Trimethoprim-sulfamethoxazole is a well-known antibiotic that inhibits folic acid synthesis, a topic of renewed interest. Since resistant strains are increasingly more common, an early and accurate discrimination of susceptibility may assure confident therapy. Two morphological assays were performed in Escherichia coli (n = 50; 27 non-susceptible) and Klebsiella pneumoniae (n = 52; 18 non-susceptible). First, the strains were incubated with the CLSI breakpoint of cotrimoxazole for 150 min, which induced cell lengthening in the susceptible strains. Second, the bacteria were incubated with mitomycin C (MMC) (0.5 mg/L) for 120 min to induce a SOS-linked cell enlargement higher than that obtained by cotrimoxazole alone. When cotrimoxazole was added 30 min before MMC, the inhibition of folic acid synthesis in the susceptible strain resulted in the suppression of MMC-induced extra elongation. In the non-susceptible strains, folic acid synthesis continued despite the antibiotic, so that the MMC-induced extra cell lengthening could not be impeded. Whereas the first assay resulted in five false negatives and four false positives of resistance, the results of the second assay matched those of the conventional antibiogram. This simple morphological procedure is performed in 2 h and 45 min and may allow a rapid selection of useful and relatively inexpensive therapy, thereby preserving the newer broad-spectrum antibiotics.

Current antibiograms cannot discern the particular effect of a specific antibiotic when the bacteria are incubated with a mixture of antibiotics. To prove that this task is achievable, Escherichia coli strains were treated with ciprofloxacin for 45 min, immobilized on a slide and stained with SYBR Gold. In susceptible strains, the nucleoid relative surface started to decrease near the MIC, being progressively condensed as the dose increased. The shrinkage level correlated with the DNA fragmentation degree. Ciprofloxacin-resistant bacilli showed no change. Additionally, E. coli strains were incubated with ampicillin for 45 min and processed similarly. The ampicillin-susceptible strain revealed intercellular DNA fragments that increased with dose, unlike the resistant strain. Co-incubation with both antibiotics revealed that ampicillin did not modify the nucleoid condensation effect of ciprofloxacin, whereas the quinolone partially decreased the background of DNA fragments induced by ampicillin. Sixty clinical isolates, with different combinations of susceptibility-resistance to each antibiotic, were co-incubated with the EUCAST breakpoints of susceptibility of ciprofloxacin and ampicillin. The morphological assay correctly categorized all the strains for each antibiotic in 60 min, demonstrating the feasible independent evaluation of a mixture of quinolone and beta-lactam. The rapid phenotypic assay may shorten the incubation times and necessary microbial mass currently required for evaluation.

Current antibiograms cannot discern the particular effect of a specific antibiotic when the bacteria are incubated with a mixture of antibiotics. To prove that this task is achievable, Escherichia coli strains were treated with ciprofloxacin for 45 min, immobilized on a slide and stained with SYBR Gold. In susceptible strains, the nucleoid relative surface started to decrease near the MIC, being progressively condensed as dose increased. The shrinkage level correlated with the DNA fragmentation degree. Ciprofloxacin-resistant bacilli showed no change. Additionally, E. coli strains were incubated with ampicillin for 45 min and processed similarly. The ampicillin-susceptible strain revealed intercellular DNA fragments that increased with dose, unlike the resistant strain. Co-incubation with both antibiotics revealed that ampicillin did not modify the nucleoid condensation effect of ciprofloxacin, whereas the quinolone partially decreased the background of DNA fragments induced by ampicillin. Sixty clinical isolates, with different combinations of susceptibility-resistance to each antibiotic, were co-incubated with the EUCAST breakpoints of susceptibility of ciprofloxacin and ampicillin. The morphological assay correctly categorized all the strains for each antibiotic in 60 min, demonstrating the feasible independent evaluation of a mixture of quinolone and beta-lactam. The rapid phenotypic assay may shorten the incubation times and necessary microbial mass currently required for evaluation.