AAC Accepted Manuscript Posted Online 26 May 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00484-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor
2
GSK1322322 in Haemophilus influenzae, Staphylococcus aureus,
3
Streptococcus pyogenes, and Streptococcus pneumoniae
4 5
Sharon Min1#, Karen Ingraham1, Jianzhong Huang1, Lynn McCloskey1, Sarah Rilling1*, Anne
6
Windau2, Jason Pizzollo1*, Deborah Butler1, Kelly Aubart1, Linda A. Miller1, Magdalena
7
Zalacain1*, David J. Holmes1 and Karen O’Dwyer1
8
1
9
GlaxoSmithKline, Collegeville, PA, USA; 2Laboratory Specialists, Inc., Westlake, OH, USA
Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area,
10 11
Running Title: Resistance to PDF inhibitor GSK1322322
12 13 14
#
corresponding author. E-mail address, [email protected]
15 16 17
*Present addresses: S.R., Janssen Research and Development, Spring House, PA, USA; J.P.,
18
Department of Biology, University of Massachusetts Amherst, Amherst, MA, USA; M.Z.,
19
Zala Drug Discovery Consulting LLC, West Chester, PA, USA
20
1
21 22
ABSTRACT The continuous emergence of multidrug resistant pathogenic bacteria is compromising
23
the successful treatment of serious microbial infections. GSK1322322, a novel peptide
24
deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated
25
safety and efficacy in human proof-of-concept clinical studies. In vitro studies were
26
performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major
27
pathogens causing respiratory tract and skin infections. Resistance to GSK1322322 occurred
28
at high frequency through loss-of-function mutations in the formyl-methionyl transferase
29
(FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4
30
strains), and via missense mutations in Streptococcus pneumoniae (6/21 strains), but these
31
mutations are associated with a severe in vitro and/or in vivo fitness cost. The overall FoR to
32
GSK1322322 was very low in Haemophilus influenzae with only one PDF mutant identified
33
in one of four strains. No target-based mutants were identified from S. pyogenes, and only
34
one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S.
35
pneumoniae, PDF mutants were only isolated from six of 21 strains tested and an additional
36
10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants
37
characterized from these three organisms (35/37), carried mutations in residues at or in close
38
proximity to one of three highly conserved motifs, which are part of the active site of the PDF
39
protein, with 30 of the 35 mutations occurring at position V71 (S. pneumoniae numbering
40
system).
41
2
42
INTRODUCTION
43
The continuous appearance and dissemination of multidrug resistance among
44
pathogenic bacteria is compromising the successful treatment of serious microbial infections
45
and alternative therapies are urgently needed. The development of new agents against
46
clinically unexploited targets provides the additional advantage of preventing potential cross-
47
resistance issues with already marketed antibiotics. One such target is peptide deformylase
48
(PDF), a highly conserved bacterial metalloprotease that hydrolyzes the N-terminal formyl
49
group from all nascent polypeptides (1-3) and plays an essential role in protein maturation. A
50
large number of structurally diverse PDF inhibitors have been identified over the years,
51
including several compounds with demonstrated in vivo efficacy and good safety profiles (4),
52
some of which have progressed to clinical trials (5, 6). GSK1322322 is a novel non-peptidic
53
PDF inhibitor from the hydrazide class, which shows good in vitro antibacterial activity (7)
54
and has demonstrated safety and efficacy in human proof-of-concept clinical studies (8-11).
55
In bacteria where protein synthesis can still initiate with unformylated Met-tRNAi,
56
therefore bypassing the need for a deformylation step, the main mechanism of resistance to
57
PDF inhibitors is loss-of-function mutations in genes involved in the formylation of the
58
initiator tRNA, mostly in the gene encoding formyl-methionyl transferase (FMT) (12-19)
59
although mutations in FolD and GlyA, two enzymes involved in the synthesis of 10-formyl-
60
tetrahydrofolate, have also been described (14, 16). These mutants are highly resistant to PDF
61
inhibitors, but they display compromised in vitro (12, 13, 15-18) and in vivo (12, 16, 20)
62
growth, and, in Staphylococcus aureus, FMT mutants also show a drastic reduction in
63
production of virulence factors and a restricted ability to produce an invasive infection (20).
64
In those organisms where formylation seems to be essential, such as Streptococcus
3
65
pneumoniae and Haemophilus influenzae, resistance to PDF inhibitors involves mutations in,
66
or increased production of, the target gene (21-23) or efflux (24).
67
This study was performed to determine the target-based frequency of resistance (FoR)
68
to PDF inhibitor GSK1322322 in major pathogens causing respiratory tract and skin
69
infections, such as H. influenzae, S. aureus, Streptococcus pyogenes, and S. pneumoniae.
70
4
71
MATERIALS AND METHODS
72
Bacterial Strains and Growth Conditions. All strains used in this study were clinical
73
isolates obtained from the GlaxoSmithKline Microbiology Department Culture Collection. S.
74
pneumoniae, S. pyogenes and S. aureus strains were cultured on Trypticase Soy Agar (TSA)
75
or Cation Adjusted Mueller-Hinton (CAMH) agar with 5% defibrinated sheep blood. H.
76
influenzae strains were cultured on Chocolate agar II plates. CAMH broth was used for S.
77
aureus, with 3% lysed horse blood added for S. pneumoniae and S. pyogenes. Haemophilus
78
Test Medium (HTM) broth was used for H. influenzae. For inoculum preparation in the
79
resistance studies, S. pneumoniae and S. pyogenes strains were grown in Todd Hewitt broth
80
with 0.5% Yeast Extract (THYB). A total of 4 strains of H. influenzae, S. aureus and S.
81
pyogenes, and 21 strains of S. pneumoniae were evaluated in these studies.
82
Antimicrobial Agents and Susceptibility Testing. PDF inhibitor GSK1322322 and the
83
pleuromutilin tiamulin were obtained from GlaxoSmithKline Pharmaceuticals (Collegeville,
84
PA, USA) and dissolved in DMSO. Minimum inhibitory concentration (MIC) endpoints were
85
determined by broth microdilution methodology according to Clinical and Laboratory
86
Standards Institute (CLSI) guidelines (25).
87
Spontaneous FoR Studies. The frequency of spontaneous resistance was calculated by
88
dividing the number of confirmed resistant colonies growing on antibiotic-containing plates
89
by the total number of colony forming units in the initial test inoculum. Colonies were defined
90
as resistant if their MIC was ≥4-fold the MIC of the wild-type strain. When necessary, the
91
total number of resistant colonies on the plates was extrapolated from a representative set
92
tested.
5
93
Preparation of plates. GSK1322322 was added to the appropriate media-containing molten
94
agar at 50oC to yield 20 mL of agar at the correct multiple of MIC for each organism. Plates
95
containing GSK1322322 at 4X and/or 10X MIC were poured and left to cool and solidify.
96
Plates containing no compound were also prepared to be used for viable counts and to observe
97
growth.
98
Preparation of inoculum. Cultures were prepared by inoculating broth with a bacterial
99
suspension in saline, made from plates derived from individual colonies (S. pneumoniae, S.
100
aureus, H. influenzae) or by dilution of overnight cultures (S. pyogenes). The cultures were
101
incubated at 35°C with (S. pneumoniae, S. aureus, H. influenzae) or without agitation (S.
102
pyogenes) in the presence of CO2 (S. pneumoniae, H. influenzae) until the broth was visibly
103
turbid. Cultures were centrifuged and cell pellets were resuspended to an appropriate cell
104
concentration in fresh media. Cell concentrations of 1-5 × 108 CFU/mL (S. pneumoniae, S.
105
pyogenes) or 1 × 109 CFU/mL (S. aureus, H. influenzae) were targeted.
106
Plating bacterial suspension. To determine the number of colony forming units (CFU)
107
present in the initial test inoculum, each organism suspension was serially diluted 1:10 and
108
three 20 μL drops from dilutions 10-4 to 10-8 were plated onto the appropriate agar plates and
109
incubated overnight at 35°C in the presence of 5% CO2 (H. influenzae, S. pneumoniae).
110
Counts were performed at the dilution that provided distinguishable colonies and an average
111
of the three samples was used to calculate the number of CFUs in the original suspension.
112
Colonies were counted after 48 h incubation at 35oC ambient air, with the aid of a magnifying
113
glass when necessary.
114
Confirmation of resistance phenotype. Colony size relative to wild-type was noted and
115
single colonies from each drug/organism combination were streaked onto new plates
6
116
containing identical drug concentrations or no drug. Plates were incubated at 35oC and
117
growth and resistance were evaluated at 24 and 48 h. In those cases where a large number of
118
mutants were obtained, only a representative set of colonies was tested. In the case of S.
119
aureus and S. pyogenes, colonies were also analyzed for production of hemolysin in blood
120
agar plates as it is known that S. aureus fmt mutants are non-hemolytic (20).
121
Genetic Characterization of Isolates with Reduced Susceptibility to GSK1322322. The
122
PCR primers used for DNA amplification of the pdf and fmt genes were designed from the
123
appropriate regions of the corresponding publically available genomic sequences using the
124
Lasergene PrimerSelect software. Although S. pneumoniae and S. pyogenes contain a second
125
pdf gene, def2, this was not amplified as it has been reported in the literature that def2 encodes
126
an inactive protein (21). In addition, PCR amplification of folD and glyA, encoding
127
bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase and serine
128
hydroxymethyltransferase, respectively, was also performed with selected S. aureus and S.
129
pneumoniae mutants. The PCR templates were prepared by boiling cells collected from
130
overnight plates. PCR products were purified using the QIAquick PCR purification kit, and
131
sequenced using the AB v3.1 BigDye-terminator cycle sequencing kit (Applied Biosystems).
132
Sequencing reactions were purified using the Performa® DTR V3 96-Well Short Plate Kit
133
(Edge Bio) and analyzed in a Genetic Analyzer 3730XL (Applied Biosystems). In order to
134
identify specific mutations responsible for the resistance phenotype, alignment of the DNA
135
sequences of the pdf, fmt, folD and glyA genes and promoter regions from mutant strains and
136
their corresponding parent organisms was carried out using Lasergene MegAlign Software
137
(DNASTAR).
7
138
Growth Curves of S. pyogenes Strains. S. pyogenes cells, grown on TSA with 5% sheep
139
blood at 35oC for approximately 20 h, were resuspended in 6 mL of THYB and adjusted to an
140
OD600 (1 cm path length) of 0.01. The cultures were incubated at 35oC without agitation and
141
growth was monitored at different time points by optical density at 600 nm and viable
142
bacterial counts.
143
Competitive Growth Studies of S. pneumoniae Parent and Mutant Strains. For the
144
preparation of early log phase cultures, a suspension equivalent to a 0.5 McFarland standard
145
from an overnight agar plate was diluted 50 fold into 4 mL of THYB and incubated for
146
approximately 4 h, shaking, in the presence of 5% CO2, to an OD600 of approximately 0.05.
147
Competitive growth studies were carried out in THYB media at 35oC in 5% CO2 with
148
continuous shaking, using initial inocula from early log phase cultures to give a 1:1 mutant to
149
wild-type ratio. At specific time points, samples were serially diluted and plated with and
150
without selective concentrations of GSK1322322 in order to calculate mutant and wild-type
151
CFU/mL as described above. At the end of each growth cycle (4 hours), cultures were diluted
152
into fresh media to start another cycle.
8
153
RESULTS
154
H. influenzae
155
Studies were performed to determine both the overall and the target-based FoR in four strains
156
of H. influenzae at 4X and 10X the GSK1322322 MIC. FoR was low in this species and
157
mutants with reduced susceptibility to GSK1322322 were not isolated in three of the four
158
strains tested, resulting in a FoR of 16-fold increase in the GSK1322322 MIC observed relative to
162
the parent strain.
163
S. aureus
164
Mutation in the S. aureus fmt gene,is associated with a number of phenotypic features which
165
include lack of hemolysin production, high level resistance to PDF inhibitors, a small colony
166
phenotype, and hypersensitivity to pleuromutilins (20). Therefore, in order to easily identify
167
FMT mutants, FoR studies were performed in four methicillin-resistant hemolytic S. aureus
168
strains. The overall FoR was high in all strains at both 4X and 10X the GSK1322322 MIC,
169
with similar frequencies ranging between 5 × 10-7 and 6 × 10-8 (Table 2). In order to
170
determine the target-based FoR, colony size, susceptibility to GSK1322322 and tiamulin, and
171
hemolytic phenotype were evaluated in a representative number of colonies. In every case,
172
non-hemolytic isolates were highly resistant to GSK1322322 (MICs >64 μg/mL) and
173
displayed a small colony phenotype, and were classified as FMT-like mutants, although not
174
all colonies were hypersensitive to tiamulin. Only FMT-like isolates were identified from the
175
resistance study with S. aureus X31360. DNA sequencing analysis of a number of these
9
176
isolates, including those not hypersensitive to tiamulin, confirmed that they carried mutations
177
in the fmt gene that should lead to inactive FMT proteins, with premature stop codons,
178
frameshifts, or amino acid substitutions known to affect its catalytic mechanism (26). Ten
179
isolates from S. aureus WCUH29 and one each from S. aureus PVL-1 and T6466 were
180
identified with a hemolytic phenotype, elevated GSK1322322 MICs, and wild-type tiamulin
181
MICs. Their genotypic characterization showed that most of them carried a mutation in
182
amino acid V59 of the PDF protein. The GSK1322322 MIC increased 32-fold in isolates
183
carrying mutation V59A and >64-fold in those with a PDF V59D substitution (Table 3).
184
Low-level resistance was associated with changes in the promoter region in two of the
185
mutants, and one isolate had no obvious mutation in fmt, folD, glyA, or pdf genes, or in their
186
promoter regions (Table 3). Resistance to GSK1322322 through target-based mutations
187
occurred in these S. aureus strains at a frequency ranging from 2 × 10-8 to
1
Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor
2
GSK1322322 in Haemophilus influenzae, Staphylococcus aureus,
3
Streptococcus pyogenes, and Streptococcus pneumoniae
4 5
Sharon Min1#, Karen Ingraham1, Jianzhong Huang1, Lynn McCloskey1, Sarah Rilling1*, Anne
6
Windau2, Jason Pizzollo1*, Deborah Butler1, Kelly Aubart1, Linda A. Miller1, Magdalena
7
Zalacain1*, David J. Holmes1 and Karen O’Dwyer1
8
1
9
GlaxoSmithKline, Collegeville, PA, USA; 2Laboratory Specialists, Inc., Westlake, OH, USA
Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area,
10 11
Running Title: Resistance to PDF inhibitor GSK1322322
12 13 14
#
corresponding author. E-mail address, [email protected]
15 16 17
*Present addresses: S.R., Janssen Research and Development, Spring House, PA, USA; J.P.,
18
Department of Biology, University of Massachusetts Amherst, Amherst, MA, USA; M.Z.,
19
Zala Drug Discovery Consulting LLC, West Chester, PA, USA
20
1
21 22
ABSTRACT The continuous emergence of multidrug resistant pathogenic bacteria is compromising
23
the successful treatment of serious microbial infections. GSK1322322, a novel peptide
24
deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated
25
safety and efficacy in human proof-of-concept clinical studies. In vitro studies were
26
performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major
27
pathogens causing respiratory tract and skin infections. Resistance to GSK1322322 occurred
28
at high frequency through loss-of-function mutations in the formyl-methionyl transferase
29
(FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4
30
strains), and via missense mutations in Streptococcus pneumoniae (6/21 strains), but these
31
mutations are associated with a severe in vitro and/or in vivo fitness cost. The overall FoR to
32
GSK1322322 was very low in Haemophilus influenzae with only one PDF mutant identified
33
in one of four strains. No target-based mutants were identified from S. pyogenes, and only
34
one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S.
35
pneumoniae, PDF mutants were only isolated from six of 21 strains tested and an additional
36
10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants
37
characterized from these three organisms (35/37), carried mutations in residues at or in close
38
proximity to one of three highly conserved motifs, which are part of the active site of the PDF
39
protein, with 30 of the 35 mutations occurring at position V71 (S. pneumoniae numbering
40
system).
41
2
42
INTRODUCTION
43
The continuous appearance and dissemination of multidrug resistance among
44
pathogenic bacteria is compromising the successful treatment of serious microbial infections
45
and alternative therapies are urgently needed. The development of new agents against
46
clinically unexploited targets provides the additional advantage of preventing potential cross-
47
resistance issues with already marketed antibiotics. One such target is peptide deformylase
48
(PDF), a highly conserved bacterial metalloprotease that hydrolyzes the N-terminal formyl
49
group from all nascent polypeptides (1-3) and plays an essential role in protein maturation. A
50
large number of structurally diverse PDF inhibitors have been identified over the years,
51
including several compounds with demonstrated in vivo efficacy and good safety profiles (4),
52
some of which have progressed to clinical trials (5, 6). GSK1322322 is a novel non-peptidic
53
PDF inhibitor from the hydrazide class, which shows good in vitro antibacterial activity (7)
54
and has demonstrated safety and efficacy in human proof-of-concept clinical studies (8-11).
55
In bacteria where protein synthesis can still initiate with unformylated Met-tRNAi,
56
therefore bypassing the need for a deformylation step, the main mechanism of resistance to
57
PDF inhibitors is loss-of-function mutations in genes involved in the formylation of the
58
initiator tRNA, mostly in the gene encoding formyl-methionyl transferase (FMT) (12-19)
59
although mutations in FolD and GlyA, two enzymes involved in the synthesis of 10-formyl-
60
tetrahydrofolate, have also been described (14, 16). These mutants are highly resistant to PDF
61
inhibitors, but they display compromised in vitro (12, 13, 15-18) and in vivo (12, 16, 20)
62
growth, and, in Staphylococcus aureus, FMT mutants also show a drastic reduction in
63
production of virulence factors and a restricted ability to produce an invasive infection (20).
64
In those organisms where formylation seems to be essential, such as Streptococcus
3
65
pneumoniae and Haemophilus influenzae, resistance to PDF inhibitors involves mutations in,
66
or increased production of, the target gene (21-23) or efflux (24).
67
This study was performed to determine the target-based frequency of resistance (FoR)
68
to PDF inhibitor GSK1322322 in major pathogens causing respiratory tract and skin
69
infections, such as H. influenzae, S. aureus, Streptococcus pyogenes, and S. pneumoniae.
70
4
71
MATERIALS AND METHODS
72
Bacterial Strains and Growth Conditions. All strains used in this study were clinical
73
isolates obtained from the GlaxoSmithKline Microbiology Department Culture Collection. S.
74
pneumoniae, S. pyogenes and S. aureus strains were cultured on Trypticase Soy Agar (TSA)
75
or Cation Adjusted Mueller-Hinton (CAMH) agar with 5% defibrinated sheep blood. H.
76
influenzae strains were cultured on Chocolate agar II plates. CAMH broth was used for S.
77
aureus, with 3% lysed horse blood added for S. pneumoniae and S. pyogenes. Haemophilus
78
Test Medium (HTM) broth was used for H. influenzae. For inoculum preparation in the
79
resistance studies, S. pneumoniae and S. pyogenes strains were grown in Todd Hewitt broth
80
with 0.5% Yeast Extract (THYB). A total of 4 strains of H. influenzae, S. aureus and S.
81
pyogenes, and 21 strains of S. pneumoniae were evaluated in these studies.
82
Antimicrobial Agents and Susceptibility Testing. PDF inhibitor GSK1322322 and the
83
pleuromutilin tiamulin were obtained from GlaxoSmithKline Pharmaceuticals (Collegeville,
84
PA, USA) and dissolved in DMSO. Minimum inhibitory concentration (MIC) endpoints were
85
determined by broth microdilution methodology according to Clinical and Laboratory
86
Standards Institute (CLSI) guidelines (25).
87
Spontaneous FoR Studies. The frequency of spontaneous resistance was calculated by
88
dividing the number of confirmed resistant colonies growing on antibiotic-containing plates
89
by the total number of colony forming units in the initial test inoculum. Colonies were defined
90
as resistant if their MIC was ≥4-fold the MIC of the wild-type strain. When necessary, the
91
total number of resistant colonies on the plates was extrapolated from a representative set
92
tested.
5
93
Preparation of plates. GSK1322322 was added to the appropriate media-containing molten
94
agar at 50oC to yield 20 mL of agar at the correct multiple of MIC for each organism. Plates
95
containing GSK1322322 at 4X and/or 10X MIC were poured and left to cool and solidify.
96
Plates containing no compound were also prepared to be used for viable counts and to observe
97
growth.
98
Preparation of inoculum. Cultures were prepared by inoculating broth with a bacterial
99
suspension in saline, made from plates derived from individual colonies (S. pneumoniae, S.
100
aureus, H. influenzae) or by dilution of overnight cultures (S. pyogenes). The cultures were
101
incubated at 35°C with (S. pneumoniae, S. aureus, H. influenzae) or without agitation (S.
102
pyogenes) in the presence of CO2 (S. pneumoniae, H. influenzae) until the broth was visibly
103
turbid. Cultures were centrifuged and cell pellets were resuspended to an appropriate cell
104
concentration in fresh media. Cell concentrations of 1-5 × 108 CFU/mL (S. pneumoniae, S.
105
pyogenes) or 1 × 109 CFU/mL (S. aureus, H. influenzae) were targeted.
106
Plating bacterial suspension. To determine the number of colony forming units (CFU)
107
present in the initial test inoculum, each organism suspension was serially diluted 1:10 and
108
three 20 μL drops from dilutions 10-4 to 10-8 were plated onto the appropriate agar plates and
109
incubated overnight at 35°C in the presence of 5% CO2 (H. influenzae, S. pneumoniae).
110
Counts were performed at the dilution that provided distinguishable colonies and an average
111
of the three samples was used to calculate the number of CFUs in the original suspension.
112
Colonies were counted after 48 h incubation at 35oC ambient air, with the aid of a magnifying
113
glass when necessary.
114
Confirmation of resistance phenotype. Colony size relative to wild-type was noted and
115
single colonies from each drug/organism combination were streaked onto new plates
6
116
containing identical drug concentrations or no drug. Plates were incubated at 35oC and
117
growth and resistance were evaluated at 24 and 48 h. In those cases where a large number of
118
mutants were obtained, only a representative set of colonies was tested. In the case of S.
119
aureus and S. pyogenes, colonies were also analyzed for production of hemolysin in blood
120
agar plates as it is known that S. aureus fmt mutants are non-hemolytic (20).
121
Genetic Characterization of Isolates with Reduced Susceptibility to GSK1322322. The
122
PCR primers used for DNA amplification of the pdf and fmt genes were designed from the
123
appropriate regions of the corresponding publically available genomic sequences using the
124
Lasergene PrimerSelect software. Although S. pneumoniae and S. pyogenes contain a second
125
pdf gene, def2, this was not amplified as it has been reported in the literature that def2 encodes
126
an inactive protein (21). In addition, PCR amplification of folD and glyA, encoding
127
bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase and serine
128
hydroxymethyltransferase, respectively, was also performed with selected S. aureus and S.
129
pneumoniae mutants. The PCR templates were prepared by boiling cells collected from
130
overnight plates. PCR products were purified using the QIAquick PCR purification kit, and
131
sequenced using the AB v3.1 BigDye-terminator cycle sequencing kit (Applied Biosystems).
132
Sequencing reactions were purified using the Performa® DTR V3 96-Well Short Plate Kit
133
(Edge Bio) and analyzed in a Genetic Analyzer 3730XL (Applied Biosystems). In order to
134
identify specific mutations responsible for the resistance phenotype, alignment of the DNA
135
sequences of the pdf, fmt, folD and glyA genes and promoter regions from mutant strains and
136
their corresponding parent organisms was carried out using Lasergene MegAlign Software
137
(DNASTAR).
7
138
Growth Curves of S. pyogenes Strains. S. pyogenes cells, grown on TSA with 5% sheep
139
blood at 35oC for approximately 20 h, were resuspended in 6 mL of THYB and adjusted to an
140
OD600 (1 cm path length) of 0.01. The cultures were incubated at 35oC without agitation and
141
growth was monitored at different time points by optical density at 600 nm and viable
142
bacterial counts.
143
Competitive Growth Studies of S. pneumoniae Parent and Mutant Strains. For the
144
preparation of early log phase cultures, a suspension equivalent to a 0.5 McFarland standard
145
from an overnight agar plate was diluted 50 fold into 4 mL of THYB and incubated for
146
approximately 4 h, shaking, in the presence of 5% CO2, to an OD600 of approximately 0.05.
147
Competitive growth studies were carried out in THYB media at 35oC in 5% CO2 with
148
continuous shaking, using initial inocula from early log phase cultures to give a 1:1 mutant to
149
wild-type ratio. At specific time points, samples were serially diluted and plated with and
150
without selective concentrations of GSK1322322 in order to calculate mutant and wild-type
151
CFU/mL as described above. At the end of each growth cycle (4 hours), cultures were diluted
152
into fresh media to start another cycle.
8
153
RESULTS
154
H. influenzae
155
Studies were performed to determine both the overall and the target-based FoR in four strains
156
of H. influenzae at 4X and 10X the GSK1322322 MIC. FoR was low in this species and
157
mutants with reduced susceptibility to GSK1322322 were not isolated in three of the four
158
strains tested, resulting in a FoR of 16-fold increase in the GSK1322322 MIC observed relative to
162
the parent strain.
163
S. aureus
164
Mutation in the S. aureus fmt gene,is associated with a number of phenotypic features which
165
include lack of hemolysin production, high level resistance to PDF inhibitors, a small colony
166
phenotype, and hypersensitivity to pleuromutilins (20). Therefore, in order to easily identify
167
FMT mutants, FoR studies were performed in four methicillin-resistant hemolytic S. aureus
168
strains. The overall FoR was high in all strains at both 4X and 10X the GSK1322322 MIC,
169
with similar frequencies ranging between 5 × 10-7 and 6 × 10-8 (Table 2). In order to
170
determine the target-based FoR, colony size, susceptibility to GSK1322322 and tiamulin, and
171
hemolytic phenotype were evaluated in a representative number of colonies. In every case,
172
non-hemolytic isolates were highly resistant to GSK1322322 (MICs >64 μg/mL) and
173
displayed a small colony phenotype, and were classified as FMT-like mutants, although not
174
all colonies were hypersensitive to tiamulin. Only FMT-like isolates were identified from the
175
resistance study with S. aureus X31360. DNA sequencing analysis of a number of these
9
176
isolates, including those not hypersensitive to tiamulin, confirmed that they carried mutations
177
in the fmt gene that should lead to inactive FMT proteins, with premature stop codons,
178
frameshifts, or amino acid substitutions known to affect its catalytic mechanism (26). Ten
179
isolates from S. aureus WCUH29 and one each from S. aureus PVL-1 and T6466 were
180
identified with a hemolytic phenotype, elevated GSK1322322 MICs, and wild-type tiamulin
181
MICs. Their genotypic characterization showed that most of them carried a mutation in
182
amino acid V59 of the PDF protein. The GSK1322322 MIC increased 32-fold in isolates
183
carrying mutation V59A and >64-fold in those with a PDF V59D substitution (Table 3).
184
Low-level resistance was associated with changes in the promoter region in two of the
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mutants, and one isolate had no obvious mutation in fmt, folD, glyA, or pdf genes, or in their
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promoter regions (Table 3). Resistance to GSK1322322 through target-based mutations
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occurred in these S. aureus strains at a frequency ranging from 2 × 10-8 to
