Molecular basis for trimethoprim and sulphonamide resistance in Gram negative pathogens

Resistance to antibiotics is becoming an important global public health problem. Infectious diseases are still among the major causes of disease and death in many parts of the world and the possibilities to treat bacterial infections are now threatened due to the spread of antibiotic resistance. Trimethoprim and sulphonamides are widely accessible and affordable antibiotics which act by inhibiting the folic acid synthesis in bacteria. Trimethoprim and its combination with the sulphonamide sulphamethoxazole, are considered first- and second-line treatment for many infections, such as uncomplicated and severe urinary tract infections.

Gram negative bacteria usually become resistant to these drugs by acquiring genes encoding additional enzymes which are not inhibited by the drugs. Such enzymes are encoded by dfr- and sul-genes which can efficiently be spread among bacteria. The majority of the dfr-genes are carried as gene cassettes in a genetic element called integron. Integrons can insert and express gene cassettes conferring resistance to various different antibiotics and are thus important tools for spread of resistance in pathogenic bacteria.

In the present studies, a material of 105 Gram negative bacterial strains from urinary samples has been thoroughly investigated. Additional studies have also been performed on other selected materials of E. coli bacteria. The presence of integrons, dfr-genes and sulgenes has been mapped. A subset of detected integrons has been sequenced to explore the carriage of antibiotic resistance gene cassettes in these elements. In strains where no known integron, dfr- or sul-gene could be detected, further studies by means of shotgun cloning experiments were performed to understand the underlying mechanisms for resistance and the spread of such determinants. Two multiplex PCR screening methods for the five most frequently occurring dfr-genes have also been developed and tested in these studies.

Gene cassettes in the integrons studied in these strains only contained genes encoding resistance to trimethoprim and two rarely used antibiotics of the aminoglycoside type. The acquisition of other antibiotic resistance determinants must hence be explained by additional genetic dissemination mechanisms in these isolates. The new su13 gene was in the present studies for the first time described in pathogens from humans. The gene was also shown to be present in several isolates from healthy pigs and a spread of this gene from animals to humans was suggested. As many as four new dfr-genes, dfr2d, dfrA22, dfrA24 and dfrA26, were described in the investigated material. The first two of these are carried as gene cassettes in integrons while the third is spread by an unknown mechanism and the fourth was shown to be connected to the recently described elements called the common regions.

Common regions have been found to capture and spread a large number of various antibiotic resistance genes unrelated to gene cassettes. As confirmed by the description of dfrA26 in these studies, almost all dfr-genes now fall into the two categories as disseminated either by integrons or by common regions. The importance of the association of resistance to trimethoprim and probably also sulphonamides with common regions can be expected to increase in the future.

List of scientific papers

I. Grape M, Sundstrom L, Kronvall G (2003). Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources. J Antimicrob Chemother. 52(6): 1022-4.
https://pubmed.ncbi.nlm.nih.gov/14585855

II. Grape M, Sundstrom L, Kronvall G (2003). New dfr2 gene as a single-gene cassette in a class 1 integron from a trimethoprim-resistant Escherichia coli isolate. Microb Drug Resist. 9(4): 317-22.
https://pubmed.ncbi.nlm.nih.gov/15000737

III. Grape M, Farra A, Kronvall G, Sundstrom L (2005). Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram-negative bacteria. Clin Microbiol Infect. 11(3): 185-92.
https://pubmed.ncbi.nlm.nih.gov/15715715

IV. Grape M, Sundström L, Kronvall G (2006). Identification of new dfr-genes in trimethoprim-resistant integron-negative Gram negative pathogens. [Manuscript]

V. Grape M, Motakefi A, Pavuluri S, Kahlmeter G (2006). Standard and real-time multiplex PCR methods for detection of trimethoprim resistance dfr-genes in large collections of bacteria. [Manuscript]