Vancomycin-Resistant Enterococci in Humans and Imported Chickens in Japan

The phenotypes and genotypes of 22 VanA-type vancomycin-resistantenterococci that had been isolated in Japan were examined. TheVanA resistance determinant was plasmid mediated in each ofthe 22 strains. Of the 22 strains, 8 were isolated from differentpatients and 11 and 3 were obtained from different samples ofchickens imported from Thailand and France, respectively. Threeof the strains that were isolated from patients and the 11 strainsisolated from the Thai chickens showed high-level vancomycinresistance (MICs, 512 to 1,024 µg/ml) and low-level teicoplaninresistance (MICs, 0.5 to 4 µg/ml). Each of these strainshad three amino acid substitutions in the N-terminal regionof the deduced VanS sequence. L50 was converted to V, E54 wasconverted to Q, and Q69 was converted to H compared to the vanSgene sequence of Tn1546.

INTRODUCTION

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Introduction

Since the first reports of infections and colonizations by vancomycin-resistantenterococci (VRE) in France (10) and the United Kingdom (15),there have been reports of VRE in many locations in the UnitedStates and the European Union. The major factors contributingto the dissemination of VRE in the United States and Europeare now evident. In the United States, the major factor is likelythe excessive use of glycopeptide antibiotics in the healthcare environment, resulting in a selective increase in VRE inhuman intestines (8, 11). In Europe, it is strongly suggestedthat the use of avoparcin as a growth promoter in animal feedhas resulted in the selective increase in VRE in animal intestines,and the VRE subsequently appear in the human community (9, 13,16, 17). In Japan, vancomycin injections have been used forthe treatment of methicillin-resistant Staphylococcus aureusinfections since November 1991 and infection or colonizationdue to VRE is rare (6, 7, 9). Until 2000, a total of eight patientswith VRE were detected in four hospitals. We have conductedan investigation of VRE in imported and domestic chickens everyyear since 1998 with the support of the Japanese Ministry ofHealth, Labor, and Welfare (9). VRE have never been isolatedfrom domestic chickens. However, VRE are isolated at a highfrequency in samples from imported chickens. VRE isolation frequenciesof about 20% and 30 to 50% have been observed in Thailand andFrance, respectively, where avoparcin has been used in animalfeed (9). We compared the phenotypes and genotypes of VRE isolatesfrom humans with those of isolates from imported chickens inJapan.

Glycopeptide resistance and pulsed-field gel electrophoresis (PFGE) analysis of VRE strains.

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Glycopeptide resistance and...

Twenty-two VRE strains were examined in this study. Of thesestrains, eight strains were isolated from eight different patientsat four hospitals in Japan up until 2000 and 11 and 3 strainswere obtained from chickens imported from Thailand and France,respectively, in investigations carried out in 1998 and 1999(Table 1).

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TABLE 1. Characteristics of VRE isolated from humans and imported chickens in Japan

The levels of glycopeptide resistance were examined in the VREstrains (Table 1). Of the eight strains isolated from the patients,five strains from patients at two hospitals showed high-levelvancomycin and teicoplanin resistance, with MICs of 256 to 512µg/ml and 64 to 128 µg/ml, respectively, and theother three strains from the patients at two hospitals showedhigh-level vancomycin resistance, with MICs of 512 to 1,024µg/ml, and low-level teicoplanin resistance, with MICsof 0.5 to 4 µg/ml. Of the strains isolated from the importedchickens, all four strains isolated from French chicken samplesshowed high-level vancomycin and teicoplanin resistance, withMICs of 512 µg/ml and 64 to 128 µg/ml, respectively.All 11 strains isolated from Thai chicken samples showed high-levelvancomycin resistance, with MICs of 256 to 1,024 µg/ml,and relatively low-level teicoplanin resistance, with MICs of2 to 8 µg/ml.

The DNA from each of the VRE strains was analyzed by PCR forthe presence of vancomycin resistance gene(s) with vanA-, vanB-,and vanC-specific primers (4, 5, 12). All strains gave riseto the expected 732-bp product with the primer specific forthe vanA gene, indicating that the strains were VanA-type VRE(data not shown).

Banding patterns obtained by PFGE of SmaI-digested genomic DNAwere used to compare the VRE strains. Figure 1 shows the PFGEpatterns of VRE strains isolated from Thai chickens and VREstrains KV12, KV21, and CV1 isolated from patients, which showedhigh-level vancomycin resistance and low-level teicoplanin resistance,and also VRE strain FN1 (6) (Fig. 1; Table 1). Of these strains,two (KV12 and KV21) isolated from patients at a hospital andthree (TV9, TV10, and TV11) isolated from Thai chickens, respectively,differed by one or two bands, indicating that strains of eachgroup were related. Other strains showed different patterns,indicating that these VRE strains are different (Fig. 1)

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FIG. 1. PFGE of SmaI-digested chromosomal DNAs isolated from VRE strains. ${lambda}$ , a bacteriophage ${lambda}$ DNA ladder used as a molecular size marker. Lanes 1 to 16, VRE strains CV1, KV12, KV21, FN1, TV1, TV2, TV3, TV4, TV5, TV6, TV7, TV8, TV9, TV10, TV11, and GV2, respectively.

Conjugative transfer of vancomycin resistance.

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Conjugative transfer of...

Each of the 22 VRE strains harbored several plasmids. The EcoRIrestriction profiles of total plasmid DNAs isolated from theVRE strains were different (data not shown). The vanA probehybridized to a specific EcoRI fragment (4.1 kb) of plasmidDNA in each of the strains that is specific to the prototypeVanA-type gene encoded on Tn1546 (5), with the exception ofthe plasmid DNAs of one strain (FV2), where the probe hybridizedto a 5-kbp EcoRI fragment (data not shown). The transferabilityof the vancomycin resistance of the VRE strains was examinedbetween each of the strains and recipient strain Enterococcusfaecalis FA2-2 (Rif^r Fus^r) (3) by filter mating. The vancomycinresistances of 5 of the 11 strains from Thai chickens and 2of the 3 strains from the patients, which were high-level vancomycinresistant and low-level teicoplanin resistant, and the vancomycinresistance of one of the three strains from French chickens,which were high-level vancomycin and teicoplanin resistant,were transferred to the recipient E. faecalis FA2-2 (Rif^r Fus^r)strain at about 10^-4 to 10^-5 cells per donor cell by filtermating (Table 1). After repeated transfer experiments done byshort mating between E. faecalis FA2-2 (Rif^r Fus^r) and E. faecalisJH2SS (Str^r Spc^r) (14), the transferable plasmid from each ofthe strains was identified from the transconjugant (Fig. 2).The plasmid DNA of the transconjugants of two strains (KV12,and KV21) isolated from patients at a hospital and three strains(TV9, TV10, and TV11) isolated from Thai chickens, respectively,exhibited the same EcoRI restriction profiles (Fig. 2). ThevanA probe hybridized to a specific EcoRI fragment (4.1 kb)(5) of conjugative plasmid DNA of each the transconjugants,with the exception of the plasmid DNA isolated from strain FV2.These results indicated that the VanA determinants, which areencoded on transposons such as Tn1546, are carried on a conjugativeplasmid and can transfer between enterococci and also to differentreplicons by transposition.

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FIG. 2. Agarose gel electrophoresis of restriction endonuclease-digested conjugative plasmid DNA and hybridization with a vanA-specific probe. (A) Agarose gel electrophoresis of EcoRI-digested plasmid DNA isolated from each the transconjugants. (B) The gel was Southern blotted and hybridized to a vanA-specific probe. Lanes 1 to 9, plasmid pMG2 and plasmid DNAs isolated from transconjugants of VRE strains KV12, KV21, TV1, TV9, TV10, TV11, TV5, and FV2, respectively. Lanes 10, HindIII-digested lambda DNA.

DNA sequence analysis of the vanS gene of the vancomycin determinants encoded on the plasmids.

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Dna sequence analysis of...

A nationwide survey was conducted to investigate VRE in broilerfarms in Japan in December 1996 (18). Three VanA-type VRE strainswere isolated from droppings obtained from 3 of 35 broiler farmsin 24 prefectures in Japan that used avoparcin (18). The threeVanA-type VRE isolates showed high-level vancomycin resistanceand relatively low-level teicoplanin resistance, and the VanAdeterminant of E. faecalis GV2, which is one of the three isolates,was analyzed in detail (7). The glycopeptide resistance of E.faecalis GV2 is mediated by conjugative plasmid pMG2 (85 kb).The nucleotide sequences of the genes for the VanA determinantencoded on pMG2 are completely identical to those of the prototypeVanA-type gene encoded on Tn1546, with the exception of thegene for glycopeptide sensor protein VanS (1). There are threeamino acid substitutions in the N-terminal region of the deducedsequence encoded by vanS (Table 1).

Nucleotide sequence analysis of the vanS gene of the VRE strainswas performed by sequencing PCR products with vanS-specificprimers (Table 2). To analyze the van determinant correspondingto vanS of the VanA-type determinant encoded on Tn1546 (2),one or two pairs of PCR primers specific for vanS were usedfor PCR amplifications. The pairs of PCR primers were also usedto sequence the amplified genes. The nucleotide sequence andthe deduced amino acid residues of the vanS gene of the VREstrains isolated from Thai chickens and from three patientsat two hospitals revealed three amino acid substitutions inthe N-terminal region of the deduced VanS sequence. L50 wasconverted to V, E54 was converted to Q, and Q69 was convertedto H compared to the vanS gene sequence of Tn1546 (Table 1).

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TABLE 2. Nucleotide sequences of PCR primers for vanS of the VanA-type determinant encoded on Tn1546

The VRE strains gave rise to the expected PCR products of 1,744,1,856, and 926 bp with the primers specific for open readingframe (ORF) 1, both ORFs 1 and 2, and ORF2, respectively, whichare located on Tn1546 and encode a transposase (ORF1) and aresolvase (ORF2) (data not shown) (12). These results suggestedthat the VanA-type determinants of VRE strains were encodedon transposon Tn1546 or a closely related transposon.

Concluding comments.

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Concluding comments.

The VRE strains isolated from French chickens exhibited high-levelvancomycin and teicoplanin resistance and could not be distinguishedfrom the representative VanA-type VRE (2, 5, 12). The VRE strainsisolated from Thai chickens exhibited high-level vancomycinresistance and relatively low-level teicoplanin resistance,had three amino acid substitutions in the vanS gene of the VanA-typedeterminant, and were distinguished by these characters fromthe representative VanA-type VRE. The VRE strains isolated fromthree patients at the two hospitals showed the same characteristicsas the VRE strains from the Thai chickens. It is very rare thatidentical substitutions would occur independently in each ofthe strains. Japan imports about 600,000 tons of chicken a year,while the nation's domestic chicken production is about 1.2million tons a year. About 100,000 tons (16.6%) of the importedchicken come from Thailand. Although isolation of VRE is rarein enterococci from inside or outside the health care environment,these results imply that the VanA-type vancomycin resistancedeterminant, which resides on transposons such as Tn1546 andcan be transferred between enterococci by a conjugative plasmid,can transfer from chickens to humans.

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of HealthLabor and Welfare and by grants from the Japanese Ministry ofEducation, Science and Culture.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, Gunma University School of Medicine, Showa-machi 3-39-22, Maebashi, Gunma 371-8511, Japan. Phone: 81-27-220-7990. Fax: 81-27-220-7996. E-mail: yasuike{at}sb.gunma-u.ac.jp.

REFERENCES

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