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Antimicrobial Agents and Chemotherapy, August 2004, p. 3141-3146, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.3141-3146.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Lack of Evidence that DNA in Antibiotic Preparations Is a Source of Antibiotic Resistance Genes in Bacteria from Animal or Human Sources

Susanna K. P. Lau, Patrick C. Y. Woo, Amanda P. C. To, Alexson T. K. Lau, and Kwok-yung Yuen^*

Department of Microbiology, The University of Hong Kong, Queen Mary Hospital, Hong Kong

Received 31 July 2003/ Returned for modification 2 January 2004/ Accepted 5 April 2004

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Although DNA encoding antibiotic resistance has been discovered in antibiotic preparations, its significance for the development of antibiotic resistance in bacteria is unknown. No phylogenetic evidence was obtained for recent horizontal transfer of antibiotic resistance genes from antibiotic-producing organisms to bacteria from human or animal sources.

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Although the use of antibiotics has been the major weapon in combating infectious diseases, the rapid development of antibiotic resistance has resulted in treatment failures and outbreaks of infections caused by antibiotic-resistant bacteria (2, 5, 10, 11, 15). Numerous studies have proven that the use of an antibiotic is closely related to the rate of resistance to that antibiotic (3, 4). Traditionally, the role of antibiotics in antibiotic-induced antimicrobial resistance is to provide selective pressures to resistant clones. In 1990, Chakrabarty et al. reported the detection of nucleic acids in various antibiotics and the capacity of such nucleic acids to transform bacteria to drug resistance (1). Subsequently, Webb and Davies demonstrated that DNA encoding antibiotic resistance genes that are present in bacteria used in the production of antibiotics can be recovered in antibiotic preparations (9). It was proposed that antimicrobial resistance genes may be coadministered with antibiotics to humans or animals and taken up by bacteria in the hosts, contributing to the rapid development of antibiotic resistance. However, the importance of this second mechanism in the present widespread antibiotic resistance was unknown. In this study, we examined by a comprehensive phylogenetic analysis whether recent horizontal transfer of antibiotic resistance genes from antibiotic-producing organisms to human or animal bacteria has occurred. The phylogenetic relationships of the genes among different bacteria were also studied.

All antibiotic resistance genes that were present in the corresponding antibiotic-producing and non-antibiotic-producing organisms were included in this study (Medline search from 1966 to June 2003 and GenBank search from 1988 to June 2003) (Table 1). Amino acid sequences were used for phylogenetic analysis to minimize the effect of codon usage bias among different bacteria after gene acquisition. All were published sequences downloaded from GenBank (http://www.ncbi.nlm.nih.gov) and were aligned by multiple-sequence alignment with the CLUSTAL W program (8). For phylogenetic tree construction, all sequences found in the corresponding antibiotic-producing organisms and at least 10 sequences from human or animal bacterial isolates closest to those from antibiotic-producing organisms by BLAST search were included. Phylogenetic tree construction was performed with ClustalX version 1.81 (6) and the neighbor-joining method with GrowTree (Genetics Computer Group, Inc., San Diego, Calif.).

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TABLE 1. Antibiotic resistance genes present in both antibiotic-producing organisms and human or animal bacteria

A total of 13 antibiotic resistance genes that were present in both the corresponding antibiotic-producing and non-antibiotic-producing organisms were found (Table 1). Phylogenetic tree construction was performed on the first 10 genes. For the last three genes, fewer than 10 sequences were found in non-antibiotic-producing organisms and they were analyzed by multiple-sequence alignment alone.

The erm sequences of the macrolide and lincosamide producers were distant from those of human or clinical bacterial isolates (amino acid identities of less than 40%). Recent horizontal transfer of erm genes from the antibiotic producers to human or animal bacteria was not observed. In contrast, the sequences within the human or animal bacteria were more closely related among themselves and showed evidence of frequent horizontal transfer, even between gram-positive and gram-negative bacteria. In particular, transfer among human gut bacteria was very common. There also appeared to be two clusters of genes that evolved independently, one generally from human or animal bacteria and the other generally from environmental bacteria, including the antibiotic producers (Fig. 1).

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FIG. 1. Phylogenetic tree showing the relationships of erm genes found in the macrolide- and lincosamide-producing organisms (in bold) and those from other bacterial isolates. The tree was inferred from 290 amino acids. Bootstrap values were calculated from 1,000 iterations. The scale bars indicate the estimated number of substitutions per 10 amino acids using the Jukes-Cantor correction. Different classes of erm genes are denoted by vertical lines, and names are on the right. Names and accession numbers are given as cited in the GenBank database.

The tetracycline resistance ribosomal protection protein of the tetracycline producer Streptomyces rimosus was also distantly related to those of human or clinical bacterial isolates (amino acid identities of less than 50%), indicating no evidence of recent horizontal gene transfer from the antibiotic producer to human or clinical bacteria. In contrast, closely related genes among human or clinical bacteria were observed. Horizontal gene transfer appeared to have occurred among distantly related bacteria and the female genital tract flora and pathogens (Fig. 2).

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FIG. 2. Phylogenetic tree showing the relationships of tetracycline resistance ribosomal protection proteins of the tetracycline producer S. rimosus (in bold) and those from other bacterial isolates. The tree was inferred from 880 amino acids. Bootstrap values were calculated from 1,000 iterations. The scale bars indicate the estimated number of substitutions per 10 amino acids using the Jukes-Cantor correction. Different classes of tetracycline resistance ribosomal protection proteins are denoted by vertical lines, and names are on the right. Names and accession numbers are given as cited in the GenBank database.

Phylogenetic studies of the other 11 genes showed patterns similar to those reported in detail for erm and tetracycline resistance ribosomal protection protein. The results of their phylogenetic analysis are summarized in Table 1. The maximum amino acid identities of genes among different non-antibiotic-producing bacterial isolates were close to 100% for most genes, but those between antibiotic-producing and human or animal bacteria ranged from <28 to <77%.

From the present study, recent horizontal transfer of antibiotic resistance genes from bacteria that are used for antibiotic production to human or animal bacteria was not observed. Although our results suggest that DNA in antibiotic preparations has not been a very active and widespread mechanism in the dissemination of antibiotic resistance genes, the possibility of its being a minor player cannot be excluded. Nevertheless, our results suggest that DNA decontamination of antibiotic preparations is probably unnecessary. On the other hand, the fact that horizontal transfer was observed among human gastrointestinal tract bacteria, which is in line with our previous findings (7, 12), suggests that this is one of the other possible mechanisms of antibiotic-induced antibiotic resistance (13, 14).

 
This work was partly supported by the University Development Fund, University Research Grant Council, and the Committee of Research and Conference Grants, The University of Hong Kong.

 
* Corresponding author. Mailing address: Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, Hong Kong. Phone: (852) 28554892. Fax: (852) 28551241. E-mail: hkumicro{at}hkucc.hku.hk.

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Antimicrobial Agents and Chemotherapy, August 2004, p. 3141-3146, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.3141-3146.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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