- Volume 4, Issue 7, 2018

Antimicrobial resistance (AMR) is a global public-health emergency, which threatens the advances made by modern medical care over the past century. The World Health Organization has recently published a global priority list of antibiotic-resistant bacteria, which includes extended-spectrum β-lactamase-producing Enterobacteriaceae and carbapenemase-producing Enterobacteriaceae. In this review, we highlight the mechanisms of resistance and the genomic epidemiology of these organisms, and the impact of AMR.

Xanthomonas fragariae is a quarantine organism in Europe, causing angular leaf spots on strawberry plants. It is spreading worldwide in strawberry-producing regions due to import of plant material through trade and human activities. In order to resolve the population structure at the strain level, we have employed high-resolution molecular typing tools on a comprehensive strain collection representing global and temporal distribution of the pathogen. Clustered regularly interspaced short palindromic repeat regions (CRISPRs) and variable number of tandem repeats (VNTRs) were identified within the reference genome of X. fragariae LMG 25863 as a potential source of variation. Strains from our collection were whole-genome sequenced and used in order to identify variable spacers and repeats for discriminative purpose. CRISPR spacer analysis and multiple-locus VNTR analysis (MLVA) displayed a congruent population structure, in which two major groups and a total of four subgroups were revealed. The two main groups were genetically separated before the first X. fragariae isolate was described and are potentially responsible for the worldwide expansion of the bacterial disease. Three primer sets were designed for discriminating CRISPR-associated markers in order to streamline group determination of novel isolates. Overall, this study describes typing methods to discriminate strains and monitor the pathogen population structure, more especially in the view of a new outbreak of the pathogen.

Serratia grimesii BXF1 is a bacterium with the ability to modulate the development of several eukaryotic hosts. Strain BXF1 was isolated from the pinewood nematode, Bursaphelenchus xylophilus, the causative agent of pine wilt disease affecting pine forests worldwide. This bacterium potentiates Bursaphelenchus xylophilus reproduction, acts as a beneficial pine endophyte, and possesses fungal and bacterial antagonistic activities, further indicating a complex role in a wide range of trophic relationships. In this work, we describe and analyse the genome sequence of strain BXF1, and discuss several important aspects of its ecological role. Genome analysis indicates the presence of several genes related to the observed production of antagonistic traits, plant growth regulation and the modulation of nematode development. Moreover, most of the BXF1 genes are involved in environmental and genetic information processing, which is consistent with its ability to sense and colonize several niches. The results obtained in this study provide the basis to a better understanding of the role and evolution of strain BXF1 as a mediator of interactions between organisms involved in a complex disease system. These results may also bring new insights into general Serratia and Enterobacteriaceae evolution towards multitrophic interactions.

Pathogen monitoring is becoming more precise as sequencing technologies become more affordable and accessible worldwide. This transition is especially apparent in the field of food safety, which has demonstrated how whole-genome sequencing (WGS) can be used on a global scale to protect public health. GenomeTrakr coordinates the WGS performed by public-health agencies and other partners by providing a public database with real-time cluster analysis for foodborne pathogen surveillance. Because WGS is being used to support enforcement decisions, it is essential to have confidence in the quality of the data being used and the downstream data analyses that guide these decisions. Routine proficiency tests, such as the one described here, have an important role in ensuring the validity of both data and procedures. In 2015, the GenomeTrakr proficiency test distributed eight isolates of common foodborne pathogens to participating laboratories, who were required to follow a specific protocol for performing WGS. Resulting sequence data were evaluated for several metrics, including proper labelling, sequence quality and new single nucleotide polymorphisms (SNPs). Illumina MiSeq sequence data collected for the same set of strains across 21 different laboratories exhibited high reproducibility, while revealing a narrow range of technical and biological variance. The numbers of SNPs reported for sequencing runs of the same isolates across multiple laboratories support the robustness of our cluster analysis pipeline in that each individual isolate cultured and resequenced multiple times in multiple places are all easily identifiable as originating from the same source.

A better understanding of the genomic changes that facilitate the emergence and spread of drug-resistant Mycobacterium tuberculosis strains is currently required. Here, we report the use of the MinION nanopore sequencer (Oxford Nanopore Technologies) to sequence and assemble an extensively drug-resistant (XDR) isolate, which is part of a modern Beijing sub-lineage strain, prevalent in Western Province, Papua New Guinea. Using 238-fold coverage obtained from a single flow-cell, de novo assembly of nanopore reads resulted into one contiguous assembly with 99.92 % assembly accuracy. Incorporation of complementary short read sequences (Illumina) as part of consensus error correction resulted in a 4 404 064 bp genome with 99.98 % assembly accuracy. This assembly had an average nucleotide identity of 99.7 % relative to the reference genome, H37Rv. We assembled nearly all GC-rich repetitive PE/PPE family genes (166/168) and identified variants within these genes. With an estimated genotypic error rate of 5.3 % from MinION data, we demonstrated identification of variants to include the conventional drug resistance mutations, and those that contribute to the resistance phenotype (efflux pumps/transporter) and virulence. Reference-based alignment of the assembly allowed detection of deletions and insertions. MinION sequencing provided a fully annotated assembly of a transmissible XDR strain from an endemic setting and showed its utility to provide further understanding of genomic processes within Mycobacterium tuberculosis.

The genus Escherichia is composed of Escherichia albertii, E. fergusonii, five cryptic Escherichia clades and E. coli sensu stricto. Furthermore, the E. coli species can be divided into seven main phylogroups termed A, B1, B2, C, D, E and F. As specific lifestyles and/or hosts can be attributed to these species/phylogroups, their identification is meaningful for epidemiological studies. Classical phenotypic tests fail to identify non-sensu stricto E. coli as well as phylogroups. Clermont and colleagues have developed PCR assays that allow the identification of most of these species/phylogroups, the triplex/quadruplex PCR for E. coli phylogroup determination being the most popular. With the growing availability of whole genome sequences, we have developed the ClermonTyping method and its associated web-interface, the ClermonTyper, that allows a given strain sequence to be assigned to E. albertii, E. fergusonii, Escherichia clades I–V, E. coli sensu stricto as well as to the seven main E. coli phylogroups. The ClermonTyping is based on the concept of in vitro PCR assays and maintains the principles of ease of use and speed that prevailed during the development of the in vitro assays. This in silico approach shows 99.4 % concordance with the in vitro PCR assays and 98.8 % with the Mash genome-clustering tool. The very few discrepancies result from various errors occurring mainly from horizontal gene transfers or SNPs in the primers. We propose the ClermonTyper as a freely available resource to the scientific community at: http://clermontyping.iame-research.center/.