h at 35°C. The remaining enrichment cultures were incubated for an additional 24 h, and each plated on a second set of agar media. Suspect colonies were screened biochemically on triple sugar iron (TSI) and lysine iron (LI) agars, and confirmed serologically using polyvalent and single grouping somatic (O) and flagellar (H) antisera. All bacteriological media were obtained from Difco Laboratories (Detroit, MI) except MKTBG (CM343), and LI agar which were supplied by Oxoid Ltd (U.K.). RV was prepared in our laboratory (Vassiliadis et al., 1981), and stored in the refrigerator for up to four weeks. A semi-quantitative score scheme was used to assess the sensitivity and specificity of each selective enrichment condition after 24 and 48 h of incubation. The scheme was applied to 129 of the 251 positive samples identified in this study. Growth of Salmonella on BSA and BGS inoculated from the 24 and 48 h enrichment cultures was estimated according to the following scale: 1 = suspect salmonellae in the first quadrant; 2 = salmonellae in quadrants 1 and 2; 3 = salmonellae in three quadrants; 4 = salmonellae in all quadrants. For example, the presence of suspect colonies in the third quadrant and confluent growth in quadrants 1 and 2 would be recorded as three positive quadrants on the assumption that salmonellae were present in quadrants 1 and 2. The index score for each enrichment condition after 24 and 48 h of incubation consisted of the sum of the corresponding BSA and BGS scores for the 129 positive samples. Similarly, the selectivity of each enrichment condition was assessed semi-quantitatively by esti-
123
mating the number of non-salmonellae colonies on BSA and BGS agar plates. The following scale of selcctivity was applied to Sahnonella positive samples: I = 0-25% of surface growth is presumptively non-salmonellae; 2 = 26-50%, 3 = 51-75%, 4 = 76-100% prevalence of non-salmonellae; 5 = presence of non-salmonellae but absence of suspect Sahnonella colonies; 6 = absence of bacterial growth. The index score for each enrichment condition was calculated as previously described for Sahnonella. The selectivity of each enrichment condition was further evaluated in terms of percent incidence of black (non-salmonellae) colonial mimics on BSA plates for 514 of the 963 test samples in this study.
Results and Discussion
Of 797 high moisture foods tested, 171 (21.5%) yielded Salmonella by one or more analytical conditions (Table 1), whereas 80 (48.2%) of the 166 low moisture foods tested were found to be contaminated (Table II). The combined results of the five enrichment conditions (TBG35, TBG43, MKTBG43, RV43 and SC35) identified 247 (98.4%) of the 251 contaminated foods after 24 h of incubation, and 250 (99.6%) after 48 h of selective enrichment (Table I11). Our findings concur with earlier reports (Fricker, 1984; Beckers et al., 1985; de Fatima and Gomes, 1985; Kalapothaki ct al., 1986) on the inability of extended (>/48 h) incubation of enrichment media to increase the recovery of Sahnonella from foods and environmental samples. However, the existence of conflicting reports on the merits of extended enrichment is noteworthy (Harvey and Price, 1981; Yde and Ghysels, 1984; Fricker et al., 1985). A detailed examination of the productivity of enrichment media as affected by incubation time and temperature is revealing (Table iV). The sensitivities of TBG43 and MKTBG43 for both high and low moisture foods compared favorably, and exceeded that obtained with other media after 24 h of incubation. The foods responsible for the seven false-negative results with TBG43 (24 h) consisted of raw poultry (3), frozen liquid eggs (1), chocolate (1), cocoa beans (1), and one sample of pork liver (data not shown). Similarly, MKTBG43 (24 h) failed to detect Salmonella in contaminated samples of raw poultry (2), cocoa beans (1), liquid frozen eggs (1), and egg noodles (1). The high levels of background flora generally encountered in raw poultry (World Health Organization, 1988), and the inhibitory anthocyanins found in cocoa products (Gabis and Langlois, 1967; Zapatka et al., 1977) probably hindered the successful isolation of salmonellae under these usually effective conditions of enrichment. The lower productivity of RV43likely arose, in part, from our choice of a 1:10 transfer volume to minimize the number of experimental variables in our study. The limited selectivity of this enrichment medium, and the need for a 1:100 inoculum to broth ratio for optimal recovery have been reported (Vassiliadis et al., 1981; van Schothorst and Renaud, 1983). It is noteworthy that the common denominator of the three enrichment conditions showing greatest recovery was the high temperature (43°C) of incubation. The propensity of enrichment at elevated temperatures (41-43°C) for the effective repression of background flora and the
124 TABLE l Detraction of Sabmmella in high moisture foods
Food
Serovar
N u m b e r of samples Tested
Positive "
Poultry: Whole chicken
60
47
Cut-up chicken
21
15
Chicken giblets
54
19
Others b
33
18
Pork: Raw sat~age
99
10
Giblo~s
50
7
Oth~:rs c
45
5
Beef Other meats a Fish/shellfish
43 19 189
2 4 8
Egg and egg products
183
36
1
0
797
171
Cheese
Total
S. agona ( I ): S. albany ( 1 ); S. anatum ( 1 ); S. brandenburg (2): S. haardt (2); S. hadar (7): S. heidelberg (2); S. indiana (2): S. mfantis (9): S. montel'ideo ( 1 ); S. muenchen ( 1 ); S. ohio ( I ); S. saint-paul (3): S. schwarzengnmd (2); S. stanlt:v (2); S. thompson (5): S, o'phbnurium (5). S. blockley (I): S. haard¢ (!); S. hadar (2): S. heidelberg (11: S. i:~diana (11: S. infantis (2); S. muenchen (2): S. stanley (1): S. thompson (21: S. typhimurium (2). S. albato' ( I ); S. eimsbuettel (I); S. hadar (3); S. heidelberg (31; S, infilntis (4); S. m u o w h e n (I); S. thompson (11; S. typhimtoqum (31: Sahnonella 1,4,12:r:- ( I ); Sabnonella 1,4,12:z:- ( 1). S. agona ( 1); S. albany ( I ); S. arizonae ( 1 ); S. enteritidis (2); S. haardt (I); S, infimtis (21; S. monte~ideo (I); S. muenchen (2): S. saint-paul (4); S. senftenberg (2); S. o,ph#nurium ( 1). S, brandenburg (6); S. heidelberg (1): S. saint-paul (11; S. nvrthington (2). S. infantis ( 1 ); S. hmdon ( I ); S. montet'ideo ( I ); S. muenster (I); S. nienstedten (I); S. ohio (I); Sahnonella O.R.:eh:1,5 ( I ), S. brandenburg (I); S. b~diana (1): S. hlfantis (!); S. t vphinmrium (2). S. brandenburg (I); S. hadar (I). S. hcittingfoss (11; S. stanley (2); S. circhow (!). S. aberdeen (I); S. #zfantis (I); S. Iohbruegge (1); S. montet'ideo ( 1 ); S. potsdam ( 1); S. stanley ( 1 ); S. sunsdt.all ( I ); Sahnonella I:-:- ( 1 ). S. alachua (I); S. brandenburg (I); S. cerro (6); S. enteritidis ~lh"~" S. ,~,,~.~,~,t";'4"lh .... (6); S. monterideo (181; S. saint-paul (2): S. schwarzengnmd ( I ).
" Positive by one or more analytical condition. b Turkey cut-up (2), giblets (1), carcasses (4), and burgers (5); whole cluck (10); quail (2); cornish hen (6); stewing hen (2); chicken nuggets (11. " Minced pork (22); retail cuts (I 1 ); fermented sausage (61; pork pie ( I ), burgers (2), and blood pudding (3). d Froglegs (11); lamb chops (5) and stew (3).
125 TABLE
Ii
Dclecli(ul of ,~'tlhllollt'lltl Food
ill low mt~inltire foods
Numl~er of s a m p l e s Tested
Posilivc "
9 311
7 I0
Spices •
22
111
Coco n u t Egg P o w d e r Animal Feed
12 9 64
3 4 44
Tea Gelatin Milk Products d Others "
"~ 2 ii 5
2 II 11 0
Total
I 6fl
NO
Pasla Chocolate
b
St? I'( IV{I
r
S. in lbnti.~' (5): S. m h a m l a k a (2). S. mtpoli (4): S. .s~'t~lh',h,'t;e (2): S. typhimurium (2); S. w a n g a t a (2).
S. anatttm ( I '; ~'. .t,,ice ( I ): S. .qhxstrup (3); S. ma~h'lia (.); S. morehead (2);
S. new-hrunswick ( 1 ): S. samliego ( I ). S. setdh,nherg (3). S. monteHdeo (I); S. reading (2); S. senftenberg (I). S. ak,ona ( 1 ); S. hinza (2); S. hredc,ey (4): S. california ( 1 ): S. c('rro (2!; S. do'pool ( 1 ): S. eim.~huettel ( 1 ): S. johanncshurg (3); S. kentucky (21; S. mhamhzka (I): S. molltel'id(,o (9); S. m'wington I I ); S. orion ( I ); S. samfi,:eo ( I ): S..~chwatzengrund (2); S. sen.ftcnlwrg (4): S. tak,wmy (2); S. tennessee (3); S. typhimurium (2); Sahnonelh~ I, 3, 19:-:- ( I ). Sahmmelh~ il 9. 12:s,m,t:- (2).
~' Positive by one or more analytical condition. h ( ' h o c o l a t c (16); cocoa powder (8); beans (6). " Black pepper ( 11 ); chili powder (7); chili pepner ( I ): thyme ( I ): sesame seeds ( 1); turmeric ( I ). d Nonfat dry milk (7); whey powder (4). ~' Yeast (4); dict drink mix (1).
facilitated isolation of Sahnonella on plating media has been reviewed extensively (D'Aoust, 1989). In contrast, TBG35 and SC3.~ incubated tbr 24 h were distinctly less productive and identified, respectively, 220 (87.7%) and 209 (83.3%) of the 251
T A B L E !I1 Productivity of extended (48 h) selective enrichment Total number s a m p l e s
Nnmher samples positive (el)
Tested
Positive "
24 h
48 I1
ttigh moisture Low moisture
797 166
171 80
169 (98.8) 78 (97.5)
1711 (99.4) 80 (100.0)
Total
963
251
247 (98.4)
251) (99,6)
Food
" Based on combined results of five enrichment conditions and two periods of incubation.
126 "FABLE IV
Effect of enrichment media on detection of Sahnom'lla spp. in contaminated foods Enrichment media
Number of positive samples ( ~ ) Rec~wery. patterns (24 h / 4 8 h)
ltigh moisture (171) ~': TBG35 TBG43 MKTBG4~ RV43 SC3~ Low noisture (80) ": TBG~ 5 TBG43 MKTBG4 ~ RVa3 SCb Total (251 ) ": TBG3s TBG.~3 MKTBG43 RV43
S('3s
+/+
+/-
-/+
-/-
142 183.0) 163 (95.3) 164 (95.9) 158 (92.4) 132 (77.2}
6 (3.5) I 10.6) 2 (I.2) 0 (0.0) 6 (3.5}
~) (5.3) 3 (I.8) I (0.6) 4 (2.3) 10 (5.8)
14 (8.2) 4 (2.3) 4 (2.3) 9 (5.3) 23 (13.5)
71 (88.8) 77196.3) 76195.0) 68 (85.11) 71) {87.5)
I (I.3) I) (0.0) 1) (0.0) I} (0.0) 1 11.3)
313.8) 2 (2.5) 1 (1.3) 3 (3.8) 3 (3.8)
5 (6.3) I (1.3) 3 (3.8) 9 (11.3) 6 17.51
213 {84.9} 240(95.6) 241) (95.6) 226 (90.0) 2(}2 (80.5)
7 {2.S) I (0.4) 2 (0.8) 11(I).11) 712.8)
12(4.8) 5 (2.0) 2 (0.8) 7 (2.8) 13 (5.2)
19 (7.6) 5 (2.0) 7 (2.8) 18 (7,2) 29 (11.6)
" Samples positive by one or more analytical condition.
contaminated samples (Table IV). Enrichment at a non-selective temperature 135°C), and the limited bacteriostatic action of the selenite component in SC35 (Leifson, 1936; North and Bartram, 1953) contributed to the low productivity of this medium in the present (Table IV) and in earlier studies (Andrews et al., 1979; Rengel and Mendoza, 1984; D'Aoust, 1989). The concern for maximum recovery of foodborne salmonellae has favored the use of two enrichment conditions in standard cultural procedures (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984; International Organization for Standardization, 1990). Prolonged (48 h) incubation of TBG35 and SC35 increased method sensitivity by 4.8-5.2% compared to that obtained with the standard (24 h) period of enrichment. Smaller gains in productivity were obtained with enrichment media incubated for 48 h at 43°C (Table IV). Conceivably, the greater recoveries with TBG35 and SC35 after 48 h of incubation did not result from enhanced growth of salmonellae during the extended period of enrichment, but rather arose from differences in the death rate kinetics of Sahnonella and competitive microflora in the enrichment cultures. Inoculation of TBG35 and SC35 broths of low selectivity with a portion of pre-enrichment (non-selective) culture containing high numbers of non-salmonellae predictably engendered an unfavorable ratio of salmonellae to non-salmonellae upon enrichment for 24 h at a growth permissive (35°C) temperature (Rhodes et al., 1985). Enrichment for an additional 24 h likely did not
127
produce a net increase in the number of Sahnonella cells as previously demonstrated with RV37 (Beckers et al., 19881, but rather precipitated the death of sensitive competitive microflora from stress conditions created by nutrient deficiency, suboptimal pH, and/or accumulation of antibacterial metabolites in the enrichment cultures. Such interactive factors increased the prominence of Sabnonella in TBG35 and SC35 cultures to levels more easily recoverable on plating media of moderate selectivity. Inoculation of BSA and BGS with 48 h enrichment cultures did not markedly increase the levels of recovery obtained with 24 h enrichment cultures. BSA and BGS identificd, rcspectively, 1090 and 1059 positive samples after 24 h of enrichment, compared to 1118 and 1078 positive samples after 48 h of incubation. These findings are consistent with earlier reports on the superiority of BSA for the detection of foodborne Sahnonella (Andrews et al., 1979; Rengel and Mendoza, 1984; D'Aoust, 1984). Synergism between the selective agents in this medium, and its saccharide-independent identification of presumptive isolates has earned BSA a prominent role in standard cultural procedures (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984; International Organization for Stan-
TABLE V
.~
Sensitivity ,,)f plating media under different enrichment conditions :' Enrichment media
Number of isolations (c,~,) b BSA
ttigh moisture 11711 ~' TBG35 TBG~3 MKTBGa3 RV43 SC35 Low moisture (80) ~' TBGss TBG43 MKTBGa3 RV43 SC35 Total (2511 " TBG35 TBG43 MKTBG43 RV43 SC35
BGS
24 h
48 h
142 (83.0) 155 (90.6) 153 (89.5) 152 (88.9) 125 (73.1)
144 (84.2) 160 (93.6) 156 (91.2) 156 191.2) 133 (77.8)
136 (79.5) 16(1 (93.6) 164 (95.9) 149 (87. I ) 119 (69.6)
137180.11 163 (95.3) 163 (95.3) 148 (86.5) 119 (69.6)
72 (90.0) 77 (96.3) 76 (95.0) 67 (83.8) 71 (88.8)
73 (91.3) 79 (98.8) 77 (96.3) 69 (86.3) 71 (88.8)
64 (80.0) 73 (91.31 73 (0!.31 63 (78.8) 58 (72.5)
64 (80.0) 75 (93.8) 75 (93.8) 67 (83.8) 67 (83.8)
214 (85.3) 232 (92.4) 229 191.21 219 (87.3) 196 (78.1)
217 (86.5) 239 (95.2) 233 (92.8) 225 (89.6) 204 (81.3)
200 (79.7) 233 (92.8) 237 (94.4) 212 (84.5) 177 (70.5)
2(11 (80.11 238 (94.8) 238 (94.8) 215 (85.7) 186 (74. I )
" Recoveries from 24 and 48 h enrichment cultures. h Percent of total positive samples. c Samples positive by one or more analytical condition.
24 h
48 h
128,
dardization, 199(I). Inability of any enrichment-plating combination to identify, all contaminated samples (Tables IV and V) corroborates earlier statements on the need for multiple enrichment and plating media to achieve optimal recovery of Salmonella in foods (Gabis and Sillikcr, 1974; Bailey et al., 1981; Catsaras and Sery, 1981; D'Aoust. 1984). Semi-quantitative estimates of Sahnonella and nonsalmonellae populations on isolation media andcrlined the interplay between the selectivity of enrichment conditk)ns and succe:;sful recovery of Sahnonella on BSA and BGS (Table VI). The number of suspect Sahnonella colonies was highest with TGB43 and MKTBG43, and lowest with SC35 after 24 and 48 h of selective enrichment. Similarly, index scores for competitive flora demonstrated the ability of TBG4~ and MKTBG43 to effectively repress the growth of non-salmonellae thereby providing for a more luxuriant growth of Salmonella on agar media. Our use of a 1:10 transfer volume with RV43 presumably undermined the limited selectivity of this medium, and produced high levels of competitive flora on corresponding BSA and BGS media. RV43 also yielded a substantial number of Sahnonelkt mimics on BSA following 24 and 48 h of selective enrichment (Table V1). The present study reiteratcd the greater selectivity of enrichment at elevated (43°C) than at lower (35°C) temperatures (Table IV), and emphasized the use of multiple enrichment and plating media for grcatcr method sensitivity. In contrast to earlier reports (Harvey and Price, 1981; Yde and Ghysels, 1984; Fricker ct al., 1985), our results failed to show a substantial increase in the recovery of foodborne salmonellae with prolonged (48 h) enrichment (Table II1). Use of this approach would not only increase the analytical effort necessitated by the sequential plating of 24 and 48 h enrichment cultures, but would not respond to current food industry needs for more rapid and cost-efficient methods of detection.
TABLE VI Semi-quantitative growth charactcrisli~:x on plating media " Enrichment
Index score i,
media
Sahmmella ~
TBG35 TBG43 MKTBG43 RV43 SC35 " t, c d
Mimicry on BSA d Competitive flora ¢
24 I1
4/4 h
24 h
48 h
702 919 938 798 757
796 967 953 818 772
895 575 536 g51 818
892 612 617 816 817
24 h
4E h
4.5 2.9 2.7 12.8 5.3
2.7 1.9 1.4 8.6 2.3
Data relate to 129 of the 251 positive samples identified in this study. Scores for growth on BSA and BGS were combined. Perfect scores for & d m o m , lla and competitive flora are 11}32 and 258, respectively. Percent occurrence of BSA plates with black (non salmonellae) colonies.
129
References Andrews. W.ll. (1989) Methods for recovering injured "'classical" enteric pathogenic bacteria (,~ahnonella, Shigelht, and enleropathogenic Escherichia coil) from foods. In: B. Ray (Ed.), Injured Index and Pathogenic Bacteria: Occurrence and Detection in Foods, Water and Feeds, CRC Press, Boca Raton. Florida, pp. 55-113. Andrews. W.tt., W~lson, C.R., Poelma. P.L. and Romero, A. (1979) Relative productivity of five selective plating agars for Ihe recove~ of Sahm,nella from selected fl~od types. J. Assoc. Off. Anal. ('hem. 62, 320-326. Bailey, J.S., Cox, N.A. and Thomson, J.E. (1981) Efficiency of selenite cysfine and TT enrichment broths for the detection of Salmonella. J. Appl. Bacleriol. 51.4(19-414. Beckers, lt.J., Tips, P.D., Socnloro. P.S.S., Delfgou-van Asch. E.II.M. and Peters, R. (1988) The efficacy of enzyme immunoassays for the detection of salmonellas. Food Microbiol. 5, 147-156. Beckers, li.J., van Leusden, F.M.. Roberts, D., Pietzsch. O., Price, T.H., van Schothorst, M., Tips, P.D., Vassiliadis, P. and Kampelmacher, E.H. (1985~ Collaborative study on the isolation of Sahnonella from artificially contaminated milk powder. J. Appl. Baeleriol. 59. 35-40. Catsaras, M. and Sery, C. (1981) Enrichment and isolation media for Sahmmella. Sciences des Aliments I, 477-488. D'Aoust, J.-Y. (1984) Effective enrichment-plating conditions for detection of Sahmmella in foods. J. Food Pint. 47, 588-590. D'Aoust, J.-Y. (1989) Sabnonella. t n: M.P. Doyle (Ed.), Foodborne Bacterial Pat hogens, Marcel Dekker Inc., New York, NY, pp. 327-445. D'Aoust, J.-Y. (t991) Pathogenicity of fi~odborne Sahnonella. int. J. Food Microbiol. 12, 17-40, D'Aoust. J.-Y., Sewell, A. and Jean, A. (19911) Limited sensitivity of short (6 h) selective enrichment for detection of fi~odborne Sahmmella. J. Food Prot. 53, 562-565. de Fatima, M. and Gomes, F.F. (1985) Isolation of Sabnonelht from liver and evaluation of enrichment broths, time and temperature of incubation. Rev. Microbiol. (Sao Paulo) 16. 211-221. Fricker, C.R. (1984) A comparison of methods for the isolation of salmonellae from sewage sludge. Zbl. Bakt. ltyg. I. Abt. Orig. B 179, 170-178. Fricker, C.F,., Quail, E., McGibbon, L. and Girdwood, R.W.A. (1985)An evaluation of commercially available dehydrated Rappaport-Vassiliadis medium for the isolation of salmonellae from poultry. J, tlyg. 95, 337-344. Gabis, D.A. and Langlois, B.E. (1967) Effect of cocoa powder used in chocolate milk on bacterial growth. J. Dairy Sci. 5(1, 945. Gabis, D.A, and Silliker, J.lt. (1974) ICMSF methods studies. V1, The influence of selective enrichment media and incubation temperatures on the detection of Sahnonella in dried fl~ods and feeds. Can. J. Microbiol. 20, 1509-151 I. Harvey, R.W.S. and Price, T.H. (!981) Comparison of selenite F, Muller-Kauffmann tetrathionate and Rappaport's medium for Sahnonella isolation from chicken giblets after pre-enrichment in buffered peptone water. J. Hyg. 87, 219-224. Health Protection Branch (1978) Methods for the isolation and identification of Sahnonella from foods. Method MF HPB-20, Health and Welfare Canada, Ottawa. International Organization for Standardization (1990) International Standard 6579. Microbiology-General guidance on methods for the detection of Sahnonella. ISO, Geneva. Kalapothaki, V., Trichopoulos, D., Papadakis, J., Mavrommati, Ch. and Vassiliadis, P. (1986) A comparison of the efficiency of two forms of Rappaport-Vassiliadis enrichment medium, int. J. Food Microbiol. 3, 89-97. Leifson, E. (1936) New selenite enrichment media for the isolation of typhoid and paratyphoid (Sahnonella) bacilli. Am. J. Hyg. 24, 423-432. North, W.R. and Bartram, M.T. (1953). The efficiency of selenite broth of different compositions in the isolation of Sahnonella. Appl. Microbiol. 1, 130-134. Rengel, A. and Mendoza, S. (1984) Isolation of Sabnonella from raw chicken in Venezuela. J. Food Prot. 47, 213-216.
130 Rhodes, P., Ouesnel, L.B. and Collard. P. (1985) Growth kinetics of mixed culture in salmonella enrichment media. J. Appl. Bactcriol. 59, 231-237. U.S. Food and Drug Administration (1984) Bacteriological Analytical Manual (6th edilion). Association of Official Analytical Chemists. Washington, DC. wm Schothorst, M. and Renaud, A.M. (19831 Dynamics of Sahnonella isolation with Rappaport's medium (R I(J). J. Appl. Bacleriol. 54. 2(1q~215. Vassiliadis, P.. Trichopouios, D., Pai0adakis, J.. Kalapolhaki. V., Zavitsanos, X. and Serie, Ch. (19811 Sahnrmelhl isolation with Rappaporl's enrichment medium of different composition. Zbl. Bakt. ltyg. i. Abt. ()rig. B 173, 382-389. World Health Organization (Iq88) Salmoncllosis control: the role of animal and product hygiene. Technical Report Series no. 774. World !!ealth Org;mization. Geneva, 83 pp. Yde, M. and Ghysels, G. (19841 Performance of several enrichment media in the isolation of salmonellae from liquid egg products. J. Food Prot. 47, 217-210. Zapatka, F.A., Varney, G.W. and Sinskey, A.J. ( 19771 Neutralization of the bactericidal effect ~f ct)coa powder on salmonellae by casein. J. Appl. Bacteriol. 42.21-25.
12 I
'i~) 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1605/92/$115.00 FOOD 1111466
Efficacy of prolonged (48 h) selective enrichment for the detection of foodborne Salmonella J.-Y. D ' A o u s t , A . M . Seweil and A. Jean Food Directorate. fh'alth and Welfare Canada, Sir l'¥ederick G. Bant#lg Research Centre, Ottawa, Ontario, Canada (Received 25 September 1991; accepted 26 December 1991)
The effect of prolonged (48 h) incubation on the productivity of five enrichment-temperature conditions (tetralhi~mate brilliant green, 35 and 43°C; Muller-Kauffman tctrathionate brilliant green, 43°C; Rappaport-Vassiliadis, 43°C; selenite cystine, 35°C) was compared to homologous results obtained under standard (24 h) conditions of selective enrichment. Of 797 high moisture and 166 low moisture foods tested, 171 121.5%) and 80 (48.2%), respectively, were found to contain salmonellae by one or more analytical condition. Combined results of the five enrichment conditions after 24 and 48 h of incubation identified 247 (98.4%) and 250 (99.6%) of the 251 contaminated samples identified in this study. Our results are at variance with earlier reports on the greater method sensitivity with extended ( >/48 h) periods of selective enrichment. The productivities of individual enrichment conditions after each period of incubation varied markedly where reco,,ery rates with TBGa3 and MKTBG4s exceeded that obtained with SC35 and TBG35. Our findings also underline the determinant role of enrichment at an elevated temperature (43°C), and use of multiple enrichment and plating media for the optimal recovery of foodborne Salmonella. Key words: Salmonella; Incubation; Selective enrichment; Foodborne pathogen
Introduction The ubiquitous distribution of Salmonella in the natural environment and in the global food chain, and the growing evidence that iow numbers of this pathogen can cause human illness, predicate the need for highly sensitive methods of detection (D'Aoust, 1989; D'Aoust, 1991). Research efforts into the development of abbreviated analytical procedures have demonstrated greater recovery of foodborne Salmonella with overnight 116-24 h) than with short (6 h) periods of pre-enrichment and selective enrichment (Andrews, 1989; D'Aoust et al., 1990). Although standard cultural methods provide for an 18-24 h period of selective enrichment (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984; International Organization for Standardization, 1990), isolated reports have shown Correspondence address: J.-Y. D'Aoust, Food Directorate, Health and Welfare Canada, Sir Frederick G. Banting Research Centre, Tunney's Pasture, Ottawa, Ontario, Canada KIA 0L2.
122
increased method sensitivity with extended ( > 48 h) enrichment (Harvcy and Price, 1981; Yde and Ghysels, 1984; Fricker et al., 1985). The present study compares the productivity of five selective c~richment conditions after 24 and 48 h of incubation.
Materials and Methods
Naturally-contaminated high moisture (797) and low moisture (166) foods were obtained as a result of monitoring or compliance activities of Canadian government agencies, or were purchased at local retail outlets. Most fish and shellfish were imported from Asiatic and from South and Central Americar~ countries, whereas raw meat and poultry samples were generally obtained from local shops. Cultural analysis of each test sample (100 g) consisted of pre-enrichment in 9 volumes of appropriate nonselective broth medium (Health Protection Branch, 1978) for 24 h at 35°C. Poultry carcasses were thoroughly rinsed in 1 liter of nutrient broth, and the entire rinse solution incubated as previously described. Replicate portions (1 ml) of each pre-enrichment culture were inoculated separately into the following enrichment media: tetrathionate brilliant green incubated at 35°C (TBG35) and a separate tube of broth incubated at 43°C (TBG43); selenite cystine at 35°C (SC35); Muller-Kauffman tetrathionate brilliant green at 43°C (MKTBG43); Rappaport-Vassiliadis at 43°C (RV43). Following incubation for 24 h, each of the five enrichment cultures was streaked in quadrants on bismuth sulfite (BSA) and brilliant green sulfa (BGS) agar media; plates were incubated for 16-18 h at 35°C. The remaining enrichment cultures were incubated for an additional 24 h, and each plated on a second set of agar media. Suspect colonies were screened biochemically on triple sugar iron (TSI) and lysine iron (LI) agars, and confirmed serologically using polyvalent and single grouping somatic (O) and flagellar (H) antisera. All bacteriological media were obtained from Difco Laboratories (Detroit, MI) except MKTBG (CM343), and LI agar which were supplied by Oxoid Ltd (U.K.). RV was prepared in our laboratory (Vassiliadis et al., 1981), and stored in the refrigerator for up to four weeks. A semi-quantitative score scheme was used to assess the sensitivity and specificity of each selective enrichment condition after 24 and 48 h of incubation. The scheme was applied to 129 of the 251 positive samples identified in this study. Growth of Salmonella on BSA and BGS inoculated from the 24 and 48 h enrichment cultures was estimated according to the following scale: 1 = suspect salmonellae in the first quadrant; 2 = salmonellae in quadrants 1 and 2; 3 = salmonellae in three quadrants; 4 = salmonellae in all quadrants. For example, the presence of suspect colonies in the third quadrant and confluent growth in quadrants 1 and 2 would be recorded as three positive quadrants on the assumption that salmonellae were present in quadrants 1 and 2. The index score for each enrichment condition after 24 and 48 h of incubation consisted of the sum of the corresponding BSA and BGS scores for the 129 positive samples. Similarly, the selectivity of each enrichment condition was assessed semi-quantitatively by esti-
123
mating the number of non-salmonellae colonies on BSA and BGS agar plates. The following scale of selcctivity was applied to Sahnonella positive samples: I = 0-25% of surface growth is presumptively non-salmonellae; 2 = 26-50%, 3 = 51-75%, 4 = 76-100% prevalence of non-salmonellae; 5 = presence of non-salmonellae but absence of suspect Sahnonella colonies; 6 = absence of bacterial growth. The index score for each enrichment condition was calculated as previously described for Sahnonella. The selectivity of each enrichment condition was further evaluated in terms of percent incidence of black (non-salmonellae) colonial mimics on BSA plates for 514 of the 963 test samples in this study.
Results and Discussion
Of 797 high moisture foods tested, 171 (21.5%) yielded Salmonella by one or more analytical conditions (Table 1), whereas 80 (48.2%) of the 166 low moisture foods tested were found to be contaminated (Table II). The combined results of the five enrichment conditions (TBG35, TBG43, MKTBG43, RV43 and SC35) identified 247 (98.4%) of the 251 contaminated foods after 24 h of incubation, and 250 (99.6%) after 48 h of selective enrichment (Table I11). Our findings concur with earlier reports (Fricker, 1984; Beckers et al., 1985; de Fatima and Gomes, 1985; Kalapothaki ct al., 1986) on the inability of extended (>/48 h) incubation of enrichment media to increase the recovery of Sahnonella from foods and environmental samples. However, the existence of conflicting reports on the merits of extended enrichment is noteworthy (Harvey and Price, 1981; Yde and Ghysels, 1984; Fricker et al., 1985). A detailed examination of the productivity of enrichment media as affected by incubation time and temperature is revealing (Table iV). The sensitivities of TBG43 and MKTBG43 for both high and low moisture foods compared favorably, and exceeded that obtained with other media after 24 h of incubation. The foods responsible for the seven false-negative results with TBG43 (24 h) consisted of raw poultry (3), frozen liquid eggs (1), chocolate (1), cocoa beans (1), and one sample of pork liver (data not shown). Similarly, MKTBG43 (24 h) failed to detect Salmonella in contaminated samples of raw poultry (2), cocoa beans (1), liquid frozen eggs (1), and egg noodles (1). The high levels of background flora generally encountered in raw poultry (World Health Organization, 1988), and the inhibitory anthocyanins found in cocoa products (Gabis and Langlois, 1967; Zapatka et al., 1977) probably hindered the successful isolation of salmonellae under these usually effective conditions of enrichment. The lower productivity of RV43likely arose, in part, from our choice of a 1:10 transfer volume to minimize the number of experimental variables in our study. The limited selectivity of this enrichment medium, and the need for a 1:100 inoculum to broth ratio for optimal recovery have been reported (Vassiliadis et al., 1981; van Schothorst and Renaud, 1983). It is noteworthy that the common denominator of the three enrichment conditions showing greatest recovery was the high temperature (43°C) of incubation. The propensity of enrichment at elevated temperatures (41-43°C) for the effective repression of background flora and the
124 TABLE l Detraction of Sabmmella in high moisture foods
Food
Serovar
N u m b e r of samples Tested
Positive "
Poultry: Whole chicken
60
47
Cut-up chicken
21
15
Chicken giblets
54
19
Others b
33
18
Pork: Raw sat~age
99
10
Giblo~s
50
7
Oth~:rs c
45
5
Beef Other meats a Fish/shellfish
43 19 189
2 4 8
Egg and egg products
183
36
1
0
797
171
Cheese
Total
S. agona ( I ): S. albany ( 1 ); S. anatum ( 1 ); S. brandenburg (2): S. haardt (2); S. hadar (7): S. heidelberg (2); S. indiana (2): S. mfantis (9): S. montel'ideo ( 1 ); S. muenchen ( 1 ); S. ohio ( I ); S. saint-paul (3): S. schwarzengnmd (2); S. stanlt:v (2); S. thompson (5): S, o'phbnurium (5). S. blockley (I): S. haard¢ (!); S. hadar (2): S. heidelberg (11: S. i:~diana (11: S. infantis (2); S. muenchen (2): S. stanley (1): S. thompson (21: S. typhimurium (2). S. albato' ( I ); S. eimsbuettel (I); S. hadar (3); S. heidelberg (31; S, infilntis (4); S. m u o w h e n (I); S. thompson (11; S. typhimtoqum (31: Sahnonella 1,4,12:r:- ( I ); Sabnonella 1,4,12:z:- ( 1). S. agona ( 1); S. albany ( I ); S. arizonae ( 1 ); S. enteritidis (2); S. haardt (I); S, infimtis (21; S. monte~ideo (I); S. muenchen (2): S. saint-paul (4); S. senftenberg (2); S. o,ph#nurium ( 1). S, brandenburg (6); S. heidelberg (1): S. saint-paul (11; S. nvrthington (2). S. infantis ( 1 ); S. hmdon ( I ); S. montet'ideo ( I ); S. muenster (I); S. nienstedten (I); S. ohio (I); Sahnonella O.R.:eh:1,5 ( I ), S. brandenburg (I); S. b~diana (1): S. hlfantis (!); S. t vphinmrium (2). S. brandenburg (I); S. hadar (I). S. hcittingfoss (11; S. stanley (2); S. circhow (!). S. aberdeen (I); S. #zfantis (I); S. Iohbruegge (1); S. montet'ideo ( 1 ); S. potsdam ( 1); S. stanley ( 1 ); S. sunsdt.all ( I ); Sahnonella I:-:- ( 1 ). S. alachua (I); S. brandenburg (I); S. cerro (6); S. enteritidis ~lh"~" S. ,~,,~.~,~,t";'4"lh .... (6); S. monterideo (181; S. saint-paul (2): S. schwarzengnmd ( I ).
" Positive by one or more analytical condition. b Turkey cut-up (2), giblets (1), carcasses (4), and burgers (5); whole cluck (10); quail (2); cornish hen (6); stewing hen (2); chicken nuggets (11. " Minced pork (22); retail cuts (I 1 ); fermented sausage (61; pork pie ( I ), burgers (2), and blood pudding (3). d Froglegs (11); lamb chops (5) and stew (3).
125 TABLE
Ii
Dclecli(ul of ,~'tlhllollt'lltl Food
ill low mt~inltire foods
Numl~er of s a m p l e s Tested
Posilivc "
9 311
7 I0
Spices •
22
111
Coco n u t Egg P o w d e r Animal Feed
12 9 64
3 4 44
Tea Gelatin Milk Products d Others "
"~ 2 ii 5
2 II 11 0
Total
I 6fl
NO
Pasla Chocolate
b
St? I'( IV{I
r
S. in lbnti.~' (5): S. m h a m l a k a (2). S. mtpoli (4): S. .s~'t~lh',h,'t;e (2): S. typhimurium (2); S. w a n g a t a (2).
S. anatttm ( I '; ~'. .t,,ice ( I ): S. .qhxstrup (3); S. ma~h'lia (.); S. morehead (2);
S. new-hrunswick ( 1 ): S. samliego ( I ). S. setdh,nherg (3). S. monteHdeo (I); S. reading (2); S. senftenberg (I). S. ak,ona ( 1 ); S. hinza (2); S. hredc,ey (4): S. california ( 1 ): S. c('rro (2!; S. do'pool ( 1 ): S. eim.~huettel ( 1 ): S. johanncshurg (3); S. kentucky (21; S. mhamhzka (I): S. molltel'id(,o (9); S. m'wington I I ); S. orion ( I ); S. samfi,:eo ( I ): S..~chwatzengrund (2); S. sen.ftcnlwrg (4): S. tak,wmy (2); S. tennessee (3); S. typhimurium (2); Sahnonelh~ I, 3, 19:-:- ( I ). Sahmmelh~ il 9. 12:s,m,t:- (2).
~' Positive by one or more analytical condition. h ( ' h o c o l a t c (16); cocoa powder (8); beans (6). " Black pepper ( 11 ); chili powder (7); chili pepner ( I ): thyme ( I ): sesame seeds ( 1); turmeric ( I ). d Nonfat dry milk (7); whey powder (4). ~' Yeast (4); dict drink mix (1).
facilitated isolation of Sahnonella on plating media has been reviewed extensively (D'Aoust, 1989). In contrast, TBG35 and SC3.~ incubated tbr 24 h were distinctly less productive and identified, respectively, 220 (87.7%) and 209 (83.3%) of the 251
T A B L E !I1 Productivity of extended (48 h) selective enrichment Total number s a m p l e s
Nnmher samples positive (el)
Tested
Positive "
24 h
48 I1
ttigh moisture Low moisture
797 166
171 80
169 (98.8) 78 (97.5)
1711 (99.4) 80 (100.0)
Total
963
251
247 (98.4)
251) (99,6)
Food
" Based on combined results of five enrichment conditions and two periods of incubation.
126 "FABLE IV
Effect of enrichment media on detection of Sahnom'lla spp. in contaminated foods Enrichment media
Number of positive samples ( ~ ) Rec~wery. patterns (24 h / 4 8 h)
ltigh moisture (171) ~': TBG35 TBG43 MKTBG4~ RV43 SC3~ Low noisture (80) ": TBG~ 5 TBG43 MKTBG4 ~ RVa3 SCb Total (251 ) ": TBG3s TBG.~3 MKTBG43 RV43
S('3s
+/+
+/-
-/+
-/-
142 183.0) 163 (95.3) 164 (95.9) 158 (92.4) 132 (77.2}
6 (3.5) I 10.6) 2 (I.2) 0 (0.0) 6 (3.5}
~) (5.3) 3 (I.8) I (0.6) 4 (2.3) 10 (5.8)
14 (8.2) 4 (2.3) 4 (2.3) 9 (5.3) 23 (13.5)
71 (88.8) 77196.3) 76195.0) 68 (85.11) 71) {87.5)
I (I.3) I) (0.0) 1) (0.0) I} (0.0) 1 11.3)
313.8) 2 (2.5) 1 (1.3) 3 (3.8) 3 (3.8)
5 (6.3) I (1.3) 3 (3.8) 9 (11.3) 6 17.51
213 {84.9} 240(95.6) 241) (95.6) 226 (90.0) 2(}2 (80.5)
7 {2.S) I (0.4) 2 (0.8) 11(I).11) 712.8)
12(4.8) 5 (2.0) 2 (0.8) 7 (2.8) 13 (5.2)
19 (7.6) 5 (2.0) 7 (2.8) 18 (7,2) 29 (11.6)
" Samples positive by one or more analytical condition.
contaminated samples (Table IV). Enrichment at a non-selective temperature 135°C), and the limited bacteriostatic action of the selenite component in SC35 (Leifson, 1936; North and Bartram, 1953) contributed to the low productivity of this medium in the present (Table IV) and in earlier studies (Andrews et al., 1979; Rengel and Mendoza, 1984; D'Aoust, 1989). The concern for maximum recovery of foodborne salmonellae has favored the use of two enrichment conditions in standard cultural procedures (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984; International Organization for Standardization, 1990). Prolonged (48 h) incubation of TBG35 and SC35 increased method sensitivity by 4.8-5.2% compared to that obtained with the standard (24 h) period of enrichment. Smaller gains in productivity were obtained with enrichment media incubated for 48 h at 43°C (Table IV). Conceivably, the greater recoveries with TBG35 and SC35 after 48 h of incubation did not result from enhanced growth of salmonellae during the extended period of enrichment, but rather arose from differences in the death rate kinetics of Sahnonella and competitive microflora in the enrichment cultures. Inoculation of TBG35 and SC35 broths of low selectivity with a portion of pre-enrichment (non-selective) culture containing high numbers of non-salmonellae predictably engendered an unfavorable ratio of salmonellae to non-salmonellae upon enrichment for 24 h at a growth permissive (35°C) temperature (Rhodes et al., 1985). Enrichment for an additional 24 h likely did not
127
produce a net increase in the number of Sahnonella cells as previously demonstrated with RV37 (Beckers et al., 19881, but rather precipitated the death of sensitive competitive microflora from stress conditions created by nutrient deficiency, suboptimal pH, and/or accumulation of antibacterial metabolites in the enrichment cultures. Such interactive factors increased the prominence of Sabnonella in TBG35 and SC35 cultures to levels more easily recoverable on plating media of moderate selectivity. Inoculation of BSA and BGS with 48 h enrichment cultures did not markedly increase the levels of recovery obtained with 24 h enrichment cultures. BSA and BGS identificd, rcspectively, 1090 and 1059 positive samples after 24 h of enrichment, compared to 1118 and 1078 positive samples after 48 h of incubation. These findings are consistent with earlier reports on the superiority of BSA for the detection of foodborne Sahnonella (Andrews et al., 1979; Rengel and Mendoza, 1984; D'Aoust, 1984). Synergism between the selective agents in this medium, and its saccharide-independent identification of presumptive isolates has earned BSA a prominent role in standard cultural procedures (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984; International Organization for Stan-
TABLE V
.~
Sensitivity ,,)f plating media under different enrichment conditions :' Enrichment media
Number of isolations (c,~,) b BSA
ttigh moisture 11711 ~' TBG35 TBG~3 MKTBGa3 RV43 SC35 Low moisture (80) ~' TBGss TBG43 MKTBGa3 RV43 SC35 Total (2511 " TBG35 TBG43 MKTBG43 RV43 SC35
BGS
24 h
48 h
142 (83.0) 155 (90.6) 153 (89.5) 152 (88.9) 125 (73.1)
144 (84.2) 160 (93.6) 156 (91.2) 156 191.2) 133 (77.8)
136 (79.5) 16(1 (93.6) 164 (95.9) 149 (87. I ) 119 (69.6)
137180.11 163 (95.3) 163 (95.3) 148 (86.5) 119 (69.6)
72 (90.0) 77 (96.3) 76 (95.0) 67 (83.8) 71 (88.8)
73 (91.3) 79 (98.8) 77 (96.3) 69 (86.3) 71 (88.8)
64 (80.0) 73 (91.31 73 (0!.31 63 (78.8) 58 (72.5)
64 (80.0) 75 (93.8) 75 (93.8) 67 (83.8) 67 (83.8)
214 (85.3) 232 (92.4) 229 191.21 219 (87.3) 196 (78.1)
217 (86.5) 239 (95.2) 233 (92.8) 225 (89.6) 204 (81.3)
200 (79.7) 233 (92.8) 237 (94.4) 212 (84.5) 177 (70.5)
2(11 (80.11 238 (94.8) 238 (94.8) 215 (85.7) 186 (74. I )
" Recoveries from 24 and 48 h enrichment cultures. h Percent of total positive samples. c Samples positive by one or more analytical condition.
24 h
48 h
128,
dardization, 199(I). Inability of any enrichment-plating combination to identify, all contaminated samples (Tables IV and V) corroborates earlier statements on the need for multiple enrichment and plating media to achieve optimal recovery of Salmonella in foods (Gabis and Sillikcr, 1974; Bailey et al., 1981; Catsaras and Sery, 1981; D'Aoust. 1984). Semi-quantitative estimates of Sahnonella and nonsalmonellae populations on isolation media andcrlined the interplay between the selectivity of enrichment conditk)ns and succe:;sful recovery of Sahnonella on BSA and BGS (Table VI). The number of suspect Sahnonella colonies was highest with TGB43 and MKTBG43, and lowest with SC35 after 24 and 48 h of selective enrichment. Similarly, index scores for competitive flora demonstrated the ability of TBG4~ and MKTBG43 to effectively repress the growth of non-salmonellae thereby providing for a more luxuriant growth of Salmonella on agar media. Our use of a 1:10 transfer volume with RV43 presumably undermined the limited selectivity of this medium, and produced high levels of competitive flora on corresponding BSA and BGS media. RV43 also yielded a substantial number of Sahnonelkt mimics on BSA following 24 and 48 h of selective enrichment (Table V1). The present study reiteratcd the greater selectivity of enrichment at elevated (43°C) than at lower (35°C) temperatures (Table IV), and emphasized the use of multiple enrichment and plating media for grcatcr method sensitivity. In contrast to earlier reports (Harvey and Price, 1981; Yde and Ghysels, 1984; Fricker ct al., 1985), our results failed to show a substantial increase in the recovery of foodborne salmonellae with prolonged (48 h) enrichment (Table II1). Use of this approach would not only increase the analytical effort necessitated by the sequential plating of 24 and 48 h enrichment cultures, but would not respond to current food industry needs for more rapid and cost-efficient methods of detection.
TABLE VI Semi-quantitative growth charactcrisli~:x on plating media " Enrichment
Index score i,
media
Sahmmella ~
TBG35 TBG43 MKTBG43 RV43 SC35 " t, c d
Mimicry on BSA d Competitive flora ¢
24 I1
4/4 h
24 h
48 h
702 919 938 798 757
796 967 953 818 772
895 575 536 g51 818
892 612 617 816 817
24 h
4E h
4.5 2.9 2.7 12.8 5.3
2.7 1.9 1.4 8.6 2.3
Data relate to 129 of the 251 positive samples identified in this study. Scores for growth on BSA and BGS were combined. Perfect scores for & d m o m , lla and competitive flora are 11}32 and 258, respectively. Percent occurrence of BSA plates with black (non salmonellae) colonies.
129
References Andrews. W.ll. (1989) Methods for recovering injured "'classical" enteric pathogenic bacteria (,~ahnonella, Shigelht, and enleropathogenic Escherichia coil) from foods. In: B. Ray (Ed.), Injured Index and Pathogenic Bacteria: Occurrence and Detection in Foods, Water and Feeds, CRC Press, Boca Raton. Florida, pp. 55-113. Andrews. W.tt., W~lson, C.R., Poelma. P.L. and Romero, A. (1979) Relative productivity of five selective plating agars for Ihe recove~ of Sahm,nella from selected fl~od types. J. Assoc. Off. Anal. ('hem. 62, 320-326. Bailey, J.S., Cox, N.A. and Thomson, J.E. (1981) Efficiency of selenite cysfine and TT enrichment broths for the detection of Salmonella. J. Appl. Bacleriol. 51.4(19-414. Beckers, lt.J., Tips, P.D., Socnloro. P.S.S., Delfgou-van Asch. E.II.M. and Peters, R. (1988) The efficacy of enzyme immunoassays for the detection of salmonellas. Food Microbiol. 5, 147-156. Beckers, li.J., van Leusden, F.M.. Roberts, D., Pietzsch. O., Price, T.H., van Schothorst, M., Tips, P.D., Vassiliadis, P. and Kampelmacher, E.H. (1985~ Collaborative study on the isolation of Sahnonella from artificially contaminated milk powder. J. Appl. Baeleriol. 59. 35-40. Catsaras, M. and Sery, C. (1981) Enrichment and isolation media for Sahmmella. Sciences des Aliments I, 477-488. D'Aoust, J.-Y. (1984) Effective enrichment-plating conditions for detection of Sahmmella in foods. J. Food Pint. 47, 588-590. D'Aoust, J.-Y. (1989) Sabnonella. t n: M.P. Doyle (Ed.), Foodborne Bacterial Pat hogens, Marcel Dekker Inc., New York, NY, pp. 327-445. D'Aoust, J.-Y. (t991) Pathogenicity of fi~odborne Sahnonella. int. J. Food Microbiol. 12, 17-40, D'Aoust. J.-Y., Sewell, A. and Jean, A. (19911) Limited sensitivity of short (6 h) selective enrichment for detection of fi~odborne Sahmmella. J. Food Prot. 53, 562-565. de Fatima, M. and Gomes, F.F. (1985) Isolation of Sabnonelht from liver and evaluation of enrichment broths, time and temperature of incubation. Rev. Microbiol. (Sao Paulo) 16. 211-221. Fricker, C.R. (1984) A comparison of methods for the isolation of salmonellae from sewage sludge. Zbl. Bakt. ltyg. I. Abt. Orig. B 179, 170-178. Fricker, C.F,., Quail, E., McGibbon, L. and Girdwood, R.W.A. (1985)An evaluation of commercially available dehydrated Rappaport-Vassiliadis medium for the isolation of salmonellae from poultry. J, tlyg. 95, 337-344. Gabis, D.A. and Langlois, B.E. (1967) Effect of cocoa powder used in chocolate milk on bacterial growth. J. Dairy Sci. 5(1, 945. Gabis, D.A, and Silliker, J.lt. (1974) ICMSF methods studies. V1, The influence of selective enrichment media and incubation temperatures on the detection of Sahnonella in dried fl~ods and feeds. Can. J. Microbiol. 20, 1509-151 I. Harvey, R.W.S. and Price, T.H. (!981) Comparison of selenite F, Muller-Kauffmann tetrathionate and Rappaport's medium for Sahnonella isolation from chicken giblets after pre-enrichment in buffered peptone water. J. Hyg. 87, 219-224. Health Protection Branch (1978) Methods for the isolation and identification of Sahnonella from foods. Method MF HPB-20, Health and Welfare Canada, Ottawa. International Organization for Standardization (1990) International Standard 6579. Microbiology-General guidance on methods for the detection of Sahnonella. ISO, Geneva. Kalapothaki, V., Trichopoulos, D., Papadakis, J., Mavrommati, Ch. and Vassiliadis, P. (1986) A comparison of the efficiency of two forms of Rappaport-Vassiliadis enrichment medium, int. J. Food Microbiol. 3, 89-97. Leifson, E. (1936) New selenite enrichment media for the isolation of typhoid and paratyphoid (Sahnonella) bacilli. Am. J. Hyg. 24, 423-432. North, W.R. and Bartram, M.T. (1953). The efficiency of selenite broth of different compositions in the isolation of Sahnonella. Appl. Microbiol. 1, 130-134. Rengel, A. and Mendoza, S. (1984) Isolation of Sabnonella from raw chicken in Venezuela. J. Food Prot. 47, 213-216.
130 Rhodes, P., Ouesnel, L.B. and Collard. P. (1985) Growth kinetics of mixed culture in salmonella enrichment media. J. Appl. Bactcriol. 59, 231-237. U.S. Food and Drug Administration (1984) Bacteriological Analytical Manual (6th edilion). Association of Official Analytical Chemists. Washington, DC. wm Schothorst, M. and Renaud, A.M. (19831 Dynamics of Sahnonella isolation with Rappaport's medium (R I(J). J. Appl. Bacleriol. 54. 2(1q~215. Vassiliadis, P.. Trichopouios, D., Pai0adakis, J.. Kalapolhaki. V., Zavitsanos, X. and Serie, Ch. (19811 Sahnrmelhl isolation with Rappaporl's enrichment medium of different composition. Zbl. Bakt. ltyg. i. Abt. ()rig. B 173, 382-389. World Health Organization (Iq88) Salmoncllosis control: the role of animal and product hygiene. Technical Report Series no. 774. World !!ealth Org;mization. Geneva, 83 pp. Yde, M. and Ghysels, G. (19841 Performance of several enrichment media in the isolation of salmonellae from liquid egg products. J. Food Prot. 47, 217-210. Zapatka, F.A., Varney, G.W. and Sinskey, A.J. ( 19771 Neutralization of the bactericidal effect ~f ct)coa powder on salmonellae by casein. J. Appl. Bacteriol. 42.21-25.
