Antimicrobial resistance in urinary pathogens and culture-independent detection of trimethoprim resistance in urine from patients with urinary tract infection.

BACKGROUND: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture. METHODS: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94). RESULTS: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC90 > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%). CONCLUSIONS: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing.

dfrA trimethoprim resistance genes found in Gram-negative bacteria: compilation and unambiguous numbering.

To track the spread of antibiotic resistance genes, accurate identification of individual genes is essential. Acquired trimethoprim resistance genes encoding trimethoprim-insensitive homologues of the sensitive dihydrofolate reductases encoded by the folA genes of bacteria are increasingly found in genome sequences. However, naming and numbering in publicly available records (journal publications or entries in the GenBank non-redundant DNA database) has not always been unambiguous. In addition, the nomenclature has evolved over time. Here, the changes in nomenclature and the most commonly encountered problems and pitfalls affecting dfrA gene identification arising from historically incorrect or inaccurate numbering are explained. The complete set of dfrA genes/DfrA proteins found in Gram-negative bacteria for which readily searchable sequence information is currently available has been compiled using less than 98% identity for both the gene and the derived protein sequence as the criteria for assignment of a new number. In most cases, trimethoprim resistance has been demonstrated. The gene context, predominantly in a gene cassette or near the ori end of CR1 or CR2, is also covered. The RefSeq database that underpins the programs used to automatically identify resistance genes in genome data sets has been curated to assign all sequences listed to the correct number. This led to the assignment of corrected or new gene numbers to several mis-assigned sequences. The unique numbers assigned for the dfrA/DfrA set are now listed in the RefSeq database, which we propose provides a way forward that should end future duplication of numbers and the confusion that causes.

Capture of a novel, antibiotic resistance encoding, mobile genetic element from Escherichia coli using a new entrapment vector.

AIMS: Antimicrobial resistance genes (ARGs) are often associated with mobile genetic elements (MGEs), which facilitate their movement within and between bacterial populations. Detection of mobility is therefore important to understand the dynamics of MGE dissemination and their associated genes, especially in resistant clinical isolates that often have multiple ARGs associated with MGEs. Therefore, this study aimed to develop an entrapment vector to capture active MGEs and ARGs in clinical isolates of Escherichia coli. METHODS AND RESULTS: We engineered an entrapment vector, called pBACpAK, to capture MGEs in clinical E. coli isolates. It contains a cI-tetA positive selection cartridge in which the cI gene encodes a repressor that inhibits the expression of tetA. Therefore, any disruption of cI, for example, by insertion of a MGE, will allow tetA to be expressed and result in a selectable tetracycline-resistant phenotype. The pBACpAK was introduced into clinical E. coli isolates and grown on tetracycline-containing agar to select for clones with the insertion of MGEs into the entrapment vector. Several insertion sequences were detected within pBACpAK, including IS26, IS903B and ISSbo1. A novel translocatable unit (TU), containing IS26 and dfrA8 was also captured, and dfrA8 was shown to confer trimethoprim resistance when it was cloned into E. coli DH5α. CONCLUSIONS: The entrapment vector, pBACpAK was developed and shown to be able to capture MGEs and their associated ARGs from clinical E. coli isolates. We have captured, for the first time, a TU encoding antibiotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that a TU and associated resistance gene has been captured from clinical E. coli isolates using an entrapment vector. The pBACpAK has the potential to be used not only as a tool to capture MGEs in clinical E. coli isolates, but also to study dynamics, frequency and potentiators of mobility for MGEs.

High-Order Epistasis in Catalytic Power of Dihydrofolate Reductase Gives Rise to a Rugged Fitness Landscape in the Presence of Trimethoprim Selection.

Evolutionary fitness landscapes of several antibiotic target proteins have been comprehensively mapped showing strong high-order epistasis between mutations, but understanding these effects at the biochemical and structural levels remained open. Here, we carried out an extensive experimental and computational study to quantitatively understand the evolutionary dynamics of Escherichia coli dihydrofolate reductase (DHFR) enzyme in the presence of trimethoprim-induced selection. To facilitate this, we developed a new in vitro assay for rapidly characterizing DHFR steady-state kinetics. Biochemical and structural characterization of resistance-conferring mutations targeting a total of ten residues spanning the substrate binding pocket of DHFR revealed distinct changes in the catalytic efficiencies of mutated DHFR enzymes. Next, we measured biochemical parameters (Km, Ki, and kcat) for a mutant library carrying all possible combinations of six resistance-conferring DHFR mutations and quantified epistatic interactions between them. We found that the high-order epistasis in catalytic power of DHFR (kcat and Km) creates a rugged fitness landscape under trimethoprim selection. Taken together, our data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, and suggest new strategies for developing mutant specific inhibitors.

Pathogenic Nocardia cyriacigeorgica and Nocardia nova Evolve To Resist Trimethoprim-Sulfamethoxazole by both Expected and Unexpected Pathways.

Nocardia spp. are Gram-positive opportunistic pathogens that affect largely immunocompromised patients, leading to serious pulmonary or systemic infections. Combination therapy using the folate biosynthesis pathway inhibitors trimethoprim (TMP) and sulfamethoxazole (SMX) is commonly used as an antimicrobial therapy. Not surprisingly, as antibiotic therapies for nocardiosis can extend for many months, resistance to TMP-SMX has emerged. Using experimental evolution, we surveyed the genetic basis of adaptation to TMP-SMX across 8 strains of Nocardia nova and 2 strains of Nocardia cyriacigeorgica By employing both continuous experimental evolution to provide longitudinal information on the order of changes and characterization of resistant endpoint isolates, we observe changes that are consistent with modifications of two enzymes of the folate biosynthesis pathway: dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) (FolP), with the mutations often being clustered near the active site of the enzymes. While changes to DHFR and DHPS might be expected, we also noted that mutations in a previously undescribed homolog of DHPS (DHPS2 or FolP2) that was annotated as being "nonfunctional" were also sufficient to generate TMP-SMX resistance, which serves as a cautionary tale for the use of automated annotation by investigators and for the future discovery of drugs against this genus. Additionally, folP2 overlapped glucosyl-3-phosphoglycerate synthase. Remarkably, an adaptive frameshift mutation within the overlapping region resulted in a new in-frame fusion to the downstream gene to produce a potentially new bifunctional enzyme. How a single potentially bifunctional DHPS2 enzyme might confer resistance is unclear. However, it highlights the unexpected ways in which adaptive evolution finds novel solutions for selection.

Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance.

Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a >2,000-fold increased trimethoprim resistance on overexpression in E. coli Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.

Prediction of antibiotic resistance from antibiotic resistance genes detected in antibiotic-resistant commensal Escherichia coli using PCR or WGS.

Objectives: To assess the effectiveness of bioinformatic detection of resistance genes in whole-genome sequences in correctly predicting resistance phenotypes. Methods: Genomes of a collection of well-characterized commensal Escherichia coli were sequenced using Illumina HiSeq technology and assembled with SPAdes. Antibiotic resistance genes identified by PCR, SRST2 analysis of reads and ResFinder analysis of SPAdes assemblies were compared with known resistance phenotypes. Results: Generally, the antibiotic resistance genes detected using bioinformatic methods were concordant, but only ARG-ANNOT included sat2 . However, the presence or absence of genes was not always predictive of the phenotype. In one strain, trimethoprim resistance was due to a known mutation in the chromosomal folA gene. In cases where the copy number was low, the aadA5 gene downstream of dfrA17 did not confer streptomycin or spectinomycin resistance. Resistance genes were found in the genomes that were not detected previously by PCRs targeting a limited gene set and gene cassettes in class 1 or class 2 integrons. In one isolate, the aadA1 gene cassette in the estX - aadA1 cassettes pair was outside an integron context and was not expressed. The qnrS1 gene, conferring reduced susceptibility to fluoroquinolones, and the bla CMY-2 gene, encoding an ESBL, were each detected in a single isolate and mphA (macrolide resistance) was present in six isolates surrounded by IS 26 and IS 6100 . Conclusions: WGS analysis detected more genes than PCR. Some were not expressed, causing inconsistencies with the experimentally determined phenotype. An unpredicted chromosomal folA mutation causing trimethoprim resistance was found.

Identification of the Molecular Mechanism of Trimethoprim Resistance in Listeria monocytogenes.

Trimethoprim with sulfamethoxazole is a therapeutic agent combination used to treat infections caused by the facultative intracellular foodborne pathogen Listeria monocytogenes. The aim of this study was to assess the frequency of resistance of L. monocytogenes arising due to exposure to trimethoprim and subsequently investigate the molecular mechanisms of resistance. After exposure of a culture of L. monocytogenes ATCC 13932 to trimethoprim at 10-fold the minimal inhibitory concentration spontaneous resistant mutants were recovered, giving a frequency of resistance development of 6.85 ± 0.92 × 10-8. The isolates exhibited a 32-64-fold decrease in susceptibility compared with the parental strain. These results indicate the capacity of L. monocytogenes to develop low-level resistance toward trimethoprim after exposure to the drug. The trimethoprim resistance genes (dhfr) and their promoter regions from all trimethoprim-resistant isolates were amplified and sequenced, leading to the identification of four single amino acid substitutions (Met20-Val, Pro21-Leu, Thr46-Asn, Val95-Leu) and two double substitutions (Met20-Ile+Thr46-Asn and Thr46-Asn+Leu85-Phe) in DHFR. Of the identified mutations, the Thr46-Asn substitution has not been previously reported as the mechanism of resistance to trimethoprim in other bacteria; thus this substitution seems to be unique to L. monocytogenes. The expression of the mutated L. monocytogenes dhfr genes in Escherichia coli led to decreased susceptibility of the heterological host, therefore proving that the identified point mutations in dhfr serve as the molecular mechanism of acquired resistance of L. monocytogenes to trimethoprim.

Factors that cause trimethoprim resistance in Streptococcus pyogenes.

The use of trimethoprim in treatment of Streptococcus pyogenes infections has long been discouraged because it has been widely believed that this pathogen is resistant to this antibiotic. To gain more insight into the extent and molecular basis of trimethoprim resistance in S. pyogenes, we tested isolates from India and Germany and sought the factors that conferred the resistance. Resistant isolates were identified in tests for trimethoprim or trimethoprim-sulfamethoxazole (SXT) susceptibility. Resistant isolates were screened for the known horizontally transferable trimethoprim-insensitive dihydrofolate reductase (dfr) genes dfrG, dfrF, dfrA, dfrD, and dfrK. The nucleotide sequence of the intrinsic dfr gene was determined for resistant isolates lacking the horizontally transferable genes. Based on tentative criteria, 69 out of 268 isolates (25.7%) from India were resistant to trimethoprim. Occurring in 42 of the 69 resistant isolates (60.9%), dfrF appeared more frequently than dfrG (23 isolates; 33.3%) in India. The dfrF gene was also present in a collection of SXT-resistant isolates from Germany, in which it was the only detected trimethoprim resistance factor. The dfrF gene caused resistance in 4 out of 5 trimethoprim-resistant isolates from the German collection. An amino acid substitution in the intrinsic dihydrofolate reductase known from trimethoprim-resistant Streptococcus pneumoniae conferred resistance to S. pyogenes isolates of emm type 102.2, which lacked other aforementioned dfr genes. Trimethoprim may be more useful in treatment of S. pyogenes infections than previously thought. However, the factors described herein may lead to the rapid development and spread of resistance of S. pyogenes to this antibiotic agent.

Within-population distribution of trimethoprim resistance in Escherichia coli before and after a community-wide intervention on trimethoprim use.

A 2-year prospective intervention on the prescription of trimethoprim reduced the use by 85% in a health care region with 178,000 inhabitants. Here, we performed before-and-after analyses of the within-population distribution of trimethoprim resistance in Escherichia coli. Phylogenetic and population genetic methods were applied to multilocus sequence typing data of 548 consecutively collected E. coli isolates from clinical urinary specimens. Results were analyzed in relation to antibiotic susceptibility and the presence and genomic location of different trimethoprim resistance gene classes. A total of 163 E. coli sequence types (STs) were identified, of which 68 were previously undescribed. The isolates fell into one of three distinct genetic clusters designated BAPS 1 (E. coli phylogroup B2), BAPS 2 (phylogroup A and B1), and BAPS 3 (phylogroup D), each with a similar frequency before and after the intervention. BAPS 2 and BAPS 3 were positively and BAPS 1 was negatively associated with trimethoprim resistance (odds ratios of 1.97, 3.17, and 0.26, respectively). In before-and-after analyses, trimethoprim resistance frequency increased in BAPS 1 and decreased in BAPS 2. Resistance to antibiotics other than trimethoprim increased in BAPS 2. Analysis of the genomic location of different trimethoprim resistance genes in isolates of ST69, ST58, and ST73 identified multiple independent acquisition events in isolates of the same ST. The results show that despite a stable overall resistance frequency in E. coli before and after the intervention, marked within-population changes occurred. A decrease of resistance in one major genetic cluster was masked by a reciprocal increase in another major cluster.

Crystal structures of Klebsiella pneumoniae dihydrofolate reductase bound to propargyl-linked antifolates reveal features for potency and selectivity.

Resistance to the antibacterial antifolate trimethoprim (TMP) is increasing in members of the family Enterobacteriaceae, driving the design of next-generation antifolates effective against these Gram-negative pathogens. The propargyl-linked antifolates are potent inhibitors of dihydrofolate reductases (DHFR) from several TMP-sensitive and -resistant species, including Klebsiella pneumoniae. Recently, we have determined that these antifolates inhibit the growth of strains of K. pneumoniae, some with MIC values of 1 µg/ml. In order to further the design of potent and selective antifolates against members of the Enterobacteriaceae, we determined the first crystal structures of K. pneumoniae DHFR bound to two of the propargyl-linked antifolates. These structures highlight that interactions with Leu 28, Ile 50, Ile 94, and Leu 54 are necessary for potency; comparison with structures of human DHFR bound to the same inhibitors reveal differences in residues (N64E, P61G, F31L, and V115I) and loop conformations (residues 49 to 53) that may be exploited for selectivity.

Complete sequence of a conjugative incn plasmid harboring blaKPC-2, blaSHV-12, and qnrS1 from an Escherichia coli sequence type 648 strain.

We sequenced a novel conjugative blaKPC-2-harboring IncN plasmid, pYD626E, from an Escherichia coli sequence type 648 strain previously identified in Pittsburgh, Pennsylvania. pYD626E was 72,800 bp long and carried four ß-lactamase genes, blaKPC-2, blaSHV-12, blaLAP-1, and blaTEM-1. In addition, it harbored qnrS1 (fluoroquinolone resistance) and dfrA14 (trimethoprim resistance). The plasmid profile and clinical history supported the in vivo transfer of this plasmid between Klebsiella pneumoniae and Escherichia coli.

Holistic understanding of trimethoprim resistance in Streptococcus pneumoniae using an integrative approach of genome-wide association study, resistance reconstruction, and machine learning.

Antimicrobial resistance (AMR) is a public health threat worldwide. Next-generation sequencing (NGS) has opened unprecedented opportunities to accelerate AMR mechanism discovery and diagnostics. Here, we present an integrative approach to investigate trimethoprim (TMP) resistance in the key pathogen Streptococcus pneumoniae. We explored a collection of 662 S. pneumoniae genomes by conducting a genome-wide association study (GWAS), followed by functional validation using resistance reconstruction experiments, combined with machine learning (ML) approaches to predict TMP minimum inhibitory concentration (MIC). Our study showed that multiple additive mutations in the folA and sulA loci are responsible for TMP non-susceptibility in S. pneumoniae and can be used as key features to build ML models for digital MIC prediction, reaching an average accuracy within ±1 twofold dilution factor of 86.3%. Our roadmap of in silico analysis-wet-lab validation-diagnostic tool building could be adapted to explore AMR in other combinations of bacteria-antibiotic. IMPORTANCE: In the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on S. pneumoniae clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic folA mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the sulA locus produces trimethoprim (TMP) resistance in Streptococcus pneumoniae. These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility.

Trimethoprim resistance of dihydrofolate reductase variants from clinical isolates of Pneumocystis jirovecii.

Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

Novel erm(T)-carrying multiresistance plasmids from porcine and human isolates of methicillin-resistant Staphylococcus aureus ST398 that also harbor cadmium and copper resistance determinants.

This study describes three novel erm(T)-carrying multiresistance plasmids that also harbor cadmium and copper resistance determinants. The plasmids, designated pUR1902, pUR2940, and pUR2941, were obtained from porcine and human methicillin-resistant Staphylococcus aureus (MRSA) of the clonal lineage ST398. In addition to the macrolide-lincosamide-streptogramin B (MLSB) resistance gene erm(T), all three plasmids also carry the tetracycline resistance gene tet(L). Furthermore, plasmid pUR2940 harbors the trimethoprim resistance gene dfrK and the MLSB resistance gene erm(C), while plasmids pUR1902 and pUR2941 possess the kanamycin/neomycin resistance gene aadD. Sequence analysis of approximately 18.1 kb of the erm(T)-flanking region from pUR1902, 20.0 kb from pUR2940, and 20.8 kb from pUR2941 revealed the presence of several copies of the recently described insertion sequence ISSau10, which is probably involved in the evolution of the respective plasmids. All plasmids carried a functional cadmium resistance operon with the genes cadD and cadX, in addition to the multicopper oxidase gene mco and the ATPase copper transport gene copA, which are involved in copper resistance. The comparative analysis of S. aureus RN4220 and the three S. aureus RN4220 transformants carrying plasmid pUR1902, pUR2940, or pUR2941 revealed an 8-fold increase in CdSO4 and a 2-fold increase in CuSO4 MICs. The emergence of multidrug resistance plasmids that also carry heavy metal resistance genes is alarming and requires further surveillance. The colocalization of antimicrobial resistance genes and genes that confer resistance to heavy metals may facilitate their persistence, coselection, and dissemination.

The BpeEF-OprC efflux pump is responsible for widespread trimethoprim resistance in clinical and environmental Burkholderia pseudomallei isolates.

Trimethoprim-sulfamethoxazole (co-trimoxazole) is the primary drug used for oral eradication therapy of Burkholderia pseudomallei infections (melioidosis). Here, we demonstrate that trimethoprim resistance is widespread in clinical and environmental isolates from northeast Thailand and northern Australia. This resistance was shown to be due to BpeEF-OprC efflux pump expression. No dihydrofolate reductase target mutations were involved, although frequent insertion of ISBma2 was noted within the putative folA transcriptional terminator. All isolates tested remained susceptible to trimethoprim-sulfamethoxazole, suggesting that resistance to trimethoprim alone in these strains probably does not affect the efficacy of co-trimoxazole therapy.

Construction of mobilizable mini-Tn7 vectors for bioluminescent detection of gram-negative bacteria and single-copy promoter lux reporter analysis.

We describe the construction of mini-Tn7-based broad-host-range vectors encoding lux genes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn7-lux integration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.

Trimethoprim resistance in surface and wastewater is mediated by contrasting variants of the dfrB gene.

Trimethoprim (TMP) is a low-cost, widely prescribed antibiotic. Its effectiveness is increasingly challenged by the spread of genes coding for TMP-resistant dihydrofolate reductases: dfrA, and the lesser-known, evolutionarily unrelated dfrB. Despite recent reports of novel variants conferring high level TMP resistance (dfrB10 to dfrB21), the prevalence of dfrB is still unknown due to underreporting, heterogeneity of the analyzed genetic material in terms of isolation sources, and limited bioinformatic processing. In this study, we explored a coherent set of shotgun metagenomic sequences to quantitatively estimate the abundance of dfrB gene variants in aquatic environments. Specifically, we scanned sequences originating from influents and effluents of municipal sewage treatment plants as well as river-borne microbiomes. Our analyses reveal an increased prevalence of dfrB1, dfrB2, dfrB3, dfrB4, dfrB5, and dfrB7 in wastewater microbiomes as compared to freshwater. These gene variants were frequently found in genomic neighborship with other resistance genes, transposable elements, and integrons, indicating their mobility. By contrast, the relative abundances of the more recently discovered variants dfrB9, dfrB10, and dfrB13 were significantly higher in freshwater than in wastewater microbiomes. Moreover, their direct neighborship with other resistance genes or markers of mobile genetic elements was significantly less likely. Our findings suggest that natural freshwater communities form a major reservoir of the recently discovered dfrB gene variants. Their proliferation and mobilization in response to the exposure of freshwater communities to selective TMP concentrations may promote the prevalence of high-level TMP resistance and thus limit the future effectiveness of antimicrobial therapies.

Prospective screening of novel antibacterial inhibitors of dihydrofolate reductase for mutational resistance.

Resistance to trimethoprim (TMP) resulting from point mutations in the enzyme drug target dihydrofolate reductase (DHFR) drives the development of new antifolate inhibitors effective against methicillin-resistant Staphylococcus aureus (MRSA). For the past several years we have used structure-based design to create propargyl-linked antifolates that are highly potent antibacterial agents. In order to focus priority on the development of lead compounds with a low propensity to induce resistance, we prospectively evaluated resistance profiles for two of these inhibitors in an MRSA strain. By selection with the lead inhibitors, we generated resistant strains that contain single point mutations F98Y and H30N associated with TMP resistance and one novel mutation, F98I, in DHFR. Encouragingly, the pyridyl propargyl-linked inhibitor selects mutants at low frequency (6.85 × 10(-10) to 1.65 × 10(-9)) and maintains a low MIC (2.5 µg/ml) and a low mutant prevention concentration (1.25 µg/ml), strongly supporting its position as a lead compound. Results from this prospective screening method inform the continued design of antifolates effective against mutations at the Phe 98 position. Furthermore, the method can be used broadly to incorporate ideas for overcoming resistance early in the development process.