A Novel Trimethoprim Resistance Gene, dfrA35, Characterized from Escherichia coli from Calves

The presence of dfrA35 associated with ISCR2 in Escherichia coli from animals, as well as its presence in other E. coli strains from different sources and countries and in Acinetobacter, highlights the global spread of this gene and its potential for further dissemination. The genetic link of ISCR2-dfrA35 with other antibiotic and disinfectant resistance genes showed that multidrug-resistant E. coli may be selected and maintained by the use of either one of several antimicrobials.

sensitive target enzyme by upregulation of gene expression or gene duplication, and (v) the acquisition of alternative DHPS (sul) and DHFR (dfr) genes with integrons, plasmids, and transposons (6,7). To date, three different alternative sul genes (sul1, sul2, and sul3) and 40 different types of alternative dfr genes have been described in Gram-positive and Gram-negative bacteria (8)(9)(10). However, the trimethoprim resistance mechanism remained unknown for 8 of 56 trimethoprim-resistant E. coli strains isolated from rectal swabs of healthy veal calves in 2017 in Switzerland (11). We selected two genetically diverse E. coli strains (MF2156 and PF9285) from two different farms based on rep-PCR profile to further investigate the nature of the trimethoprim resistance in these strains. The whole-genome sequences of both strains were screened for DHFR homologs followed by proof of functionality.
Identification and localization of a new DHFR. Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Inc., Venlo, The Netherlands), and purified using the AMPure XP PCR purification system (Beckman Coulter Life Sciences, Indianapolis, IN). DNA was sequenced on an Illumina HiSeq platform (2 ϫ 150 paired ends) (Eurofins, Constance, Germany) and on a R9.4 SpotON flow cell and library kit (Oxford Nanopore Technologies, Oxford, United Kingdom) to obtain long reads and facilitate genome assembly. Genome assembly and read mapping of the Illumina reads against the MinION scaffolds were performed as previously described (12). Analyses of the complete genome using RESFinder 3.1 (Center for Genomic Epidemiology, DTU, Denmark) (20% coverage, 30% identity) confirmed the absence of any known acquired dfr gene in both strains MF2156 and PF9285. Comparison of the chromosomal folA gene with that of the trimethoprim-susceptible E. coli strain K-12 MG1655 (GenBank accession no. NC_000913) showed no mutation in this gene in both strains, suggesting the presence of an alternative mechanism. Search for a possible new acquired dfr gene within the complete genomes of MF2156 and PF9285 using blastx (https://blast.ncbi .nlm.nih.gov/) and DfrA1 (NCBI accession no. CAA25445) as the reference revealed the presence of a 177-amino-acid DHFR homologue (513 bp) in both strains. This putative new DHFR shared the closest amino acid identity (49.4%) with DfrA20 from Pasteurella multocida (GenBank accession no. CAE53424) (13) and was next closely related to Dfr proteins DfrD, DfrG, and DfrK, which have been identified so far only in Gram-positive bacteria (Fig. 1). The new gene was named dfrA36 following the nomenclature used for Gram-negative bacteria (9).
The dfrA36 gene was located on a 22,977-bp fragment integrated in the same location of the chromosome in both E. coli strains MF2156 and PF9285. Both ends of the integrated element were identified by comparative analysis of sequences of strains MF2156 and PF9285 with those from E. coli strain PSU078, which shared an identical flanking region (GenBank accession no. CP012112). The fragment was delimited by a truncated ISCR2 (ΔISCR2) interrupting a putative restriction endonuclease subunit S gene on one side and by a sul2 gene interrupting a putative Tat pathway signal sequence protein gene on the other side. The fragment contains an ISCR2 element carrying the dfrA36 gene, the florfenicol/chloramphenicol export gene floR, and the sulfonamide resistance gene sul2 (floR-ISCR2-dfrA36-sul2) and a 14,231-bp Tn21-like element (Fig. 2). Given the comparison to PSUO78, it is likely that the dfrA36 region and the Tn21-like element were mobilized into the chromosome either simultaneously or separately by homologous recombination. The dfrA36 gene was integrated with a gene of the Rrf2 transcriptional regulator family into the phosphoglucosamine mutase gene glm of a floR-ISCR2-sul2 element, which has been previously reported in Stenotrophomonas maltophilia (14,15). Integration of the dfrA36-rrf2 fragment split the glm gene into two pieces, generating a duplication of the ACGT integration sequence (Fig. 2). The dfrA36-rrf2 has likely been trapped by the gene-capturing machinery described for ISCR elements and mobilized by rolling circle transposition, but dfrA36 does not seem to be a single cassette as many other dfr genes are (14,16,17). The Tn21-like element contained the characteristic features defining the Tn21 subgroup with a transposase gene, tnpA, a resolvase gene, tnpR, in the same orientation, the res sites preceding tnpR, New dfrA36 Gene in E. coli and two 38-bp inverted repeats at both ends (18,19). It also contained a class I integron In290 (INTEGRALL database), including the integrase gene intI1, the sulfonamide resistance gene sul1, the streptomycin/spectinomycin resistance gene aadA1 [ant(3Љ)-Ia], the gentamicin/tobramycin/kanamycin resistance gene aadB [ant(2Љ)-Ia], the quaternary ammonium compound (QAC) efflux transporter gene qacEΔ1, as well as the transposase genes of IS1326 and of a new transposase gene related to IS3. Duplication of ISCR2 sequences suggests that insertion and movement of the different pieces of the element may have also occurred by homologous recombination (Fig. 2).
As expected by its chromosomal location and genetic context, transferability of the dfrA36 gene could not be observed experimentally by filter mating using strains MF1256 and PF9285 as donors and the rifampin-, sodium azide-resistant strain E. coli J53dR as the recipient (20). Selection was performed on Mueller-Hinton (MH) agar plates containing trimethoprim (30 g/ml) with either sodium azide (100 g/ml) or rifampin (50 g/ml) for 48 h at 37°C.
The ligated vector insert DNA was transformed into chemically competent cells of E. coli One Shot TOP10 (Thermo Fisher Scientific). Transformants obtained on LB plates containing 50 g/ml kanamycin after 24 h of incubation at 37°C were tested for the presence of the dfrA36 gene by PCR using Taq polymerase and internal primers dfrA36int-F (5=-GCATTTACCGGCCGATATGC-3=) and dfrA36int-R (5=-ACACGCAGCACCTC TTCATT-3=) (annealing temperature, 56°C; extension time, 30 s). The resulting dfrA36containing plasmids, pT2156c19 and pT9285c17, were isolated using the PureLink Quick plasmid miniprep kit (Thermo Fisher Scientific) and analyzed by Sanger sequencing to confirm the veracity of the sequence and that dfrA36 was inserted in the opposite direction of the TOPO promoter P lac and was therefore under the control of its own promoter. The MIC of trimethoprim for the parent strains MF2156 and PF9285, recipient strain TOP10, and transformant strains TOP10/pT2156c19 and TOP10/pT9285c17 was determined on a microtiter plate using the 2-fold-dilution technique (concentration range, 1 to 512 g/ml) following CLSI guidelines (21). The MIC for trimethoprim increased in E. coli TOP10 expressing dfrA36 to 128 g/ml compared to the nontransformed TOP10 strain (Յ0.25 g/ml), almost reaching the level of the MIC observed in the parent strains MF2156 and PF9285 (256 g/ml). The MIC of 13 other antibiotics was determined using EUVSEC Sensititre plates (Thermo Fisher Scientific), showing an association between the other antibiotic resistance genes found on strains MF2156 and PF9285 and their phenotype ( Table 1).
Spread of dfrA36 in association with ISCR2 and sul2. PCR screening of 8 additional strains from calves with no known trimethoprim resistance gene revealed dfrA36 in 5 of them. The gene was also located in these 5 strains on an ISCR2-dfrA36-sul2 fragment, as determined by Taq PCR using primers ISCR2-F (5=-CGCCTGCATTGAAGAC CCTA-3=) and Sul2-F (5=-TGTCTGTTTCGCGCAAATCC-3=) (annealing temperature, 56°C; extension time, 3 min). Searching for DfrA36 in the NCBI database using blastp revealed its presence in 26 other E. coli strains as well as in an Acinetobacter sp. strain originating from different sources (cattle, dogs, a horse, and humans) and countries, which indicates that the dfrA36 gene has potential for dissemination among bacterial species even if transfer could not be demonstrated experimentally. In 18 of them, the available sequences allowed us to determine that dfrA36 was also linked to sul2, and 6 sequences revealed the presence of the ISCR2-dfrA36-sul2 element (see Table S1 in the supplemental material).
This study identified a novel functional trimethoprim resistance gene (dfrA36) in E. coli from calves. This gene appeared to be widespread in other E. coli strains as well as in Acinetobacter, where it was also mainly directly flanked by ISCR2 and/or sul2. In the two analyzed E. coli strains from cattle, the ISCR2-dfrA36-sul2 sequence was also linked to floR and was part of a larger multiple antibiotic and QAC resistance element. The dissemination of this element may further jeopardize the efficacy of antibiotics and disinfectants in both veterinary and human medicine.
Accession number(s). The complete chromosome of E. coli strain PF9285 containing the 22,977-bp fragment carrying dfrA36 and flanking regions (positions 989354 to 1012331) has been deposited in GenBank under accession no. CP038791 (BioProject no. PRJNA530748).