In Vitro and Intracellular Activities of Peptide Deformylase Inhibitor GSK1322322 against Legionella pneumophila Isolates Jacques Dubois,a Maïtée Dubois,a Jean-François Martel,a Kelly Aubart,b Deborah Butlerb M360, Sherbrooke, Québec, Canadaa; Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USAb
T
he opportunistic Gram-negative bacterium Legionella pneumophila, which can grow free living or as a facultative intracellular organism in amoebae or human alveolar macrophages, is the major causative agent of Legionnaires’ disease, a severe form of pneumonia (1). Although at least 15 serogroups have been identified, L. pneumophila serogroup 1 accounts for more than 80% of the cases worldwide (2). Except in the event of a Legionnaires’ disease outbreak, L. pneumophila is treated empirically as part of the therapy used for hospitalized pneumonia, and given the severity of the disease, it is important to ensure that the antibacterial agent to be utilized shows good activity against this pathogen. GSK1322322 is a novel peptide deformylase (PDF) inhibitor with good safety and pharmacokinetic properties (3–5) and is currently in phase II development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections. GSK1322322 has demonstrated good antibacterial activity against other causative agents of bacterial pneumonia, such as Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae (6), so it was therefore important to investigate its potency against this atypical pathogen. In order to assess the in vitro activity of GSK1322322, we performed susceptibility studies against 50 L. pneumophila strains: 25 from serogroup 1 and 5 each from serogroups 2, 3, 4, 5, and 6. L. pneumophila isolates were collected from 1992 to 2013, mostly from the human respiratory tract, and grown on buffered charcoal yeast extract (BCYE) agar to produce pure cultures. MIC endpoints were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines (7). MIC plates, containing approximately 5 ⫻ 105 CFU/ml in buffered yeast extract (BYE) broth (with Legionella BCYE growth supplement), were incubated at 35°C in aerobic conditions for 48 h. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited visible growth after the appropriate incubation time. GSK1322322 showed variable activities against this panel of 50 L. pneumophila strains, with MICs ranging from 1 to 16 g/ml and MIC50 and MIC90 values of 8 and 16 g/ml, respectively (Table 1). No significant differences were observed in the activities of GSK1322322 against strains from different serogroups; 8 g/ml was the most frequent MIC in all sets (Table 1). Azithromycin and levofloxacin showed more potent extracellular activity, with MIC50/MIC90 values of 0.06/0.5 g/ml and 0.008/0.016 g/ml,
January 2015 Volume 59 Number 1
respectively (Table 1). The results of this study indicate that GSK1322322 is substantially less active in vitro against L. pneumophila serogroups 1 to 6 than azithromycin or levofloxacin, the drugs normally used for the treatment of legionellosis. The GSK1322322 MIC for S. aureus ATCC 29213 tested in MuellerHinton broth was identical to or within a 2-fold dilution of the MIC obtained using BYE, indicating that there is no drug inactivation by the test medium. As L. pneumophila is a facultative intracellular bacterium that multiplies within phagocytic cells, the ability of GSK1322322 to effectively inhibit growth of L. pneumophila in human monocytes was investigated. In these studies, human mononuclear cells (U937) (8), cultured at a final concentration of approximately 104 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum, were infected in a suspension with 104 to 105 of each of the 50 L. pneumophila strains grown to logarithmic phase by incubating them together for 1 h in a shaking incubator. Infected cultures were transferred to flat-bottomed wells and maintained thereafter without shaking at 37°C in 5% CO2 and 95% air for 7 days. After 24 h, the infected cultures were washed three times with drug-free broth to remove extracellular bacteria, and GSK1322322 (1 to 16 g/ml), azithromycin (0.03 to 0.05 g/ml), or erythromycin (0.06 to 1 g/ml) was added to the wells at a concentration equal to the corresponding MIC-strain combination. At day 3, the cultures were washed, split into two groups, and incubated for an additional 4 days with or without antibiotic. The numbers of CFU were determined in duplicate using BCYE agar at time zero and then every 24 h until day 7. The results indicate that the MICs of GSK1322322 not only prevented the growth of L. pneumophila inside macrophages but
Received 31 July 2014 Returned for modification 11 September 2014 Accepted 19 October 2014 Accepted manuscript posted online 27 October 2014 Citation Dubois J, Dubois M, Martel J-F, Aubart K, Butler D. 2015. In vitro and intracellular activities of peptide deformylase inhibitor GSK1322322 against Legionella pneumophila isolates. Antimicrob Agents Chemother 59:707–710. doi:10.1128/AAC.04006-14. Address correspondence to Deborah Butler, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.04006-14
Antimicrobial Agents and Chemotherapy
aac.asm.org
707
Downloaded from http://aac.asm.org/ on June 9, 2015 by UNIV OF CONNECTICUT
GSK1322322, a novel peptide deformylase inhibitor currently in development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia, showed poor in vitro activity against a panel of 50 Legionella pneumophila strains, with MICs ranging from 1 to 16 g/ml and an MIC90 of 16 g/ml, but very potent intracellular activity, with the minimum extracellular concentrations capable of inhibiting intracellular proliferation (MIECs) ranging from 0.12 to 2 g/ml and 98% of the strains being inhibited by concentrations of
T
he opportunistic Gram-negative bacterium Legionella pneumophila, which can grow free living or as a facultative intracellular organism in amoebae or human alveolar macrophages, is the major causative agent of Legionnaires’ disease, a severe form of pneumonia (1). Although at least 15 serogroups have been identified, L. pneumophila serogroup 1 accounts for more than 80% of the cases worldwide (2). Except in the event of a Legionnaires’ disease outbreak, L. pneumophila is treated empirically as part of the therapy used for hospitalized pneumonia, and given the severity of the disease, it is important to ensure that the antibacterial agent to be utilized shows good activity against this pathogen. GSK1322322 is a novel peptide deformylase (PDF) inhibitor with good safety and pharmacokinetic properties (3–5) and is currently in phase II development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections. GSK1322322 has demonstrated good antibacterial activity against other causative agents of bacterial pneumonia, such as Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae (6), so it was therefore important to investigate its potency against this atypical pathogen. In order to assess the in vitro activity of GSK1322322, we performed susceptibility studies against 50 L. pneumophila strains: 25 from serogroup 1 and 5 each from serogroups 2, 3, 4, 5, and 6. L. pneumophila isolates were collected from 1992 to 2013, mostly from the human respiratory tract, and grown on buffered charcoal yeast extract (BCYE) agar to produce pure cultures. MIC endpoints were determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines (7). MIC plates, containing approximately 5 ⫻ 105 CFU/ml in buffered yeast extract (BYE) broth (with Legionella BCYE growth supplement), were incubated at 35°C in aerobic conditions for 48 h. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited visible growth after the appropriate incubation time. GSK1322322 showed variable activities against this panel of 50 L. pneumophila strains, with MICs ranging from 1 to 16 g/ml and MIC50 and MIC90 values of 8 and 16 g/ml, respectively (Table 1). No significant differences were observed in the activities of GSK1322322 against strains from different serogroups; 8 g/ml was the most frequent MIC in all sets (Table 1). Azithromycin and levofloxacin showed more potent extracellular activity, with MIC50/MIC90 values of 0.06/0.5 g/ml and 0.008/0.016 g/ml,
January 2015 Volume 59 Number 1
respectively (Table 1). The results of this study indicate that GSK1322322 is substantially less active in vitro against L. pneumophila serogroups 1 to 6 than azithromycin or levofloxacin, the drugs normally used for the treatment of legionellosis. The GSK1322322 MIC for S. aureus ATCC 29213 tested in MuellerHinton broth was identical to or within a 2-fold dilution of the MIC obtained using BYE, indicating that there is no drug inactivation by the test medium. As L. pneumophila is a facultative intracellular bacterium that multiplies within phagocytic cells, the ability of GSK1322322 to effectively inhibit growth of L. pneumophila in human monocytes was investigated. In these studies, human mononuclear cells (U937) (8), cultured at a final concentration of approximately 104 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum, were infected in a suspension with 104 to 105 of each of the 50 L. pneumophila strains grown to logarithmic phase by incubating them together for 1 h in a shaking incubator. Infected cultures were transferred to flat-bottomed wells and maintained thereafter without shaking at 37°C in 5% CO2 and 95% air for 7 days. After 24 h, the infected cultures were washed three times with drug-free broth to remove extracellular bacteria, and GSK1322322 (1 to 16 g/ml), azithromycin (0.03 to 0.05 g/ml), or erythromycin (0.06 to 1 g/ml) was added to the wells at a concentration equal to the corresponding MIC-strain combination. At day 3, the cultures were washed, split into two groups, and incubated for an additional 4 days with or without antibiotic. The numbers of CFU were determined in duplicate using BCYE agar at time zero and then every 24 h until day 7. The results indicate that the MICs of GSK1322322 not only prevented the growth of L. pneumophila inside macrophages but
Received 31 July 2014 Returned for modification 11 September 2014 Accepted 19 October 2014 Accepted manuscript posted online 27 October 2014 Citation Dubois J, Dubois M, Martel J-F, Aubart K, Butler D. 2015. In vitro and intracellular activities of peptide deformylase inhibitor GSK1322322 against Legionella pneumophila isolates. Antimicrob Agents Chemother 59:707–710. doi:10.1128/AAC.04006-14. Address correspondence to Deborah Butler, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.04006-14
Antimicrobial Agents and Chemotherapy
aac.asm.org
707
Downloaded from http://aac.asm.org/ on June 9, 2015 by UNIV OF CONNECTICUT
GSK1322322, a novel peptide deformylase inhibitor currently in development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia, showed poor in vitro activity against a panel of 50 Legionella pneumophila strains, with MICs ranging from 1 to 16 g/ml and an MIC90 of 16 g/ml, but very potent intracellular activity, with the minimum extracellular concentrations capable of inhibiting intracellular proliferation (MIECs) ranging from 0.12 to 2 g/ml and 98% of the strains being inhibited by concentrations of
