Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges

111

A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gm r ) genes in different non-clinical environments has been performed. We were interested to find out whether Gm r 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 Gmmodifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6P)-II/Ib, ant(2Q)-I, aph(2Q)-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 Gm r 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, K-and L-Proteobacteria. Gm r genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to K-, L-and Q-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPL plasmids. Molecular detection of Gm r genes, and broad host range plasmidspecific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gm r 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 Gm r genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gm r genes was not obvious.

Section: Discussionmentioning

confidence: 99%

Gentamicin resistance genes in environmental bacteria: prevalence and transfer

Heuer¹

2002

111

“…A modi¢cation of the triparental approach was applied by Top et al [34] to isolate mobilising plasmids from di¡erent polluted soils and activated sludges. E. coli CM330 with the IncQ plasmid pMOL155 (tra 3 , mob þ ) carrying a czc cassette not expressed in this strain was used as donor of the mobilisable plasmid.…”

Section: Exogenous Plasmid Isolation In Triparental Matingsmentioning

confidence: 99%

“…Mobilising plasmids were also isolated by van Elsas et al [35] when bacterial communities obtained from the rhizosphere of young wheat plants served as donor in triparental matings according to the protocol described by Top et al [34]. Amongst a group of other plasmids, plasmid pIPO2 was isolated in R. eutropha based on its mobilising capacity.…”

Section: Exogenous Plasmid Isolation In Triparental Matingsmentioning

confidence: 99%

The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: insights gained from different methodological approaches

Smalla¹

2002

The pool of mobile genetic elements (MGE) in microbial communities consists of plasmids, bacteriophages and other elements that are either self-transmissible or use mobile plasmids and phages as vehicles for their dissemination. By facilitating horizontal gene exchange, the horizontal gene pool (HGP) promotes the evolution and adaptation of microbial communities. Efforts to characterise MGE from bacterial populations resident in a variety of ecological habitats have revealed a surprisingly vast and seemingly untapped diversity. MGE, conferring such selectable traits as mercury or antibiotic resistance and degradative functions, have been readily acquired from diverse microbial communities. To circumvent the need to isolate microbial hosts, polymerase chain reaction (PCR)-based detection methods have frequently been used to assess the prevalence of MGE-specific sequences resident in the 'microbial community' HGP. As studies continue to reveal novel and distinct MGE, sequencing of newly isolated MGE from diverse habitats is essential for the continued development of DNA probes, PCR primers as well as for gene array and proteomics-based approaches. This minireview highlights insight gained from different methodological approaches, biased albeit largely toward plasmids in Gram-negative bacteria, used to study the HGP of naturally occurring microbial communities from various aquatic and terrestrial habitats.

“…(rep P probe) [14] were done as described elsewhere [15]. Membranes were washed at high stringency [15].…”

Section: Confirmation and Identification Of Transconjugantsmentioning

confidence: 99%

Transfer of the catabolic plasmid RP4::Tn4371 to indigenous soil bacteria and its effect on respiration and biphenyl breakdown

Rore¹

1994

The catabolic plasmid RP4::Tn4371, containing the genes for biphenyl and 4-chlorobiphenyl catabolism, was transferred from Enterobacter agglomerans DMK3 to indigenous bacteria in biphenyl amended sandy soil, although the donor survived for only 3 days. Proliferation of transconjugants above a detectable level required presence of the concomitant pollutant biphenyl. Different Gram-negative bacterial species, tentatively identified as Pseudomonas chlororaphis, Pseudomonas corrugata and Comamonas sp., had taken up the plasmid and were able to express the biphenyl degradation genes. These genera have, until now, not been reported to express the biphenyl degradative genes. The soil had a high natural catabolic capacity for biphenyl. Notwithstanding this, out of 8 trials in which DMK3 was inoculated, 2 were found not to respond but 6 had an increased biphenyl degradation and concomitant increased oxygen consumption. The results indicate that seeding of RP4::Tn4371 generates a variety of transconjugants and holds potential for increasing the soil biodegradation capacity towards chlorinated biphenyl analogues.