versão impressa ISSN 0001-3714
Rev. Microbiol. v.30 n.4 São Paulo out./dez. 1999
Fabiana Cristina Pimenta1, Sirdéia Maura Perrone Furlanetto1, Leonard W. Mayer2, Jorge Timenetsky1, Manoel Armando Azevedo dos Santos1*
1Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil. 2Division of Vector-Borne Infection Diseases, Center for Disease Control, Atlanta, USA
Submitted: May 06, 1998; Returned to authors for corrections: December 02, 1998; Approved: August 26, 1999
A total of 30 strains of Listeria monocytogenes isolated from different foods (16 of differents kinds of sausage, 14 cheese,) purchased at groceries of São Paulo City were ribotyped and analysed for the presence and expression of hemolysin gene and production of phosphatidylinositol-specific phosphalipase C - PI-PLC enzyme. The L. monocytogenes strains were differentiated into six ribotype classes. A total of 13 (43.3%) from these strains belong to the same ribotype (ribotype I), and was coincident to the ribotype of the standard L. monocytogenes prototype strain (ATCC-15313). The hemolytic activity was observed in 29 (96.7%) strains when incubated at 37oC, but not at 4oC. The direct colony hybridization method for hemolysin gene detection showed a positive reaction whit all the 30 L. monocytogenes strains, while showed negative reaction with other Listeria spp. The PI-PLC was produced by 27 (90%) of the strains analysed. There was no correlation between the six identified ribotypes and the virulence factors (hemolysin and PI-PLC) studied.
Key words: Listeria monocytogenes, ribotyping, virulence factors, food
Several food-borne outbreaks have highlighted the importance of the L. monocytogenes to the public health (8, 9, 11, 18, 25). Establishing new methods to detect this pathogen in food (21) is very important because different food can be contaminated with this bacteria (11). Characterization using biochemical tests relies on expression of phenotypes that may not discriminate between species or strains within a same species (2). Alternative methods ideally should be based on the detection of L.monocytogenes virulence genes or gene products. One marker should be the production of a hemolysin, listeriolysin O (LLO), which is required for intracellular survival of invading bacteria in mammalian host (10). The gene encoding LLO had been named hlyA. L.monocytogenes produces other hemolysins besides LLO like phosphatidylinositol-specific phospholipase C - PI-PLC (17) and phosphatidylcholine-specific phospholipase C - PC-PLC (5). Unlike LLO, which lyses host cells by forming pores in the membrane, the phospholipases disrupt host membranes by hydrolyzing membrane lipids (24).
Methods for the characterization of Listeria spp. have been developed, and isoenzyme analysis shows some promise as a typing method (3). Nucleic acid hybridization has contributed significantly to the progress of bacterial classification and identification. One possible disadvantage of DNA probes is that each probe will detect only one taxonomic entity or clones harboring a given gene (14). Species specific probes for L monocytogenes have been developed, and used in colony hybridization assay (6, 7, 22, 23). The DNA fingerprint reveals DNA restriction fragment length polymorphisms, and has already been used in epidemiological investigations (2), but, with no success. Ribotyping, based on restriction fragment length polymorphism in the chromosomal DNA containing rRNA genes has also been used as important tool.
The purpose of this study was to type L.monocytogenes strains, isolated from foods in São Paulo city, by using a probe complementary to ribosomal RNA (plasmid pKK3535). Haemolytic activity was analysed in blood agar plates and the presence of the hemolysin gene detected by using the plasmid pIP5 as a probe. We also examined the enzymatic activity of phosphotidylinositol-specific phospholipase C, in order to examine the eventually relationship between the ribotypes and virulence associated genes
MATERIALS AND METHODS
A total of 30 strains of L.monocytogenes, serovars 1/2b and 4b, isolated from different kinds of food (16 different kinds of sausagesand 14 cheeses) purchased at groceries of São Paulo city were studied. The non-monocytogenes Listeria and L. monocytogenes type strains were from the American Type Culture Collection. The bacteria were maintained at room temperature in a solid medium (trypitic soy agar supplemented with 0.6% of yeast extract). Host strain E.coli (ED 8654-pKK3535), and E.coli DH5a -pIP5-[F-, endA1 hsd R17 (r-k m+k) supE44 thi-1l- l-recA1 gyrA96 relA1 (arg F-lac ZYA) U169 f80d lac DAM15] were maintained under selective pressure with ampicilin (100µg/ml).
Preparation and labeling of DNA probe
Plasmid pKK3535 (4) was isolated from a culture of E.coli (ED 8654 pKK3535), and the plasmid pIP5 (23) was isolated from a culture of E.coli DH5a (pIP5), grown overnight at 30oC in 2xYT broth contaning ampicilin (100µg/ml) as described by Ish-Horowicz and Burke (15). The plasmid pKK3535 was labeled using digoxigenin-dUTP (1) by nick-translation (Genius Kit, Boehring Manheim Biochemicals, Indianapolis, IN) following the manufacturer's instructions. The plasmid pIP5 was used as a probe after linearization with restriction endonuclease KpnI (Promega) (23), and labeled as described above.
Total DNA of Listeria monocytogenes strains was extracted from one liter 48h culture in 2xYT using the procedure of Sambrook et al. (25). DNA samples (0.5-2ug) were cleaved by restriction endonucleases Hind III (Promega) according to the manufacturer's instructions, electrophoresed on 0.7% agarose gel, in Tris-acetate buffer (0.04M Tris acetate, 0.001M EDTA, pH 6.7), using 1 Kb DNA ladder (Gibco) as a marker, and stained in ethidium bromide (1.0mg/ml) for 20 min. Gels were then photographed. The DNA was transfered to nylon membrane by the capillary transfer method (27), and the fixed DNA (14) was prehybridized at 37oC in hybridization solution [25ml 20xSSC; 30ml formamide; 1ml NaCl a 10%; 0.2ml SDS a 10%; 5g blocking reagent (Genius Kit-Boehringer Mannheim); 43.8ml dd water]. The solution was replaced by 2.5ml of hybridization solution containing the labeled probe pKK3535. After 24 h incubation, the membrane was washed and positive reactions were colorimetrically visualized according to Genius Kit manufacturer's recommendations.
The strains were stabbed on 5% horse blood agar plates (19), incubated at 37oC for 48h, and at 4oC for 30 days. The production of clear zones around the colonies indicated beta-hemolytic activity.
Bacterial colonies, from blood agar plates, were transferred to the nylon membrane, which were prepared for hibridization according to Peterkin et al. (23). The treated membrane, carrying DNA, was prehybridized at 68oC for 1 h in a bag containing hybridization solution. This solution was replaced by fresh hybridization solution containing the labeled pIP5 and after 24h incubation, the membrane was washed, and positive reactions were colorimetrically visualized according to Genius Kit manufacturer's recommendations.
Detection of PI-PLC activity
PI-PLC activity were determinated as described by Notermans et al. (21). The plates were observed up to 5 days of incubation at 37°C, for turbid halos around colonies.
The 30 L. monocytogenes strains were differentiated in six ribotypes. The schematic representation of the ribotype patterns can be observed in Fig. 1. The type I (rI) presented 5 fragments: 2.3; 3.3; 3.6; 7.3 and 7.4Kb, the type II (rII) with 4 fragments: 2.3; 3.3; 6.9 and 7.4Kb, the type III (rIII) with 6 fragments: 2.3; 3.3; 4.0; 4.7; 7.3 and 7.4Kb, the type IV (rIV) with 4 fragments: 2.0; 2.3; 2.7 and 7.4Kb, the type V(rV) with 5 fragments: 2.3; 4.0; 4.7; 7.3 and 7.4Kb and the type VI(rVI) with 3 fragments: 2.3; 3.3 and 6.9Kb. All ribotypes presented a 2.3Kb common band, and another band of 7.4Kb was observed in five ribotypes (I, II, III, IV and V). Interestingly, a total of 13 (43.4%) of the 30 strains typed as a ribotype I were identical to the profile of the standard L.monocytogenes strain (ATCC-15313). The probe used was successful to differentiate L. monocytogenes strains from other Listeria spp, which lacked the 2.3Kb band. The hemolytic activity was observed in 29 (96.7%) strains when they were incubated at 37oC in blood agar, but at 4oC the listeriolysin was not produced (Table 1). The hemolytic activity using the direct colony hybridization method showed a positive reaction with all the L.monocytogenes strains. The phosphatidylinositol-specific phospholipase C activity was detected in 27 strains (Table 1).
Figure 1: Schematic representation of six L. monocytogenes ribotype patterns.
DISCUSSION AND CONCLUSION
Epidemiological tracking of food-borne pathogens requires methods of analysis that allow discrimination between phenotypically indistinguishable strains within a species (2).
Grimont and Grimont (14) analyzed the DNA from 41 different bacterial species, and demonstrated that patterns of hybridized fragments could be used to identify biochemically indistinguishable strains. Baloga and Harlandar (2) studied 28 strains of L.monocytogenes isolated from food implicated in 276205
food-borne illness and from patients with listeriosis. The authors determinated the fingerprints, ribotypes, and the resulting subtypes were compared with multiloccus enzyme electrophoresis classification schemes. They observed that the serotypes were distributed among several distinct fingerprints and ET categories.
The mechanisms of pathogenicity of L. monocytogenes have been studied, and usually is associated with hemolysin production (12). There have been a number of reports suggesting the possibility of the temperature regulation of virulence genes expression in L. monocytogenes, but the results have been conflicting (13, 17). The hemolytic activity of Listeria spp., and the level of listeriolysin produced may be dependent on enrichment procedures, selective media, temperature and virulence of the bacteria (8, 21).
In our study, hemolytic activity was detected in 29 L. monocytogenes strains when incubated at 37oC, but, this activity was not observed when incubated at 4oC. Ours data agree with the Girard et al. (13) and Leimeister-Wachter et al. (17) results, which observed higher levels of the hemolysin production in different temperatures (between 20 to 37oC), when compared to 4oC.
Leimeister-Wachter et al. (17) found that low temperature have no significant effect on the L. monocytogenes virulence factors production. A likely explanation for the absence of hemolytic activity in one strain could be an alteration in the environment or in the regulatory gene (17, 20).
The most recent developments in methodologies for the detection of L. monocytogenes have involved gene probes. A commercial hybridization assay based on 16S rRNA sequences (Gene Trak) was developed for the detection of Listeria spp. in dairy products and environmental samples (16). Some researchers developed probes specifically for L. monocytogenes (7, 23). Those probes are used to detect the listeriolysin gene (hlyA), and are used in colony hybridization assays. In our study, the direct colony hybridization method gave positive reaction with all the 30 L. monocytogenes strains analysed, while showed a negative reaction with other Listeria spp, as expected.
The pfrA gene is a positively acting factor that transcriptionally activates the expression of the pic, hlyA and other genes (17). In this study, the PI-PLC activity was expressed only by the majority of the strains. Probably, the negative PI-PLC activity in three tested strains should be explained by the absence of or mutation on the pic or prfA genes as suggested by Notermans et al. (21)
Our data permit us to conclude that there were no correlation between the six identified ribotypes and some L. monocytogenes virulence factors (hemolysin and PI-PLC).
Caracterização molecular de Listeria monocytogenes isolada de alimentos
Foram estudadas 30 cepas de Listeria monocytogenes isoladas a partir de diferentes alimentos (16 diferentes tipos de linguiça, 14 de queijo), adquiridos em supermercados da cidade de São Paulo. As cepas foram classificadas através da ribotipagem e analisadas quanto à presença e expressão do gene da hemolisina e à produção da enzima fosfolipase C fosfatidilinositol-específica PI-PLC. As cepas de L. monocytogenes foram diferenciadas em 6 ribotipos. As cepas do tipo I possuiam o mesmo perfil da amostra padrão de L.monocytogenes (ATCC 15313), sendo 13 (43,3%) das cepas estudadas correspondentes a ele. A atividade hemolítica foi observada em 29 (96,7%) das cepas, quando incubadas a 37°C em agar sangue, mas não a 4°C. O método da hibridização direta de colônias, utilizando sonda para hemolisina, revelou resultado positivo para todas as cepas. A PI-PLC foi produzida por 90% das amostras analisadas. Deste modo, foi possível concluir que não há relação entre os seis ribotipos de L. monocytogenes identificados nesse estudo e os fatores de virulência estudados (hemolisina e PI-PLC).
Palavras-chave: Listeria monocytogenes, ribotipagem, fatores de virulência, alimentos.
1. Altwegg, M.; Mayer, L.W. Bacterial molecular epidemiology based on a non-radiactive probe complementary to ribosomal RNA. Res. Microbiol., 140: 325-333,1989.
2. Baloga, A.O.; Harlander, S.K. Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys. Appl. Environ. Microbiol., 57: 2324-2331, 1991.
3. Bibb, W.F.; Schwartz, B.; Gellin, B.G.; Plikaytis, B.D.; Weaver, R.E. Analysis of Listeria monocytogenes by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations. Int. J. Food Microbiol., 8: 233-239, 1990.
4. Brosius, J.; Ullrich, A.; Raker, M.A.; Gray, A.; Dull, J.T.; Gutell, R.R.; Noller, H.F. Construction and fine mapping of recombinant plasmids contaning the rrnB ribossomal RNA operon of E.coli. Plasmid, 6: 112-118,1981.
5. Camilli, A.; Tilney, L.G.; Portnoy, D.A. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol. Microbiol., 8: 143-147, 1993.
6. Datta, A.R.; Wentez, B.A.; Hill, W.E. Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization. Appl. Environ. Microbiol., 53: 2256-2259, 1987.
7. Datta, A.R.; Wentz, B.A.; Shook, D.; Trucksess, M.W. Synthetic oligodeoxyribonucleotide probes for detection of Listeria monocytogenes. Appl. Environ. Microbiol., 54: 2933-2937, 1988.
8. Faber, J.M.; Peterkin, P.I. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev., 55: 476-511, 1991.
9. Fleming, D.W.; Cochi, S.L.; Mac Donald, K.L.; Brondum, M.; Hayes, P.S.; Plikaytis, B.D.; Holmes, M.B.; Audurier, A.; Broome, C.V.; Reingold, A.L. Pasteurized milk as a vehicle of infection in a outbreak of listeriosis. N. Engl. J. Med., 312: 404-407, 1985.
10. Gaillard, J.; Berche, P.; Sansonetti, P.J. Transposon mutagenesis as a tool to study the role of the hemolysin in the virulence of Listeria monocytogenes. Infect. Immun., 55: 2822-2829, 1986.
11. Gellin, B.G.; Broome, C.V. Listeriosis. J. Am. Med. Assoc., 261: 1313-1320, 1989.
12. Gellin, B.G.; Broome, C.V.; Bibb, W.F.; Weaver, R.E.; Gaventa, S.; Mascola, L. The Epidemiology of listeriosis in the Unided States-1986. Listeriosis Study Group. Am. J. Epidemiol., 133: 392-401, 1991.
13. Girard, K.F.; Sbarra, A.J.; Bardawil, W.A. Serology of Listeria monocytogenes. J. Bacteriol., 85: 349-55, 1963.
14. Grimont, F.; Grimont, P.A.D. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur, 137B: 165-175,1986.
15. Isch-Horowicz, D.; Burke, J.F. Rapid and efficient consmic cloning. Nucleic. Acids Res., 9: 2989-2998,1981.
16. Klinger, J.D.; Johnson, A.; Croan, D.; Flynn, K.; Whippie, K. Kimball, M.; Lawrie, J.; Curiale, M. Comparative studies of nucleic acid hibridization assay for Listeria in foods. J. Assoc. Off. Anal. Chem., 71: 669-673, 1988.
17. Leimeister-Wacher, M.; Domann,E.; Chakraborty, T. Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is coordinately expressed with listeriolysin in Listeria monocytogenes. Mol. Microbiol., 5:361-6, 1991.
18. Linnan, M.J.; Mascola, L.; Mantell, C.; Bercroft, D.; Dove, B.; Farmer, K.; Tonkin, S.; Yeasts, N.; Stamp, R.; Mickleson, K. Epidemic perinatal listeriosis. Pediactric. Infect. Dis., 3: 30-34, 1984.
19. Lovett, J. Listeria isolation. In: Bacteriological Analytical Manual. 6ed. FOOD and Drug Administration (US)., Washington, 1987, S.9.
20. Njoku-Obi, A.N.; Jenkins, E.M.; Njoku-Obi, J.C.; Adms, J.; Covington,V. Production and nature of Listeria monocytogenes hemolysins. J. Bacterial., 86: 1-8, 1963.
21. Notermans, S.H.W.; Dufrenne, J.; Leimester-Wachter, M.; Domann, E.; Chakraborty, T. Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species. Appl. Environ. Microbiol., 57: 2666-2670, 1991.
22. Peterkin, P.I.; Idziak, E.S.; Sharpe, A.N. Screening DNA probes using the hydrophobic grid-membrane filter. Food Microbiol., 6: 281-284, 1989.
23. Peterkin, P.I.; Idziak, E.S.; Sharpe, A.N. Detection of Listeria monocytogenes by direct colony hibridization on hydrophobic gride-membrane filters by using a chromogen-labeled DNA probe. Appl. Environ. Microbiol., 57: 586-91, 1991.
24. Salyers, A.A.; Whitt, D.D. Listeria monocytogenes. In: Bacterial Pathogenesis. A Molecular Approach. ASM, Washington., 1994, 418 p.
25. Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning. A laboratory manual. Sec. Edition. CHH 1989.
26. Schwartz, B.; Hexter, D.; Broome, C.V.; Hightower, A.W.; Hirschorn, R.B.; Porter, J.D.; Hayes, B.S.; Bibb, W.F.; Lorber, B.; Faris, D.G. Investion of an outbreak of listeriosis: new hypothesis for the etiology of epidemic Listeria monocytogenes infectios. J. Infect. Dis., 159: 680 -685, 1989.
27. Southern, E.M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Microbiol., 27: 1571-1576, 1989.
* Corresponding author. Mailing address: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo. Av. Prof. Lineu Prestes, 1374, CEP 05508-900, São Paulo, SP, Brasil.