AbstractThe rapid emergence of antibiotic-resistant bacteria is

AbstractThe rapid emergence of antibiotic-resistant bacteria is rendering many antibiotics ineffective against the simplest infections. Many of these bacteria have gained resistance through the transfer of antibiotic-resistant genes often encoded on plasmids, which are small, extrachromosomal pieces of DNA. Exploiting these antibiotic-resistance conferring plasmids could therefore be very effective since their removal would re-sensitise bacteria to treatment with antibiotics. This review considers three potential strategies targeting plasmid maintenance or transfer to combat antibiotic resistance; inhibition of plasmid conjugation, inhibition of plasmid replication and employing CRISPR-Cas.Keywords: antibiotic resistance, relaxase, plasmid incompatibility, curing vectors, CRISPR-Cas IntroductionThe rise of antibiotic resistance in pathogenic bacteria is posing a worldwide health crisis. In Europe, approximately 25,000 deaths a year are due to multidrug-resistant bacterial infections (Blair, et al., 2015). This matter is worsened due to the fact that over the last two decades, there has been a 56% decrease in the number of antibiotics approved by the FDA (DeNap & Hergenrother, 2005).Resistance to antibiotics may arise through chromosomal mutations; however, this is a rare event occurring at a rate of 10-6 to 10-10 per organism. The acquisition of antibiotic resistance is therefore more likely to result from gaining plasmids harbouring resistance genes. Plasmids are commonly found in bacterial cells and are small, extrachromsomal pieces of DNA, able to replicate independently of the genome (Clewell, 2014). Plasmids often encode genes that are useful under atypical conditions, such as resistance to metal ions, antiseptic compounds and most significantly antibiotics (Clewell, 2014). The antibiotic resistance crisis is largely down to the ability of these plasmid-mediated antibiotic-resistance genes to transfer easily between cells of the same bacterial type as well as across different genus and species (DeNap & Hergenrother, 2005). For example, it is considered that methicillin-resistant Staphylococcus aureus (MRSA), the most common drug-resistant bacteria found in hospitals gained its resistance to vancomycin through plasmid transfer from vancomycin-resistant enterococci (VRE) (DeNap & Hergenrother, 2005). Therefore, the importance of plasmids in bacterial resistance to antibiotics makes them ideal targets to be exploited. Multiple strategies targeting plasmid maintenance and transfer have been proposed, as illustrated in Figure 1.Figure 1 – Overview of the possible strategies targeting plasmid maintenance and transfer to combat antibiotic resistance:A – Inhibit the plasmid transfer process of conjugation.B – Inhibit plasmid replication by targeting the plasmid incompatibility mechanism.C – Employ CRISPR-Cas to target and remove antibiotic-resistance genes present in plasmids. Inhibition of plasmid conjugationOne promising method for combatting antibiotic resistance is to target the plasmid conjugation process. All conjugative plasmids contain the relaxase, a protein responsible for cleavage of the origin of transfer (oriT) sequence, producing a transferrable DNA strand (Smillie, et al., 2010). A type IV coupling protein involved in the connection between the relaxosome and the transport channel aids the transfer of the relaxase protein bound to the DNA into the recipient cell (Smillie, et al., 2010). The relaxase protein is finally involved in recircularisation and replication of the plasmid DNA in the recipient cell (Ramsay, et al., 2016).The search for conjugation inhibitors involved a high-throughput conjugation assay based on the transfer of a plasmid containing the lux gene from a donor to recipient strain. The donor cells contained both the plasmids pSU2007 : : Tn lux and pUC18 : : lacIq, and therefore the lux operon was repressed, resulting in no luminescence (Fernandez-Lopez, et al., 2005). However following conjugation, pSU2007 : : Tn lux but not pUC18 : : lacIq was transferred, allowing expression of the lux gene and luminescence in the recipient cell (Fernandez-Lopez, et al., 2005). A total of 224 common chemicals were screened and those which successfully reduced light production by 95% or more were considered conjugation inhibitors. This resulted in the identification of two inhibitors, the fatty acids linoleic acid and dehydrocrepenynic acid (Fernandez-Lopez, et al., 2005). However, the targets of these fatty acids were not identified and inhibition of plasmid conjugation was only successful between those with similar DNA replication and transfer machinery. Nonetheless, this study was fundamental in demonstrating the possible application of small compounds for inhibiting plasmid conjugation and transfer of antibiotic-resistance genes.In a subsequent study, synthetic 2-alkynoic fatty acids with varying chain lengths as depicted in Figure 2, were instead considered as compounds for the inhibition of plasmid conjugation. Synthetic 2-hexadecynoic acid (2-HDA) was identified as an effective conjugation inhibitor, successfully preventing the spread of the highly infective IncF plasmid R1drd19 (Getino, et al., 2015). This study makes positive advancements from previous findings, since synthetic 2-alkynoic fatty acids are more stable and easier to obtain than natural unsaturated fatty acids, such as linoleic acid, which must be obtained from natural sources (Getino, et al., 2015).Figure 2 – Chemical structure of 2-alkynoic fatty acids.Despite this however, 2-HDA has been demonstrated as being toxic for fungi, protozoa, gram positive bacteria, some gram negative bacteria and eukaryotic cells (Getino, et al., 2016). Therefore, a further study identified more suitable conjugation inhibitors by screening bioactive compounds isolated from aquatic microorganisms. This resulted in the discovery of the partially purified natural compounds, Tanzawaic acids (TZAs) A and B (Getino, et al., 2016). The TZAs were successful in inhibiting the conjugation of the IncW plasmid R388 by nearly 100-fold at 0.4 mM concentration, as well as inhibiting other plasmids to a lesser extent (Getino, et al., 2016). TZAs may therefore be a more appropriate compound for inhibiting plasmid conjugation due to them having greatly reduced toxicity in comparison to synthetic compounds.In contrast to the studies discussed so far, research has been carried out to investigate the effect of intrabodies in inhibiting conjugative transfer. The relaxase enzyme plays a major role in plasmid conjugation as shown in Figure 3 and therefore, Garcillan-Barcia and colleagues set out to identify intrabodies using a fluorescence-based assay that target these relaxase enzymes. Intrabodies are engineered single-chain antibodies which can be targeted to intracellular antigens (Lo, et al., 2008). Both scFv-P4.E7 and scFv-P1.F2 were found to be conjugation inhibitors, successfully preventing transfer of 

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