A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (TcR) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of TcR streptomycetes originated from bulk and rhizosphere soil. Fewer TcR streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene–carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which TcR streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.

The prevalence of selected streptomycin (Sm)-resistance genes, i.e. aph (3''), aph (6)-1d, aph (6)-1c, ant (3'') and ant (6), was assessed in a range of pristine as well as polluted European habitats. These habitats included bulk and rhizosphere soils, manure from farm animals, activated sludge from wastewater treatment plants and seawater. The methods employed included assessments of the prevalence of the genes in habitat-extracted DNA by PCR, followed by hybridisation with specific probes, Sm-resistant culturable bacteria and exogenous isolation of plasmids carrying Sm-resistance determinants. The direct DNA-based analysis showed that aph (6)-1d genes were most prevalent in the habitats examined. The presence of the other four Sm-modifying genes was demonstrated in 58% of the tested habitats. A small fraction of the bacterial isolates (8%) did not possess any of the selected Sm-modifying genes. These isolates were primarily obtained from activated sludge and manure. The presence of Sm-modifying genes in the isolates often coincided with the presence of IncP plasmids. Exogenous isolation demonstrated the presence of plasmids of 40-200 kb in size harbouring Sm-resistance genes from all the environments tested. Most plasmids were shown to carry the ant (3'') gene, often in combination with other Sm-resistance genes, such as aph (3'') and aph (6)-1d. The most commonly found Sm-modifying gene on mobile genetic elements was ant (3''). Multiple Sm-resistance genes on the same genetic elements appeared to be the rule rather than the exception. It is concluded that Sm-resistance genes are widespread in the environmental habitats studied and often occur on mobile genetic elements and ant (3'') was most often encountered.

Streptomycin resistant streptomycetes were isolated from four diverse sites. Two sites in Germany were sampled and included an agricultural experimental plot of apple trees which had a history of streptomycin application (AR) and a control site without antibiotic application (CR). Two sites in the UK were sampled; a pastureland site which had sewage injection (DW) and an agricultural site (CW). The actinomycete counts indicated a high proportion of streptomycin resistant isolates in the CW soil. Streptomycetes were identified by partial sequencing of the 16S rDNA. PCR product of the hypervariable gamma region of 16S rDNA allowed analysis by denaturing gradient gel electrophoresis to assess the diversity within the isolates. The streptomycin and sewage sludge treated sites showed decreased diversity within streptomycete populations. Isolates were screened for the streptomycin resistance gene, strA, and flanking biosynthesis gene, strB1. Distribution of these genes indicated the prevalence of str genes in the streptomycin treated soil. Evidence of horizontal gene transfer was recorded in isolates identified as Streptomyces platensis recovered from CR and AR sites which had acquired a streptomycin resistance gene homologous to that found in Streptomyces griseus. Members of the latter species were the most abundant streptomycin resistant streptomycetes isolated from all soils.

A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gmr) genes in different non-clinical environments has been performed. We were interested to find out whether Gmr genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6′)-II/Ib, ant(2″)-I, aph(2″)-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gmr genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, α- and β-Proteobacteria. Gmr genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to α-, β- and γ-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPβ plasmids. Molecular detection of Gmr genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gmr genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gmr genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gmr genes was not obvious.

The phylogenetic relationships of a collection of streptomycete soil isolates and type strains were resolved by sequence analysis of trpB,a housekeeping gene involved in tryptophan biosynthesis. The analysis confirmed that two isolates were recipients in a gene transfer event, demonstrated by phylogenetic incongruency between trpB and strB1 trees. One strain had acquired the entire streptomycin biosynthetic cluster, whilst the other contained only strRAB1, the resistance gene and two flanking genes from the cluster. Sequence analysis of trpB, as part of a polyphasic approach, was a useful tool in determining intra-generic relationships within the genus Streptomyces.

ABSTRACT Streptomyces strains isolated from soil were found to possess various numbers of genes from the streptomycin biosynthesis cluster. The strains missing genes from the cluster also lacked the ability to produce streptomycin. Two of the isolates which contain only part of the cluster are apparently recipients of a gene transfer event. The implications for the role of gene transfer in antibiotic evolution are discussed.

Phylogenetic analysis was used to evaluate the hypothesis of gene transfer in streptomycetes, many of which are antibiotic producers. The diversity and possible origins of streptomycin‐resistance genes was investigated for a population of Streptomyces strains isolated from a site in Brazil where antibiotic production had previously been implicated. The analysis provides compelling evidence for the transfer of these genes. Examination of other Streptomyces‐type strains also reveals a scattered distribution of streptomycin producers with respect to the overall phylogeny. These results suggest that horizontal gene transfer may be an important factor in the evolution of antibiotic genes in streptomycetes.