Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.     

Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea

Background

Cabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F₂ population of 4000 individuals derived from the same parental lines.

Results

We confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99–77 and the highly susceptible line 99–91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F₂ population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes’ sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.

Conclusions

This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.     

DNA Sequencing Reveals the Midgut Microbiota of Diamondback Moth,Plutella xylostella (L.) and a Possible Relationship with Insecticide Resistance

Background

Insect midgut microbiota is important in host nutrition, development and immune response. Recent studies indicate possible links between insect gut microbiota and resistance to biological and chemical toxins. Studies of this phenomenon and symbionts in general have been hampered by difficulties in culture-based approach. In the present study, DNA sequencing was used to examine the midgut microbiota of diamondback moth (DBM), Plutella xylostella (L.), a destructive pest that attacks cruciferous crops worldwide. Its ability to develop resistance to many types of synthetic insecticide and even Bacillus thuringiensis toxins makes it an important species to study.

Methodology/Principal Findings

Bacteria of the DBM larval midgut in a susceptible and two insecticide (chlorpyrifos and fipronil) resistant lines were examined by Illumina sequencing sampled from an insect generation that was not exposed to insecticide. This revealed that more than 97% of the bacteria were from three orders: Enterobacteriales, Vibrionales and Lactobacillales. Both insecticide-resistant lines had more Lactobacillales and the much scarcer taxa Pseudomonadales and Xanthomonadales with fewer Enterobacteriales compared with the susceptible strain. Consistent with this, a second study observed an increase in the proportion of Lactobacillales in the midgut of DBM individuals from a generation treated with insecticides.

Conclusions/Significance

This is the first report of high-throughput DNA sequencing of the entire microbiota of DBM. It reveals differences related to inter- and intra-generational exposure to insecticides. Differences in the midgut microbiota among susceptible and insecticide-resistant lines are independent of insecticide exposure in the sampled generations. While this is consistent with the hypothesis that Lactobacillales or other scarcer taxa play a role in conferring DBM insecticide resistance, further studies are necessary to rule out other possibilities. Findings constitute the basis for future molecular work on the functions of insect midgut microbiota taxa and their possible role in conferring host resistance to toxins.     

How slug herbivory of juvenile hybrid willows alters chemistry,growth and subsequent susceptibility to diverse plant enemies

Background and Aims

Selective feeding by herbivores, especially at the seedling or juvenile phase, has the potential to change plant traits and ultimately the susceptibility of surviving plants to other enemies. Moreover, since hybridization is important to speciation and can lead to introgression of traits between plant species, differential feeding (herbivore-induced mortality) can influence the expression of resistance traits of hybrids and ultimately determine the consequences of hybridization. While it would be expected that herbivore-induced mortality would lead to greater resistance, there may be trade-offs whereby resistance to one herbivore increases susceptibility to others. The hypothesis was tested that the exotic slug, Arion subfuscus, causes non-random survival of hybrid willows and alters plant: (1) susceptibility to slugs; (2) secondary and nutritional chemistry, and growth; and (3) susceptibility to other phytophages.

Methods

Two populations of plants, control and selected, were created by placing trays of juvenile willows in the field and allowing slugs access to only some. When ≤10 individuals/tray remained (approx. 85 % mortality), ‘selected’ and undamaged ‘control’ trays were returned to a common area. Traits of these populations were then examined in year 1 and in subsequent years.

Key Results

The selected population was less palatable to slugs. Surprisingly, foliar concentrations of putative defence traits (phenolic glycosides and tannins) did not differ between treatments, but the selected population had higher foliar nitrogen and protein, lower carbon to nitrogen ratio and greater above-ground biomass, indicating that vigorously growing plants were inherently more resistant to slugs. Interestingly, selected plants were more susceptible to three phytophages: an indigenous pathogen (Melampsora epitea), a native herbivorous beetle (Chrysomela knabi) and an exotic willow leaf beetle (Plagiodera versicolora).

Conclusions

This exotic slug changed the population structure of F₂ hybrid willows in unanticipated ways. Defence expression remained unchanged, while nutritional and growth traits changed. These changes caused plants to be more susceptible to other plant enemies. Other exotic herbivore species are anticipated to have similar direct and indirect effects on native plant populations.     

Mapping in the era of sequencing: high density genotyping and its application for mapping TYLCV resistance in Solanum pimpinellifolium

Background

A RIL population between Solanum lycopersicum cv. Moneymaker and S. pimpinellifolium G1.1554 was genotyped with a custom made SNP array. Additionally, a subset of the lines was genotyped by sequencing (GBS).

Results

A total of 1974 polymorphic SNPs were selected to develop a linkage map of 715 unique genetic loci. We generated plots for visualizing the recombination patterns of the population relating physical and genetic positions along the genome.This linkage map was used to identify two QTLs for TYLCV resistance which contained favourable alleles derived from S. pimpinellifolium. Further GBS was used to saturate regions of interest, and the mapping resolution of the two QTLs was improved. The analysis showed highest significance on Chromosome 11 close to the region of 51.3 Mb (qTy-p11) and another on Chromosome 3 near 46.5 Mb (qTy-p3). Furthermore, we explored the population using untargeted metabolic profiling, and the most significant differences between susceptible and resistant plants were mainly associated with sucrose and flavonoid glycosides.

Conclusions

The SNP information obtained from an array allowed a first QTL screening of our RIL population. With additional SNP data of a RILs subset, obtained through GBS, we were able to perform an in silico mapping improvement to further confirm regions associated with our trait of interest. With the combination of different ~ omics platforms we provide valuable insight into the genetics of S. pimpinellifolium-derived TYLCV resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1152) contains supplementary material, which is available to authorized users.