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Genomic typing of Listeria monocytogenes strains by automated laser fluorescence analysis of amplified fragment length polymorphism fingerprint patterns
International Journal of Food Microbiology 49 (1999) 95–102
Genomic typing of Listeria monocytogenes strains by automated laser fluorescence analysis of amplified fragment length polymorphism fingerprint patterns H.J.M. Aarts*, L.E Hakemulder, A.M.A. Van Hoef DLO-State Institute for Quality Control of Agricultural Products ( RIKILT-DLO), P.O. Box 230, NL-6700 AE Wageningen, Netherlands Received 27 October 1998; received in revised form 14 April 1999; accepted 15 April 1999
Abstract The genetic relationship between isolates of Listeria monocytogenes belonging to different serotypes was determined and the suitability of automated laser fluorescent analysis (ALFA) of amplified fragment length polymorphism (AFLP) fingerprints was assessed by genomic typing of 106 L. monocytogenes isolates belonging to serotypes 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 1, and 7. Digitised AFLP fingerprints were obtained that showed approximately 50 clearly distinguishable selectively amplified EcoRI /MseI bands for each strain. The coefficient of similarity between the profiles was determined by simple matching (Ssm ). Based on these coefficients of similarity the investigated strains clustered in two genomic groups. The first group consisted of strains belonging to serotype 1 / 2a, 1 / 2c, 3a and 4a, while the second group was comprised of strains belonging to serotypes 1 / 2b, 3b, 4ab, 4b, 4e and 1. The average simple matching coefficient of similarity between strains of the second group was 92%, which was 4% higher than within group 1. Hence, the serotypes which are responsible for the majority of the listeriosis cases, 1 / 2a, 1 / 2b and 4b, fall into two distinct genetic groups, in concordance with their flagellar antigen type. The discriminatory power of AFLP in combination with automation of the analysis of the fingerprint profiles by ALFA makes AFLP–ALFA highly suitable for typing L. monocytogenes. 1999 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Genomic fingerprinting; AFLP; Automatic laser fluorescent analysis
1. Introduction Listeria monocytogenes is an opportunistic foodborne pathogen that causes listeriosis. Infection can manifest itself by fever, vomiting and diarrhoea, but *Corresponding author. Tel.: 131-317-475-604; fax: 131-317417-717. E-mail address: [email protected] (H.J.M. Aarts)
also by more severe symptoms such as meningitis, abortions and sepsis. This is in agreement with the infection process that starts with colonisation of the gastrointestinal tract then translocates to the liver and spleen and resulting finally in infection of other tissues, such as the placenta. The progression of infection is dependent on a number of factors such as the intestinal microbial population (Lammerding et al., 1992), the immune competence of the patient
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00057-4
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(Jones, 1990), but also on the virulence and the ability to adapt to diverse ecological niches (McLauchlin, 1997). Serotypes 1 / 2a, 1 / 2b and 4b are responsible for the majority of the listeriosis cases. Because serotyping does not allow a higher level of discrimination between L. monocytogenes strains, alternative typing methods are necessary to obtain insight into the potential pathogenicity of a strain. More discriminatory typing methods based on the detection of DNA polymorphisms have been applied, but most of these methods are either labourious (pulse field gel electrophoresis (PFGE), bacterial restriction endonucleases DNA analysis (BRENDA)) or difficult to reproduce between experiments (random amplified polymorphic DNA (RAPD)). Efficient high resolution typing methods are not only necessary to allow determination of the relative importance and distribution of certain L. monocytogenes strains with respect to virulence and the ability to adapt to diverse ecological niches, but also for epidemiological studies that are directed at prevention of contamination. L. monocytogenes poses an increasing health risk, which in part is due to increasing consumption of ready-to-eat food products and the introduction of increasing numbers of food products from regions with different dietary habits. L. monocytogenes can be present in meat, shellfish, vegetables, unpasteurised milk and soft cheese and poses a risk if food containing these products is stored at refrigeration temperature and is not properly heated before consumption, as L. monocytogenes is psychrotrophic (Seelinger and Jones, 1986). Amplified fragment length polymorphism (AFLP) is a robust and highly reproducible DNA fingerprinting technique (Vos et al., 1995), that is based on the selective amplification of genomic restriction fragments to generate fingerprint patterns consisting of large number of bands. AFLP combines the reliability of RFLP analysis and the power of PCR and has successfully been applied for the typing and classification of bacterial strains (Folkertsma et al., 1996; Huys et al., 1996; Janssen et al., 1996; Lin et al., 1996). Application of AFLP is hampered by the manual analysis of the fingerprint patterns, which is difficult and not suitable for comparisons in space and time. This can be circumvented by automated laser fluorescence analysis (ALFA; Grundmann et al., 1995) of the fingerprint patterns in combination with computerised data processing and analysis. The suitability of AFLP–ALFA for typing Listeria was
investigated by analysing the AFLP profiles of 106 L. monocytogenes and two Listeria innocua on an automated sequencer.
2. Materials and methods
2.1. Listeria strains and DNA isolation The 106 L. monocytogenes strains and two L. innocua strains used in this study were either isolated from either different food products or cheese producing environments or were reference strains (Table 1). The strains were serotyped by the National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands. This study included 14 strains of serotype 1 / 2a, 44 strains of serotype 1 / 2b, seven strains of serotype 1 / 2c, one strain of serotype 3a, two strains of serotype 3b, one strain of serotype 3c, one strain of serotype 4a, three strains of serotype 4ab, 22 strains of serotype 4b, one strain of serotype 4c, one strain of serotype 4d, five strains of serotype 4e, one strain of serotype 1, three strains of serotype 7 and two L. innocua serotype 6a strains (Table 1). For DNA isolation, cultures were grown overnight at 378C in Listeria enrichment broth (no. 10985, Merck, Darmstadt, Germany). Cells were collected from 1 ml of culture by centrifugation and genomic DNA was isolated according to Boom et al. (1990) with modification as described by Aarts et al. (1998).
2.2. AFLP– ALFA The AFLP technique was performed essentially as described by Vos et al. (1995). Bacterial DNA (100 ng) was digested for 1 h at 378C with four units of EcoRI (Boehringer, Mannheim, Germany) and four units of MseI (Life-Technologies, Bethesda, MD, USA). Adapters were ligated to the restriction fragments by adding 5 ml of ligation mix containing 2.5 pmol of EcoRI adapter (Vos et al., 1995), 25 pmol of MseI adapter (Vos et al., 1995) and 0.5 units of T4 DNA–ligase (Life-Technologies), with incubation for 3 h at 378C. The ligation mix was diluted tenfold with TE (10 mM Tris, 1 mm EDTA)-buffer (pH 7.0) and 5 ml was used in a selective amplification step using a Cy5E (Amersham Pharmacia, Roosendaal, The Netherlands) labeled EcoRI Primer E01 ( 5 E00 1 A; 59-GACTGCGTACCAATTCA-39) and
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Table 1 Listeria monocytogenes and Listeria innocua strains used in the investigation Strain
Serotype
Source
Strain
Serotype
Source
Strain
Serotype
Source
1 19 20 21 22 23 24 25 27 28 29 30 32 34 35 36 37 38 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 59 60
1 / 2a 1 / 2b 1 / 2b 1 / 2c 4b 7 7 3b 3b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4e 4e 4ab 4ab 4ab 4e 4e 4b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b
Cheese Barsella cheese Barsella cheese CPE a Cheese Egg-yolk Barsella cheese Barsella cheese Barsella cheese Ham Ham Egg-yolk Cheese Egg-yolk CPE a CPE a CPE a CPE a Ham Ham Cockle Cockle Spisula Spisula Spisula Spisula Spisula Egg-yolk Egg-yolk Ham Cheese Cheese Cheese Cheese Cheese Cheese Cheese
61 62 63 64 65 66 67 68 70 71 72 73 74 76 77 78 79 80 84 87 85 88 89 90 91 94 96 97 98 102 104 103 105 106 107 108 109
1 / 2b 1 / 2b 1 / 2a 1 / 2b 1 / 2b 1 / 2b 1 / 2a 1 / 2a 1 / 2a 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2a 1 / 2a 1 / 2b 1 / 2b 1 / 2b 1 / 2a
Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese
110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 136 138 141 142 143 145 146 147 148 149 150 152 159
1 / 2b 1 / 2a 1 / 2b 1 / 2b 4b ? 1 / 2a 1 / 2b 1 / 2b 4b 4b 1 / 2a 1 / 2c 1 / 2c 1 / 2a 1 / 2c 1 / 2c 1 / 2c 1 / 2a 4b 1 L.i. 6a 4b 1 / 2a 3a 3b 3c 4a 4b 4c 4d 4e 7 L.i. 6a 4b
Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Pork Pork Pork Pork Pork Pork Pork Cheese LUW b RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c
a
Cheese producing environment. Collection strain obtained from the Agricultural University Wageningen, Wageningen, The Netherlands. c Collection strain obtained from the National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands. b
nonlabeled M02 (M00 1 C; 59-GATGAGTCCTGAGTAAC-39) MseI primer. AFLP was performed in a volume of 20 ml containing standard Taq polymerase buffer, 0.4 units of Taq polymerase (Perkin-Elmer, Foster City, USA), 0.2 mM dNTPs, 5 ng of primer E01 and 50 ng of primer M02. Thermocycling was done on a PTC 200 (MJ Research, Watertown, USA) or a PE9600 (PerkinElmer) using the following temperature / time parameters: 10 cycles of (928C, 1 min; 658C, 1 min, during cycle 2–10 the annealing temperature was lowered
each cycle with 18C; 728C, 1.5 min), 22 cycles of (928C, 30 s; 568C, 30 s; 728C, 1 min). After the AFLP reaction, an equal amount of loading buffer was added to the reaction mixtures. The samples were denatured for 3 min at 958C and immediately thereafter transferred to ice. The AFLP fragments (4 ml) were analysed for 700 min, at 60 mA, 1500 V and 25 W at 558C on an automatic laser fluorescence (ALF)-Express sequencer (Amersham Pharmacia) using 6% ReadyMix denaturing polyacrylamide gels (Amersham Pharmacia). The electro-
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phoresis buffer was 0.6 3 TBE (0.6 M Tris-base, 0.5 M boric acid, 6 mM EDTA). Gels were normalised with a 50 base pair (bp) ladder (50–500 bp, Amersham Pharmacia). Fluorescent signals were determined using a sampling interval of 2 s.
2.3. Numerical analysis of AFLP banding patterns ALF-Win 1.0 ‘‘alx’’-files were converted into ‘‘tif’’-files and imported in the software package ImageMaster 1D Elite software (Amersham Pharmacia). Lanes were set at 95% of the actual width of the lanes, and the rubber band algorithm, which is part of the software, was used to subtract the background. The band detection parameters were 10 (minimum slope), 1 (noise reduction) and 1 (% maximum peak). The band width was fixed at a value of 5, which corresponds to approximately 4 nucleotides. The molecular weights of the bands were calculated using the cubic spline calibration curve and the ‘‘between standards’’ option. The 50 bp ladder (Amersham Pharmacia) was used as a standard. Matching of the bands within one gel was done by position to a synthetic reference lane consisting of all bands present in the analysed lanes. Per gel matching was done automatically with a vector value of 5. The matching results were, if necessary, corrected manually. Based on a matrix containing 108 strains and 99 bands (having a length between 100 and 350 nucleotides) the simple matching coefficient of similarity (Ssm ) between pairs of lanes was determined by: Ssm 5 (a 1 b) /(a 1 b 1 c), in which a was the number of positive matches, b the number of negative matches and c the number of nonmatches.
3. Results
3.1. AFLP– ALFA AFLP–ALFA was performed for 106 L. monocytogenes strains and two L. innocua strains in separate runs on an ALF-Express automatic DNA sequencer. DNA from these strains was isolated and fragmented with the unfrequent cutting restriction enzyme EcoRI and the frequent cutting restriction enzyme MseI. After adapter ligation, a subset of the restriction fragments, approximately 1 / 16 of the
total, were amplified by a PCR reaction using primers with one selective nucleotide (primer combination M02 / E01), of which primer E01 was labeled with the fluorophore Cy5E. After PCR, the reactions were subjected to electrophoresis during which the fluorescent labeled bands pass a fixed laser beam and the emitted signals were collected by photodetectors every 2 s. Fluorograms corresponding to approximately 50 bands per lane were obtained. The deduced digitised ALFA–AFLP image of one gel is shown in Fig. 1.
3.2. Similarity coefficients for AFLP profiles of L. monocytogenes strains A matrix containing all the investigated strains and the presence or absence of 99 AFLP bands within the molecular weight region of 100 to 350 nucleotides was used to determine the Ssm values between pairs of profiles. Within and between serotypes the average Ssm was determined. The results of these analyses are given in Table 2. At the 70% level two major clusters of serotypes were formed (not included were serotypes of which only one strain was investigated and serotype 7 strains). The first genetic group (A) consisted of strains belonging to serotype 1 / 2a or 1 / 2c with an average overall similarity of Ssm of 8865% on average. At 9663%, the 1 / 2c strains exhibited the highest within serotype similarity, whereas 1 / 2a strains were mutually less homogenous and were all, except for strains 67 and 68, characterised by a unique profile. The within serotype similarity for serotype 1 / 2a was 886 5% on average. The second genetic group (B) consisted of strains belonging to serotypes 1 / 2b, 3b, 4ab, 4b and 4e with an average similarity coefficient of 9267%. The lowest within serotype similarity was found for serotype 4b. The other members of group B exhibited high values of both within and between simple matching coefficients of similarity.
3.3. Strain, serotype and group specific AFLP fragments Despite a substantial number of ‘‘listeria’’ common bands, the majority of the investigated strains had a unique AFLP profile. Although a simple matching coefficient of 9663% was found between the 44 investigated 1 / 2b strains, 20 different 1 / 2b
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Fig. 1. Digital electrophoresis image of AFLP fragments of Listeria monocytogenes run on an ALF-Express automatic DNA sequencer. Strain number and serotype corresponding with Table 1 are indicated above the lanes. Arrows indicate serotype specific fragments. Band 4 is present in serotypes 4a, 4ab, 4b, 4c, 4d and 4e. Marker lanes are indicated with Marker 50 bp.
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Table 2 Average simple matching (Ssm ) coefficient of similarity within and between different serotypes of Listeria monocytogenes (L.m.) or Listeria innocua (L.i.) Serotype
Genomic group
L.m. 1/2a (n514)
L.m. 1/2b (n544)
L.m. 1/2c (n57)
L.m. 3a (n51)
Mean
Mean
Mean
Mean
SD
A L.m. 1/2a L.m. 1/2b L.m. 1/2c L.m. 3a L.m. 3b L.m. 3c L.m. 4a L.m. 4ab L.m. 4b L.m. 4c L.m. 4d L.m. 4e L.m. 1 L.m. 7 L.i. 6a L.i. 6a
L.m. 1/2a L.m. 1/2b L.m. 1/2c L.m. 3a L.m. 3b L.m. 3c L.m. 4a L.m. 4ab L.m. 4b L.m. 4c L.m. 4d L.m. 4e L.m. 1 L.m. 7 L.i. 6a L.i. 6a
A B A A B ? A B B ? B B B ? ? ?
0.88
Genomic group
B
A B A A B ? A B B ? B B B ? ? ?
0.72 0.89 0.70 0.72 0.91 0.88 0.71 0.90 0.86
SD
B 0.05
0.71 0.96
A 0.30 0.03
? 0.06 0.07 0.01 0.07 0.08 0.07 0.07 0.08 0.09
0.68 0.75 0.68 0.70 0.76 0.75 0.73 0.75 0.73 1.00
SD
0.87 0.68 0.96
0.00
0.01 0.03
0.67 0.93 0.65 0.71 0.95 0.94 0.70 0.94 0.89 0.73 1.00
SD
A 0.03 0.02 0.03
B 0.03 0.02 0.03
L.m. 3b (n52)
0.88 0.71 0.87 1.00
0.02
0.03 0.08
profiles were found. When the similarity coefficient drops to 86%, as was found for the 4b strains, almost each strain had its own characteristic profile. The members of the two genetic groups could be easily be identified by a number of group specific bands, which are indicated in Fig. 1. At the serotype level, specific bands could only be identified for a small number of serotypes. Strains belonging to serotype 1 / 2a and 1 / 2c differed by the presence or absence of band ‘‘1 / 2c’’ (see Fig. 1) which had a length of 163
0.70 0.94 0.65 0.70 0.96 0.92 0.70 0.96 0.90 0.74 0.96 0.95
SD
B 0.04 0.01 0.01
B 0.03 0.02 0.02
Mean
L.m. 3c (n51)
0.70 0.89 0.67 0.70 0.98
0.65 0.81 0.64 0.63 0.81 0.76 0.60 0.79 0.77 0.67 0.76 0.79 1.00
SD
? 0.04 0.12 0.01
B 0.03 0.03 0.02 0.02 0.13 0.01 0.01 0.03 0.08 0.01 0.02 0.03
Mean
L.m. 4a (n51)
0.70 0.89 0.86 0.75 0.91 1.00
0.00
0.03 0.06
0.02
0.83 0.79 0.83 0.84 0.79 0.78 0.83 0.78 0.78 0.74 0.76 0.78 0.7 0.80
SD
A 0.03 0.02 0.01 0.00
? 0.02 0.01 0.01
Mean
L.m. 4ab (n53)
0.84 0.72 0.86 0.93 0.71 0.74 1.00
0.68 0.77 0.70 0.71 0.78 0.74 0.70 0.77 0.74 0.77 0.78 0.76 0.73 0.69 1.00
SD
B 0.03 0.02 0.01 0.01 0.00
? 0.08 0.08 0.11 0.07 0.09 0.13 0.07 0.10 0.09 0.03 0.13 0.10 0.01 0.14
Mean
0.69 0.95 0.68 0.69 0.97 0.91 0.70 0.95
0.03 0.02 0.07 0.01 0.02 0.01 0.04
? 0.03 0.01 0.01 0.01
0.01 0.03
0.02 0.02
0.60 0.71 0.66 0.62 0.70 0.63 0.61 0.64 0.67 0.60 0.67 0.67 0.63 0.60 0.77 1.00
0.02 0.01 0.01 0.00
0.01 0.03
0.02 0.01
bp. All serotype 1 / 2b strains had the ‘‘1 / 2b’’ band, with a length of 92 bp, which was absent from the profiles of the other strains.
4. Discussion AFLP in combination with ALFA is an useful alternative that can be used for rapid and reliable genotyping. AFLP reactions, gel-analysis, fluorescent
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band detection and analysis of the fluorograms can be standardised creating the possibility to store the profiles in an AFLP database for comparison with future strains. The present study describes the use of AFLP– ALFA for typing L. monocytogenes strains and shows that AFLP is more discriminating than serotyping. In most cases, this higher level of discrimination enabled identification at the strain level, resulting in unique characteristic profiles (Fig. 1). Furthermore, it was shown that L. monocytogenes strains belonging to serotypes 1 / 2a, 1 / 2b and 4b fall into two distinct genetic groups, based on the simple matching coefficients of similarity. Group A comprised serotypes 1 / 2a, 1 / 2c, 3a and 4a and group B comprised serotypes 1 / 2b, 3b, 4ab, 4b, 4d, 4e and 1. Similar results were also found by Bibb et al. (1990) by multilocus enzyme electrophoresis, by Graves et al. (1994) using ribotyping, by Moore and Datta (1994) using PFGE, by Comi et al. (1997) using PCR-restriction enzyme analysis of the entire iap gene and by Allerberger et al. (1997) who studied L. monocytogenes strains using RAPD–ALFA. The serotype of a strain is determined by antigenic variation in a limited number of proteins, making it unlikely that this will result in large variations in genomic fingerprinting patterns. The findings presented here correlates with variation in the flagellar (H) antigen type (Seelinger and Jones, 1986). The members of group B all possess the H-antigens A, B and C. H-antigen C is absent in the members of group A with the exception of Strain 145 having serotype 4a (Seelinger and Jones, 1986). In a study which analysed 62 different serotypes of Salmonella by AFLP, no correlation was observed between the presence of certain antigens and AFLP banding patterns (Aarts et al., 1998). The heterogeneity that was observed within serotype 1 / 2a was also found with other DNA fingerprinting techniques, such as repetitive element sequence-based (ERIC (enterobacterial repetitive intergenic consensus)– and REP (repetitive extragenic ˇ et al., palindrome)–PCR) DNA fingerprinting (Jersek 1996) and PFGE analysis (Buchrieser et al., 1993; Moore and Datta, 1994). The other members of group A, serotype 1 / 2c, were found to be highly homogeneous. This confirms the results obtained by Niederhauser et al. (1994), who used low resolution RAPD fingerprinting for the analysis of 19 L.
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monocytogenes 1 / 2c strains and found identical RAPD profiles for all strains. While serotype 1 / 2c appears to be a homogeneous genetic group, further genomic analysis is necessary to determine whether the other serotypes, in particular serotype 1 / 2a, can be considered a homogeneous genetically related group. In conclusion it was shown that L. monocytogenes strains can be identified by AFLP fingerprinting with subsequent automatic profile analysis and that the obtained results match the results obtained with other phenotypic or genotypic typing methods. The use of ALFA and appropriate software significantly reduced postrun analysis time and improved the quality of the results. The data show that the discriminatory power of AFLP is sufficient to adequately support the genomic typing of L. monocytogenes. By standardisation and automation of the experimental procedures and data-analysis, labour input is reduced making AFLP–ALFA a cost effective procedure. Furthermore, highly reproducible fingerprints are obtained that allow for incorporation in a database, facilitating typing studies in space and time to support epidemiological studies.
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Lammerding, A.M., Glass, K.A., Gendron-Fitzpatrick, A., Doyle, M.P., 1992. Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice. Appl. Environ. Microbiol. 58, 3991– 4000. Lin, J.J., Kuo, J., Ma, J., 1996. A PCR-based DNA fingerprinting technique — AFLP for molecular typing of bacteria. Nucleic Acids Res. 24, 3649–3650. McLauchlin, J., 1997. Animal and human listeriosis: a shared problem? Vet. J. 153, 3–5. Moore, M.A., Datta, A.R., 1994. DNA fingerprinting of Listeria monocytogenes strains by pulsed-field gel electrophoresis. Food Microbiol. 11, 31–38. ¨ Niederhauser, Ch., Hofelein, Ch., Allmann, M., Burkhalter, P., ¨ Luthy, J., Candrian, U., 1994. Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes. J. Appl. Bacteriol. 77, 574–582. Seelinger, H.P.R., Jones, D., 1986. Listeria. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, Vol. 2, Williams and Wilkins, Baltimore, pp. 1235–1245. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijtens, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acid Res. 23, 4407–4414.
Genomic typing of Listeria monocytogenes strains by automated laser fluorescence analysis of amplified fragment length polymorphism fingerprint patterns H.J.M. Aarts*, L.E Hakemulder, A.M.A. Van Hoef DLO-State Institute for Quality Control of Agricultural Products ( RIKILT-DLO), P.O. Box 230, NL-6700 AE Wageningen, Netherlands Received 27 October 1998; received in revised form 14 April 1999; accepted 15 April 1999
Abstract The genetic relationship between isolates of Listeria monocytogenes belonging to different serotypes was determined and the suitability of automated laser fluorescent analysis (ALFA) of amplified fragment length polymorphism (AFLP) fingerprints was assessed by genomic typing of 106 L. monocytogenes isolates belonging to serotypes 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 1, and 7. Digitised AFLP fingerprints were obtained that showed approximately 50 clearly distinguishable selectively amplified EcoRI /MseI bands for each strain. The coefficient of similarity between the profiles was determined by simple matching (Ssm ). Based on these coefficients of similarity the investigated strains clustered in two genomic groups. The first group consisted of strains belonging to serotype 1 / 2a, 1 / 2c, 3a and 4a, while the second group was comprised of strains belonging to serotypes 1 / 2b, 3b, 4ab, 4b, 4e and 1. The average simple matching coefficient of similarity between strains of the second group was 92%, which was 4% higher than within group 1. Hence, the serotypes which are responsible for the majority of the listeriosis cases, 1 / 2a, 1 / 2b and 4b, fall into two distinct genetic groups, in concordance with their flagellar antigen type. The discriminatory power of AFLP in combination with automation of the analysis of the fingerprint profiles by ALFA makes AFLP–ALFA highly suitable for typing L. monocytogenes. 1999 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Genomic fingerprinting; AFLP; Automatic laser fluorescent analysis
1. Introduction Listeria monocytogenes is an opportunistic foodborne pathogen that causes listeriosis. Infection can manifest itself by fever, vomiting and diarrhoea, but *Corresponding author. Tel.: 131-317-475-604; fax: 131-317417-717. E-mail address: [email protected] (H.J.M. Aarts)
also by more severe symptoms such as meningitis, abortions and sepsis. This is in agreement with the infection process that starts with colonisation of the gastrointestinal tract then translocates to the liver and spleen and resulting finally in infection of other tissues, such as the placenta. The progression of infection is dependent on a number of factors such as the intestinal microbial population (Lammerding et al., 1992), the immune competence of the patient
0168-1605 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00057-4
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(Jones, 1990), but also on the virulence and the ability to adapt to diverse ecological niches (McLauchlin, 1997). Serotypes 1 / 2a, 1 / 2b and 4b are responsible for the majority of the listeriosis cases. Because serotyping does not allow a higher level of discrimination between L. monocytogenes strains, alternative typing methods are necessary to obtain insight into the potential pathogenicity of a strain. More discriminatory typing methods based on the detection of DNA polymorphisms have been applied, but most of these methods are either labourious (pulse field gel electrophoresis (PFGE), bacterial restriction endonucleases DNA analysis (BRENDA)) or difficult to reproduce between experiments (random amplified polymorphic DNA (RAPD)). Efficient high resolution typing methods are not only necessary to allow determination of the relative importance and distribution of certain L. monocytogenes strains with respect to virulence and the ability to adapt to diverse ecological niches, but also for epidemiological studies that are directed at prevention of contamination. L. monocytogenes poses an increasing health risk, which in part is due to increasing consumption of ready-to-eat food products and the introduction of increasing numbers of food products from regions with different dietary habits. L. monocytogenes can be present in meat, shellfish, vegetables, unpasteurised milk and soft cheese and poses a risk if food containing these products is stored at refrigeration temperature and is not properly heated before consumption, as L. monocytogenes is psychrotrophic (Seelinger and Jones, 1986). Amplified fragment length polymorphism (AFLP) is a robust and highly reproducible DNA fingerprinting technique (Vos et al., 1995), that is based on the selective amplification of genomic restriction fragments to generate fingerprint patterns consisting of large number of bands. AFLP combines the reliability of RFLP analysis and the power of PCR and has successfully been applied for the typing and classification of bacterial strains (Folkertsma et al., 1996; Huys et al., 1996; Janssen et al., 1996; Lin et al., 1996). Application of AFLP is hampered by the manual analysis of the fingerprint patterns, which is difficult and not suitable for comparisons in space and time. This can be circumvented by automated laser fluorescence analysis (ALFA; Grundmann et al., 1995) of the fingerprint patterns in combination with computerised data processing and analysis. The suitability of AFLP–ALFA for typing Listeria was
investigated by analysing the AFLP profiles of 106 L. monocytogenes and two Listeria innocua on an automated sequencer.
2. Materials and methods
2.1. Listeria strains and DNA isolation The 106 L. monocytogenes strains and two L. innocua strains used in this study were either isolated from either different food products or cheese producing environments or were reference strains (Table 1). The strains were serotyped by the National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands. This study included 14 strains of serotype 1 / 2a, 44 strains of serotype 1 / 2b, seven strains of serotype 1 / 2c, one strain of serotype 3a, two strains of serotype 3b, one strain of serotype 3c, one strain of serotype 4a, three strains of serotype 4ab, 22 strains of serotype 4b, one strain of serotype 4c, one strain of serotype 4d, five strains of serotype 4e, one strain of serotype 1, three strains of serotype 7 and two L. innocua serotype 6a strains (Table 1). For DNA isolation, cultures were grown overnight at 378C in Listeria enrichment broth (no. 10985, Merck, Darmstadt, Germany). Cells were collected from 1 ml of culture by centrifugation and genomic DNA was isolated according to Boom et al. (1990) with modification as described by Aarts et al. (1998).
2.2. AFLP– ALFA The AFLP technique was performed essentially as described by Vos et al. (1995). Bacterial DNA (100 ng) was digested for 1 h at 378C with four units of EcoRI (Boehringer, Mannheim, Germany) and four units of MseI (Life-Technologies, Bethesda, MD, USA). Adapters were ligated to the restriction fragments by adding 5 ml of ligation mix containing 2.5 pmol of EcoRI adapter (Vos et al., 1995), 25 pmol of MseI adapter (Vos et al., 1995) and 0.5 units of T4 DNA–ligase (Life-Technologies), with incubation for 3 h at 378C. The ligation mix was diluted tenfold with TE (10 mM Tris, 1 mm EDTA)-buffer (pH 7.0) and 5 ml was used in a selective amplification step using a Cy5E (Amersham Pharmacia, Roosendaal, The Netherlands) labeled EcoRI Primer E01 ( 5 E00 1 A; 59-GACTGCGTACCAATTCA-39) and
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Table 1 Listeria monocytogenes and Listeria innocua strains used in the investigation Strain
Serotype
Source
Strain
Serotype
Source
Strain
Serotype
Source
1 19 20 21 22 23 24 25 27 28 29 30 32 34 35 36 37 38 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 59 60
1 / 2a 1 / 2b 1 / 2b 1 / 2c 4b 7 7 3b 3b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4e 4e 4ab 4ab 4ab 4e 4e 4b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b
Cheese Barsella cheese Barsella cheese CPE a Cheese Egg-yolk Barsella cheese Barsella cheese Barsella cheese Ham Ham Egg-yolk Cheese Egg-yolk CPE a CPE a CPE a CPE a Ham Ham Cockle Cockle Spisula Spisula Spisula Spisula Spisula Egg-yolk Egg-yolk Ham Cheese Cheese Cheese Cheese Cheese Cheese Cheese
61 62 63 64 65 66 67 68 70 71 72 73 74 76 77 78 79 80 84 87 85 88 89 90 91 94 96 97 98 102 104 103 105 106 107 108 109
1 / 2b 1 / 2b 1 / 2a 1 / 2b 1 / 2b 1 / 2b 1 / 2a 1 / 2a 1 / 2a 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2b 1 / 2a 1 / 2a 1 / 2b 1 / 2b 1 / 2b 1 / 2a
Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese
110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 136 138 141 142 143 145 146 147 148 149 150 152 159
1 / 2b 1 / 2a 1 / 2b 1 / 2b 4b ? 1 / 2a 1 / 2b 1 / 2b 4b 4b 1 / 2a 1 / 2c 1 / 2c 1 / 2a 1 / 2c 1 / 2c 1 / 2c 1 / 2a 4b 1 L.i. 6a 4b 1 / 2a 3a 3b 3c 4a 4b 4c 4d 4e 7 L.i. 6a 4b
Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Cheese Pork Pork Pork Pork Pork Pork Pork Cheese LUW b RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c RIVM c
a
Cheese producing environment. Collection strain obtained from the Agricultural University Wageningen, Wageningen, The Netherlands. c Collection strain obtained from the National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands. b
nonlabeled M02 (M00 1 C; 59-GATGAGTCCTGAGTAAC-39) MseI primer. AFLP was performed in a volume of 20 ml containing standard Taq polymerase buffer, 0.4 units of Taq polymerase (Perkin-Elmer, Foster City, USA), 0.2 mM dNTPs, 5 ng of primer E01 and 50 ng of primer M02. Thermocycling was done on a PTC 200 (MJ Research, Watertown, USA) or a PE9600 (PerkinElmer) using the following temperature / time parameters: 10 cycles of (928C, 1 min; 658C, 1 min, during cycle 2–10 the annealing temperature was lowered
each cycle with 18C; 728C, 1.5 min), 22 cycles of (928C, 30 s; 568C, 30 s; 728C, 1 min). After the AFLP reaction, an equal amount of loading buffer was added to the reaction mixtures. The samples were denatured for 3 min at 958C and immediately thereafter transferred to ice. The AFLP fragments (4 ml) were analysed for 700 min, at 60 mA, 1500 V and 25 W at 558C on an automatic laser fluorescence (ALF)-Express sequencer (Amersham Pharmacia) using 6% ReadyMix denaturing polyacrylamide gels (Amersham Pharmacia). The electro-
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phoresis buffer was 0.6 3 TBE (0.6 M Tris-base, 0.5 M boric acid, 6 mM EDTA). Gels were normalised with a 50 base pair (bp) ladder (50–500 bp, Amersham Pharmacia). Fluorescent signals were determined using a sampling interval of 2 s.
2.3. Numerical analysis of AFLP banding patterns ALF-Win 1.0 ‘‘alx’’-files were converted into ‘‘tif’’-files and imported in the software package ImageMaster 1D Elite software (Amersham Pharmacia). Lanes were set at 95% of the actual width of the lanes, and the rubber band algorithm, which is part of the software, was used to subtract the background. The band detection parameters were 10 (minimum slope), 1 (noise reduction) and 1 (% maximum peak). The band width was fixed at a value of 5, which corresponds to approximately 4 nucleotides. The molecular weights of the bands were calculated using the cubic spline calibration curve and the ‘‘between standards’’ option. The 50 bp ladder (Amersham Pharmacia) was used as a standard. Matching of the bands within one gel was done by position to a synthetic reference lane consisting of all bands present in the analysed lanes. Per gel matching was done automatically with a vector value of 5. The matching results were, if necessary, corrected manually. Based on a matrix containing 108 strains and 99 bands (having a length between 100 and 350 nucleotides) the simple matching coefficient of similarity (Ssm ) between pairs of lanes was determined by: Ssm 5 (a 1 b) /(a 1 b 1 c), in which a was the number of positive matches, b the number of negative matches and c the number of nonmatches.
3. Results
3.1. AFLP– ALFA AFLP–ALFA was performed for 106 L. monocytogenes strains and two L. innocua strains in separate runs on an ALF-Express automatic DNA sequencer. DNA from these strains was isolated and fragmented with the unfrequent cutting restriction enzyme EcoRI and the frequent cutting restriction enzyme MseI. After adapter ligation, a subset of the restriction fragments, approximately 1 / 16 of the
total, were amplified by a PCR reaction using primers with one selective nucleotide (primer combination M02 / E01), of which primer E01 was labeled with the fluorophore Cy5E. After PCR, the reactions were subjected to electrophoresis during which the fluorescent labeled bands pass a fixed laser beam and the emitted signals were collected by photodetectors every 2 s. Fluorograms corresponding to approximately 50 bands per lane were obtained. The deduced digitised ALFA–AFLP image of one gel is shown in Fig. 1.
3.2. Similarity coefficients for AFLP profiles of L. monocytogenes strains A matrix containing all the investigated strains and the presence or absence of 99 AFLP bands within the molecular weight region of 100 to 350 nucleotides was used to determine the Ssm values between pairs of profiles. Within and between serotypes the average Ssm was determined. The results of these analyses are given in Table 2. At the 70% level two major clusters of serotypes were formed (not included were serotypes of which only one strain was investigated and serotype 7 strains). The first genetic group (A) consisted of strains belonging to serotype 1 / 2a or 1 / 2c with an average overall similarity of Ssm of 8865% on average. At 9663%, the 1 / 2c strains exhibited the highest within serotype similarity, whereas 1 / 2a strains were mutually less homogenous and were all, except for strains 67 and 68, characterised by a unique profile. The within serotype similarity for serotype 1 / 2a was 886 5% on average. The second genetic group (B) consisted of strains belonging to serotypes 1 / 2b, 3b, 4ab, 4b and 4e with an average similarity coefficient of 9267%. The lowest within serotype similarity was found for serotype 4b. The other members of group B exhibited high values of both within and between simple matching coefficients of similarity.
3.3. Strain, serotype and group specific AFLP fragments Despite a substantial number of ‘‘listeria’’ common bands, the majority of the investigated strains had a unique AFLP profile. Although a simple matching coefficient of 9663% was found between the 44 investigated 1 / 2b strains, 20 different 1 / 2b
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Fig. 1. Digital electrophoresis image of AFLP fragments of Listeria monocytogenes run on an ALF-Express automatic DNA sequencer. Strain number and serotype corresponding with Table 1 are indicated above the lanes. Arrows indicate serotype specific fragments. Band 4 is present in serotypes 4a, 4ab, 4b, 4c, 4d and 4e. Marker lanes are indicated with Marker 50 bp.
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Table 2 Average simple matching (Ssm ) coefficient of similarity within and between different serotypes of Listeria monocytogenes (L.m.) or Listeria innocua (L.i.) Serotype
Genomic group
L.m. 1/2a (n514)
L.m. 1/2b (n544)
L.m. 1/2c (n57)
L.m. 3a (n51)
Mean
Mean
Mean
Mean
SD
A L.m. 1/2a L.m. 1/2b L.m. 1/2c L.m. 3a L.m. 3b L.m. 3c L.m. 4a L.m. 4ab L.m. 4b L.m. 4c L.m. 4d L.m. 4e L.m. 1 L.m. 7 L.i. 6a L.i. 6a
L.m. 1/2a L.m. 1/2b L.m. 1/2c L.m. 3a L.m. 3b L.m. 3c L.m. 4a L.m. 4ab L.m. 4b L.m. 4c L.m. 4d L.m. 4e L.m. 1 L.m. 7 L.i. 6a L.i. 6a
A B A A B ? A B B ? B B B ? ? ?
0.88
Genomic group
B
A B A A B ? A B B ? B B B ? ? ?
0.72 0.89 0.70 0.72 0.91 0.88 0.71 0.90 0.86
SD
B 0.05
0.71 0.96
A 0.30 0.03
? 0.06 0.07 0.01 0.07 0.08 0.07 0.07 0.08 0.09
0.68 0.75 0.68 0.70 0.76 0.75 0.73 0.75 0.73 1.00
SD
0.87 0.68 0.96
0.00
0.01 0.03
0.67 0.93 0.65 0.71 0.95 0.94 0.70 0.94 0.89 0.73 1.00
SD
A 0.03 0.02 0.03
B 0.03 0.02 0.03
L.m. 3b (n52)
0.88 0.71 0.87 1.00
0.02
0.03 0.08
profiles were found. When the similarity coefficient drops to 86%, as was found for the 4b strains, almost each strain had its own characteristic profile. The members of the two genetic groups could be easily be identified by a number of group specific bands, which are indicated in Fig. 1. At the serotype level, specific bands could only be identified for a small number of serotypes. Strains belonging to serotype 1 / 2a and 1 / 2c differed by the presence or absence of band ‘‘1 / 2c’’ (see Fig. 1) which had a length of 163
0.70 0.94 0.65 0.70 0.96 0.92 0.70 0.96 0.90 0.74 0.96 0.95
SD
B 0.04 0.01 0.01
B 0.03 0.02 0.02
Mean
L.m. 3c (n51)
0.70 0.89 0.67 0.70 0.98
0.65 0.81 0.64 0.63 0.81 0.76 0.60 0.79 0.77 0.67 0.76 0.79 1.00
SD
? 0.04 0.12 0.01
B 0.03 0.03 0.02 0.02 0.13 0.01 0.01 0.03 0.08 0.01 0.02 0.03
Mean
L.m. 4a (n51)
0.70 0.89 0.86 0.75 0.91 1.00
0.00
0.03 0.06
0.02
0.83 0.79 0.83 0.84 0.79 0.78 0.83 0.78 0.78 0.74 0.76 0.78 0.7 0.80
SD
A 0.03 0.02 0.01 0.00
? 0.02 0.01 0.01
Mean
L.m. 4ab (n53)
0.84 0.72 0.86 0.93 0.71 0.74 1.00
0.68 0.77 0.70 0.71 0.78 0.74 0.70 0.77 0.74 0.77 0.78 0.76 0.73 0.69 1.00
SD
B 0.03 0.02 0.01 0.01 0.00
? 0.08 0.08 0.11 0.07 0.09 0.13 0.07 0.10 0.09 0.03 0.13 0.10 0.01 0.14
Mean
0.69 0.95 0.68 0.69 0.97 0.91 0.70 0.95
0.03 0.02 0.07 0.01 0.02 0.01 0.04
? 0.03 0.01 0.01 0.01
0.01 0.03
0.02 0.02
0.60 0.71 0.66 0.62 0.70 0.63 0.61 0.64 0.67 0.60 0.67 0.67 0.63 0.60 0.77 1.00
0.02 0.01 0.01 0.00
0.01 0.03
0.02 0.01
bp. All serotype 1 / 2b strains had the ‘‘1 / 2b’’ band, with a length of 92 bp, which was absent from the profiles of the other strains.
4. Discussion AFLP in combination with ALFA is an useful alternative that can be used for rapid and reliable genotyping. AFLP reactions, gel-analysis, fluorescent
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band detection and analysis of the fluorograms can be standardised creating the possibility to store the profiles in an AFLP database for comparison with future strains. The present study describes the use of AFLP– ALFA for typing L. monocytogenes strains and shows that AFLP is more discriminating than serotyping. In most cases, this higher level of discrimination enabled identification at the strain level, resulting in unique characteristic profiles (Fig. 1). Furthermore, it was shown that L. monocytogenes strains belonging to serotypes 1 / 2a, 1 / 2b and 4b fall into two distinct genetic groups, based on the simple matching coefficients of similarity. Group A comprised serotypes 1 / 2a, 1 / 2c, 3a and 4a and group B comprised serotypes 1 / 2b, 3b, 4ab, 4b, 4d, 4e and 1. Similar results were also found by Bibb et al. (1990) by multilocus enzyme electrophoresis, by Graves et al. (1994) using ribotyping, by Moore and Datta (1994) using PFGE, by Comi et al. (1997) using PCR-restriction enzyme analysis of the entire iap gene and by Allerberger et al. (1997) who studied L. monocytogenes strains using RAPD–ALFA. The serotype of a strain is determined by antigenic variation in a limited number of proteins, making it unlikely that this will result in large variations in genomic fingerprinting patterns. The findings presented here correlates with variation in the flagellar (H) antigen type (Seelinger and Jones, 1986). The members of group B all possess the H-antigens A, B and C. H-antigen C is absent in the members of group A with the exception of Strain 145 having serotype 4a (Seelinger and Jones, 1986). In a study which analysed 62 different serotypes of Salmonella by AFLP, no correlation was observed between the presence of certain antigens and AFLP banding patterns (Aarts et al., 1998). The heterogeneity that was observed within serotype 1 / 2a was also found with other DNA fingerprinting techniques, such as repetitive element sequence-based (ERIC (enterobacterial repetitive intergenic consensus)– and REP (repetitive extragenic ˇ et al., palindrome)–PCR) DNA fingerprinting (Jersek 1996) and PFGE analysis (Buchrieser et al., 1993; Moore and Datta, 1994). The other members of group A, serotype 1 / 2c, were found to be highly homogeneous. This confirms the results obtained by Niederhauser et al. (1994), who used low resolution RAPD fingerprinting for the analysis of 19 L.
101
monocytogenes 1 / 2c strains and found identical RAPD profiles for all strains. While serotype 1 / 2c appears to be a homogeneous genetic group, further genomic analysis is necessary to determine whether the other serotypes, in particular serotype 1 / 2a, can be considered a homogeneous genetically related group. In conclusion it was shown that L. monocytogenes strains can be identified by AFLP fingerprinting with subsequent automatic profile analysis and that the obtained results match the results obtained with other phenotypic or genotypic typing methods. The use of ALFA and appropriate software significantly reduced postrun analysis time and improved the quality of the results. The data show that the discriminatory power of AFLP is sufficient to adequately support the genomic typing of L. monocytogenes. By standardisation and automation of the experimental procedures and data-analysis, labour input is reduced making AFLP–ALFA a cost effective procedure. Furthermore, highly reproducible fingerprints are obtained that allow for incorporation in a database, facilitating typing studies in space and time to support epidemiological studies.
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