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Occurrence of Listeria monocytogenes in food in Chile
International Journal of Food Microbiology 70 Ž2001. 175–178 www.elsevier.comrlocaterijfoodmicro
Short communication
Occurrence of Listeria monocytogenes in food in Chile Ana Marıa ´ Cordano a,) , Joselyne Rocourt b a
b
Seccion de Chile, Casilla 48, Santiago, Chile ´ Microbiologıa ´ de Alimentos, Instituto de Salud Publica ´ Laboratoire des Listeria, Centre Collaborateur de l’OMS pour la listeriose d’origine alimentaire, Institut Pasteur, Paris, France Received 9 June 2000; received in revised form 25 March 2001; accepted 20 April 2001
Abstract Out of 2145 food samples analysed 77 were found contaminated with Listeria monocytogenes in Santiago, Chile. Samples were: 603 ice-cream Ž3.5% contaminated., 256 soft cheese Ž0.8%., 155 hard cheese Ž0%., 229 baby milk bottles Ž0%., 634 processed meat products Ž3.6%. and 268 crustaceous shellfish Ž11.6%.. Three different isolation media were used: for 318 samples, Modified McBride Agar ŽMMA., Lithium chloride Phenylethanol Moxalactam agar, and Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar; for 1827 samples MMA was replaced by Listeria Selective Agar Oxford Formulation. Isolates were classified as follow: serovar 1r2a Ž25 isolates., serovar 4b Ž20., serovar 1r2b Ž19., serovar 3b Ž7., serovar 1r2c Ž2., untypable Ž4.. A high variety of phagovars was detected although 52% of strains was untypable. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Chilean food; Serotyping; Phagetyping
1. Introduction Listeriosis has emerged as a major foodborne disease since the 80s. However, no data have been published on listeriosis and the presence of Listeria in food in Chile. This study was therefore undertaken to evaluate the prevalence of this pathogen in various foodstuffs of common consumption in Santiago, including mostly foods known to be at risk for listeriosis ŽRocourt and Bille, 1997., for eventual inclusion of Listeria monocytogenes in future Food Regulations.
) Corresponding author. Tel.: q56-2-350-7378; fax: q56-2350-7589. E-mail address: [email protected] ŽA.M. Cordano..
2. Material and methods 2.1. Food samples Out of the food samples received from the Metropolitan Environmental Health Service ŽChile. for routine control at the Instituto de Salud Publica ´ de Chile, 2145 samples of different brands and batches were selected for search of L. monocytogenes over the period between 1990 and 1997 ŽTable 1.. Samples were collected in industries, markets, restaurants and hospitals. Six-hundred and three were ice-creams, 256 were soft cheeses, 155 were hard cheeses, 229 were baby milk bottles, 634 were processed meat products and 268 were crustaceous shellfish.
0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 Ž 0 1 . 0 0 5 3 3 - 5
176
A.M. Cordano, J. Rocourtr International Journal of Food Microbiology 70 (2001) 175–178
Table 1 L. monocytogenes isolated from different types of food; Santiago, Chile, 1990–1997 Type of food Ice-cream Soft cheese Hard cheese Baby milk bottles Processed meat productsa Shellfish Total
Number of samples
Positives Ž%.
603 256 155 229 634 268
21 Ž3.5. 2 Ž0.8. 0 0 23 Ž3.6. 31 Ž11.6.
2145
77 Ž3.6.
a Included 443 sausages Ž20 contaminated., 160 pates ´ Ž2. and 31 hams Ž1..
2.2. Bacteriological method The method followed was that recommended by the BAM of the FDA ŽLovett and Hitchins, 1988; Hitchins, 1992, 1995.. Twenty-five grams of each sample were homogenized in Enrichment broth— Tryptic Soy Broth plus Yeast Extract—both Difco, Detroit, MI Žcatalogue nos. 0370-17-3 and 0127-179. plus selective agents, incubated 48 h at 308C and plated simultaneously on three different isolation media. Modified McBride Agar ŽMMA. and Lithium chloride Phenylethanol Moxalactam agar ŽLPM. were prepared with Phenylethanol agar ŽDifco catalogue no. 0504-17-2. as base agar, and each formula was completed with the relevant ingredients ŽSigma or
Table 2 L. monocytogenes isolated from 1827 food samples comparing Oxford, LPM and Palcam agars Type of food
Number of samples
Positives
Isolation on: Oxforda
LPM b
Palcamc
Ice-cream
449
9 1 4 1 4 1 20
q q q y q y 18
q q y q y y 11
q y q q y q 15
Soft cheese Hard cheese Baby milk bottles Processed meat products
188 113 192 634
2 0 0 5 1 3 3 3 4 4 23
q y y q q q y q y y 12
q y y q q y q y q y 13
q y y q y q q y y q 15
Shellfish
251
8 7 1 1 2 11 30
q q y q y y 16
q y q y q y 11
q q q y y q 27
a
Listeria Selective Agar Oxford Formulation. Lithium Chloride Phenylethanol Moxalactam agar. c Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar. b
A.M. Cordano, J. Rocourtr International Journal of Food Microbiology 70 (2001) 175–178
Merck, Darmstadt, Germany.. Oxford and Palcam agars were Oxoid ŽBasingstoke, UK catalogue nos. CM856 and CM877, Selective Supplements catalogue nos. SR140E and SR150E, respectively.. Sodium pyruvate ŽMerck 106619. was added to the enrichment broth for frozen products in the second group of samples. In a first group of 318 samples Ž68 soft cheeses, 42 hard cheeses, 37 baby milk bottles and 17 frozen crab meat., the isolation media compared were MMA, LPM and Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar ŽPalcam.. In a second group of 1827 food samples, following the modification in the Bacteriological Analytical Manual ŽBAM. of the protocol of the Food and Drug Administration ŽFDA. ŽHitchins, 1992., MMA was replaced by Listeria Selective Agar Oxford Formulation ŽOxford. ŽTable 2.. Four typical colonies from each of the isolation media were reisolated on Tryptic Soy Extract agar ŽDifco, 0369-17-6. plus Yeast Extract, identified, serotyped and phage-typed ŽLovett and Hitchins, 1988; Rocourt et al., 1985; Seeliger and Hohne, 1979..
3. Results and discussion L. monocytogenes was obtained from 77 out of the 2145 food samples tested ŽTable 1.. Out of the first group of 318 samples, one out of 17 frozen crab meat and one out of 154 ice-cream samples developed L. monocytogenes on all three media. Out of the second group of samples tested on Oxford, LPM and Palcam, 75 isolates were recovered; detailed results are mentioned in Table 2. L. monocytogenes was found in various types of food ŽTable 1.. Out of 603 ice-cream samples, 68 were cones scooped out individually for each buyer Žfive contaminated, 7.4%. and 535 were packed at the factories Ž16 contaminated, 3%.. For a single manufacturer, L. monocytogenes was obtained from three different ice-cream flavors Žvanilla, chocolate and pistachio., whose strains were serotype 4b, 1r2a and 1r2b. For the 634 processed meat products, 521 were precooked ready-to-eat products Ž11 contami-
177
nated, 2.1%. and 113 were raw sausages Ž12 contaminated, 10.6%.. From a total of 268 shellfish samples analysed 209 were cooked shellfish Ž14 contaminated, 6.7%., and 59 were raw shrimps Ž17 contaminated 28.8%.. Comparing the results obtained on Oxford, LPM and Palcam agars, for recovering L. monocytogenes for ice-cream samples, Oxford seems to be slightly better while Palcam agar seems to be clearly better for shellfish. In processed meat products a similar, but rather low, number of isolates was recovered from each medium ŽTable 2.. These results, and the fact that no isolation medium gave all positive results, emphasize the usefulness of using different isolation media. Strains were characterized by five different serovars, with a high proportion Ž59.7%. of serogroup 1 as previously described in food ŽFarber and Peterkin, 1991.. Strains serovar 4b Ž20 isolates., 1r2a Ž25. and 1r2b Ž19., frequently associated to human listeriosis ŽFarber and Peterkin, 1991., were detected in dairy and meat food; serotype 1r2c Ž2. was found in meat and shellfish while serotype 3b Ž7. was found exclusively in shrimps. More than half of the isolates were untypable by phages, the rest showed a great diversity of phagovars Ždata not shown.. In a few cases, strains of the same serovar and phagovar were found: four isolates 1r2a, 4477 Žtwo from different flavor ice-cream of the same origin and two from sausages of different origin. and six isolates 1r2a, 4477:19:387:1806 from strawberry ice-cream from the same source, which could suggest a longlasting contamination in the plant. This study has demonstrated the presence of numerous varieties of L. monocytogenes in Chilean food. It should be stressed that most types of food analysed were ready-to-eat, requiring no thermal treatment; furthermore, some of them are kept refrigerated for periods of time, providing conditions for Listeria multiplication. Raw products analysed showed a markedly higher percentage of contamination by L. monocytogenes; although cooking is required before consumption for this kind of products, they represent a high risk of contamination for other foods at different stages of handling. It would seem advisable to consider the health hazard of L. monocytogenes for the Chilean population, particularly for high-risk groups.
178
A.M. Cordano, J. Rocourtr International Journal of Food Microbiology 70 (2001) 175–178
Acknowledgements Special thanks to Prof. Dr. Rafael Virgilio for the helpful critical review of the manuscript, and to Carmen Virgilio for editing the manuscript.
References Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55, 476–511. Hitchins, A.D., 1992. Listeria monocytogenes. Chapter 10. Bacteriological Analytical Manual ŽBAM.. AOAC, Arlington, VA.
Hitchins, A.D., 1995. Listeria monocytogenes. Chapter 10. Bacteriological Analytical Manual ŽBAM.. AOAC, Arlington, VA. Lovett, J., Hitchins, A.D., 1988. Listeria isolation. Chapter 29. Bacteriological Analytical Manual ŽBAM.. AOAC, Arlington, VA, Revised October 13, 1988. Rocourt, J., Bille, J.S., 1997. Foodborne listeriosis. World Heallth Stat. Q. 50 Ž1–2., 67–73. Rocourt, J., Audurier, A., Courtieu, A.L., Durst, J., Ortel, S., Schrettenbrunner, A., Taylor, A.G., 1985. A multi-centre study on the phage-typing of Listeria monocytogenes. Zbl. Bakt. Hyg. A 259, 489–497. Seeliger, H.P.R., Hohne, K., 1979. Serotyping of Listeria monocytogenes and related species. In: Bergan, T., Norris, J. ŽEds.., Methods in Microbiology. Academic Press, New York, NY, pp. 33–48.
Short communication
Occurrence of Listeria monocytogenes in food in Chile Ana Marıa ´ Cordano a,) , Joselyne Rocourt b a
b
Seccion de Chile, Casilla 48, Santiago, Chile ´ Microbiologıa ´ de Alimentos, Instituto de Salud Publica ´ Laboratoire des Listeria, Centre Collaborateur de l’OMS pour la listeriose d’origine alimentaire, Institut Pasteur, Paris, France Received 9 June 2000; received in revised form 25 March 2001; accepted 20 April 2001
Abstract Out of 2145 food samples analysed 77 were found contaminated with Listeria monocytogenes in Santiago, Chile. Samples were: 603 ice-cream Ž3.5% contaminated., 256 soft cheese Ž0.8%., 155 hard cheese Ž0%., 229 baby milk bottles Ž0%., 634 processed meat products Ž3.6%. and 268 crustaceous shellfish Ž11.6%.. Three different isolation media were used: for 318 samples, Modified McBride Agar ŽMMA., Lithium chloride Phenylethanol Moxalactam agar, and Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar; for 1827 samples MMA was replaced by Listeria Selective Agar Oxford Formulation. Isolates were classified as follow: serovar 1r2a Ž25 isolates., serovar 4b Ž20., serovar 1r2b Ž19., serovar 3b Ž7., serovar 1r2c Ž2., untypable Ž4.. A high variety of phagovars was detected although 52% of strains was untypable. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Chilean food; Serotyping; Phagetyping
1. Introduction Listeriosis has emerged as a major foodborne disease since the 80s. However, no data have been published on listeriosis and the presence of Listeria in food in Chile. This study was therefore undertaken to evaluate the prevalence of this pathogen in various foodstuffs of common consumption in Santiago, including mostly foods known to be at risk for listeriosis ŽRocourt and Bille, 1997., for eventual inclusion of Listeria monocytogenes in future Food Regulations.
) Corresponding author. Tel.: q56-2-350-7378; fax: q56-2350-7589. E-mail address: [email protected] ŽA.M. Cordano..
2. Material and methods 2.1. Food samples Out of the food samples received from the Metropolitan Environmental Health Service ŽChile. for routine control at the Instituto de Salud Publica ´ de Chile, 2145 samples of different brands and batches were selected for search of L. monocytogenes over the period between 1990 and 1997 ŽTable 1.. Samples were collected in industries, markets, restaurants and hospitals. Six-hundred and three were ice-creams, 256 were soft cheeses, 155 were hard cheeses, 229 were baby milk bottles, 634 were processed meat products and 268 were crustaceous shellfish.
0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 Ž 0 1 . 0 0 5 3 3 - 5
176
A.M. Cordano, J. Rocourtr International Journal of Food Microbiology 70 (2001) 175–178
Table 1 L. monocytogenes isolated from different types of food; Santiago, Chile, 1990–1997 Type of food Ice-cream Soft cheese Hard cheese Baby milk bottles Processed meat productsa Shellfish Total
Number of samples
Positives Ž%.
603 256 155 229 634 268
21 Ž3.5. 2 Ž0.8. 0 0 23 Ž3.6. 31 Ž11.6.
2145
77 Ž3.6.
a Included 443 sausages Ž20 contaminated., 160 pates ´ Ž2. and 31 hams Ž1..
2.2. Bacteriological method The method followed was that recommended by the BAM of the FDA ŽLovett and Hitchins, 1988; Hitchins, 1992, 1995.. Twenty-five grams of each sample were homogenized in Enrichment broth— Tryptic Soy Broth plus Yeast Extract—both Difco, Detroit, MI Žcatalogue nos. 0370-17-3 and 0127-179. plus selective agents, incubated 48 h at 308C and plated simultaneously on three different isolation media. Modified McBride Agar ŽMMA. and Lithium chloride Phenylethanol Moxalactam agar ŽLPM. were prepared with Phenylethanol agar ŽDifco catalogue no. 0504-17-2. as base agar, and each formula was completed with the relevant ingredients ŽSigma or
Table 2 L. monocytogenes isolated from 1827 food samples comparing Oxford, LPM and Palcam agars Type of food
Number of samples
Positives
Isolation on: Oxforda
LPM b
Palcamc
Ice-cream
449
9 1 4 1 4 1 20
q q q y q y 18
q q y q y y 11
q y q q y q 15
Soft cheese Hard cheese Baby milk bottles Processed meat products
188 113 192 634
2 0 0 5 1 3 3 3 4 4 23
q y y q q q y q y y 12
q y y q q y q y q y 13
q y y q y q q y y q 15
Shellfish
251
8 7 1 1 2 11 30
q q y q y y 16
q y q y q y 11
q q q y y q 27
a
Listeria Selective Agar Oxford Formulation. Lithium Chloride Phenylethanol Moxalactam agar. c Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar. b
A.M. Cordano, J. Rocourtr International Journal of Food Microbiology 70 (2001) 175–178
Merck, Darmstadt, Germany.. Oxford and Palcam agars were Oxoid ŽBasingstoke, UK catalogue nos. CM856 and CM877, Selective Supplements catalogue nos. SR140E and SR150E, respectively.. Sodium pyruvate ŽMerck 106619. was added to the enrichment broth for frozen products in the second group of samples. In a first group of 318 samples Ž68 soft cheeses, 42 hard cheeses, 37 baby milk bottles and 17 frozen crab meat., the isolation media compared were MMA, LPM and Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar ŽPalcam.. In a second group of 1827 food samples, following the modification in the Bacteriological Analytical Manual ŽBAM. of the protocol of the Food and Drug Administration ŽFDA. ŽHitchins, 1992., MMA was replaced by Listeria Selective Agar Oxford Formulation ŽOxford. ŽTable 2.. Four typical colonies from each of the isolation media were reisolated on Tryptic Soy Extract agar ŽDifco, 0369-17-6. plus Yeast Extract, identified, serotyped and phage-typed ŽLovett and Hitchins, 1988; Rocourt et al., 1985; Seeliger and Hohne, 1979..
3. Results and discussion L. monocytogenes was obtained from 77 out of the 2145 food samples tested ŽTable 1.. Out of the first group of 318 samples, one out of 17 frozen crab meat and one out of 154 ice-cream samples developed L. monocytogenes on all three media. Out of the second group of samples tested on Oxford, LPM and Palcam, 75 isolates were recovered; detailed results are mentioned in Table 2. L. monocytogenes was found in various types of food ŽTable 1.. Out of 603 ice-cream samples, 68 were cones scooped out individually for each buyer Žfive contaminated, 7.4%. and 535 were packed at the factories Ž16 contaminated, 3%.. For a single manufacturer, L. monocytogenes was obtained from three different ice-cream flavors Žvanilla, chocolate and pistachio., whose strains were serotype 4b, 1r2a and 1r2b. For the 634 processed meat products, 521 were precooked ready-to-eat products Ž11 contami-
177
nated, 2.1%. and 113 were raw sausages Ž12 contaminated, 10.6%.. From a total of 268 shellfish samples analysed 209 were cooked shellfish Ž14 contaminated, 6.7%., and 59 were raw shrimps Ž17 contaminated 28.8%.. Comparing the results obtained on Oxford, LPM and Palcam agars, for recovering L. monocytogenes for ice-cream samples, Oxford seems to be slightly better while Palcam agar seems to be clearly better for shellfish. In processed meat products a similar, but rather low, number of isolates was recovered from each medium ŽTable 2.. These results, and the fact that no isolation medium gave all positive results, emphasize the usefulness of using different isolation media. Strains were characterized by five different serovars, with a high proportion Ž59.7%. of serogroup 1 as previously described in food ŽFarber and Peterkin, 1991.. Strains serovar 4b Ž20 isolates., 1r2a Ž25. and 1r2b Ž19., frequently associated to human listeriosis ŽFarber and Peterkin, 1991., were detected in dairy and meat food; serotype 1r2c Ž2. was found in meat and shellfish while serotype 3b Ž7. was found exclusively in shrimps. More than half of the isolates were untypable by phages, the rest showed a great diversity of phagovars Ždata not shown.. In a few cases, strains of the same serovar and phagovar were found: four isolates 1r2a, 4477 Žtwo from different flavor ice-cream of the same origin and two from sausages of different origin. and six isolates 1r2a, 4477:19:387:1806 from strawberry ice-cream from the same source, which could suggest a longlasting contamination in the plant. This study has demonstrated the presence of numerous varieties of L. monocytogenes in Chilean food. It should be stressed that most types of food analysed were ready-to-eat, requiring no thermal treatment; furthermore, some of them are kept refrigerated for periods of time, providing conditions for Listeria multiplication. Raw products analysed showed a markedly higher percentage of contamination by L. monocytogenes; although cooking is required before consumption for this kind of products, they represent a high risk of contamination for other foods at different stages of handling. It would seem advisable to consider the health hazard of L. monocytogenes for the Chilean population, particularly for high-risk groups.
178
A.M. Cordano, J. Rocourtr International Journal of Food Microbiology 70 (2001) 175–178
Acknowledgements Special thanks to Prof. Dr. Rafael Virgilio for the helpful critical review of the manuscript, and to Carmen Virgilio for editing the manuscript.
References Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55, 476–511. Hitchins, A.D., 1992. Listeria monocytogenes. Chapter 10. Bacteriological Analytical Manual ŽBAM.. AOAC, Arlington, VA.
Hitchins, A.D., 1995. Listeria monocytogenes. Chapter 10. Bacteriological Analytical Manual ŽBAM.. AOAC, Arlington, VA. Lovett, J., Hitchins, A.D., 1988. Listeria isolation. Chapter 29. Bacteriological Analytical Manual ŽBAM.. AOAC, Arlington, VA, Revised October 13, 1988. Rocourt, J., Bille, J.S., 1997. Foodborne listeriosis. World Heallth Stat. Q. 50 Ž1–2., 67–73. Rocourt, J., Audurier, A., Courtieu, A.L., Durst, J., Ortel, S., Schrettenbrunner, A., Taylor, A.G., 1985. A multi-centre study on the phage-typing of Listeria monocytogenes. Zbl. Bakt. Hyg. A 259, 489–497. Seeliger, H.P.R., Hohne, K., 1979. Serotyping of Listeria monocytogenes and related species. In: Bergan, T., Norris, J. ŽEds.., Methods in Microbiology. Academic Press, New York, NY, pp. 33–48.