This hypothesis is supported by the unchanged cell morphology learn more of L. monocytogenes in the exponential phase of growth, both in the absence of PBP3 [8] and when this protein is overexpressed. Effect of overexpression of PBP3 on the susceptibility of L. monocytogenes to β-lactams To determine whether PBP3 plays a role in β-lactam resistance, L. monocytogenes pAKB and L. monocytogenes pAKB-lmo1438 were tested for their susceptibility to penicillins, cephalosporins, monobactams

and carbapenems using an antibiotic disk sensitivity assay. This preliminary assay did not reveal any significant changes in the sensitivity to β-lactams caused by the overproduction of PBP3 – the diameters of the zones of bacterial growth inhibition surrounding the filter disks were identical after 24 h incubation. However, after 48 h incubation, partial 3-Methyladenine manufacturer autolysis of the bacterial growth in the presence of subinhibitory concentrations of penicillin G, ampicillin, amoxicillin, mezlocillin and imipenem was observed (data not shown). Penicillin G, ampicillin and amoxicillin were then chosen for MIC determination using the E-test. This assay confirmed the results of the antibiotic disk tests, namely that both strains were equally susceptible to the β-lactams tested and that in the case of the strain overexpressing PBP3, a zone of partial autolysis of the bacterial

lawn was observed at an antibiotic concentration three to four times lower than the MIC. The results for ampicillin are presented in Figure 4A. A survival assay was also performed

for L. monocytogenes pAKB and see more L. monocytogenes pAKB-lmo1438 by culturing these strains in broth Erastin chemical structure supplemented with a lethal dose of penicillin G. The optical density of the L. monocytogenes pAKB-lmo1438 culture decreased at a faster rate than that of the control strain, which correlated with the more rapid elimination of viable bacteria from the culture (Figure 4B). Keeping in mind that in the constructed strain an increased level of PBP4 expression was also observed, which was found to contribute to the susceptibility of L. monocytogenes to β-lactams [8, 23], the changes in the susceptibility of L. monocytogenes pAKB-lmo1438 to β-lactam antibiotics may be not only an effect of PBP3 overexpression. Thus, it seems probable that the altered susceptibility of this strain to β-lactams is the effect of overexpression of PBP3, PBP4 or both of these proteins. Regardless of the reason for the altered susceptibility, it may definitely be concluded that overexpression of PBP3 (accompanied by increased levels of PBP4) leads to minor changes in the susceptibility of L. monocytogenes to β-lactams without any change in the MIC values. Together with the lack of changes in β-lactam MIC values in the case of the lmo1438 mutant strain reported by Guinane et al. [8], this result demonstrates that the role of PBP3 is non-essential in the β-lactam resistance of L. monocytogenes. These findings concerning the β-lactam antibiotic resistance of L.