Genetic Characterization of mcr-1-Positive Multidrug-Resistant Salmonella enterica Serotype Typhimurium Isolated From Intestinal Infection in Children and Pork Offal in China

With the rapid emergence of plasmid-mediated colistin resistance gene mcr-1, the increased resistance of Salmonella has attracted extensive attention. This study reports on 11 multidrug-resistant Salmonella enterica serovar Typhimurium strains harboring mcr-1 in China. They all presented resistance to colistin, and additionally, one that was isolated from a child’s stool sample was also resistant to ceftriaxone and azithromycin. We screened 1454 strains of Salmonella for mcr-1 gene through PCR, and these strains are all preserved in our laboratory. Antimicrobial sensitivity analysis was carried out for the screened mcr-1 positive strains. Genetic polymorphism analysis of S. Typhimurium was performed by using the Pulsed-Field Gel Electrophoresis (PFGE). The plasmids harboring mcr-1 were identified by S1-PFGE and southern blotting. Plasmid conjugation assays were used to analyze the transferability of colistin resistance. The plasmids harboring mcr-1 were characterized by sequencing and bioinformatic analysis. Eleven S. Typhimurium strains harboring mcr-1 with colistin resistance (MICs 4μg/ml) were detected, which were isolated from children and pig offal in China. All of them were multidrug-resistant strains. PFGE results revealed that the strains isolated from different samples or locations have identical genotypes. S1-PFGE and southern blotting experiments showed that three plasmids of different sizes (33, 60, and 250 kb) all carried the mcr-1 gene. The plasmid conjugation assays revealed that Salmonella acquired mcr-1 harboring plasmids by horizontal transfer. Sequencing and plasmid type analysis revealed that these plasmids were types IncX4, IncI2, and IncHI2. Among them, IncX4 and IncI2 plasmids had extremely similar backbones and contained one resistant gene mcr-1. IncHI2 plasmid contained multiple resistant genes including blaCTX–M, oqxB, sul, aph, aadA, and blaTEM. We identified 11 mcr-1 harboring S. Typhimurium strains in China and described their characteristics. Our findings indicate that the mcr-1 gene can effectively spread among intestinal bacteria by horizontal transfer of three types of plasmids. Moreover, the IncHI2 plasmid can also mediate the transfer of other drug resistance genes. These results reveal that constant surveillance of mcr-1 harboring S Typhimurium is imperative to prevent the spread of colistin resistance.

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces

Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces .

Why do plasmids manipulate the expression of bacterial phenotypes?

Conjugative plasmids play an important role in bacterial evolution by transferring niche-adaptive traits between lineages, thus driving adaptation and genome diversification. It is increasingly clear, however, that in addition to this evolutionary role, plasmids also manipulate the expression of a broad range of bacterial phenotypes. In this review, we argue that the effects that plasmids have on the expression of bacterial phenotypes may often represent plasmid adaptations, rather than mere deleterious side effects. We begin by summarizing findings from untargeted omics analyses, which give a picture of the global effects of plasmid acquisition on host cells. Thereafter, because many plasmids are capable of both vertical and horizontal transmission, we distinguish plasmid-mediated phenotypic effects into two main classes based upon their potential fitness benefit to plasmids: (i) those that promote the competitiveness of the host cell in a given niche and thereby increase plasmid vertical transmission, and (ii) those that promote plasmid conjugation and thereby increase plasmid horizontal transmission. Far from being mere vehicles for gene exchange, we propose that plasmids often act as sophisticated genetic parasites capable of manipulating their bacterial hosts for their own benefit. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Evaporation-induced hydrodynamics promote conjugation-mediated plasmid transfer in microbial populations

Conjugative plasmids bestow important traits to microbial communities, such as virulence, antibiotic resistance, pollutant biotransformation, and biotechnology-relevant functions. While the biological mechanisms and determinants of plasmid conjugation are well established, the underlying physical and ecological driving forces remain unclear. Microbial communities often inhabit unsaturated environments, such as soils and host surfaces (e.g., skin, teeth, leaves, roots), where water evaporation and associated small-scale hydrodynamic processes frequently occur at numerous air-water and solid-water interfaces. Here, we hypothesized that evaporation can induce water flows with profound effects on the spatial distribution and surface deposition of cells, and consequently on the extent of plasmid conjugation. Using droplet experiments with an antibiotic resistance-encoding plasmid, we show that evaporation-induced water flows reduce cell-cell distances and significantly increase the extent of plasmid conjugation. Counterintuitively, we found that evaporation results in lower expression levels of conjugation-related genes. This negative relationship between the extent of plasmid conjugation and the expression of conjugation-related genes could be attributed to increased conjugation efficiency during evaporation. This study provides new insights into the physical and ecological determinants of plasmid conjugation, with important implications for understanding the spread and proliferation of plasmid-encoded traits.

The dilution effect limits plasmid horizontal transmission in multispecies bacterial communities

By transferring ecologically important traits between species, plasmids drive genomic divergence and evolutionary innovation in their bacterial hosts. Bacterial communities are often diverse and contain multiple coexisting plasmids, but the dynamics of plasmids in multi-species communities are poorly understood. Here, we show, using experimental multi-species communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to the ‘dilution effect’; this is an epidemiological phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species. In addition, plasmid horizontal transmission was also affected by plasmid diversity, such that the rate of plasmid conjugation was reduced from co-infected host cells carrying both plasmids. In diverse microbial communities, plasmid spread may be limited by the dilution effect and plasmid–plasmid interactions, reducing the rate of horizontal transmission.

A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection

The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.

Characteristics of colistin-resistant Escherichia coli from pig farms in Central China

The emergence and dissemination of colistin resistance in Enterobacteriaceae mediated by plasmid-borne mcr genes in recent years now pose a threat to public health. In this study, we isolated and characterized colistin-resistant and/or mcr-positive E. coli from pig farms in Central China. Between 2018 and 2019, 594 samples were collected and recovered 445 E. coli isolates. Among them, 33 with colistin resistance phenotypes and 37 that were positive for mcr genes were identified, including 34 positive for mcr-1, one positive for mcr-3, and two positive for both mcr-1 and mcr-3. An insertion of nine bases (“CTGGATACG”) into mcr-1 in four mcr-positive isolates led to gene dysfunction, and therefore did not confer the colistin resistance phenotype. Antimicrobial susceptibility testing revealed that 37 mcr-positive isolates showed severe drug resistance profiles, as 50% of them were resistant to 20 types of antibiotics. Multilocus sequence typing revealed a heterogeneous group of sequence types in mcr-positive isolates, among which ST10 (5/37), ST156 (5/37), and ST617 (4/37) were the predominant types. Plasmid conjugation assays showed that mcr-carrying plasmids of 25 mcr-positive isolates were conjugated with E. coli recipient, with conjugation frequencies ranging from 1.7 × 10-6 to 4.1 × 10-3 per recipient. Conjugation of these mcr genes conferred a colistin resistance phenotype upon the recipient bacterium. PCR typing of plasmids harbored in the 25 transconjugants determined six types of plasmid replicons, including IncX4 (14/25), FrepB (4/25), IncI2 (3/25), IncHI2 (2/25), FIB (1/25), and IncI1 (1/25). This study contributes to the current understanding of antibiotic resistance and molecular characteristics of colistin-resistant E. coli in pig farms.

The dilution effect limits plasmid horizontal transmission in multispecies bacterial communities

By transferring ecologically important traits between species, plasmids drive genomic divergence and evolutionary innovation in their bacterial hosts. Bacterial communities are often diverse and contain multiple coexisting plasmids, but the dynamics of plasmids in multispecies communities are poorly understood. Here, we show, using experimental multispecies communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to the dilution effect; an epidemiological phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species. In addition, plasmid horizontal transmission was also affected by plasmid diversity, such that the rate of plasmid conjugation was reduced from coinfected host cells carrying both plasmids. In diverse microbial communities, plasmid spread may be limited by the dilution effect and plasmid-plasmid interactions reducing the rate of horizontal transmission.

Antioxidant molecules as a source of mitigation of antibiotic resistance genes dissemination

Escherichia coli is the most commonly identified human pathogen, and a prominent microorganism of the gut microbiota. Acquired resistance to antibiotics in that species is mainly driven by horizontal gene transfer, and mainly by plasmid acquisition. The main concern nowadays corresponds to the acquisition of extended-spectrum ß-lactamases of the CTX-M-type in E. coli, a worldwide observed phenomenon. Plasmids encoding CTX-M enzymes are of different scaffolds, and conjugate at different frequencies. Here we showed that the conjugation rates of several plasmid types encoding broad-spectrum ß-lactamases are increased when the E. coli donor strain is exposed to sub-inhibitory concentrations of diverse orally-given antibiotics, including fluoroquinolones such as ciprofloxacin and levofloxacin, but also trimethoprim, and nitrofurantoin. This study provided insights into underlying mechanisms leading to increase plasmid conjugation frequency in relation with DNA synthesis inhibitors-type antibiotics, involving reactive oxygen species (ROS) production and probably increased expression of genes involved in the SOS response. Furthermore, we showed that some antioxidant molecules currently approved for unrelated clinical uses such as edaravone, p-Coumaric acid and N-acetylcysteine may antagonize the inducibility effect of antibiotics in term of increased plasmid conjugation rates. These results suggest that several antioxidative molecules might be used in combination with those “inducer” antibiotics to mitigate the unwanted increased resistance plasmid dissemination.