The presence and diversity of IncP-1 plasmids in the influent of a Danish wastewater treatment plant was studied by PCR amplification of the trfA gene in community DNA followed by sequencing. Three sets of PCR primers were designed to amplify a 281 bp fragment of trfA from all currently sequenced IncP-1 plasmids. A neighbor-joining tree, based on a multiple alignment of 72 obtained sequences together with homologous sequences of previously published IncP-1 plasmids, revealed that all established subgroups of IncP-1 plasmids, alpha, beta, gamma and delta, were present in the wastewater treatment plant influent. Also sequences representing the recently described fifth subgroup, the epsilon subgroup, were detected in the wastewater. Thus, these results confirm the presence of at least five phylogenetically distinct subgroups of IncP-1 plasmids and represent the first time that sequences associated with plasmids of all of these five subgroups have been detected in a single setting. Additionally, the results confirm that wastewater constitutes a reservoir for the conjugative IncP-1 plasmids, which often harbor multiple antibiotic resistance genes. (c) 2009 Elsevier Inc. All rights reserved.