Molecular study of vancomycin-resistant enterococci isolated from humans and from food in a cattle-rearing area of France

Fabien Garnier*, Karine Gambarotto, François Denis and Marie-Cécile Ploy

Laboratoire de Bactériologie-Virologie-Hygiène, CHU Dupuytren, EA 3175, 2, avenue Martin Luther King, 87042 Limoges cedex, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: Study possible links between vancomycin-resistant enterococci strains isolated from human stool samples and from pork or poultry food products.

Methods: One hundred and eleven vancomycin-resistant enterococci strains (15 VanA and 96 VanC) were isolated from human stool samples and from pork or poultry food products. Characterization of the Tn1546-like element of the 15 VanA strains was realized by restriction analysis of PCR products and polymorphism study. The 96 strains of VanC phenotype (75 Enterococcus gallinarum and 21 Enterococcus casseliflavus) were analysed by pulsed-field gel electrophoresis (PFGE).

Results: In the study described here, polymorphism of the Tn1546-like element enabled the establishment of five groups. Groups III, IV and V were found only in human strains. Groups I and II were found to occur in strains isolated from humans and from food, suggesting a possible transfer of the Tn1546-like element. The isolates from Group I harboured the whole Tn1546 element. In Group II, the five strains possessed a novel Tn1546-like element, characterized by a single-nucleotide difference in the vanX gene and a deletion upstream from the nucleotide 164. Analysis by PFGE of the 75 E. gallinarum strains revealed 20 different patterns. One pattern was shared by isolates from pork food and human samples. None of the 21 E. casseliflavus strains tested was found to share similar PFGE patterns.

Conclusions: Results tend to support the possible transfer of the Tn1546-like element between strains of VanA phenotype. Concerning VanC phenotype strains, the transfer was not demonstrated.

Keywords: glycopeptide resistance , VRE , Tn1546-like element


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Glycopeptide resistance in enterococci is a major cause of concern.1,2 Currently, six operons (vanA to vanG) conferring glycopeptide resistance have been described in enterococci.3 The vanA operon (vanRSHAXYZ), which is the most commonly encountered in Europe, is located on the transposon Tn1546.4

The use of avoparcin in Europe, as a growth promoter in animal feed until 1997, has been shown to create a reservoir for vancomycin-resistant enterococci (VRE) in animals and is a possible route for VRE transmission from animals to humans through the food chain.5

In two previous studies in a cattle-rearing area in the centre of France (Limousin), we reported a high incidence of VRE from human stools and from pork and poultry food products.6,7 Using PFGE, the 15 VanA strains isolated were found to be genetically unrelated.7

To investigate further the possible links between VRE strains isolated from humans or from food, we characterized the Tn1546-like element of the VanA strains and typed the VanC strains by PFGE.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates

The 111 VRE strains studied were isolated, after an enrichment broth step, from stool samples collected from hospitalized patients, from outpatients and from meat samples taken from pork and poultry products obtained in Limousin, a cattle-rearing area located in the centre of France.6,7 The strains comprised 75 Enterococcus gallinarum (46 from humans and 29 from food); 21 Enterococcus casseliflavus (seven from humans and 14 from food); 14 VanA phenotype Enterococcus faecium (nine from humans and five from food); one VanA phenotype Enterococcus durans (from food).

PCR procedures

Total DNA was prepared by the cetyldimethylethyl-ammonium bromide method.8 PCRs were performed using the DyNAzyme EXT (FINNZYMES OY, Espoo, Finland) and a 2400 thermal cycler (Perkin-Elmer Applied Biosystems, Les Ulis, France), as recommended by manufacturers, with annealing temperatures according to the primer pairs used (Figure 1, Table 1). Long PCR fragments were analysed by digestion with the ClaI restriction enzyme (Roche Diagnostics GmbH, Mannheim, Germany) as previously described.1



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Figure 1. (a) Map of the Tn1546 prototype. Open arrows represent coding sequences. Horizontal arrows represent primers described in this study; (b) table of the PCR primers.

 

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Table 1. VRE Groups based on detection of Tn1546-like elements using the following primer pair combinations in the long-PCR and in the presence or absence of vanX mutation

 
The vanRSHAX genes were analysed by PCR using primers P2 and P3 (Figure 1, Table 1) and restriction analysis with the DdeI enzyme (Roche Diagnostics GmbH) as described previously.1 Sequences with mutation (G to T) at position 8324 in the vanX gene lacked a restriction site and subsequently produced one less fragment after digestion.1

Pulsed-field gel electrophoresis

Plugs were prepared and digested as previously described6 except for E. casseliflavus strains, for which digestion by SmaI was carried out at a temperature of 27.5°C. Parameters for electrophoresis were 3.5 V/cm at 14°C for 21 h with pulse time ramps from 0.1–5 s for E. gallinarum strains or from 0.1–15 s over a period of 20 h for E. casseliflavus strains.

DNA sequencing

PCR fragments were sequenced by using the ABI PRISM dRhodamine terminator protocol, as recommended by the manufacturer (Perkin–Elmer Applied Biosystems). Products were analysed with an ABI PRISM 310 automated DNA sequencing apparatus (Perkin–Elmer Applied Biosystems).

The nucleotide sequence analysis was carried out using the BLASTN program on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Characterization of Tn1546-like elements

The DdeI restriction analysis of the PCR products obtained with primers P2 and P3 (Figure 1) showed five strains (Table 1), two from humans (LIM 30 and LIM 32) and three from food (LIM 40, LIM 41 and LIM 43), which possessed the G to T mutation in the vanX gene at position 8324 (Table 1).

For six strains, three of human origin (LIM 34, LIM 35 and LIM 38) and three isolated from food (LIM 39, LIM 42 and LIM 44) (Table 1), the ClaI restriction analysis of PCR products obtained with the primer P1 suggested that these strains contained the whole Tn1546 element (data not shown).1

The remaining nine isolates were then studied using the primer pairs P2-P1 and P1-P3 to characterize the 5' and 3' ends, respectively, of the Tn1546-like element (Figure 1). With the pair P2-P1, a PCR product of the expected size was obtained for all strains except for LIM 33, of human origin, for which the fragment was longer by about 900 bp (Table 1). The sequence analysis of this longer fragment showed the addition of an IS1216V element4 at nucleotide position 8710 in the vanX-vanY inter-region, associated with a deletion of five base pairs upstream from nucleotide 8710. Using the primer pair P1-P3, no product was amplified for the nine strains, suggesting the presence of deletions or mutations in the P1 binding region of the left end of the Tn1546-like element. Subsequently, four other primers, P4, P5, P6 and P7, were selected (Figure 1). All strains yielded a PCR fragment of the expected size with the P4-P3 primer pair, suggesting a deletion between the 5' end of the transposon and orf2. A combination of PCRs using different primer pairs, P5-P3, P6-P3 and P7-P3 suggested that different deletions in the 5' region occurred depending on the strain type (Table 1).

On the basis of these results, Tn1546-like elements from the 15 different VanA strains were classed in five different groups (Table 1). Group I consisted of the six isolates that contained the whole Tn1546 element. Group II consisted of five strains harbouring a deletion upstream from nucleotide 164 and a mutation in the vanX gene. The same deletion and mutation combination has been reported on previous occasions, such Tn1546 variants being classed as type 2 by Jensen9 and as Group D by Palepou et al.1 Both, however, contained an IS1216V-IS3-like element that we did not find in any strain of Group II. This new type of Tn1546-like element was recently described in an environmental Enterococcus mundii strain.2 Here we describe its first detection in strains isolated from humans and from food. In addition, the mutation in the vanX gene was previously described as a marker of the porcine origin of the Tn1546-like element.9 However, in our study, occurrence of this mutation appeared to be a poor marker since it was detected in only 50% (3/6) of strains from pork meat samples. Group III contained two strains, with a deletion of the entire fragment upstream from nucleotide 1148. Group IV consisted of a single strain isolated from humans characterized by the addition of an IS1216V element in the vanX-vanY inter-region and a sequence deletion upstream from nucleotide 1148, as described above.2 A final strain with a deletion of the region upstream from orf2 comprised Group V.

Groups I and II consisted of strains isolated from humans or from food, indicating that indistinguishable Tn1546-like elements could be present in bacteria from different hosts, although these strains seemed distinct based on PFGE analysis. Two hypotheses may therefore be proposed to account for these results. First, the Tn1546-like element could come from a common reservoir of environmental enterococci. A possible flow of such a transposable element through different environmental enterococcal species and populations has been described recently.4 Alternatively, horizontal gene transfer could occur between the bacteria of humans and food in the gastrointestinal tracts as was shown with gnotobiotic mice.5

VanC phenotype strains

The 75 E. gallinarum strains isolated from human or from food were compared by PFGE. A PFGE type was assigned to each strain in accordance with the criteria of Tenover et al.10 A number was assigned to the 20 different patterns identified (data not shown). Among these 20 patterns, 10 were specific to strains isolated from humans and nine to strains isolated from food, whereas one pattern was displayed by strains from both humans (five) and food (11). This latter result could suggest a possible transfer of E. gallinarum strains from food to humans, but human contamination of food during the production process could not be excluded.

The 21 E. casseliflavus strains were also analysed by PFGE. A different pattern was obtained for each strain (data not shown).

In conclusion, the study revealed a degree of diversity within Tn1546-like elements of vanA VRE in samples taken from humans and from food in this area of France, confirming previously published reports.6,7 However, a new type of Tn1546-like element was identified in strains with distinct PFGE patterns from humans as well as from food (Group II). These results tend to support the hypothesis of a possible transfer of Tn1546-like elements between strains for which the precise origin remains unknown (human, animal or environmental). Concerning VanC phenotype, the low number of E. casseliflavus strains studied and possible human contamination during the production process for E. gallinarum strains prevent conclusions regarding a possible transfer being made.

An epidemiological study of VRE incidence in livestock reared in this part of France could clarify the situation concerning VRE colonization in farm animal species.


    Footnotes
 
* Corresponding author. Tel: +33-555-05-63-48; Fax: +33-555-05-67-22; Email: fabien.garnier{at}unilim.fr


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Palepou, M. F. I., Adebiyi, A. M. A., Tremlett, C. H. et al. (1998). Molecular analysis of diverse elements mediating VanA glycopeptide resistance in enterococci. Journal of Antimicrobial Chemotherapy 42, 605–12.[Abstract]

2 . Guardabassi, L. & Dalsgaard, A. (2004). Occurrence, structure, and mobility of Tn1546-like element in environmental isolates of vancomycin-resistant enterococci. Applied and Environmental Microbiology 70, 984–90.[Abstract/Free Full Text]

3 . Depardieu, F., Bonora, M. G., Reynolds, P. E. et al. (2003). The vanG glycopeptide resistance operon from Enterococcus faecalis revisited. Molecular Microbiology 50, 931–48.[CrossRef][ISI][Medline]

4 . Arthur, M., Molinas, C., Depardieu, F. et al. (1993). Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. Journal of Bacteriology 175, 117–27.[Abstract]

5 . Moubareck, C., Bourgeois, N., Courvalin, P. et al. (2003). Multiple antibiotic resistance gene transfer from animal to human enterococci in the digestive tract of gnotobiotic mice. Antimicrobial Agents and Chemotherapy 47, 2993–6.[Abstract/Free Full Text]

6 . Gambarotto, K., Ploy, M. C., Turlure, P. et al. (2000). Prevalence of vancomycin-resistant enterococci in fecal samples from hospitalized patients and nonhospitalized controls in a cattle-rearing area of France. Journal of Clinical Microbiology 38, 620–4.[Abstract/Free Full Text]

7 . Gambarotto, K., Ploy, M. C., Dupron, F. et al. (2001). Occurrence of vancomycin-resistant enterococci in pork and poultry products from a cattle-rearing area of France. Journal of Clinical Microbiology 39, 2354–5.[Abstract/Free Full Text]

8 . Ausubel, F. M., Brent, R., Kingston, R. E., et al. (1992). Current Protocols in Molecular Biology, vol 1, pp. 2–33. Wiley, New York, NY, USA.

9 . Jensen, L. B. (1998). Differences in the occurrence of two base pair variants of Tn1546 from vancomycin-resistant enterococci from humans, pigs, and poultry. Antimicrobial Agents and Chemotherapy 42, 2463–4.[Free Full Text]

10 . Tenover, F. C., Arbeit, R. D., Goering, R. V. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Free Full Text]





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