Trimethoprim resistance in normal flora from India

Abstract

Resistance to antibiotics is an increasing problem in the developing world. Resistance genes in the normal gut flora may act as a reservoir from which pathogens may acquire resistance. Trimethoprim is an important drug in the treatment of a wide range of infections but resistance has increased in recent years particularly in the developing world.A survey was conducted in Vellore, south India, to determine the rate of carriage of antibiotic resistant enterobacteria in the normal gut flora of a sample of the population. Very high rates of resistance were encountered to the widely used antimicrobials, ampicillin, chloramphenicol and trimethoprim. The trimethoprim resistant strains were analyzed further, 35% were capable of plasmid-mediated transfer of trimethoprim resistance by different plasmid types, as identified by restriction endonuclease digestion. Resistance to a variety of other agents was cotransferred. Transferable trimethoprim resistance was shown, by DNA hybridization, to result from the presence of three different drug resistant dihydrofolate reductase (dhfr) genes. The dhfrV was present in 50% of transconjugants, associated with either the transposon Tn21 or only with the integrase-like open reading frame (orf) of this transposon; 31% possessed the dhfrl, associated with the integrase orf of Tn7 and 19% had dhfrlVThe dihydrofolate reductase (DHFR) enzyme type IV has only ever been identified in this area of India, in 1984. The enzyme mediates only low level resistance, as measured by conventional tests, but it is inducible, a unique property in the DHFRs. It has persisted in this area despite a seemingly poor resistance mechanism. The DHFR typelV enzymes characterized during this survey were also inducible. It was demonstrated that the induction mechanism will produce over 100-fold greater resistance if the cells are challenged prior to determination of resistance level. This mechanism was shown to be mediated by a lack of thymine and to be dependant on cell phase and density. A plasmid characterized in this study also mediated resistance to ampicillin as a result of the presence of the TEM-1 ß-lactamase. This enzyme was also inducible, a property not observed before for this enzyme or for any other plasmid mediated ß-lactamase in Gram-negative bacteria, producing increased resistance to the widely used amoxycillinclavulanic acid combination. The induction of both enzymes was cross-reactive, both trimethoprim and the ß-lactam drugs induced both mechanisms. The metabolic impact of the ß-lactams must be triggering the induction mechanism. This cannot, however, be by the same pathway as trimethoprim.The high rates of carriage of normal gut flora resistant to antimicrobials are probably the result of selection by antimicrobials which are freely available without prescription combined with poor hygiene and sanitation. The evolution of such unusual induction mechanisms may have resulted from the prevailing low level of antimicrobials or frequent exposure to low levels of drug as a consequence of self - dosing. High rates of resistance in normal flora and the evolution of such resistance mechanisms is serious because of their impact on the development of resistance and in the testing for resistance, when such induction mechanisms may disguise true resistance levels.The DNA sequence of the dhfrIV was also determined and was shown to be only distantly related to the chromosomal dhfr and other plasmid mediated dhfrs.