Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution and as reservoirs of antibiotic-resistance and virulence genes
The introduction of human fecal material into water sources represents a concern to public health since pathogens could be introduced. Not all laboratories possess the facilities to detect human enteric pathogens and thus microbial indicators are used. An ideal microbial indicator of human fecal pollution should be: (i) enumerated using simple laboratory methods, (ii) present in fecally polluted waters and absent in pristine waters, (iii) associated with the source of the contamination, (iv) able to survive and inactivate similarly to the pathogen of concern and (v) be detected in various geographical regions. No microbial indicator of fecal pollution satisfies all these characteristics. Therefore, we have developed a relatively simple culture technique to characterized bacteriophages that infect a specific type strain of Enterococcus faecalis, which we call enterophages, as markers of human fecal pollution. Enterophages were detected exclusively in domestic wastewaters in Puerto Rico and Portugal, tropical recreational waters and possess a survival and inactivation rates similar to human pathogenic enteric viruses. Enterophages possess non-contractile tails of 60 to 200nm, icosahedral capsids of 12 to 80nm and dsDNA genomes of 30 to 40kb. Given that their role as vectors of genes conferring antibiotic-resistance and virulence to the bacterial host remains largely unknown, the complete genomes of two phages infecting E. faecalis (one lytic and one lysogenic) are currently being sequenced. Lysogenic Enterococcus phages, as well as their host isolated from marine and fresh waters, dry and wet sands in Puerto Rico, were tested for tetracycline (tetM) and vancomycin-resistance (vanA and vanB) genes. The prevalence of the enterococcal surface protein (Esp), a virulence factor, was also determined given that it has received great attention as a marker of human fecal pollution. The prevalence of an integrase-encoding gene (int) specific for E. faecalis phages was determined since integrases are markers of lysogeny and are responsible for the integration of the phage genome into the bacterial host. tetM, vanA, vanB, esp and int were only detected in the bacterial fraction and enterococci, with the exception of int, which was detected in the viral fractions and lysogenic enterococci phages.