Abstract
Integrative Conjugative Elements (ICE’s) of the SXT/R391 family have largely been detected in clinical or environmental isolates of Gammaproteobacteria, particularly Vibrio and Proteus species. As wastewater treatment plants accumulate a large and diverse number of such species, we examined raw water samples taken from a municipal wastewater treatment plant initially using SXT/R391 family integrase gene-specific PCR probes to detect the presence of such elements in a directed approach. A positive amplification occurred over a full year period and a subsequent Restriction Fragment Length Polymorphism (RFLP) analysis revealed a very limited diversity in the treatment plant examined. Samples demonstrating positive amplification were cultured using Vibrio and Proteus selective media and PCR amplification tracking was utilized to monitor SXT/R391-ICE family containing strains. This screening procedure resulted in the isolation and identification of a Proteus mirabilis strain harbouring an ICE. Whole-genome sequencing of this ICE containing strain using Illumina sequencing technology revealed a novel 81 kb element that contained 75 open reading frames on annotation but contained no antibiotic or metal resistance determinants. Comparative genomics revealed the element contained a conserved ICE core with one of the insertions containing a novel bacteriophage defence mechanism. This directed isolation suggests that ICE elements are present in the environment without apparent selective pressure but may contain adaptive functions allowing survival in particular environments such as municipal wastewater which are reservoirs for large bacterial phage populations.
Highlights
Wastewater treatment plants (WWTP) accumulate large and diverse numbers of pathogens including viruses, bacteria, fungi, protozoans and helminths
Samples were plated on genera specific selective media as a significant number of SXT/R391 Integrating Conjugative Elements (ICEs) Mobile Genetic Elements (MGEs) have been detected in Proteus and Vibrio species[7]
A novel environmental SXT/R391 Type 1 family ICE, ICEPmiIre[01] was isolated from a local municipal wastewater by using selective media for Proteus and Vibrio strains and monitoring the presence of ICEs using ICE specific int primers amplified by PCR
Summary
Wastewater treatment plants (WWTP) accumulate large and diverse numbers of pathogens including viruses, bacteria, fungi, protozoans and helminths. Type 1 elements have been found in a variety of Gammaproteobacteria species including Vibrio, Providencia, Proteus, Shewanella, Actionbacillus, Alternomonas, Escherichia coli, Enterovibrio, Photobacterium damselae, Idiomarinaceae bacterium and Marinomonas[8] They are distinguished by possessing a Type 1 integrase, which allows site-specific integration into the 5′ end of the essential prfC gene[9], integration restores a functioning prfC gene and encodes a new hybrid PrfC protein once integrated[9]. Universitá La Sapienza, Rome, National Collection of Industrial Food and Marine Bacteria Addgene insert at the 3′ end of the multi-copy tRNA-Ser gene and have been found solely in Vibrio species[7,10] Another type of SXT/R391 ICE integrates into the pabA (para-aminobenzoate synthase) gene as with ICESh9511. A directed approach was used to detect and isolate ICEs of the SXT/R391 family from a municipal wastewater treatment plant and to characterise one of these isolates as the first ICE detected and isolated from Ireland
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