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Collaborative ring-trial of Dynabeads® anti-Salmonella for immunomagnetic separation of stressed Salmonella cells from herbs and spices
International
Journal
of Food Microbiology
International
Journal
of
Food Microbiology 29 (1996) 41-47
Collaborative ring-trial of Dynabeads” anti-Salmonella for immunomagnetic separation of stressed Salmonella cells from herbs and spices Lucielle
Mansfield,
Steve
Forsythe
*
Department of Life Sciences, The Nottingham Trent Unil;ersity, Clifton Lane, Nottingham, NGII SNS, UK
Received 26 January 1995; accepted 10 April 1995
Abstract Eight laboratories participated in a Salmonella detection ring-trial which compared selective enrichment by conventional broths with immunomagnetic separation (IMS) using Dynabeads” Anti-Salmonella. Laboratories analyzed six types of herbs and spices that were spiked with one of six freeze-dried Sa/monelZa species. Each herb and spice analysis comprised of 12 samples (25 g each) which had been spiked at three different levels, plus a negative control and stored for one week prior to testing. Out of a total 468 samples analyzed, 19.5 (41.7%) were positive by both methods. Eighteen samples were positive only by IMS enrichment, in comparison with 19 positive samples by conventional enrichment broths and not IMS. These results confirm the potential use of IMS as an alternative to enrichment broths for Salmonella isolation. Keywords:
Salmonella;
Immunomagnetic
separation;
Rapid
detection;
Ring-trial
1. Introduction
Salmonellae have persisted as one of the major food poisoning micro-organisms despite improvements in food hygiene by legislation and education. The illness is characterised by diarrhoea and a fever 24 h after ingestion and is self-limiting in healthy adults. Detection of salmonellae in foodstuffs has been well researched. However, the conventional US and EU methods still require a total period of 72-96 h before a result is obtained. This considerable time is due to the three
* Correponding
author. Tel.: 01159 418418. Fax: 01159 486636.
0168-1605/96/$15.00 0 1996 Elsevier Science B.V. Ail rights reserved SSDIO168-1605(95)00019-4
42
L. Mansfield, S. Forsythe / Int. J. Food Microbiology 29 (19961 41-47
incubation periods (pre-enrichment, enrichment and selection on solid media) required to isolate and confirm the presence of Salmonellu cells in a food. Therefore ,Salmonellu detection continues to be an area for methodology improvement, in particular procedures that shorten the detection and confirmation period. Immunomagnetic separation (IMS) using paramagnetic particles coated with anti-Salmonella antibodies has been described for the selective isolation of Salmonella from foods (Skjerve and Olsvik, 1991). Subsequently, commercially available Dynabeads” Anti-Salmonella (Dynal (UK) Ltd.) have been used in comparative studies of IMS with conventional enrichment broths. Mansfield and Forsythe (1993) tested the Dynabeads” Anti-Salmonella IMS technique with 60 foods, half of which were spiked with one of five different Salmonella strains. They concluded that the IMS procedure was as efficient as the three conventional enrichment broths; selenite cystine, Miilier-Kauffmann tetrathionate and Rappaport-Vassiliadis. In addition, only IMS recovered S. give from garlic granules. This would infer that sublethally injured cells might be recovered more efficiently by IMS from foodstuffs and hence reduce the number of false negative results. Cudjoe et al. (1994) evaluated the Dynabeads” Anti-Salmonella IMS procedure for 180 poultry samples and concluded that IMS was superior to the conventional Salmonella methodology. These studies have shown that IMS could be used as an alternative to conventional enrichment broths and hence eliminate the overnight enrichment incubation period. Herbs and spices can act as vectors for salmonellae (D’Aoust, 1994). These are health hazards since they are frequently added to ready-to-eat foods and cooked foods. Recently, S. javiana has been associated with a Salmonella outbreak associated with pepper and paprika powder (Anon., 1994). Salmonella detection in herbs and spices is complicated due to the presence of organosulfur compounds and essential oils which have a bactericidal affect. The Association of Official Analytical Chemists (AOAC) standard EU and US protocols attempt to overcome the antimicrobial activity by pre-enrichment in buffered peptone water either using high dilutions (such as 25 g/2500 ml BPW) or neutralisation with sulphite (Association of Official Analytical Chemists, 1984). Therefore, a collaborative ring-trial using herb and spices ensures that the Salmonella recovery is assessed using potentially stressed cells. The following study was designed to evaluate the Dynabeads” Anti-Salmonella IMS technique with stressed Salmonellu cells by eight independent laboratories.
2. Materials
and methods
General ring-trial design. Eight laboratories, which routinely tested food products for Safmonellu as part of their quality assurance programme, participated in the ring-trial. The Department of Life Sciences at The Nottingham Trent University prepared the spiked samples and acted as a reference laboratory. Each laboratory enriched the spiked herb and spice samples by immunomagnetic separation (IMS) using Dynabeads” Anti-Salmonella (Dynal (UK) Ltd.) and their accred-
L. Mansfield, S. Forsythe / Int. J. Food Microbiology 29 (1996) 41-47 Table 1 Salmonella recovery
by eight laboratories
Laboratory
Enrichment broth(s)
Selective
agar
1 2 3 4 5 6 7 8
RV = RV+SC b RV+SC b RV+SC’ RV+SC b RV RV+MT’ SC+MT
BGAd +XLD = BGAd +XLD e MLCB f + XLD MLCB f + XLD MLCB f + XLD BGA + MLCB BGA+XLD BGA + XLD
Total a Rappaport-Vassiliadis broth. b Selenite cystine broth. ’ Miiller-Kauffmann broth. d Brilliant Green Agar. e Xylose Lysine Desoxycholate agar. f Mannitol Lysine Crystal Violet Brilliant
according
to enrichment
Number
of positive
broth
and selective
Salmonella samples
43
agar procedures Total number of samples analyzed
Immunomagnetic separation
Conventional enrichment broth
20 31 33 33 37 35 13 11
20 34 32 37 40 27 12 12
48 72 60 60 72 72 48 36
213
214
468
Green
agar.
ited conventional method (Table 1 shows the credited procedure for each laboratory). Samples were not treated before spiking and therefore contained their normal flora. Preliminary studies showed the samples were not naturally contaminated by salmonellae. The samples were spiked with freeze-dried salmonellae according to Association of Official Chemists (AOAC) guidelines (Andrews, 1987). The samples were encoded with a random number, stored for one week at room temperature and posted to participating laboratories for receipt on a Monday morning. The laboratories analyzed the samples on the day of receipt. After analysis the results were sent to the reference laboratory for decoding. Laboratories were encoded by random and their identities were not revealed to any participating laboratory. The reference laboratory analyzed encoded samples at the same time as the participating laboratories. This was for confirmatory purposes only and are not included in the ring-trial data. Participating laboratories. A total of eight UK laboratories of national and multinational companies or government-funded food research institutes took part in the ring-trial. Participants which can be named were Dalgety, Rank Hovis MacDougal, McCormick Glentham, Lucas Ingredients, British Analytical Control Ltd. (of English Grain) and Chippen Campden Food & Drink Research Institute. Participation in the ring-trial does not indicate Dynabeads@ Anti-Salmonella product endorsement. Preparation of spiked samples. Salmonella urizonue, S. euling (Group O), S. enteritidis (Group D), S. give (Group E), S. typhimutium (Group B) and S. virchow (Group C> were used. These were from the culture collection in the reference
44
L. Mansfield,
S. Forsythe / Int. J. Food Microbiology
29 (1996) 41-47
laboratory. Salmonella cultures were freeze-dried after overnight growth in Tryptone Soya Broth (Oxoid CM129). The viability of the cultures was determined by standard plate counts before and after freeze-drying. The six herbs and spices were white pepper, paprika powder, garlic powder, onion powder, oregano and compound flavouring. Each herb and spice was inoculated with one of the six freeze-dried Salmonella cultures. Twelve samples of each herb and spice were prepared per laboratory. This comprised of one uninoculated negative control, five spiked at a ‘low’ level (10 cells/25 g), five spiked at a ‘high’ level (1000 ceils/25 g) and one referred to as ‘very high’ (10000 cells/25 g). The 12 samples were assigned a random number by a member of staff at the reference laboratory who did not participate in any Salmonella detection. The level of Salmonella inoculation for the ring-trial samples was determined in preliminary experiments at the reference laboratory. This was achieved by spiking 25 g quantities of the herb or spice with tenfold decreasing amounts of the freeze-dried Salmonella strain in triplicate. The samples were stored for one week before analysis with IMS and Rappaport-Vassiliadis enrichment broth procedures as described below. Pre-enrichment dilution factor. Laboratories were informed of the appropriate pre-enrichment dilution factors: white pepper l:lO, paprika l:lO, oregano l:lOO, garlic and onion 1:lO + 0.5% potassium sulphite (Association of Official Analytical Chemists, 1984). Preliminary experiments by the reference laboratory established that none of the Sulmonellu strains were sensitive to sodium or potassium sulphite. A 1:lOO dilution was recommended by the reference laboratory for compound flavouring due to its high salt concentration (20% w/w>. AI1 pre-enrichments were for 18 h at 37°C in buffered peptone water (BPW; Oxoid, CM509). Comlentionul enrichment broth and selectirje agur procedure Selenite cystine (9.0 ml, SC; Oxoid CM395 + L121), Rappaport-Vassiliadis (9.9 ml, RV; CM866, Oxoid) and Miiller-Kauffmann tetrathionate (9 ml, MT; Oxoid CM343) broths were inoculated with 1, 0.1 and 1 ml aliquots of the BPW pre-enrichment broths, respectively. SC and MT were incubated at 37°C RV at 42°C. After 24 and 48 h, a loopful was streaked on Xylose Lysine Desoxycholate (XLD; Oxoid CM469), Brilliant Green (BGA; Oxoid CM263) or Mannitol Lysine Crystal Violet Brilliant Green (MLCB; Oxoid CM783) agar plates which were incubated at 37°C overnight. Accredited procedures for each laboratory are given in Table 1. Immunomugnetic separation procedure. Salmonella cells were immunomagnetitally separated with Dynabeads” Anti-Salmonella as according to the manufacturer’s instructions (Dynal (UK) Ltd., Wirral, UK). Aliquots (1 ml) of BPW pre-enrichment broth were mixed with 20 ~1 of Dynabeads” Anti-Salmonella (Dynal (UK) Ltd.) in an Eppendorf tube and incubated for 10 min. Beads were separated using a magnetic particle concentrator (MPC-M; Dynal (UK) Ltd.) for 3 min. The beads were resuspended in 1 ml washing buffer (phosphate buffered saline, pH 7.4, containing 0.05% Tween 20>, separated and resuspended in 50 ~1 washing buffer. Aliquots (25 ~1) were streaked on XLD and BGA plates for overnight incubation at 37°C. Confirmation. Characteristic Salmonella colonies from selective agars were
L. Mansfield, S. Forsythe / Inl. J. Food Microbiology 29 (I 996) 41-47
4.5
confirmed by serology tests for 0 and H antigens and biochemical kits (API 20E, bioMerieux (UK) Ltd., Basingstoke, UK).
3. Results and discussion The participating laboratories were associated with national and multinational food companies and analyzed the herb and spice samples as part of their weekly SalmoneZla quality assurance programme. For internal quality control purposes it was not possible for all laboratories to use identical enrichment broths and selective agars. However, their documented accredited procedures were used because of the commercial importance of accurate Salmonella detection by the laboratories concerned. For confirmatory purposes the reference laboratory analyzed samples at the same time as the participants. However their results were not used as part of the ring-trial and are not presented. Two laboratories recovered salmonellae from the negative (uninoculated) controls of two herbs and spice types. These were taken as cross-contaminated samples since the reference laboratory had previous analyzed, in triplicate, the herbs and spices without recovering salmonellae and also the biochemical and serological profile of the recovered salmonellae matched the spiked Salmonella strain. Where cross-contamination occurred the results for that laboratory’s analysis of the herb or spice was excluded from the ring-trial. This left a total of 468 samples for analysis. No Salmonella cells were detected by either technique in 236/468 samples (50.4%). The large number of negative results was expected due to the use of negative (uninoculated) controls and samples spiked with freeze-dried salmonellae at low numbers (ca 10 cells/25 g). Additional factors leading to cell death include sample storage for one week prior to analysis and that the herbs and spices contain antibacterial agents, The recovery of salmonellae from each herb and spice is shown in Table 2. Both IMS and conventional enrichment broths recovered salmonellae from 195/468 (41.7%) samples by both IMS and conventional enrichment techniques. Salmonellae were recovered by IMS and not conventional enrichment broths from 18 samples. Whereas conventional enrichment broths recovered salmonellae from 19 samples which were negative by IMS. Certain herbs and spices, such as garlic, contain organosulphur compounds (Block, 1992). Hence the recommended Salmonella detection procedure is to supplement BPW (the pre-enrichment stage) with potassium sulphite as a neutralising agent (Association of Official Analytical Chemists, 1984). Recovery of salmonellae was notably low from garlic powder (Table 2). Three garlic powder samples were positive by both techniques and five samples were positive by IMS only. The low Salmonella recovery was confirmed by the reference laboratory. They recovered S. uirchow from two samples by both techniques, seven samples by IMS alone. No samples were positive by conventional enrichment broths alone. It is plausible that potassium sulphite did not fully protect the Salmonella cells during the pre-enrichment stage and that sublethally injured cells were still present which were unable to survive the inhibitory factors in the enrichment broths.
L. Mansfield, S. Forsythe / Int. J. Food Microbiology 29 (1996) 41-47
46 Table 2 Comparison conventional
of Salmonella recoveries enrichment techniques
Herb and spice a
Salmonella strain
from
herbs
S. S. S. S. S. S.
Total number Percentage (%)
_ _
” Herbs and h Number of ’ Number of ’ Number of ’ Number of
typhimurium enteritidis ealing rirchow gice arizonae
spices
by immunomagnetic
Number of Salmonella samples detected by immunomagnetic separation and conventional enrichment techniques Positivepositive h
White peper Paprika Onion powder Garlic Oregano Compound flavouring
and
Positivenegative ’
Negativepositive d
separation
and
Total number of samples
Negativenegative e
58 24 21 3 35 54
0 5 4 5 4 0
5 4 6 0 3 1
21 27 53 X8 42 5
195 41.7
18 3.8
19 4.1
236 50.4
84 60 84 96 84 60 468 100
spices are presented in order of analysis. samples positive by both techniques, false negative results by conventional enrichment broth techniques. false negative results by immunomagnetic separation technique. samples from which Salmonella were not recovered by either techniques.
Therefore IMS recovered sublethally injured Salmonella cells which were not resuscitated in standard enrichment broths. The results confirm a previous report of the preferential recovery of Salmonellu from garlic using IMS (Mansfield and Forsythe, 1993). A ring-trial analyses the efficiency of a procedure as used by a number of independent laboratories. However inter-laboratory variations exist due differences in staff experience and laboratory management. The enrichment and selection procedures of the participating laboratories is given in Table 1. All laboratories except one used Rappaport-Vassiliadis as one of the enrichment media. Only five laboratories used selenite cystine and two used Miiller-Kauffman tetrathionate broth. BGA and XLD were the most commonly used selective agars. Six laboratories used two enrichment broths and hence this could increase the chance of Salmonella recovery by conventional methods in contrast to the single IMS procedure. Nevertheless salmonellae were recovered from a total of 213 samples with IMS compared with 214 by conventional enrichment techniques. Recovery of Salmonella cells varied between laboratories using identical enrichment and selection media (Table 1). Therefore the results do not indicate that any particular combination of enrichment broths and selective agars was the most effective for Salmonella recovery. In conclusion, in a ring-trial involving eight independent laboratories Salmonellu cells were recovered from herb and spice samples almost as frequently with IMS as with conventional enrichment broths (213 and 214 samples, respectively). Salmonellae were recovered from 18 samples which were negative when tested
L. Mansfield, S. Forsythe /Int. J. Food Microbiology 29 (1996) 41-47
47
using conventional enrichment broths, whereas salmonellae were detected in 19 samples using conventional enrichment broths which were negative with IMS. The IMS procedure takes 30 min to capture Salmonella cells from the pre-enrichment broth. Hence, the IMS procedure can be used as an alternative to the conventional overnight enrichment period and therefore reduce the standard Salmonella detection period.
Acknowledgements
L.M. and S.F. are grateful to Pete Maguire (Dynal (UK) Ltd) for his support of this ring-trial and to all the participants for their co-operation.
References Andrews, W.H. (1987) Recommendations for preparing test samples for AOAC collaborative studies of microbiological procedures for foods. J. Assoc. Offic. Anal. Chem. 70. 931-936. Anon. (1994) Salmonella jauiana in Europe. Commun. Dis. Report (PHLS) 4, 61. Association of Official Analytical Chemists (1984). Bacteriological Analytical Manual, 6th edn. AOAC, Arlington, VA. Block, E. (1992) The organosulfur chemistry of the genus Allium - implications for the organic chemistry of sulfur. Angeur. Chem. Int. (ed. Eng.) 31, 1135-1178. Cudjoe, K., Krona, R. and Olsen, E. (1994) IMS: a new selective enrichment technique for detection of Salmonella in foods. Int. J. Food Microbial. 23, 159-165. D’Aoust, J.-Y. (1994) Salmonella and the international food trade. Int. J. Food Microbial. 24, 11-31. Mansfield, L.P. and Forsythe, S.J. (1993) Immunomagnetic separation as an alternative to enrichment broths for Salmonella detection. Lett. Appl. Microbial. 16, 122-125. Parmar, N., Easter, M.C. and Forsythe, S.J. (1992) The detection of Salmonella enteritidis and S. typhimurium using immunomagnetic separation and conductance microbiology. Lett. Appl. Microbiol. 1.5, 175-178. Skjerve, E. and Olsvik, 0. (1991) Immunoseparation of Salmonella from foods. Int. J. Food Microbial. 14. 11-18.
Journal
of Food Microbiology
International
Journal
of
Food Microbiology 29 (1996) 41-47
Collaborative ring-trial of Dynabeads” anti-Salmonella for immunomagnetic separation of stressed Salmonella cells from herbs and spices Lucielle
Mansfield,
Steve
Forsythe
*
Department of Life Sciences, The Nottingham Trent Unil;ersity, Clifton Lane, Nottingham, NGII SNS, UK
Received 26 January 1995; accepted 10 April 1995
Abstract Eight laboratories participated in a Salmonella detection ring-trial which compared selective enrichment by conventional broths with immunomagnetic separation (IMS) using Dynabeads” Anti-Salmonella. Laboratories analyzed six types of herbs and spices that were spiked with one of six freeze-dried Sa/monelZa species. Each herb and spice analysis comprised of 12 samples (25 g each) which had been spiked at three different levels, plus a negative control and stored for one week prior to testing. Out of a total 468 samples analyzed, 19.5 (41.7%) were positive by both methods. Eighteen samples were positive only by IMS enrichment, in comparison with 19 positive samples by conventional enrichment broths and not IMS. These results confirm the potential use of IMS as an alternative to enrichment broths for Salmonella isolation. Keywords:
Salmonella;
Immunomagnetic
separation;
Rapid
detection;
Ring-trial
1. Introduction
Salmonellae have persisted as one of the major food poisoning micro-organisms despite improvements in food hygiene by legislation and education. The illness is characterised by diarrhoea and a fever 24 h after ingestion and is self-limiting in healthy adults. Detection of salmonellae in foodstuffs has been well researched. However, the conventional US and EU methods still require a total period of 72-96 h before a result is obtained. This considerable time is due to the three
* Correponding
author. Tel.: 01159 418418. Fax: 01159 486636.
0168-1605/96/$15.00 0 1996 Elsevier Science B.V. Ail rights reserved SSDIO168-1605(95)00019-4
42
L. Mansfield, S. Forsythe / Int. J. Food Microbiology 29 (19961 41-47
incubation periods (pre-enrichment, enrichment and selection on solid media) required to isolate and confirm the presence of Salmonellu cells in a food. Therefore ,Salmonellu detection continues to be an area for methodology improvement, in particular procedures that shorten the detection and confirmation period. Immunomagnetic separation (IMS) using paramagnetic particles coated with anti-Salmonella antibodies has been described for the selective isolation of Salmonella from foods (Skjerve and Olsvik, 1991). Subsequently, commercially available Dynabeads” Anti-Salmonella (Dynal (UK) Ltd.) have been used in comparative studies of IMS with conventional enrichment broths. Mansfield and Forsythe (1993) tested the Dynabeads” Anti-Salmonella IMS technique with 60 foods, half of which were spiked with one of five different Salmonella strains. They concluded that the IMS procedure was as efficient as the three conventional enrichment broths; selenite cystine, Miilier-Kauffmann tetrathionate and Rappaport-Vassiliadis. In addition, only IMS recovered S. give from garlic granules. This would infer that sublethally injured cells might be recovered more efficiently by IMS from foodstuffs and hence reduce the number of false negative results. Cudjoe et al. (1994) evaluated the Dynabeads” Anti-Salmonella IMS procedure for 180 poultry samples and concluded that IMS was superior to the conventional Salmonella methodology. These studies have shown that IMS could be used as an alternative to conventional enrichment broths and hence eliminate the overnight enrichment incubation period. Herbs and spices can act as vectors for salmonellae (D’Aoust, 1994). These are health hazards since they are frequently added to ready-to-eat foods and cooked foods. Recently, S. javiana has been associated with a Salmonella outbreak associated with pepper and paprika powder (Anon., 1994). Salmonella detection in herbs and spices is complicated due to the presence of organosulfur compounds and essential oils which have a bactericidal affect. The Association of Official Analytical Chemists (AOAC) standard EU and US protocols attempt to overcome the antimicrobial activity by pre-enrichment in buffered peptone water either using high dilutions (such as 25 g/2500 ml BPW) or neutralisation with sulphite (Association of Official Analytical Chemists, 1984). Therefore, a collaborative ring-trial using herb and spices ensures that the Salmonella recovery is assessed using potentially stressed cells. The following study was designed to evaluate the Dynabeads” Anti-Salmonella IMS technique with stressed Salmonellu cells by eight independent laboratories.
2. Materials
and methods
General ring-trial design. Eight laboratories, which routinely tested food products for Safmonellu as part of their quality assurance programme, participated in the ring-trial. The Department of Life Sciences at The Nottingham Trent University prepared the spiked samples and acted as a reference laboratory. Each laboratory enriched the spiked herb and spice samples by immunomagnetic separation (IMS) using Dynabeads” Anti-Salmonella (Dynal (UK) Ltd.) and their accred-
L. Mansfield, S. Forsythe / Int. J. Food Microbiology 29 (1996) 41-47 Table 1 Salmonella recovery
by eight laboratories
Laboratory
Enrichment broth(s)
Selective
agar
1 2 3 4 5 6 7 8
RV = RV+SC b RV+SC b RV+SC’ RV+SC b RV RV+MT’ SC+MT
BGAd +XLD = BGAd +XLD e MLCB f + XLD MLCB f + XLD MLCB f + XLD BGA + MLCB BGA+XLD BGA + XLD
Total a Rappaport-Vassiliadis broth. b Selenite cystine broth. ’ Miiller-Kauffmann broth. d Brilliant Green Agar. e Xylose Lysine Desoxycholate agar. f Mannitol Lysine Crystal Violet Brilliant
according
to enrichment
Number
of positive
broth
and selective
Salmonella samples
43
agar procedures Total number of samples analyzed
Immunomagnetic separation
Conventional enrichment broth
20 31 33 33 37 35 13 11
20 34 32 37 40 27 12 12
48 72 60 60 72 72 48 36
213
214
468
Green
agar.
ited conventional method (Table 1 shows the credited procedure for each laboratory). Samples were not treated before spiking and therefore contained their normal flora. Preliminary studies showed the samples were not naturally contaminated by salmonellae. The samples were spiked with freeze-dried salmonellae according to Association of Official Chemists (AOAC) guidelines (Andrews, 1987). The samples were encoded with a random number, stored for one week at room temperature and posted to participating laboratories for receipt on a Monday morning. The laboratories analyzed the samples on the day of receipt. After analysis the results were sent to the reference laboratory for decoding. Laboratories were encoded by random and their identities were not revealed to any participating laboratory. The reference laboratory analyzed encoded samples at the same time as the participating laboratories. This was for confirmatory purposes only and are not included in the ring-trial data. Participating laboratories. A total of eight UK laboratories of national and multinational companies or government-funded food research institutes took part in the ring-trial. Participants which can be named were Dalgety, Rank Hovis MacDougal, McCormick Glentham, Lucas Ingredients, British Analytical Control Ltd. (of English Grain) and Chippen Campden Food & Drink Research Institute. Participation in the ring-trial does not indicate Dynabeads@ Anti-Salmonella product endorsement. Preparation of spiked samples. Salmonella urizonue, S. euling (Group O), S. enteritidis (Group D), S. give (Group E), S. typhimutium (Group B) and S. virchow (Group C> were used. These were from the culture collection in the reference
44
L. Mansfield,
S. Forsythe / Int. J. Food Microbiology
29 (1996) 41-47
laboratory. Salmonella cultures were freeze-dried after overnight growth in Tryptone Soya Broth (Oxoid CM129). The viability of the cultures was determined by standard plate counts before and after freeze-drying. The six herbs and spices were white pepper, paprika powder, garlic powder, onion powder, oregano and compound flavouring. Each herb and spice was inoculated with one of the six freeze-dried Salmonella cultures. Twelve samples of each herb and spice were prepared per laboratory. This comprised of one uninoculated negative control, five spiked at a ‘low’ level (10 cells/25 g), five spiked at a ‘high’ level (1000 ceils/25 g) and one referred to as ‘very high’ (10000 cells/25 g). The 12 samples were assigned a random number by a member of staff at the reference laboratory who did not participate in any Salmonella detection. The level of Salmonella inoculation for the ring-trial samples was determined in preliminary experiments at the reference laboratory. This was achieved by spiking 25 g quantities of the herb or spice with tenfold decreasing amounts of the freeze-dried Salmonella strain in triplicate. The samples were stored for one week before analysis with IMS and Rappaport-Vassiliadis enrichment broth procedures as described below. Pre-enrichment dilution factor. Laboratories were informed of the appropriate pre-enrichment dilution factors: white pepper l:lO, paprika l:lO, oregano l:lOO, garlic and onion 1:lO + 0.5% potassium sulphite (Association of Official Analytical Chemists, 1984). Preliminary experiments by the reference laboratory established that none of the Sulmonellu strains were sensitive to sodium or potassium sulphite. A 1:lOO dilution was recommended by the reference laboratory for compound flavouring due to its high salt concentration (20% w/w>. AI1 pre-enrichments were for 18 h at 37°C in buffered peptone water (BPW; Oxoid, CM509). Comlentionul enrichment broth and selectirje agur procedure Selenite cystine (9.0 ml, SC; Oxoid CM395 + L121), Rappaport-Vassiliadis (9.9 ml, RV; CM866, Oxoid) and Miiller-Kauffmann tetrathionate (9 ml, MT; Oxoid CM343) broths were inoculated with 1, 0.1 and 1 ml aliquots of the BPW pre-enrichment broths, respectively. SC and MT were incubated at 37°C RV at 42°C. After 24 and 48 h, a loopful was streaked on Xylose Lysine Desoxycholate (XLD; Oxoid CM469), Brilliant Green (BGA; Oxoid CM263) or Mannitol Lysine Crystal Violet Brilliant Green (MLCB; Oxoid CM783) agar plates which were incubated at 37°C overnight. Accredited procedures for each laboratory are given in Table 1. Immunomugnetic separation procedure. Salmonella cells were immunomagnetitally separated with Dynabeads” Anti-Salmonella as according to the manufacturer’s instructions (Dynal (UK) Ltd., Wirral, UK). Aliquots (1 ml) of BPW pre-enrichment broth were mixed with 20 ~1 of Dynabeads” Anti-Salmonella (Dynal (UK) Ltd.) in an Eppendorf tube and incubated for 10 min. Beads were separated using a magnetic particle concentrator (MPC-M; Dynal (UK) Ltd.) for 3 min. The beads were resuspended in 1 ml washing buffer (phosphate buffered saline, pH 7.4, containing 0.05% Tween 20>, separated and resuspended in 50 ~1 washing buffer. Aliquots (25 ~1) were streaked on XLD and BGA plates for overnight incubation at 37°C. Confirmation. Characteristic Salmonella colonies from selective agars were
L. Mansfield, S. Forsythe / Inl. J. Food Microbiology 29 (I 996) 41-47
4.5
confirmed by serology tests for 0 and H antigens and biochemical kits (API 20E, bioMerieux (UK) Ltd., Basingstoke, UK).
3. Results and discussion The participating laboratories were associated with national and multinational food companies and analyzed the herb and spice samples as part of their weekly SalmoneZla quality assurance programme. For internal quality control purposes it was not possible for all laboratories to use identical enrichment broths and selective agars. However, their documented accredited procedures were used because of the commercial importance of accurate Salmonella detection by the laboratories concerned. For confirmatory purposes the reference laboratory analyzed samples at the same time as the participants. However their results were not used as part of the ring-trial and are not presented. Two laboratories recovered salmonellae from the negative (uninoculated) controls of two herbs and spice types. These were taken as cross-contaminated samples since the reference laboratory had previous analyzed, in triplicate, the herbs and spices without recovering salmonellae and also the biochemical and serological profile of the recovered salmonellae matched the spiked Salmonella strain. Where cross-contamination occurred the results for that laboratory’s analysis of the herb or spice was excluded from the ring-trial. This left a total of 468 samples for analysis. No Salmonella cells were detected by either technique in 236/468 samples (50.4%). The large number of negative results was expected due to the use of negative (uninoculated) controls and samples spiked with freeze-dried salmonellae at low numbers (ca 10 cells/25 g). Additional factors leading to cell death include sample storage for one week prior to analysis and that the herbs and spices contain antibacterial agents, The recovery of salmonellae from each herb and spice is shown in Table 2. Both IMS and conventional enrichment broths recovered salmonellae from 195/468 (41.7%) samples by both IMS and conventional enrichment techniques. Salmonellae were recovered by IMS and not conventional enrichment broths from 18 samples. Whereas conventional enrichment broths recovered salmonellae from 19 samples which were negative by IMS. Certain herbs and spices, such as garlic, contain organosulphur compounds (Block, 1992). Hence the recommended Salmonella detection procedure is to supplement BPW (the pre-enrichment stage) with potassium sulphite as a neutralising agent (Association of Official Analytical Chemists, 1984). Recovery of salmonellae was notably low from garlic powder (Table 2). Three garlic powder samples were positive by both techniques and five samples were positive by IMS only. The low Salmonella recovery was confirmed by the reference laboratory. They recovered S. uirchow from two samples by both techniques, seven samples by IMS alone. No samples were positive by conventional enrichment broths alone. It is plausible that potassium sulphite did not fully protect the Salmonella cells during the pre-enrichment stage and that sublethally injured cells were still present which were unable to survive the inhibitory factors in the enrichment broths.
L. Mansfield, S. Forsythe / Int. J. Food Microbiology 29 (1996) 41-47
46 Table 2 Comparison conventional
of Salmonella recoveries enrichment techniques
Herb and spice a
Salmonella strain
from
herbs
S. S. S. S. S. S.
Total number Percentage (%)
_ _
” Herbs and h Number of ’ Number of ’ Number of ’ Number of
typhimurium enteritidis ealing rirchow gice arizonae
spices
by immunomagnetic
Number of Salmonella samples detected by immunomagnetic separation and conventional enrichment techniques Positivepositive h
White peper Paprika Onion powder Garlic Oregano Compound flavouring
and
Positivenegative ’
Negativepositive d
separation
and
Total number of samples
Negativenegative e
58 24 21 3 35 54
0 5 4 5 4 0
5 4 6 0 3 1
21 27 53 X8 42 5
195 41.7
18 3.8
19 4.1
236 50.4
84 60 84 96 84 60 468 100
spices are presented in order of analysis. samples positive by both techniques, false negative results by conventional enrichment broth techniques. false negative results by immunomagnetic separation technique. samples from which Salmonella were not recovered by either techniques.
Therefore IMS recovered sublethally injured Salmonella cells which were not resuscitated in standard enrichment broths. The results confirm a previous report of the preferential recovery of Salmonellu from garlic using IMS (Mansfield and Forsythe, 1993). A ring-trial analyses the efficiency of a procedure as used by a number of independent laboratories. However inter-laboratory variations exist due differences in staff experience and laboratory management. The enrichment and selection procedures of the participating laboratories is given in Table 1. All laboratories except one used Rappaport-Vassiliadis as one of the enrichment media. Only five laboratories used selenite cystine and two used Miiller-Kauffman tetrathionate broth. BGA and XLD were the most commonly used selective agars. Six laboratories used two enrichment broths and hence this could increase the chance of Salmonella recovery by conventional methods in contrast to the single IMS procedure. Nevertheless salmonellae were recovered from a total of 213 samples with IMS compared with 214 by conventional enrichment techniques. Recovery of Salmonella cells varied between laboratories using identical enrichment and selection media (Table 1). Therefore the results do not indicate that any particular combination of enrichment broths and selective agars was the most effective for Salmonella recovery. In conclusion, in a ring-trial involving eight independent laboratories Salmonellu cells were recovered from herb and spice samples almost as frequently with IMS as with conventional enrichment broths (213 and 214 samples, respectively). Salmonellae were recovered from 18 samples which were negative when tested
L. Mansfield, S. Forsythe /Int. J. Food Microbiology 29 (1996) 41-47
47
using conventional enrichment broths, whereas salmonellae were detected in 19 samples using conventional enrichment broths which were negative with IMS. The IMS procedure takes 30 min to capture Salmonella cells from the pre-enrichment broth. Hence, the IMS procedure can be used as an alternative to the conventional overnight enrichment period and therefore reduce the standard Salmonella detection period.
Acknowledgements
L.M. and S.F. are grateful to Pete Maguire (Dynal (UK) Ltd) for his support of this ring-trial and to all the participants for their co-operation.
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