Uneven genotypic diversity of Escherichia coli in fecal sources limits the performance of a library-dependent method of microbial source tracking on the southwestern French Atlantic coast.

To develop a library-dependent method of tracking fecal sources of contamination of beaches on the Atlantic coast of southwestern France, a library of 6368 Escherichia coli isolates was constructed from samples of feces, from 40 known human or animal sources collected in the vicinity of Arcachon Bay in 2010, and in French Basque Country, Landes, and Béarn, between 2017 and 2018. Different schemes of source identification were tested: use of the complete or filtered reference library; characterization of the isolates by genotypic or proteomic profiling based on ERIC-PCR or MALDI-TOF mass spectrometry, respectively; isolate by isolate assignment using either classifiers based on the Pearson similarity or SVM (support vector machine). With the exception of one source identification scheme, which was discarded since it used self-assignment, all tested schemes resulted in low rates of correct classification (<35%) and significant rates of incorrect classification (>15%). The heterogeneous coverage of E. coli genotypic diversity between sources and the uneven distribution of E. coli genotypes in the library likely explain the difficulties encountered in identifying the sources of fecal contamination. Shannon diversity index of sources ranged from 0 for several wildlife species sampled once to 3.03 for sewage treatment plant effluents sampled on various occasions, showing discrepancies between sources. The uneven genotypic composition of the library was attested by the value of the Pielou index (0.54), the high proportion of nondiscriminatory genotypes (>91% of the isolates), and the very low proportion of discriminatory genotypes (<3%). Since efforts made to constitute such a library are not affordable for routine analyses, the results question the relevance of developing such a method for identifying sources of fecal contamination on such a coastline.

Acipenser iridovirus-European encodes a replication factor C (RFC) sub-unit.

New genomic sequence data were acquired for the Acipenser iridovirus-European (AcIV-E), a virus whose complete genome and classification still remain to be elucidated. Here, we obtained the first full-length Major capsid protein (MCP) gene sequence for AcIV-E, as well as two additional open reading frames (ORFs) adjacent to the MCP gene. BLAST searches of the first ORF (α) resulted in no match to any gene or protein in the public databases. The other ORF (ß) was identified as a subunit of a replication factor C (RFC), known to function as a clamp loader in eukaryotes, archae and some viruses. The presence of similar RFC genes was confirmed in two distinct, yet related, viruses, the white sturgeon iridovirus and a European variant of Namao virus. The existence of an RFC gene in AcIV-E suggests a genome size larger than that of other classifiable members of the family Iridoviridae along with a mode of replication involving an interaction between a clamp loader and a proliferating nuclear cell antigen. Sequencing and comparison of the full-length RFC gene from various sturgeon samples infected with AcIV-E revealed two distinct clusters of sequences within one particular sample in which the coexistence of two lineages had previously been predicted based on analysis of the partial MCP gene sequence. These genetic data provide further evidence of the circulation of at least two concurrent AcIV-E lineages, sometimes co-infecting cultured European sturgeon.

Genetic identification of two Acipenser iridovirus-European variants using high-resolution melting analysis.

Acipenser iridovirus-European (AcIV-E) is an important pathogen of sturgeons. Two variants differing by single-nucleotide polymorphisms (SNP) in the Major Capsid Protein gene have been described, but without any indication as to their prevalence in farms. To facilitate epidemiological studies, we developed a high-resolution melting (HRM) assay to distinguish between two alleles (var1 and var2) differing by five point substitutions. The HRM assay detected as little as 100 copies of plasmids harboring cloned sequences of var1 and var2, which have melting temperatures (Tm) differing by only 1 °C. The assay was specific of AcIV-E as demonstrated by the absence of signal when testing a related, yet distinct, virus as well as DNA from an AcIV-E-negative sturgeon sample. Experiments with mixtures of two distinct plasmids revealed abnormal melting curve patterns, which showed dips just before the main melting peaks. These dips in the curves were interpreted as the dissociation of heteroduplexes fortuitously created during the PCR step. Screening AciV-E-positive field samples of Russian sturgeons from three farms revealed the presence of var2, based on the Tm. However, for a few samples, the melting curves showed patterns typical of var2 as the dominant viral genome, mixed with another minor variant which proved to be var1. In conclusion, HRM is a simple method to screen for AcIV-E var1 and var2 and can be used on a large scale in Europe to trace these two variants which likely represent two genetic lineages.