AAC Accepts, published online ahead of print on 14 April 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.00038-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Importance of multi-drug efflux pumps in the antimicrobial resistance property of
2
clinical multi-drug resistant isolates of Neisseria gonorrhoeae
3
Daniel Golparian,a William M. Shafer,b,c Makoto Ohnishi,d Magnus Unemoa*
4
WHO Collaborating Centre for Gonorrhoea and other Sexually Transmitted Infections (STIs),
5
National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine,
6
Microbiology, Örebro University Hospital, Örebro, Swedena; Department of Microbiology
7
and Immunology, Emory University School of Medicine, Atlanta, Georgia, USAb;
8
Laboratories of Bacterial Pathogenesis, Veterans Affairs Medical Center, Decatur, Georgia,
9
USAc; National Institute of Infectious Diseases, Tokyo, Japand
10 11
* Corresponding author. Mailing address: WHO Collaborating Centre for Gonorrhoea and
12
other STIs, Department of Laboratory Medicine, Microbiology, Örebro University Hospital,
13
SE-701 85 Örebro, Sweden. Phone: +46 (19) 602 1520. Fax: +46 (19) 127 416. E-mail:
14
[email protected].
15
The work was performed at the WHO Collaborating Centre for Gonorrhoea and other STIs,
16
National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine,
17
Microbiology, Örebro University Hospital, SE-701 85 Örebro, Sweden.
18
Key words: Gonorrhea; antimicrobial resistance; ceftriaxone; cefixime; efflux pump; MtrC-
19
MtrD-MtrE; MacA-MacB; NorM; treatment; efflux pump inhibitor
20
Word count: Abstract: 75; Text: 1027
21
Running title: INACTIVATION OF MtrC-MtrD-MtrE PUMP REVERTS RESISTANCE
22 23
1
24
The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae
25
that express extensively- or multi-drug resistant phenotypes has heretofore not been
26
examined. Accordingly, we assessed the consequence on antimicrobial resistance due to
27
loss of the three gonococcal efflux pumps associated with known capacity to export
28
antimicrobials (MtrC-MtrD-MtrE, MacA-MacB and NorM) in such clinical isolates. We
29
report that the MIC of several antimicrobials including seven previously and currently
30
recommended for treatment was significantly impacted.
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
2
48
Gonorrhea remains a major public health concern worldwide (1). Neisseria gonorrhoeae has
49
developed resistance to all antimicrobials introduced for treatment (2-5). Recently, treatment
50
failures with the extended-spectrum cephalosporins (ESCs) cefixime and ceftriaxone were
51
verified in several countries and some extensively-drug resistant (XDR) gonococcal strains,
52
displaying high-level ceftriaxone resistance, described (3,6-13). ESCs are the last options for
53
antimicrobial monotherapy of gonorrhea. Thus, novel antimicrobials or non-antimicrobial
54
compounds for sustained therapy of gonorrhea are essential.
55
The mechanisms of ESC resistance involve specific modifications of the target penicillin
56
binding protein 2 (PBP2), PorB1b resulting in decreased intake, and increased efflux by an
57
over-expressed MtrC-MtrD-MtrE efflux pump (3,5,7,12,14-19). The MtrC-MtrD-MtrE efflux
58
pump belongs to the family of Resistance/Nodulation/Division (RND) pumps and is
59
functionally and structurally similar to the AcrAB-TolC efflux pump in Escherichia coli and
60
MexA-MexB-OprM in Pseudomonas aeruginosa. All these three pumps contribute to
61
bacterial resistance to antimicrobials including penicillin (20) and macrolides (21). Gonococci
62
harbor two additional efflux pumps that are known to extrude antimicrobials, i.e., the MacA-
63
MacB and NorM pumps that exports macrolides (22) and fluoroquinolones (23), respectively.
64
However, detailed information regarding the potential export of ESCs and other
65
antimicrobials, especially by multi-resistant clinical isolates through those gonococcal efflux
66
pumps is limited.
67
Herein, we have genetically inactivated the MtrC-MtrD-MtrE, MacA-MacB and NorM
68
efflux pumps in XDR and multidrug-resistant (MDR) gonococcal isolates and measured the
69
effects on susceptibility to previously or currently recommended antimicrobials for treatment
70
of gonorrhea.
71
The isolates examined were the WHO reference strains WHO F, considered to be a wild-
72
type strain susceptible to all included antimicrobials, and WHO P (24), four isolates from
3
73
ESC treatment failures, including the first XDR strain (strain H041) with high-level
74
ceftriaxone resistance (6,10,12), one isolate displaying high-level azithromycin resistance
75
(25), and three isolates displaying low-level azithromycin resistance. All strains except WHO
76
F contained mtrR mutations known to increase the expression of mtrCDE (21,26) and
77
possessed the mtrCDE-, macAB- and norM-encoded pumps. None of the strains produced β-
78
lactamase or contained any erm genes (data not presented). The MICs (µg/ml) of seven
79
antimicrobials, pre- and post-inactivation of the efflux pump(s), were determined using the
80
Etest method (bioMérieux, Solna, Sweden) on Difco GC Medium Base agar (BD,
81
Diagnostics, Sparks, MD, USA) supplemented with 1% IsoVitalex (BD Diagnostics),
82
according to the manufacturer’s instructions (Table 1 and 2). The mtrD, macA and norM
83
genes were inactivated (IA) in the isolates by spot transformation (27) using 0.1 µg of
84
chromosomal DNA from genetic derivatives of strain FA19 or wild-type strain FA19 bearing
85
insertions in mtrD (strain KH14; mtrD::kan), macA (strain BR54 macA::spc) or norM (strain
86
FA19 norM::kan). Transformants resistant to kanamycin (100 µg/ml) or spectinomycin (60
87
µg/ml) were selected as previously described (22,23,28). MIC differences were considered as
88
significant if they were greater than a two-fold change in the mutants compared to parent
89
strain. Inactivation of efflux pump genes in representative transformants was confirmed using
90
PCR and pump gene-specific oligonuclucleotide primers as described (22,23,28).
91
Loss of the MtrC-MtrD-MtrE pump had the highest impact on the XDR phenotype of
92
H041 (Table 1) and on MDR phenotypes (Table 2). The H041-IAMtrCDE transformant showed
93
significantly increased susceptibility to penicillin, ceftriaxone, azithromycin, tetracycline and
94
solithromycin. The H041-IAMacAB transformant had a 4- and 2-fold increased susceptibility to
95
azithromycin and ceftriaxone, respectively, while the IANorM transformant showed
96
significantly (>32-fold) increased solithromycin susceptibility. Loss of multiple pumps did
4
97
not significantly increase susceptibility compared to mutants lacking only MtrC-MtrD-MtrE
98
(Table 1).
99
Loss of the MtrC-MtrD-MtrE pump in the three clinical ESC-resistant isolates
100
significantly increased susceptibility to ESCs, penicillin, azithromycin, ciprofloxacin, and
101
solithromycin (Table 2). Loss of the MacA-MacB or NorM pump in these strains did not
102
consistently and significantly change the MICs of antimicrobials. Since azithromycin is used
103
in the dual therapy with ceftriaxone recommended in USA (29) and Europe (30), we
104
examined four strains with low-level azithromycin resistance (MIC=2-8 µg/ml) and one strain
105
with high-level azithromycin resistance (MIC=4096 µg/ml). For all four strains with low-level
106
resistance, the susceptibility to azithromycin increased by 8-32-fold due to a loss of the MtrC-
107
MtrD-MtrE pump, whereas loss of the MacA-MacB or NorM pump had no significant effect
108
on azithromycin resistance. Similarly, loss of the MtrC-MtrD-MtrE pump in the high-level
109
resistant strain (MIC=4096 µg/ml) decreased susceptibility to azithromycin by five-fold
110
(Table 2).
111
In gonococci, the main focus of past research regarding efflux pump-mediated
112
antimicrobial resistance has been on the MtrC-MtrD-MtrE pump, which can be over-
113
expressed due to specific mutations in mtrR (promoter or coding sequence) that encodes the
114
repressor MtrR (16,17,20,31-33). Over-expression is known to affect the susceptibility of
115
macrolides, penicillins and ESCs (3,5,15,20,32). Herein, we showed that the susceptibility to
116
also ciprofloxacin and solithromycin can be affected. The MtrC-MtrD-MtrE pump also
117
exports fatty acids, bile salts and endogenous antibacterial peptides and, accordingly, an over-
118
expression might cause an enhanced fitness of the gonococcal strains (34-36). The MacA-
119
MacB and NorM pumps are known to export macrolides (22) and fluoroquinolones (23),
120
respectively. We also showed that ESCs, penicillin and solithromycin might be exported by
121
NorM and/or MacA-MacB. However, since β-lactam antimicrobials and solithromycin are not
5
122
previously described to be substrates of the MacA-MacB or NorM efflux pumps (31), we can
123
not discount the possibility that loss of these pumps have secondary effects that increase cell
124
envelope permeability to these antimicrobials.
125
In conclusion, we found that inactivation of efflux pumps in antimicrobial resistant
126
clinical gonococcal isolates influenced susceptibility to many antimicrobials, even reverting
127
strains to clinical susceptibility according to MIC breakpoints. This was most notable for the
128
MtrC-MtrD-MtrE pump in H041, the first clinical XDR isolate, with high-level resistance to
129
ceftriaxone, obtained from a ceftriaxone treatment failure (12). However, this was also
130
observed for many MDR clinical gonococcal strains (6,10,24,25), including strains that
131
resulted in ESC treatment failures. Accordingly, a specific efflux pump inhibitor (EPI) of the
132
gonococcal
133
antimicrobials, might be a future treatment option for gonorrhea. This could be a novel
134
approach to retain antimicrobials for longer use, e.g. ceftriaxone, even restore antimicrobials
135
no longer in use due to high-level resistance, e.g. penicillin, and that could also mitigate the
136
resistance emergence. Nevertheless, many challenges remain in the development of a
137
gonococcal EPI, e.g. selection of ideal target and avoidance of toxicity on human cells (37-
138
39).
MtrC-MtrD-MtrE
pump,
particularly
co-administered
with
appropriate
139 140
ACKNOWLEDGMENTS
141
This study was supported by grants from the Research Committee of Örebro County and the
142
Örebro University Hospital Research Foundation, Örebro, Sweden (M.U.), NIH grants R37
143
AI021150 and U19 AI031496, and a VA Merit Award from the Medical Research Service of
144
the Department of Veterans Affairs (W.M.S.). W.M.S. is the recipient of a Senior Research
145
Career Scientist Award from the Medical Research Service of the Department of Veterans
146
Affairs.
6
147 148
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12
276
TABLE
1
MICs
(µg/ml)
of
the
XDR
strain
H041
(Pre-IA;
12),
with
high-level
ceftriaxone
resistance,
277
of 7 antimicrobials after loss of the efflux pumps MtrC-MtrD-MtrE, MacA-MacB and NorM, inactivated separately and in all possible
278
combinations. Ceftriaxone (SIR category)
Cefixime (SIR category)
Penicillin (SIR category)
Azithromycin (SIR category)
Ciprofloxacin (SIR category)
Tetracycline (SIR category)
Solithromycin (SIR category)
Pre-IA
4 (R)
8 (R)
4 (R)
1 (R)
>32 (R)
4 (R)
0.064 (NA)
IAMtrCDE
1 (R)
4 (R)
0.5 (I)
0.064 (S)
16 (R)
1 (I)
0.004 (NA)
IAMacAB
2 (R)
4 (R)
2 (R)
0.25 (S)
>32 (R)
2 (R)
0.125 (NA)
>32 (R)
0.5 (I)
1
Importance of multi-drug efflux pumps in the antimicrobial resistance property of
2
clinical multi-drug resistant isolates of Neisseria gonorrhoeae
3
Daniel Golparian,a William M. Shafer,b,c Makoto Ohnishi,d Magnus Unemoa*
4
WHO Collaborating Centre for Gonorrhoea and other Sexually Transmitted Infections (STIs),
5
National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine,
6
Microbiology, Örebro University Hospital, Örebro, Swedena; Department of Microbiology
7
and Immunology, Emory University School of Medicine, Atlanta, Georgia, USAb;
8
Laboratories of Bacterial Pathogenesis, Veterans Affairs Medical Center, Decatur, Georgia,
9
USAc; National Institute of Infectious Diseases, Tokyo, Japand
10 11
* Corresponding author. Mailing address: WHO Collaborating Centre for Gonorrhoea and
12
other STIs, Department of Laboratory Medicine, Microbiology, Örebro University Hospital,
13
SE-701 85 Örebro, Sweden. Phone: +46 (19) 602 1520. Fax: +46 (19) 127 416. E-mail:
14
[email protected].
15
The work was performed at the WHO Collaborating Centre for Gonorrhoea and other STIs,
16
National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine,
17
Microbiology, Örebro University Hospital, SE-701 85 Örebro, Sweden.
18
Key words: Gonorrhea; antimicrobial resistance; ceftriaxone; cefixime; efflux pump; MtrC-
19
MtrD-MtrE; MacA-MacB; NorM; treatment; efflux pump inhibitor
20
Word count: Abstract: 75; Text: 1027
21
Running title: INACTIVATION OF MtrC-MtrD-MtrE PUMP REVERTS RESISTANCE
22 23
1
24
The contribution of drug efflux pumps in clinical isolates of Neisseria gonorrhoeae
25
that express extensively- or multi-drug resistant phenotypes has heretofore not been
26
examined. Accordingly, we assessed the consequence on antimicrobial resistance due to
27
loss of the three gonococcal efflux pumps associated with known capacity to export
28
antimicrobials (MtrC-MtrD-MtrE, MacA-MacB and NorM) in such clinical isolates. We
29
report that the MIC of several antimicrobials including seven previously and currently
30
recommended for treatment was significantly impacted.
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
2
48
Gonorrhea remains a major public health concern worldwide (1). Neisseria gonorrhoeae has
49
developed resistance to all antimicrobials introduced for treatment (2-5). Recently, treatment
50
failures with the extended-spectrum cephalosporins (ESCs) cefixime and ceftriaxone were
51
verified in several countries and some extensively-drug resistant (XDR) gonococcal strains,
52
displaying high-level ceftriaxone resistance, described (3,6-13). ESCs are the last options for
53
antimicrobial monotherapy of gonorrhea. Thus, novel antimicrobials or non-antimicrobial
54
compounds for sustained therapy of gonorrhea are essential.
55
The mechanisms of ESC resistance involve specific modifications of the target penicillin
56
binding protein 2 (PBP2), PorB1b resulting in decreased intake, and increased efflux by an
57
over-expressed MtrC-MtrD-MtrE efflux pump (3,5,7,12,14-19). The MtrC-MtrD-MtrE efflux
58
pump belongs to the family of Resistance/Nodulation/Division (RND) pumps and is
59
functionally and structurally similar to the AcrAB-TolC efflux pump in Escherichia coli and
60
MexA-MexB-OprM in Pseudomonas aeruginosa. All these three pumps contribute to
61
bacterial resistance to antimicrobials including penicillin (20) and macrolides (21). Gonococci
62
harbor two additional efflux pumps that are known to extrude antimicrobials, i.e., the MacA-
63
MacB and NorM pumps that exports macrolides (22) and fluoroquinolones (23), respectively.
64
However, detailed information regarding the potential export of ESCs and other
65
antimicrobials, especially by multi-resistant clinical isolates through those gonococcal efflux
66
pumps is limited.
67
Herein, we have genetically inactivated the MtrC-MtrD-MtrE, MacA-MacB and NorM
68
efflux pumps in XDR and multidrug-resistant (MDR) gonococcal isolates and measured the
69
effects on susceptibility to previously or currently recommended antimicrobials for treatment
70
of gonorrhea.
71
The isolates examined were the WHO reference strains WHO F, considered to be a wild-
72
type strain susceptible to all included antimicrobials, and WHO P (24), four isolates from
3
73
ESC treatment failures, including the first XDR strain (strain H041) with high-level
74
ceftriaxone resistance (6,10,12), one isolate displaying high-level azithromycin resistance
75
(25), and three isolates displaying low-level azithromycin resistance. All strains except WHO
76
F contained mtrR mutations known to increase the expression of mtrCDE (21,26) and
77
possessed the mtrCDE-, macAB- and norM-encoded pumps. None of the strains produced β-
78
lactamase or contained any erm genes (data not presented). The MICs (µg/ml) of seven
79
antimicrobials, pre- and post-inactivation of the efflux pump(s), were determined using the
80
Etest method (bioMérieux, Solna, Sweden) on Difco GC Medium Base agar (BD,
81
Diagnostics, Sparks, MD, USA) supplemented with 1% IsoVitalex (BD Diagnostics),
82
according to the manufacturer’s instructions (Table 1 and 2). The mtrD, macA and norM
83
genes were inactivated (IA) in the isolates by spot transformation (27) using 0.1 µg of
84
chromosomal DNA from genetic derivatives of strain FA19 or wild-type strain FA19 bearing
85
insertions in mtrD (strain KH14; mtrD::kan), macA (strain BR54 macA::spc) or norM (strain
86
FA19 norM::kan). Transformants resistant to kanamycin (100 µg/ml) or spectinomycin (60
87
µg/ml) were selected as previously described (22,23,28). MIC differences were considered as
88
significant if they were greater than a two-fold change in the mutants compared to parent
89
strain. Inactivation of efflux pump genes in representative transformants was confirmed using
90
PCR and pump gene-specific oligonuclucleotide primers as described (22,23,28).
91
Loss of the MtrC-MtrD-MtrE pump had the highest impact on the XDR phenotype of
92
H041 (Table 1) and on MDR phenotypes (Table 2). The H041-IAMtrCDE transformant showed
93
significantly increased susceptibility to penicillin, ceftriaxone, azithromycin, tetracycline and
94
solithromycin. The H041-IAMacAB transformant had a 4- and 2-fold increased susceptibility to
95
azithromycin and ceftriaxone, respectively, while the IANorM transformant showed
96
significantly (>32-fold) increased solithromycin susceptibility. Loss of multiple pumps did
4
97
not significantly increase susceptibility compared to mutants lacking only MtrC-MtrD-MtrE
98
(Table 1).
99
Loss of the MtrC-MtrD-MtrE pump in the three clinical ESC-resistant isolates
100
significantly increased susceptibility to ESCs, penicillin, azithromycin, ciprofloxacin, and
101
solithromycin (Table 2). Loss of the MacA-MacB or NorM pump in these strains did not
102
consistently and significantly change the MICs of antimicrobials. Since azithromycin is used
103
in the dual therapy with ceftriaxone recommended in USA (29) and Europe (30), we
104
examined four strains with low-level azithromycin resistance (MIC=2-8 µg/ml) and one strain
105
with high-level azithromycin resistance (MIC=4096 µg/ml). For all four strains with low-level
106
resistance, the susceptibility to azithromycin increased by 8-32-fold due to a loss of the MtrC-
107
MtrD-MtrE pump, whereas loss of the MacA-MacB or NorM pump had no significant effect
108
on azithromycin resistance. Similarly, loss of the MtrC-MtrD-MtrE pump in the high-level
109
resistant strain (MIC=4096 µg/ml) decreased susceptibility to azithromycin by five-fold
110
(Table 2).
111
In gonococci, the main focus of past research regarding efflux pump-mediated
112
antimicrobial resistance has been on the MtrC-MtrD-MtrE pump, which can be over-
113
expressed due to specific mutations in mtrR (promoter or coding sequence) that encodes the
114
repressor MtrR (16,17,20,31-33). Over-expression is known to affect the susceptibility of
115
macrolides, penicillins and ESCs (3,5,15,20,32). Herein, we showed that the susceptibility to
116
also ciprofloxacin and solithromycin can be affected. The MtrC-MtrD-MtrE pump also
117
exports fatty acids, bile salts and endogenous antibacterial peptides and, accordingly, an over-
118
expression might cause an enhanced fitness of the gonococcal strains (34-36). The MacA-
119
MacB and NorM pumps are known to export macrolides (22) and fluoroquinolones (23),
120
respectively. We also showed that ESCs, penicillin and solithromycin might be exported by
121
NorM and/or MacA-MacB. However, since β-lactam antimicrobials and solithromycin are not
5
122
previously described to be substrates of the MacA-MacB or NorM efflux pumps (31), we can
123
not discount the possibility that loss of these pumps have secondary effects that increase cell
124
envelope permeability to these antimicrobials.
125
In conclusion, we found that inactivation of efflux pumps in antimicrobial resistant
126
clinical gonococcal isolates influenced susceptibility to many antimicrobials, even reverting
127
strains to clinical susceptibility according to MIC breakpoints. This was most notable for the
128
MtrC-MtrD-MtrE pump in H041, the first clinical XDR isolate, with high-level resistance to
129
ceftriaxone, obtained from a ceftriaxone treatment failure (12). However, this was also
130
observed for many MDR clinical gonococcal strains (6,10,24,25), including strains that
131
resulted in ESC treatment failures. Accordingly, a specific efflux pump inhibitor (EPI) of the
132
gonococcal
133
antimicrobials, might be a future treatment option for gonorrhea. This could be a novel
134
approach to retain antimicrobials for longer use, e.g. ceftriaxone, even restore antimicrobials
135
no longer in use due to high-level resistance, e.g. penicillin, and that could also mitigate the
136
resistance emergence. Nevertheless, many challenges remain in the development of a
137
gonococcal EPI, e.g. selection of ideal target and avoidance of toxicity on human cells (37-
138
39).
MtrC-MtrD-MtrE
pump,
particularly
co-administered
with
appropriate
139 140
ACKNOWLEDGMENTS
141
This study was supported by grants from the Research Committee of Örebro County and the
142
Örebro University Hospital Research Foundation, Örebro, Sweden (M.U.), NIH grants R37
143
AI021150 and U19 AI031496, and a VA Merit Award from the Medical Research Service of
144
the Department of Veterans Affairs (W.M.S.). W.M.S. is the recipient of a Senior Research
145
Career Scientist Award from the Medical Research Service of the Department of Veterans
146
Affairs.
6
147 148
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149
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276
TABLE
1
MICs
(µg/ml)
of
the
XDR
strain
H041
(Pre-IA;
12),
with
high-level
ceftriaxone
resistance,
277
of 7 antimicrobials after loss of the efflux pumps MtrC-MtrD-MtrE, MacA-MacB and NorM, inactivated separately and in all possible
278
combinations. Ceftriaxone (SIR category)
Cefixime (SIR category)
Penicillin (SIR category)
Azithromycin (SIR category)
Ciprofloxacin (SIR category)
Tetracycline (SIR category)
Solithromycin (SIR category)
Pre-IA
4 (R)
8 (R)
4 (R)
1 (R)
>32 (R)
4 (R)
0.064 (NA)
IAMtrCDE
1 (R)
4 (R)
0.5 (I)
0.064 (S)
16 (R)
1 (I)
0.004 (NA)
IAMacAB
2 (R)
4 (R)
2 (R)
0.25 (S)
>32 (R)
2 (R)
0.125 (NA)
>32 (R)
0.5 (I)
