The dramatic spread of antibiotic resistance is a crisis in the treatment of infectious diseases that affect humans. Several studies suggest that wastewater treatment plants (WWTP) are reservoirs for diverse mobile antibiotic resistance elements. This review summarizes findings derived from genomic analysis of IncP-1 resistance plasmids isolated from WWTP bacteria. Plasmids that belong to the IncP-1 group are self-transmissible, and transfer to and replicate in a wide range of hosts. Their backbone functions are described with respect to their impact on vegetative replication, stable maintenance and inheritance, mobility and plasmid control. Accessory genetic modules, mainly representing mobile genetic elements, are integrated in-between functional plasmid backbone modules. These elements carry determinants conferring resistance to nearly all clinically relevant antimicrobial drug classes, to heavy metals, and quaternary ammonium compounds used as disinfectants. All plasmids analysed here contain integrons that potentially facilitate integration, exchange and dissemination of resistance gene cassettes. Comparative genomics of accessory modules located on plasmids from WWTP and corresponding modules previously identified in other bacterial genomes revealed that animal, human and plant pathogens and other bacteria isolated from different habitats share a common pool of resistance determinants.

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.

A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal meta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activatedsludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.

Broad-host-range (BHR) IncP-1 plasmids have the ability to transfer between and replicate in nearly all species of the Alpha-, Beta-and Gammaproteobacteria, but surprisingly few data are available on the stability of these plasmids in strains within their host range. Moreover, even though molecular interactions between the bacterial host and its plasmid(s) exist, no systematic study to date has compared the stability of the same plasmid among different hosts. The goal of this study was to examine whether the stability characteristics of an IncP-1 plasmid can be variable between strains within the host range of the plasmid. Therefore, 19 strains within the Alpha-, Beta-or Gammaproteobacteria carrying the IncP-1b plasmid pB10 were serially propagated in non-selective medium and the fraction of segregants was monitored through replica-picking. Remarkably, a large variation in the stability of pB10 in different strains was found, even between strains within the same genus or species. Ten strains showed no detectable plasmid loss over about 200 generations, and in two strains plasmid-free clones were only sporadically observed. In contrast, three strains, Pseudomonas koreensis R28, Pseudomonas putida H2 and Stenotrophomonas maltophilia P21, exhibited rapid plasmid loss within 80 generations. Parameter estimation after mathematical modelling of these stability data suggested high frequencies of segregation (about 0.04 per generation) or high plasmid cost (i.e. a relative fitness decrease in plasmid-bearing cells of about 15 and 40 %), which was confirmed experimentally. The models also suggested that plasmid reuptake by conjugation only played a significant role in plasmid stability in one of the three strains. Four of the 19 strains lost the plasmid very slowly over about 600 generations. The erratic decrease of the plasmid-containing fraction and simulation of the data with a new mathematical model suggested that plasmid cost was variable over time due to compensatory mutations. The findings of this study demonstrate that the ability of a so-called 'BHR' plasmid to persist in a bacterial population is influenced by strain-specific traits, and therefore observations made for one strain should not be generalized for the entire species or genus. INTRODUCTIONComparative analyses of fully sequenced bacterial genomes suggest that horizontal gene transfer has played a significant role in the adaptive evolution of microbial life (Gogarten & Townsend, 2005;Gogarten et al., 2002;Jain et al., 2002). In particular, horizontal transfer (HT) of broad-host-range (BHR) antibiotic resistance plasmids through conjugation is important to the spread of drug resistance genes (de la Cruz & Davies, 2000;Frost et al., 2005;Mazel & Davies, 1999), as these plasmids can transfer between and replicate in a broad range of taxonomically diverse species. Besides conjugation and replication, stability in the absence of selection for plasmid-encoded traits is a third characteristic that should be considered in the assessment of the long-term host r...