Bull Vet Inst Pulawy 48, 427-435, 2004
GENOTYPIC CHARACTERIZATION OF LISTERIA MONOCYTOGENES ISOLATED FROM FOODSTUFFS AND FARM ANIMALS IN POLAND ŁUKASZ WOJCIECH1, KATARZYNA KOWALCZYK1, ZDZISŁAW STARONIEWICZ2, KATARZYNA KOSEK3, JERZY MOLENDA3 AND MACIEJ UGORSKI1 2
Departments of 1Biochemistry, Pharmacology and Toxicology, Pathological Anatomy, Pathophysiology, Microbiology and Forensic Veterinary Medicine, 3 Food Hygiene and Consumer Health Care, Faculty of Veterinary Medicine, Agriculture University of Wrocław, 50-375 Wrocław, Poland e-mail:
[email protected] Received for publication May 24, 2004.
Abstract Twenty six Listeria monocytogenes strains were isolated from foodstuffs and animal clinical cases and analysed by genotyping including ITS profiling, REP- and ERIC-PCR and PFGE. Analysis of DNA banding generated by ITS profiling revealed the presence of just three different genotypes. With the use of REP-PCR, ten different DNA patterns could be discriminated among the analysed isolates. ERIC-PCR and PFGE gave similar results. With the first method, 13 different DNA patterns were found, with the latter one – 14 different genotypes. Based on data obtained by the ERIC-PCR and PFGE it was shown that the majority of food-derived L. monocytogenes represent different genotypes than clinical isolates recovered from infected animals.
Key words: Listeria monocytogenes, farm animals, food, genotyping. Listeria monocytogenes is an ubiquitous Grampositive bacterium of intracellular localisation responsible for severe infections in humans and more than 40 animal species (18). In cattle, sheep and goats, as well as in pregnant women, infants, the elderly and immunocompromised patients, clinical manifestations are often encephalitis and abortion, sometimes septicaemia. In the United States in recent years L. monocytogens caused about 500 human deaths per year (20). It has been known for a long time that silage is the main source of L. monocytogenes infections in farm animals (12, 30). However just about twenty years ago epidemiological and laboratory investigation revealed that contaminated food is an important factor in the transmission of this pathogen to humans (26). Since then it has been shown that the main source of infections is
ready-to-eat food with long shelf-life (17, 24), making L. monocytogenes one of the major causative agents (28%) of food-related deaths (20). On the other hand, it is now widely accepted that zoonotic transmission of L. monocytogenes is rare (11), even though many strains from animal and human listeriosis are not distinguishable genetically (25, 30). The only more serious source of infections seems to be dairy products coming from mastitic cows (10, 15). However, farm animals being reservoirs of L. monocytogenes can be involved indirectly in human listeriosis (4, 23). Among known L. monocytogenes serotypes mainly three (1/2a, 1/2b and 4b) are causative agents of listeriosis, which makes serotyping of limited value in epidemiological investigations. Other phenotypic methods, such as phage typing, have high discriminatory power, but limited application due to the low number of strains in serogroup ½ which can be taped this way. For these reasons molecular markers are of major importance in epidemiological studies of L. monocytogenes. Molecular methods used for typing L. monocytogens strains include restriction enzyme analysis (REA) (2, 21, 29), pulsed-field gel electrophoresis (PFGE) (5-7, 13), random amplification of polymorphic DNA (RAPD) (3, 19) and ribotyping (2, 22, 23). With genotyping methods it was suggested that: (i) clinical strains isolated from infected humans are less heterogenous genetically than food-derived ones, (ii) food-derived strains represent separate genomic groups in comparison to clinical isolates (16), (iii) contamination of meat with L. monocytogenes comes from the environment and not from the animals themselves (4). However, there are very few studies where the direct comparison of animal and food-derived isolates was performed. For this reason, the aim of the present study was to investigate the level of genetic diversity and possible relationships among L. monocytogenes strains isolated from food and animals
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with the use of REP-PCR, ERIC-PCR, ITS-profiling and PFGE.
Material and Methods Bacterial isolates and growth conditions. Twenty four L. monocytogenes strains were isolated in southwest (Lower Silesia) part of Poland. Of these, ten were of animal origin and fourteen were from poultry meat products. Two reference strains were obtained from Professor Seeliger. Food-borne L. monocytogenes strains were isolated and characterised according to the standard procedure ISO PN-EN 11290. Pre-enrichment step was modified as follows. Chicken carcasses were immersed in buffered peptone water (BPW) and kept at 370C for 4 h. Afterwards, the carcasses were removed and residual BPW was put to prolonged incubation for
the next 16 h. After that 10 ml of each sample was inoculated into 90 ml of Fraser Broth (Merck) and kept for further 48 h at 370C, and the resulted growth was recultured on Oxford Agar (BTL), and incubated 48 h at 370C. L. monocytogenes was also recovered from minced meat by using half-Fraser and Fraser Broth, Oxford Agar, and PALCAM procedure. L. monocytogens strains from the brain, liver and spleen of animals with disease symptoms were isolated by subculturing in Trypticase soy solid and liquid media with the addition of nalidixic acid and potassium telluric. Bacterial strains morphologically resembling Listeria growth were submitted to further identification by conventional procedures with the use of APILAB Plus Bacterial Identification Program (bioMérieux). For molecular typing, L. monocytogenes strains were kept in nalidixic acid broth at 40C.
Table 1 Isolates of Listeria monocytogenes used in genotyping Number
Source
Origin/Organ
1C 2F 3F 4F 5F 6F 7F 8F 9F 10F 11F 12F 13F 14F 15F 1C’ 2C 3C 4C 5C 6C 7C 8C 9C 10C 11C
clinical food food food food food food food food food food food food food food clinical clinical clinical clinical clinical clinical clinical clinical clinical clinical clinical
sheep/brain chicken/leg chicken/carcass chicken/carcass chicken/carcass chicken/carcass chicken/carcass chicken/carcass chicken/carcass chicken/carcass chicken/carcass turkey/deboned meat chicken/leg chicken/wing turkey/minced meat pig/brain sheep/liver pig/liver X sheep/brain sheep/brain X sheep/brain chinchilla/liver sheep/brain chinchilla/liver
X- reference strains (Seeliger)
REP and ERIC fingerprinting, ITS profiling. Single colonies of L. monocytogenes isolates were suspended in 3 ml of Trypticase soy broth and incubated for 48 h with moderate shaking. To isolate genomic DNA, 2 ml of the bacterial broth cultures were centrifuged, and obtained cell pellets were re-suspended in 100 µl of 2% Triton X-100 (1). Samples were left in room temperature for 10 min, after that boiled for 10 min and centrifuged for 3 min at 16 000 x g. Clear supernatants were transferred to Eppendorf tubes. One microlitre of purified DNA sample was used as a template to amplify genomic DNA sequences. In addition to the template, the reaction mixture for PCR contained: 10 mM of Tris-HCl (pH 8.8), 1.5 mM of MgCl2, 10 mM of KCl, 0.1% Triton X-100, 0.1 mM of
each primer, 1.25 mM of each deoxyribonucleotide (Pharmacia) and 1 U of Red Taq DNA genomic Polymerase (Sigma). REP- (REP-IRDT: 5’- III NCG NCG TCN GGC-3’, REP2-DT: 5’-NCG NCT TAT CNG GCC TAC-3’) and ERIC-based (ERIC-IR: 5’ATG TAA GCT CCT GGG GAT TCA-3’, ERIC-2: 5’-AAG TAA GTG ACT GGG GTG AGC G-3’) primer sequences, as well as primers for ITS profiling (G1: 5’GAA GTC GTA ACA AGG-3’, L1: 5’-CAA GGC ATC CAC CGT-3’) were published previously (9, 14, 28). Oligonucleotides were from Bionovo Inc., Legnica, Poland. For ERIC primers the cycles used were as follows: 4 min at 940C, 2 min at 460C, and 4 min at 720C (first cycle), next 2 min at 940C, 2 min at 460C, and 4 min at 720C (34 cycles). For REP and ITS profiling, DNA amplifications were performed using similar
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subjected to electrophoresis under the same conditions to assess the reproducibility of the method. Data anlaysis. Data analysis was performed, as it was described previously (8), with the use of the program CrossChecker (GelTracker Co., USA), and statistical analysis with the MVSP software package (Kovach computing Services).
conditions except for different annealing temperatures, 440C and 470C, respectively. PCR products were separated on 2% agarose gel (19 cm long) at 70 V for 6 h. Gels were stained with ethidium bromide, visualised on a UV transilluminator, and photographed using a Viller-Lormant System (Viller-Lormant, France). Each experiment was repeated at least three times. Pulsed-field gel electrophoresis. Individual bacterial colonies were expanded as it was described above. Pulsed-field gel electrophoresis was performed using GenePath Group 1 reagent kit (Bio-Rad), and DNA samples were prepared according to the conditions recommended by the manufacturer. The agarose plugs were incubated for 16 h with restriction endonuclease SmaI. PFGE was performed with a CHEF DRII system (Bio-Rad). The pulse times were linearly ramped from 2.1 to 54.4 s during a 20-h run at 6 V/cm and 140C. DNA macrorestriction fragments were resolved on 1% agarose gels (PFGE-certified agarose, Sigma-Aldrich). Lambda Ladder Pulsed-field Gel Marker (Bio-Rad) was used as size standard. The preparation and digestion of DNA from each strain was repeated, and samples were
Results ITS profiling. By ITS profiling only three different DNA patterns were observed among the 26 analysed L. monocytogenes strains (Fig. 1A). The first cluster of the isolates, representing genotype I-A (I ITS) included 12 strains (1F, 11F, 12F, 13F, 15F, 1C, 2C, 4C, 5C, 8C, 10C, 11C) and was characterised by the presence of three amplicons (332, 474 and 593 bp). Five amplicons of 248, 332, 411, 474 and 593 bp represented the second major genotype, I-B, with 11 isolates (2F, 3F, 10F, 14F, 3C, 4F, 6F, 7F, 8F, 9F and 7C). The third genotype I-C was represented by only 3 isolates (5F , 6C, 9C) and had the simplest pattern with just two DNA bands (320 and 394 bp).
1000 bp 500 bp
100 bp
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Fig. 1. (A) ITS profiling patterns of Listeria monocytogens isolates after separation in 2% (w/v) gel. The position of molecular size markers in basis pairs (100-bp ladder; Fermentas) are indicated on the right. C – animal clinical isolates, F – food derived isolates. (B) Dendrogram representing genetic relationships between L. monocytogenes isolates based on ITS-profiling analysis.
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ERIC-PCR-mediated DNA typing. With ERIC-specific primers, a maximum of seven amplification products per one isolate, ranging in length from 123 bp to 543 bp, were generated (Fig. 3). Thirteen different genotypes from E-A to E-M (E – ERIC) could be discriminated among the analysed L. monocytogenes isolates. One major cluster, consisting of 9 strains (3F, 4F, 6F, 7F, 8F, 9F, 10F, 11F, 7C), was represented by genotype E-F with the following band pattern: 170, 216, 257, 400, and 451 bp. Three isolates (1C, 4C, 5C) shared a DNA profile composed of just two amplicons: 185 and 400 bp. Again, as in the case of REP-PCR, the remaining genotypes were represented only by 1 - 2 isolates (Table 3).
REP-PCR-mediated DNA typing. With REP-PCR-mediated typing, 10 different genotypes, named R-A to R-J (R - REP), were found (Fig. 2). As many as 12 isolates, representing genotype R-B (2F, 3F, 4F, 6F, 7F, 8F, 9F, 10F, 11F, 14F, 1C and 7C), shared common amplicon profiles with bands of 220, 252, 315 and 692 bp. The second major genotype, R-D, with a simple DNA pattern composed of two amplicons (220 and 315 bp), included 5 isolates (3C, 4C, 5C, 8C and 11C). The rest of the genotypes were represented by only one isolate (Table 2). Most of them had a simple pattern with 1 - 4 amplicons. In two genotypes, R-A and R-E, 5 DNA bands were found, and in genotype R-I there were 7 DNA bands.
1000 bp 500 bp
100bp
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Fig. 2. (A) DNA fingerprints of Listeria monocytogenes isolates generated by REP-PCR and separated in a 2% (w/v) agarose gel. The position of molecular size markers (100-bp plus ladder; Fermentas) are indicated on the right. C – animal clinical isolates; F – food-derived isolates. (B) Dendrogram representing genetic relationships between L. monocytogenes isolates based on REP-PCR analysis.
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Table 2 Genotypes of Listeria monocytogenes isolates generated by the REP-PCR Genotype
Bands sizes
Strains
R-A
220, 315, 353, 418, 481
1C’
R-B
220, 252, 315, 692
R-C R-D
220, 315, 582, 692 220, 315
2F, 3F, 4F, 6F, 7F, 8F, 9F, 10F, 11F, 14F, 1C, 7C 15F 11F, 3C, 4C, 5C, 8C
R-E R-F R-G R-H R-I R-J
220, 315, 600, 659, 735 315 245, 315, 377 178, 230, 481, 582 141, 175, 203, 237, 281, 377, 437 252, 377
2C 1F 6C 12F, 13F 5C 9C
1000 bp -
500 bp
100bp
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Fig. 3. (A) DNA fingerprints of Listeria monocytogenes isolates generated by ERIC-PCR and separated in a 2% (w/v) agarose gel. The position of molecular size markers (100-bp plus ladder, Fermentas) are indicated on the right. C – animal clinical isolates; F – food-derived isolates. (B) Dendrogram representing genetic relationships between L. monocytogenes isolates based on ERIC-PCR analysis.
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Table 3 Genotypes of Listeria monocytogenes generated by the ERIC-PCR Genotype E-A E-B E-C E-D E-E E-F
Bands sizes 185, 203, 226, 400 185, 203, 226, 400, 543 185, 276, 400 185, 400 185, 400, 542 170, 216, 257, 400, 451
E-G E-H E-I E-J E-K E-L E-M
200, 257, 304, 400, 428 170, 254, 400 170, 254 170, 200 123, 151, 170,355 170, 213, 257, 355, 411, 542 216, 261
Strains 10C 8C 12F,13F 1C, 4C, 5C 2C, 3C 3F, 4F, 6F, 7F, 8F, 9F, 10F, 11F, 7C 9C 14F, 11C 2F 1C’ 15F 6C 5F
Table 4 Genotypes of Listeria monocytogenes generated by PFGE
Genotype
Strain
P-A P-B
10C 8C, 11C
P-C
1C’
P-D
12F
P-E
1C, 3C, 4C, 9C
P-F
6C
P-G
2F
P-H P-I P-J
3F, 4F, 6F, 7F, 8F, 9F, 10F, 11F 15F 5F
P-K
13F
P-L
2C, 3C
P-M
7C
P-N
14C
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145.5 kb 48.5 kb
Fig. 4. (A) Genomic DNA restriction patterns of Listeria monocytogenes isolates, performed by PFGE after digestion with SmaI. C – animal clinical isolates; F – food-derived isolates. (B) Dendrogram representing genetic relationships between L. monocytogenes isolates based on PFGE analysis.
Pulse-field gel electrophoresis. With the use of PFGE as many as fourteen different genotypes (P-A to P-N, P - PFGE) were found (Fig. 4). One cluster of 8 strains (3F, 4F, 6F, 7F, 8F, 9F, 10F, 11F) was represented by genotype P-H. Four other strains (1F, 3C, 4C and 9C) were of common genotype P-E. Two genotypes, P-B and P-L, included two strains, respectively, 8C, 11C and 2C, 5C. The remaining isolates had unique PFGE profiles.
Discussion Food products, especially ready-to-eat products with long shelf-life stored in low temperatures, are the main source of L. monocytogenes infections in humans
(17, 24). Direct transmission of L. monocytogenes from animals, including farm animals, to humans is rare and does not present any problem from the epidemiological point of view. The only more serious source of infections seems to be dairy products coming from mastitic cows (15). However, animals, especially those which are asymptomatic carriers, can be reservoirs of pathogenic L. monocytogenes strains, involved in the contamination of food products. To prove such a hypothesis, it is necessary to perform studies on the relationships between different bacterial isolates recovered form animals and food products, designed to link outbreaks to sources of contamination. Such analysis, in general, needs to be done with DNA-based molecular techniques. For this reason, the present study was performed with the use of several PCR-based DNA-
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fingerprinting methods, including REP- and ERIC-PCR, ITS profiling, and PFGE. According to our knowledge, there are few studies on animal clinical isolates of L. monocytogens with the use of these techniques (27, 16), and so far, the analysis of L. monocytogenes isolates with genotyping was not performed in Poland. REP- and ERIC-PCR were found to be more discriminatory for the typing of L. monocytogenes isolates than ITS profiling. With this last method only three different profiles of DNA were obtained. Genotype I-A included 12 isolates which were recovered from animals as well as from food products. On the other hand, among 11 strains representing genotype I-B, 9 were isolated from foodstuff. With the use of REP-PCR 10 different DNA patterns could be discriminated among the analysed 26 strains, amongst which two predominant genotypes, R-B (12 isolates) and R-D (5 isolates), were identified. Interestingly, it was found that in all samples but two, genotype R-B was represented only by food-derived strains, and genotype R-D included only clinical samples. At the level of clustering 10%, all the analysed L. monocytogenes strains could be subdivided into two genomic groups, REP-I including the majority of strains and group REP-II with just two strains (Fig. 2B). ERIC-PCR and PFGE gave similar results. With ERIC-PCR thirteen different DNA patterns could be discriminated among the analysed strains, with one predominant genotype E-F represented by 11 isolates. Among them as many as 9 isolates were recovered from foodstuff. The rest of the obtained genotypes were found only in 1 - 2 of the analysed isolates. Fourteen genotypes were identified when L. monocytogenes strains were analysed by PFGE. Again one major DNA profile including 9 isolates was present and all of them were recovered from foodstuff. Genotypes obtained by ERIC-PCR were grouped by dendrogram analysis into two distinct lineages, named ERIC-I and ERIC-II, with the level of similarity about 20% (Fig. 3B). ERIC-I (genotypes E-A to E-E) is represented mostly by clinical strains, ERIC-II (genotypes E-F to E-M) - by strains recovered from foodstuff. Similarly, isolates analysed by PFGE could be divided into two genomic groups at the clustering level below 10% (Fig. 4B). Genomic group PFGE-I was represented mostly by clinical isolates, PFGE-II by food derived strains. This data suggests that with these two methods, it was possible to discriminate the majority of food derived strains from the clinical ones at two levels. The first level was represented by genomic groups, the second - by genotypes. It is important to mention that PFGE seems to be the most appropriate method to use in molecular typing of listeriosis. A national network of data obtained by PFGE, coordinated by the Centre for Disease Control and Prevention, exists in the United States (17). So, because of its discriminatory power, the ERIC-PCR could be a complementary method useful in epidemiological studies of listeriosis. According to our knowledge, there is very few available information on the use of REP- and ERICPCR in the typing of L. monocytogenes isolates. Jersek et al. (16), using both methods, have shown that foodderived strains represent a completely different genomic
group when compared to animal clinical strains. This data is in agreement with our results, however, in our studies some food- and animal-derived L. monocytogenes strains had the same DNA profiles. These discrepancies could be partially explained by the small number (6) of animal clinical strains used in the studies. The other discrepancy between these two studies was the sizes of PCR-generated DNA fragments. The amplicons obtained by us were distinctly shorter, with sizes ranging between 123 to 735 bp, in comparison to amplicons obtained by Jersek et al. (16), with sizes from 298 to 6100 bp. As we have used the same primers, the reason of such a disagreement could be the differences in the DNA polymerases and PCR protocols used in these studies. In summary, we have shown by ERIC-PCR and PFGE that the majority of food-derived L. monocytogenes strains represent different genotypes than clinical isolates recovered from animals. This data suggests that L. monocytogenes strains of animal origin are not the common source of foodstuff contamination. In addition, we have shown that ERIC-PCR is a highly discriminatory method which seems to be suitable for epidemiological studies of L. monocytogenes. It can be used as a complementary method to the widely used pulsed-field gel electrophoresis.
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