The mechanism of microbial resistance to hexahydro-1,3,5-triethyl-s-triazine

Summary Four strains ofPseudomonas putida and two unidentifiedPseudomonas species that were resistant to hexahydro-1,3,5-triethyl-s-triazine (HHTT) were shown to be resistant to formaldehyde as well. Conjugation experiments revealed that: (a) HHTT and formaldehyde resistance was cotransferred in every case where exconjugants were recovered; (b) in every case HHTT resistance and formaldehyde resistance were expressed to the same level in the exconjugant as in the donor; (c) resistance to either HHTT or formaldehyde alone was never observed; and (d) in instances where HHTT and formaldehyde resistance in the exconjugants was unstable, the exconjugants lost resistance to both agents simultaneously and never to one agent alone. Resistant organisms (e.g.P. putida 3-T-15²) had high levels of formaldehyde dehydrogenase and this enzyme appeared to be constitutively expressed. It was concluded that resistance to HHTT was due to resistance to its degradation product, formaldehyde, via detoxification of formaldehyde by formaldehyde dehydrogenase. HHTT- and formaldehyde-sensitive organisms had barely detectable levels (most likely repressed levels) of formaldehyde dehydrogenase. Although speculative, it is possible that formaldehyde resistance may be due to a mutation resulting in derepression of the gene coding for formaldehyde dehydrogenase. While it could not be discerned whether HHTT resistance and formaldehyde resistance were carried on two separate but closely linked genes or if only one gene was involved, the evidence suggested that only one gene was involved. Similarly, it could not be determined whether HHTT and formaldehyde resistance was encoded by chromosomal or plasmid genes.     

The mechanism of action of hexahydro-1,3,5-triethyl-s-triazine

Summary The mechanism of antimicrobial action of hexahydro-1,3,5-triethyl-s-triazine (HHTT) was studied using the HHTT-resistant isolate,Pseudomonas putida 3-T-15², its HHTT-sensitive, novobiocin-cured derivative,P. putida 3-T-15² 11:21,P. putida ATCC 12633,Pseudomonas aeruginosa PA01 andEscherichia coli J53 (RP4). HHTT was oxidized byP. putida 3-T-15², while respiration ofP. putida 3-T-15² 11:21 was inhibited by HHTT. Chemical assays showed that HHTT released formaldehyde.P. putida 3-T-15² was highly resistant to formaldehyde, whileP. putida 3-T-15² 11:21 was highly sensitive to formaldehyde. Both HHTT and formaldehyde acted similarly to inhibit proline uptake in bacterial cells and to inhibit the synthesis of the inducible enzymes, -galactosidase and glucose-6-phosphate dehydrogenase. HHTT did not have uncoupler-like activity.P. putida 3-T-15² used either HHTT or ethylamine, a component of HHTT, as a nitrogen source for growth, but neither HHTT, ethylamine or formaldehyde served as a carbon and energy source for growth. We concluded that a major mechanism of antimicrobial action of HHTT was through its degradation product, formaldehyde.