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REVISTA PORTUGUESA DE
CIÊNCIAS VETERINÁRIAS
Development of a Fluorescent In Situ Hybridization protocol for the rapid detection and enumeration of Listeria monocytogenes in milk Desenvolvimento de um protocolo de “Fluorescent In Situ Hybridization” para a detecção rápida e contagem de Listeria monocytogenes em leite Manuela Oliveira, Gonçalo Andrade, Manuela Guerra e Fernando Bernardo* Laboratório de Inspecção Sanitária - CIISA, Faculdade de Medicina Veterinária, Rua Prof. Cid dos Santos, Polo Universitário do Alto da Ajuda, Lisboa, Portugal
Summary: A rapid method for the specific detection and enumeration of Listeria monocytogenes in milk was developed. This method is based on Fluorescent In Situ Hybridization (FISH), using a fluorescent 19-mer oligonucleotide probe, homologous to 16S rRNA of L. monocytogenes, and adapted from a dot-blot hybridization technique (Wang et al., 1991). The protocol was established through the hybridization of 68 reference strains, and tested against 114 Listeria cultures isolated from food and environmental samples, including L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri and L. welshimeri. The probe hybridized specifically to all L. monocytogenes serovars, and no cross hybridization was observed with any of the other strains tested. The protocol was then applied to the direct detection of L. monocytogenes in natural and artificially contaminated milk samples, and the relation between the traditional plating method and FISH cell counting was evaluated. Results show that FISH is a promising technique for the rapid detection and enumeration of L. monocytogenes in food and environmental samples, allowing positive identification in approximately seven hours, with a consumable cost of 0.45 to 3.0 euro, depending on the number of simultaneously processed samples. Resumo: Foi desenvolvido um método rápido para a detecção específica e contagem de Listeria monocytogenes em leite. Este método baseia-se na técnica Fluorescent In Situ Hybridization (FISH), e recorre a uma sonda fluorescente de 19 oligonucleótidos, homóloga ao 16S rRNA de L. monocytogenes, e adaptada de uma técnica de hibridação dot-blot (Wang et al., 1991). O protocolo foi estabelecido através da hibridação de 68 estirpes de referência, e testado em 114 isolados de Listeria obtidos a partir de amostras de alimentos e ambientais, incluindo L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri e L. welshimeri. A sonda hibridou especificamente com todas as serovariedades de L. monocytogenes, não se tendo observado hibridações cruzadas com as outras estirpes. O protocolo foi aplicado à detecção directa de L. monocytogenes em amostras de leite naturais e contaminadas artificialmente, e a relação entre o método de plaqueamento tradicional e a contagem de células por FISH foi avaliada. Os resultados demonstram que esta técnica é promissora para a detecção rápida e contagem de L. monocytogenes em amostras de alimentos e ambientais, permitindo a sua identificação positiva em aproximadamente sete horas, com um custo entre 0,45 e 3,0 euro, dependendo do número de amostras processadas simultaneamente.
* Corresponding author: e-mail:
[email protected]; telephone 213652846, fax 213652882.
Introduction Listeria monocytogenes is a foodborne pathogen able of causing either outbreaks or sporadic episodes of listeriosis. The organism has been incriminated in foodborne diseases promoted by a wide variety of food products, in many countries, since the 1980s (Sutherland and Porritt, 1997). In 1997, the worldwide incidence was two to five cases/million population (Rocourt and Cossart, 1997), and although infection can be treated successfully with antibiotics, between 20 and 40% of the cases are fatal (McLauchlin, 1996). Recently in the United States, listeriosis was estimated as the second largest identified cause of death from a foodborne disease (Mead et al., 1999), which justifies the continued analysis of the causative organism, L. monocytogenes. The development of methods for the rapid detection of L. monocytogenes in food is therefore critical for ensuring food safety. Traditional plating methods are well established, but complex and time consuming. They depend upon the obtention of pure cultures and include long steps of pre-enrichment, enrichment, selection and identification. For L. monocytogenes, positive results are only obtained after 5 days. Scientific advances over the past years resulted in the development of faster, more sensitive and more specific methods for the detection of pathogenic microorganisms. These methods, which include immunomagnetic techniques, biochemical kits (Olsen et al., 1995) and molecular techniques, such as PCR (Jaton et al., 1992; Lantz et al., 1994; Wang et al., 1997; Ingianni et al., 2001) and FISH, eliminate the need for pure cultures and give results in 3-8 hours. The FISH technique, already applied to clinical investigation of faeces (Sghir et al., 2000), urine and blood, and to the microbiological analysis of water (Kenzaka et al., 1998), soils, activated sludges (Wagner et al., 1994) and biofilms (Poulsen et al., 1993), is based on the hibridization of specific geno119
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mic sequences of a microorganism, with a fluorescent labelled-probe. It usually involves four steps: sample fixation and permeabilization; hibridization of the target sequence and the fluorescent probe; stringency washes; and detection of the hibridized cells (Amann et al., 2001). The purpose of this study was to develop a FISH protocol for the specific detection of L. monocytogenes in approximately seven hours; to evaluate its applicability for the direct detection and enumeration of this pathogen in food samples; and to evaluate the relation between the traditional plating method and FISH cell counting.
Materials and Methods Bacterial strains and culture methods In this study 68 reference strains of different bacteria species were used, including 24 Listeria strains, 17 of which correspond to different serotypes of L. monocytogenes (Table 1). Ninety-two other L. monocytogenes isolates, previously identified and serotyped (Guerra et al., 2001), were also used. Seventytwo of the isolates were recovered from food and 20 from environmental samples. Isolates belong to four serovars: 1/2a (11 isolates), 1/2b (23 isolates), 3b (2 isolates), 4b (56 isolates). Other Listeria species were also included: L. innocua (5 isolates), L. ivanovii (3 isolates), L. grayi (4 isolates), L. seeligeri (4 isolates) and L. welshimeri (6 isolates) (Table 2). All cultures were grown in Tryptone Soya Agar (TSA) (CM0131, Oxoid, Hampshire, England). Fixation of bacteria Overnight cultures grown in TSA (CM0131, Oxoid, Hampshire, England) were resuspended in 1000µl of distilled water (H2O) and centrifuged (14000 rpm, 10 mins). Pellets were resuspended in 100µl of a 4% paraformaldehyde solution (Merck Sharp & Dohme, Lisbon, Portugal). This mixtures were incubated for 20 mins, centrifuged (14000 rpm, 10 mins), and washed in 1000 µl of PBS. Bacteria were pelleted by centrifugation (14000 rpm, 10 mins), and resuspended in distilled H2O.
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FISH technique Ten-well teflon-coated slides (Heinz Herenz, Hamburg, Germany) were covered with a 2 % 3-trimethoxysilylpropylamine solution (Merck Sharp & Dohme, Lisbon, Portugal) in acetone; 10 µl of fixed bacteria were placed on the wells, air-dried and dehydrated in 70 and 96 % ethanol, for two minutes each. After drying, 10µl of hybridization buffer (0.7 M NaCl, 0.1 M Tris-HCl pH 8.0, 0.1 % SDS, 10 mM EDTA) containing 5 ng/µl of probe were added. The slides were incubated in a moisture chamber (45 ºC, 4 h), and washed in hybridization buffer (45 ºC, 15 mins). Afterwards, they were rinsed in distilled H2O and mounted in Vectashield Mounting Medium (Vector Laboratories, Inc., Burlingame, U.S.A.) containing 0.5 µg/µl of 4’-6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich Química, S.A., Sintra, Portugal). All assays were performed in duplicate. The hybridized bacteria were visualized by fluorescence microscopy at x1000 magnification (Objective HCX PLAN APD) on a Leica DMR microscope equipped with a 100 W mercury lamp and an I3 filter (Leica Microsistemas Lda, Lisboa, Portugal). Non-hybridized bacteria were located using DAPI staining and identified by observation through an A filter (Leica Microsistemas Lda, Lisboa, Portugal). Images were captured with a Leica DC200 camera. Direct detection in milk samples and bacteria quantification Tenfold dilutions from natural and artificially contaminated sheep milk inoculated with 0.5 McFarland suspensions (1.5 x 108 bacteria/ml) (bioMérieux, Marcy L’Etoile, France) of L. monocytogenes 4b CECT935, were performed. One ml of each was simultaneously plated through incorporation on Palcam Agar (CM877, Oxoid, Hampshire, England) and directly fixed for FISH as described. The plates were incubated at 37ºC for 24 h, and the colonies counted. FISH was performed as described. Cells with positive hibridization signal were counted on 20 microscope fields, and the number of cells/ml was estimated using the formula: nº cells / ml = (nº cells counted / nº fields counted x well area / field area) / volume used x dilution. Assays were repeated 5 times, in duplicate.
Oligonucleotide probes Three oligonucleotide probes were used: a Listeria oligonucleotide probe RL-2 (Wang et al., 1991); the universal bacteria probe Eub338 (Christensen et al., 1999); and a nonsense probe complementary to Eub338, Non338 (Christensen et al., 1999). Eub338 and Non338 were used as positive and negative controls of the hybridization protocol, respectively. The probes were synthesized and labelled with fluorescein at the 5’ end (MWG-Biotech, Ebersberg, Germany). 120
Results The FISH protocol revealed to be specific for L. monocytogenes detection, since all L. monocytogenes isolates showed a positive hybridization signal, and no cross-hybridization was observed (Tables 1 and 2) . Fifteen of the reference L. monocytogenes strains yielded a strong hybridization signal with the RL-2 probe. The hybridization signals of L. monocytogenes
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Table 1 – Reference strains tested by FISH with the specific protocol for L. monocytogenes Species
Serotype
Strain Code
Positive hybridisation Eub338
Non338
Citrobacter freundii
CECT401T
0
1
0
Edwardsiella tarda
CECT849T
0
1
0
Enterobacter aerogenes
CECT684T
0
1
0
Enterobacter cloacae
CECT194T
0
1
0
Enterococcus hirae
DSMZ20160T
0
1
0
Enterococcus faecium
DSMZ20477T
0
1
0
Enterococcus faecalis
DSMZ20478T
0
1
0
Enterococcus gallinarum
DSMZ20628T
0
1
0
Enterococcus durans
DSMZ20633T
0
1
0
Enterococcus avium
DSMZ20679T
0
1
0
Enterococcus casseliflavus
DSMZ20689T
0
1
0
Enterococcus pseudoavium
DSMZ5632T
0
1
0
O127a
CECT352
0
1
0
O1
CECT515NT
0
1
0
O25
CECT685
0
1
0
O111
CECT727
0
1
0
HfrK12
CECT4624
0
1
0
O157:H7
CECT4782; CDC3834
0
2
0
Klebsiella pneumoniae
CECT143T
0
1
0
Klebsiella aerogenes
NCTC418
0
1
0
Listeria grayi
CECT931T
0
1
0
Listeria innocua
CECT910T; NCTC11288
0
2
0
Listeria ivanovii
CECT913T; NCTN11846
0
2
0
1a
CECT932; CECT4031T
2
2
0
1/2a
CIP104794
1
1
0
1/2b
CECT936
1
1
0
1/2c
CECT 911
1
1
0
3b
CECT937
1
1
0
4a
CECT934
1
1
0
4b
CECT935; CECT4032
2
2
0
4c
CECT939; CIP78.39
2
2
0
6
CECT941
1
1
0
Others
NCTC11994; L7946; L7947; L2036C; ScottA
5
5
0
Listeria seeligeri
CECT917T
0
1
0
Listeria welshimeri
CECT919T
0
1
0
Morganella morganii
CECT173T
0
1
0
Pantoea agglomerans
CECT850T
0
1
0
Proteus mirabilis
CECT4168T
0
1
0
Proteus vulgaris
NCTC4175
0
1
0
Pseudomonas aeruginosa
ATCC27853
0
1
0
anatum
K84
0
1
0
derby
K1205
0
1
0
dublin
K65
0
1
0
enteritidis
CECT4300; K64
0
2
0
heidelberg
CIS154
0
1
0
infantis
K158
0
1
0
newport
K50
0
1
0
typhimurium
CECT443; NCTC74
0
2
0
Serratia marcesens
NCTC1377
0
1
0
Shigella dysenteriae
CECT584T; NCTC4837
0
2
0
Staphylococcus aureus
ATCC25923
0
1
0
Staphylococcus Epidermidis
NCTC4276
0
1
0
Streptococcus faecalis
NCTC5213
0
1
0
Streptococcus faecium
NCTC1377
0
1
0
Yersinia enterocolitica
CECT4315T
0
1
0
Escherichia coli
Listeria monocytogenes
Salmonella
RL-2
ATCC – American Type Culture Collection; CDC – Central Disease Control; CECT – Colección Española de Cultivo Tipo; CIS – WHO Collaborating Centre for Reference and Research on Salmonella, Collection de l’Institut Pasteur; DSMZ – Deutsch Sammlung für Mikroorganismen; K – Statens Seruminstitut; NCTC – National Collection Type Cultures.
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Table 2 – Bacterial strains isolated from food and environmental samples and tested with the specific FISH protocol for L. monocytogenes Origin Environment
Seagull
Species
Serotype
RL-2
Eub338
Non338
L.monocytogenes
1/2a
1
1
1
0
1/2b
4
4
4
0
4b
13
13
13
0
2
0
2
0
2
2
2
0
2
0
2
0
1/2a
1
1
1
0
1/2b
Faeces L. welshimeri Silages
L.monocytogenes
4b
L. innocua Food
Milk
Turkey
Fish
L.monocytogenes
Chicken
8
8
8
0
1
0
1
0
L. ivanovii
3
0
3
0
L. seeligeri
1
0
1
0
1/2a
1
1
1
1/2b
1
1
1
0 0
L. grayi
2
0
2
0
L. welshimeri
4
0
4
0
1/2a
1
1
1
0
1/2b
1
1
1
0
4b
35
35
35
0
1/2b
7
7
7
4b
6
6
6
0 0
L. grayi
2
0
2
0
L. innocua
2
0
2
0
L. seeligeri
3
0
3
0
1/2a
7
7
7
0
1/2b
2
2
2
0
3b
2
2
2
0
L.monocytogenes
L.monocytogenes
L.monocytogenes
Table 3 – Determination of the bacteria concentration through the conventional methods and the FISH technique (bacteria/ml) Milk Samples Natural Inoculated
Quantification method
L. monocytogenes 4b
Plating
0
FISH
0
Plating
9.0 x 10 (± 4.1 x 106)a
FISH
2.0 x 109 (± 5.0 x 108)b
7
The results correspond to the average values from 5 assays, performed in duplicate (± standard deviation). a,b
CECT941 and L. monocytogenes L2036C were positive, but weak. No cross hybridization was observed to any of the other non-L. monocytogenes strains tested. All 68 reference strains were able to hybridize with Eub338, and none hybridized with Non338 (Table 1). The applicability of the FISH technique to detect bacterial strains isolated from food and environmental samples was tested against 114 Listeria cultures, including 92 L. monocytogenes samples, corresponding to different serotypes. All L. monocytogenes cultures showed positive hybridization signal with RL-2 (Fig. 1A), and no cross hybridization was observed with any of the other Listeria strains tested (Fig. 1B and 1C). All strains were able to hybridize with Eub338, and 122
Positive Hybridisation
L. innocua
L.monocytogenes
Meat
Total
none hybridized with Non338 (Table 2). The applicability of the FISH protocol for the direct detection and enumeration of L. monocytogenes in food samples was also tested. The protocol was applied to milk samples, and it allowed the detection and enumeration of bacteria in the contaminated samples. The bacteria quantification obtained by the FISH technique and the conventional bacteriological method were studied, by comparing results obtained by colony counting and by the number of hybridized cells in 20 microscope fields. It was found that the FISH technique could count 100 x more bacteria than the plating method (Table 3).
Discussion A specific FISH protocol for the rapid detection and enumeration of L. monocytogenes was developed, using a fluorescent 19-mer oligonucleotide probe, homologous to 16S rRNA of L. monocytogenes (Wang et al., 1991). Although this probe was originally used in a dot-blot hybridization technique, its characteristics were ideal for application in FISH: it is specific for L. monocytogenes (Wang et al., 1991); its target is the 16S rRNA subunit, a conserved gene, present in
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A
B
C
Figure 1. Applicability of the FISH protocol. (A) After hybridization with RL-2, L.monocytogenes 3b isolated from chicken (F10) showed a positive hybridization signal. Clumping effect can be observed ( ). (B) L.welshimeri (R48-b1) isolated from turkey showed a negative hybridization signal. (C) The L.welshimeri (R48-b1) cells were located using DAPI staining.
a high number of copies in the cell (> 104 copies per cell), which represent enough targets so that the cell becomes visible through FISH (Amann et al., 1995); its size, 19 oligonucleotides, allows the penetration through the cell wall, reducing the time and the temperature required for hybridization; and its melting temperature, determined by the G+C content, is 48 ºC, in accordance with the recommended range of 60 ºC ≥ Tm ≥ 46 ºC (Fuchs et al., 1998; Fuchs et al., 2000). The protocol was established through the hybridization with reference strains. The first step was the fixation of the bacterial cells, which should be done in the exponential growth phase, in order to guarantee the maximum number of ribosomes (Rice et al., 1997). This step is crucial, since it allows the immobilization of the cells, facilitates the penetration of the probe, protects the ribosomes from degradation by endogenous RNase activity, and maintains the morphological structure of the cells (Amann et al., 1995). There are several protocols for the fixation of the cells. We used a paraformaldehyde solution, with an additional step of permeabilisation of the membrane through dehydration in 70 and 96 % ethanol. This additional step is sometimes necessary, in order to allow the penetration of the probe (Meier et al., 1999). The next step was the hybridization of the probe and the target sequence. The probe was diluted in a hybridization solution, to a concentration of 5 ng/µl (Wallner et al., 1993). This concentration should be determined in order to allow the maximum velocity of hybridization and the minimum of non-specific staining (Wallner et al., 1993). The composition of the hybridization solution and the hybridization temperature determined the stringency conditions necessary for the discrimination between strains with two mismatches, which include the related species L. innocua and L. ivanovii (Wang et al., 1991), allowing the specific detection of L. monocytogenes. The buffer used in the following washing step should provide the same stringency conditions as the hybridization buffer. This step is crucial for the elimination of non-bonded probe and non-specific hybrids.
This protocol was tested against 68 reference strains, including 24 Listeria strains, 17 of which correspond to different serotypes of L. monocytogenes. Only L. monocytogenes strains were able to hybridize with RL2, and no cross hybridization was observed with any of the other strains tested. Fifteen L. monocytogenes strains yielded a strong hybridization signal with RL2, and the hybridization signal of L. monocytogenes CECT941 (serovar 6) and L. monocytogenes L2036 was positive, but weak. Differences between the intensity in the hybridization signal might be related with differences in the ribosome content of the cell (Amann et al., 1995). The protocol was applied to 114 Listeria indigenous isolates, obtained from food and environmental samples, including 92 strains of L. monocytogenes, corresponding to four serovars. All L. monocytogenes cultures showed positive hybridization signals and no cross hybridization was observed to any of the other Listeria species tested. After its optimisation, the FISH protocol was applied to the direct detection in milk samples. To evaluate weather or not this technique could be implemented in food safety analysis, it was necessary to estimate the relation between CFU and the FISH cell counting. With that purpose, tenfold dilutions from natural and artificially contaminated sheep milk were simultaneously analysed by the conventional method and the FISH technique. In all assays, the sensitivity of the FISH technique was 100 x superior to the plating method, as already reported (Auty et al., 2001; Oliveira and Bernardo, 2002). This can be explained by the detection of non-viable cells that still have mRNA molecules, by the observation of individual cells, even when organised in aggregates, and by variations in the sample distribution along the total area of the slide well, which may affect the determination of the number of hybridized cells/ml. These results suggest that the FISH technique can be used for the rapid and specific detection of L. monocytogenes in food samples, although the application of this methodology to pathogen quantification needs to be optimised. This technique allows positive 123
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results in approximately 7 hours, with a consumable cost of 0.45 to 3.0 euro, depending on the number of simultaneously processed samples. Acknowledgements We thank Sergi Ferrer (Universitat Valência), Ana Tenreiro and Rogério Tenreiro (Faculdade de Ciências de Lisboa) for technical and scientific support. The work was supported by the following projects: CIISA43, POCTI/CVT/43252/2001, POCTI/ESP/ 39233/2001. M. Oliveira has a PhD grant from Fundação Ciência e Tecnologia (BD 905/2000).
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