From the experiment of incubation time, it is deduced that to discriminate with accuracy the susceptible strains from the rest it is enough, in a practical clinical approach, to assess the control 0 dose and the CLSI cut-off dose for susceptibility, incubating with the antibiotic for 60 min in case of cultures growing 24 h in agar plate, as usual in the standard clinical microbiology laboratory. If the cultures were exponentially growing in liquid medium, the incubation time with the antibiotic may be decreased for 30 min. We have observed that the greater the ageing of

the culture in agar plate, or when the culture is achieving the stationary phase of growth, INK1197 clinical trial the longer the incubation time necessary to observe the effect of the antibiotic, even several hours. To evaluate clinical strains using the technique to assess

the integrity of the cell wall, it is mandatory to simultaneously process a sensitive, an intermediate and a resistant strain as controls of the activity of the antibiotic and the efficacy of the technique. Sensitive strains from gram-negative bacteria assayed showed a background https://www.selleckchem.com/products/a-1155463.html of extracellular microgranular-fibrilar material, its concentration being dose and time dependent. This material corresponded to DNA selleck fragments released by the bacteria, since it was digested by DNase I and hybridized with a specific whole genome probe, being clearly visualized with high sensitive DNA dyes, i.e. SYBR Gold. It is interesting to note that this background of DNA fragments was practically undetectable in gram-positive strains, despite being susceptible to β-lactams or vancomycin. Farnesyltransferase Moreover, it was also undetected in the same bacteria after quinolone treatment in susceptible strains, as evidenced in our previous works with the procedure [15, 16]. This fact suggests that the release of DNA fragments could be specific to cell wall directed antibiotics

or β-lactams at least. This interesting phenomenon requires a deeper study in future works, to address the mechanisms and kinetics of production. DNA fragmentation must be a secondary effect, after cell wall damage. It could be a passive result of attack by DNases or reactive species of oxygen (ROS) liberated in the affected bacteria, or it could be active, a consequence of an apoptotic-like process triggered after cell wall damage. Considering to the first possibility, it has been recently reported that, unlike bacteriostatic antibiotics, β-lactams induce the formation of ROS in gram-negative and gram-positive microorganisms [18]. Hydroxyl radicals should attack proteins and DNA, possibly inducing DNA breakages, resulting in death of the bacteria. This response was also found with other bactericidal antibiotics, like fluoroquinolones. Possibly, the increased permeability of the cell wall that would result after impairment of peptidoglycan biosynthesis by the β-lactams, would allow the release of DNA fragments to the medium.