Isolation and characterization of bacteriophages specific to Enterococcus faecalis and Enterococcus faecium as potential markers to detect faecal contamination in water

Abstract

The aim of the study was to isolate and identify E. faecalis and E. faecium from ground and surface water samples, and evaluate the potential of using E. faeca/is and E. faecium specific phages as indicator organisms for determining faecal contamination in water. A total of 162 samples were collected from August 2015 to April 2016 and analysed for characteristics of Enterococcus species using Bile Esculin Agar (BEA) and 57 samples were positive for presumptive Enterococcus species. The samples were also analyzed to determine faecal coliform and total coliform bacterial counts and large proportion 90 (55%) of the samples were positive for either faecal or total coliform bacteria. A total of 119 presumptive isolates were picked from the 57 culture plates that were positive for Enterococcus species based on macroscopic characteristics. The isolates screened for the characteristics of Enterococcus (E. faecalis and E. faecium) using biochemical characterization and E. faecalis and E. faecium species specific PCR through amplification of the d-alanine: d-alanine ligase (dd~ gene sequences. All the isolates including the control strain were Gram positive cocci and were also catalase and oxidase negative. In addition, all the isolates grew in 6.5% (w/v) NaCl and this trait does not only identify the isolates as belonging to the genus Enterococcus but also differentiates them from organisms belonging to the genus Streptococcus. All the isolates were gamma haemolytic on sheep blood agar. A large proportion 50 (42%) of the isolates were confirmed as E. faecium while 16 (13%) were E. faeca/is. All the isolates in this study were resistant to erythromycin while large proportions (62.1 % to 97%) of these isolates were resistant to ampicillin, gentamycin, tetracycline and chloramphenicol. On the contrary, 27 (41%) of the isolates were resistant to vancomycin while only 12 (18.2%) were resistant to amoxycillin. The detection of vancomycin resistant enterococci was a cause for concern due to fact that these isolates may pose severe public health threats to humans. A total of 66 isolates comprising of 50 E. faecium and 16 E. faeca/is were subjected to cluster analysis using Statistica version 10.0 (Statsoft, US). Out of the 66 isolates analysed two major clusters (Cluster 1 and Cluster 2) were generated. Cluster 1 was subdivided into two sub-clusters (Cluster 1 A and Cluster 1 B).

Furthermore, sub-clusters 1A and 1 B, including Cluster 2, were analysed for patterns of association of isolates from different sources and/or locations. The largest sub-cluster (sub-cluster 181) contained isolates from three sampling areas. The largest proportion (58.8%) of isolates in this sub-cluster was derived from surface water samples in Mafikeng. In addition, small proportions (17.6% and 23.5%) of the isolates in this sub-cluster were obtained from surface water in Rustenburg and Zeerust respectively. The second largest sub-cluster ( sub-cluster Cluster 1 A 1) contained 12 ( 18 .2%) of the isolates analysed. The makeup of isolates in this cluster consisted of 3, 4 and 5 isolates from groundwater obtained in Rustenburg, Ventersdorp and Mafikeng respectively. Interestingly, all eight (8) isolates in sub-clusters 1A2 were obtained from groundwater in Mafikeng while all six (6) that were clustered in sub-cluster 1 B2 were obtained from surface water in Coligny. These findings indicate that isolates that clustered together share similar antibiotic resistance profiles which may have resulted from similarities in the degree of exposure to these drugs. E. faecalis and E. faecium specific bacteriophages were detected.TEM data indicated that phages possessed morphologies that were similar to those of bacteriophages belonging to the Siphoviridae family. It was also revealed that E. faecalis and E. faecium specific bacteriophages can serve as reliable indicators of faecal pollution in water.