A fluoroquinolone resistance associated mutation in gyrA Affects DNA supercoiling in Campylobacter jejuni

Abstract

The prevalence of fluoroquinolone (FQ)-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant (FQ(R)) Campylobacter, it is necessary to understand the impact of FQ(R) on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQ(R)Campylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQ(R)Campylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type (WT) allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant WT gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn), which are associated with FQ(R) in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the WT gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQ(R) mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the WT strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQ(R)Campylobacter, modulates DNA supercoiling homeostasis in FQ(R)Campylobacter.

Keywords: Campylobacter; DNA supercoiling; GyrA mutation; fitness; fluoroquinolone resistance.

Figure 1

Pairwise competitions between FQ^S and FQ^R strains in chickens using isogenic pairs of 62301R33S/62301R33R (A,B) and 11168(S)/11168(R) (C). (A) The colonization level of total Campylobacter in three groups of chickens that were inoculated with 62301R33S (solid circle), 62301R33R (open circle), or 1:1 mixture of the two stains. (B) Differential enumeration of FQ^S (solid circle) and FQ^R (open circle) Campylobacter in the group inoculated with a mixture of 62301R33S and 62301R33R. (C) A second chicken experiment shows the competition between FQ^S (solid triangle) and FQ^R (open triangle) Campylobacter in a different strain background. The chickens were inoculated with a 1:1 mixture of 11168(S) and 11168(R). In all panels, each symbol represents the colonization level of a single chicken and the median level of each group is indicated by a horizontal bar. The detection limit of the plating method was about 100 CFU/g of feces and a negative sample was arbitrarily assigned a value of zero. DAI, days after inoculation.

Figure 2

Supercoiling activities of the recombinant WT gyrase and the three recombinant mutant gyrases measured by an in vitro assay. Lane 1, DNA ladder; Lane 2, relaxed pBR322 (control); Lane 3, WT GyrA + GyrB; Lane 4, mutant GyrA (Thr-86-Ile) + GryB; Lane 5, mutant GyrA (Thr-86-Lys) + GryB; Lane 6, mutant GyrA (Asp-90-Asn) + GryB; and Lane 7, supercoiled pBR322 (control). SC indicates supercoiled DNA and R represents relaxed DNA.

Figure 3

Agarose gel electrophoresis analysis of plasmid topoisomers extracted from different strain background. The plasmid DNA was run on a 1.0% agarose gel containing 20 μg/ml chloroquine. (A) Plasmid pRY107 isolated from strain S3B derivatives. Lane 1, pRY107 from 62301S; Lane 2, pRY107 from 62301R (carrying the Thr-86-Ile mutation in GyrA); Lane 3, pRY107 from 52901-II2 (carrying the Thr-86-Lys mutation in GyrA); and Lane 4, pRY107 from 62301R37 (carrying the Asp-90-Asn mutation in GyrA). (B) Plasmid pRY107 isolated from strain 11168 and its derivatives. Lane 1, pRY107 from NCTC 11168; and Lane 2, pRY107 from 11168CT (carrying the Thr-86-Ile mutation in GyrA). The results of densitometric scanning are shown to the right of each gel image. The numbers on the left of the densitometric scanning correspond to the lane numbers of the gel image. The number below each lane indicates the percentage of the most supercoiled DNA in the total population of plasmid topoisomers as measured by densitometry.

Figure 4

Agarose gel electrophoresis showing the inhibition of DNA gyrase supercoiling activities by ciprofloxacin. In each reaction, the relaxed pBR322 was incubated with different enzymes in the presence of various concentration of ciprofloxacin. (A) Reactions with recombinant GyrA from the WT strain and GyrB; (B) reactions with mutant GyrA (Thr-86-Lys) and GyrB; (C) reactions with mutant GyrA (Asp-90-Asn) and GyrB; and (D) reactions with mutant GyrA (Thr-86-Ile) + GyrB. The numbers on top of each panel are the concentrations of ciprofloxacin (μg/ml) used in the reaction. SC indicates supercoiled DNA and R represents relaxed DNA (TopoGen). In each lane, the most supercoiled band migrates faster than the other topoisomers.