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Paragraph: Methicillin-resistant Staphylococcus aureus (MRSA) has become the most common clinically relevant multiresistant pathogen [2] causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% [3] . The prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 [4] , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 (although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010), followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 (for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010). The treatment failure of vancomycin , the therapeutic anti-MRSA agent of choice, due to the strains with elevated   minimum inhibitory concentration (MIC) values (i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism) within the susceptible range was described previously [5, 6] . Thus, the emergence of MRSA (and  -resistant S. aureus in the recent years as well [7] ) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria [8] . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs [9] . This fact should be considered during the development of effective and safe treatment options for MRSA infections. The history of clinical usage of salicylanilides (2-hydroxy-N-phenylbenzamides) dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s [10] . Nowadays, salicylanilides (SALs) are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic [11] , antibacterial [12, 13] , antimycobacterial [13] , antifungal [14] , and antiviral [15, 16] , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems (TCS) of bacteria [17] . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis [18] . Furthermore, salicylanilides have been recognised as inhibitors of some bacterial enzymes, such as sortase A from S. aureus [19] , d-alanine-d-alanine ligase [20] , or transglycosylases from S. aureus (but not from M. tuberculosis) [12] . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: (i) methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and (ii) isocitrate lyase, which is essential for the metabolism of fatty acids [21] . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges. This study is a follow-up paper to a recently published article [13] . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides, called diamides due to their skeleton (for general structure see Table 1 ), was described previously [13, 22] , and their antimycobacterial and antibacterial activities against various bacterial species were reported [13] . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA. The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect. The synthetic pathway of the series of novel diamides was described recently [13, 22] , and their structures (see Table 1 ) were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis [13, 22] . [27] ; and MRSA SA 3202 [27] (National Institute of Public Health, Prague, Czech Republic) both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected [28] . All isolates were tested for the presence of the mecA gene encoding methicillin resistance [29] . These three clinical isolates were classified as  -susceptible (but with higher MIC of   equal to 2 g/mL (VA2-MRSA) within the susceptible range for MRSA 63718) methicillinresistant S. aureus (VS-MRSA). For the MICs of  , see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus (VS-MSSA) ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates (Columbia agar base with 5% ovine blood) between experiments. (MBCs) . The MBCs (i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots (20 L) from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar (MHA) plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC [ g/mL] values of tested 5-and 4-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides (bactericidal effect of individual compounds against particular strains marked in bold). after 24 h of incubation as compared to the colony count of the starting inoculum [30] . To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions. N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC [ g/mL] MBC [ g/mL] 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 (S)-CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 (S)-CH(CH 3 ) 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 (S)-benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 (R)-CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 (S)-CH(CH 3 ) 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 (S)-CH(CH 3 ) 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br (S)-CH(CH 3 ) 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl (S)-CH(CH 3 ) 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl (S)-benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1

Time-kill assays were performed by the broth macrodilution method according to previously described methodology [30] with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth (MHB) with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL (actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL) and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots (20 L) were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time (over 24 h), and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ(log 10 CFU/mL) values of −1 (corresponding to 90% killing), −2 (99% killing), and −3 (99.9% killing) at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at least 99.9% (≥3 log 10 ) of the total count of CFU/mL in the original inoculum. Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently [13] . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 (methicillin-susceptible) as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect [31] , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study. Recently MIC values of diamides expressed as molar concentrations in mol/L were published [13] . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and  . Potential bactericidal activity of diamides was assessed using MBC assay [26] . MBC values of all tested compounds are recorded in Table 1 as well. Based on the obtained results, all compounds assessed as active according to MIC values in our previous study (1f-i) showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain. Bactericidal activity is defined as a ratio of MBC to MIC of ≤4 [32] . Table 1 bactericidal activity is expressed in bold. As mentioned above, SALs are known to exhibit a bacteriostatic effect [31] , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond (-CONH-) can cause interactions with a variety of enzymes [33] ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus (including MRSA) cell wall biosynthesis [12] . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane [12] . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents [30, 34] , the failure in the cell wall biosynthesis due to the inhibition of transglycosylases could be responsible for bactericidal activity of diamides against MRSA.

Question: What is the treatment of choice for MRSA infections?
vancomycin