Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples

Multi-Resistance Plasmids : Fitness Costs, Dynamics and Evolution

Antibiotic resistance is an escalating problem, not only due to less desirable treatment options and outcome, but also due to the economic burden to health care caused by resistant pathogens. Since the process of developing new antibiotics is slow, we need to carefully consider the usage of the antibiotics still available. Therefore it is of importance to minimize the development and spread of resistant pathogens. To do so, we need a better understanding of the mechanisms and dynamics underlying the evolution of highly resistant bacteria. In this thesis I have investigated one of the major drivers of resistance gene dissemination in Gram-negative bacteria, namely multi-resistance plasmids. We show that multi-resistance plasmids display a dynamic behavior in vivo, where genes can be readily acquired and lost again. Additionally, plasmids can be shared amongst different bacteria, especially in environments such as the human gut. Interestingly, some resistance plasmids confer a fitness disadvantage to their host displayed by decreased growth rate in absence of antibiotics. We could elucidate that two resistance genes of the multi-resistance plasmid pUUH239.2 were the cause of the lowered growth rate, namely blaCTX-M-15 and tetR/A. In contrast, other resistance genes on the plasmid were cost-free even when overexpressed and likely enable persistence in the bacterial population even under non-selective conditions. Lastly, we studied how the presence of several β-lactamase genes on a plasmid affects treatment with different combinations of β-lactam/β-lactamase inhibitors. We found that an efficient mechanism for bacteria to overcome high levels of antibiotics was by amplification of plasmid-borne resistance genes. This mechanism works as a stepping-stone for additional mutations giving rise to high-level resistance. With this work we provide insight into the mechanisms underlying resistance evolution and dissemination due to multi-resistance plasmids. Plasmids enable fast dissemination of multiple resistance genes and therefore simultaneously disable multiple treatment options. Examining the effects of resistance genes and antibiotics on strains carrying multi-resistance plasmids will enable us to understand what factors assist or inhibit plasmid spread. Hopefully, this will aid us in treatment design to prevent resistance development to effective antibiotics and have implications for resistance surveillance as well as prediction

Multi-Resistance Plasmids : Fitness Costs, Dynamics and Evolution

Antibiotic resistance is an escalating problem, not only due to less desirable treatment options and outcome, but also due to the economic burden to health care caused by resistant pathogens. Since the process of developing new antibiotics is slow, we need to carefully consider the usage of the antibiotics still available. Therefore it is of importance to minimize the development and spread of resistant pathogens. To do so, we need a better understanding of the mechanisms and dynamics underlying the evolution of highly resistant bacteria. In this thesis I have investigated one of the major drivers of resistance gene dissemination in Gram-negative bacteria, namely multi-resistance plasmids. We show that multi-resistance plasmids display a dynamic behavior in vivo, where genes can be readily acquired and lost again. Additionally, plasmids can be shared amongst different bacteria, especially in environments such as the human gut. Interestingly, some resistance plasmids confer a fitness disadvantage to their host displayed by decreased growth rate in absence of antibiotics. We could elucidate that two resistance genes of the multi-resistance plasmid pUUH239.2 were the cause of the lowered growth rate, namely blaCTX-M-15 and tetR/A. In contrast, other resistance genes on the plasmid were cost-free even when overexpressed and likely enable persistence in the bacterial population even under non-selective conditions. Lastly, we studied how the presence of several β-lactamase genes on a plasmid affects treatment with different combinations of β-lactam/β-lactamase inhibitors. We found that an efficient mechanism for bacteria to overcome high levels of antibiotics was by amplification of plasmid-borne resistance genes. This mechanism works as a stepping-stone for additional mutations giving rise to high-level resistance. With this work we provide insight into the mechanisms underlying resistance evolution and dissemination due to multi-resistance plasmids. Plasmids enable fast dissemination of multiple resistance genes and therefore simultaneously disable multiple treatment options. Examining the effects of resistance genes and antibiotics on strains carrying multi-resistance plasmids will enable us to understand what factors assist or inhibit plasmid spread. Hopefully, this will aid us in treatment design to prevent resistance development to effective antibiotics and have implications for resistance surveillance as well as prediction

The Role of Antibiotic Resistance Genes in the Fitness Cost of Multiresistance Plasmids

By providing the bacterial cell with protection against several antibiotics at once, multiresistance plasmids have an evolutionary advantage in situations where antibiotic treatments are common, such as in hospital environments. However, resistance plasmids can also impose fitness costs on the bacterium in the absence of antibiotics, something that may limit their evolutionary success. The underlying mechanisms and the possible contribution of resistance genes to such costs are still largely not understood. Here, we have specifically investigated the contribution of plasmid-borne resistance genes to the reduced fitness of the bacterial cell. The pUUH239.2 plasmid carries 13 genes linked to antibiotic resistance and reduces bacterial fitness by 2.9% per generation. This cost is fully ameliorated by the removal of the resistance cassette. While most of the plasmid-borne resistance genes individually were cost-free, even when overexpressed, two specific gene clusters were responsible for the entire cost of the plasmid: the extended-spectrum-beta-lactamase gene bla(CTX-M-15 )and the tetracycline resistance determinants tetAR. The bla(CTX-M-15 ) cost was linked to the signal peptide that exports the beta-lactamase into the periplasm, and replacement with an alternative signal peptide abolished the cost. Both the tetracycline pump TetA and its repressor TetR conferred a cost on the host cell, and the reciprocal expression of these genes is likely fine-tuned to balance the respective costs. These findings highlight that the cost of clinical multiresistance plasmids can be largely due to particular resistance genes and their interaction with other cellular systems, while other resistance genes and the plasmid backbone can be cost-free. IMPORTANCE Multiresistance plasmids are one of the main drivers of antibiotic resistance development and spread. Their evolutionary success through the accumulation and mobilization of resistance genes is central to resistance evolution. In this study, we find that the cost of the introduction of a multiresistance plasmid was completely attributable to resistance genes, while the rest of the plasmid backbone is cost-free. The majority of resistance genes on the plasmid had no appreciable cost to the host cell even when overexpressed, indicating that plasmid-borne resistance can be cost-free. In contrast, the widespread genes bla(CTX-M-15 ) and tetAR were found to confer the whole cost of the plasmid by affecting specific cellular functions. These findings highlight how the evolution of resistance on plasmids is dependent on the amelioration of associated fitness costs and point at a conundrum regarding the high cost of some of the most widespread beta-lactamase genes

De Novo Emergence of Peptides That Confer Antibiotic Resistance

The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from non-coding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes. IMPORTANCE De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes