AAC Accepted Manuscript Posted Online 11 January 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.02981-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Colistin resistance caused by inactivation of the MgrB regulator is not associated with
2
decreased virulence of ST258 KPC carbapenemase-producing Klebsiella pneumoniae
3 4
Fabio Arena,a Lucia Henrici De Angelis,a Antonio Cannatelli,a Vincenzo Di Pilato,b Marina
5
Amorese,a Marco Maria D’Andrea,a Tommaso Giani a and Gian Maria Rossolini a,c,d,e*
6 7
Department of Medical Biotechnologies, University of Siena, Siena, Italya; Department of
8
Surgery and Translational Medicine, University of Florence, Florence, Italyb; Department of
9
Experimental and Clinical Medicine, University of Florence, Florence, Italyc; Clinical
10
Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italyd; Don
11
Carlo Gnocchi Foundation, Florence, Italye
12 13
Running Head: Virulence of colistin-resistant ST258 KPC K. pneumoniae
14 15 16
* Corresponding author: Gian Maria Rossolini, Dipartimento di Medicina Sperimentale e
17
Clinica, Università di Firenze, S.O.D. Microbiologia e Virologia, Azienda Ospedaliera-
18
Universitaria
19
[email protected]
Careggi,
Via
San
Damiano,
50134
Florence,
Italy.
E-mail:
20 21 22
1
23
Abstract
24
Using the Galleria mellonella animal model, we compared the virulence of two ST258 KPC-
25
producing K. pneumoniae strains, representative of the two clades of this clonal lineage, with
26
that of isogenic colistin-resistant mgrB mutants. With both strains, the mgrB mutants did not
27
exhibit modification in virulence. In the G. mellonella model, the clade 1 strain (capsular type
28
cps-1 [wzi29, producing KPC-2]) was significantly more virulent than the clade 2 strain
29
(capsular type cps-2 [wzi154, producing KPC-3]).
2
30
Colistin is a backbone component of combination antimicrobial regimens for serious
31
infections caused by carbapenemase-producing strains of Klebsiella pneumoniae (CP-KP) (1,
32
2), which are among the most challenging antibiotic-resistant pathogens spreading globally
33
(3, 4). However, in settings of CP-KP endemicity, the emergence and rapid dissemination of
34
colistin resistance has been reported, especially among KPC-type carbapenemase-producing
35
K. pneumoniae (KPC-KP), including those of the pandemic ST258 clonal lineage (5-9).
36
Colistin resistance is mostly due to modification of the antibiotic target (the lipid A moiety of
37
the bacterial lipopolysaccharide (LPS), which can be mediated by different mutational events
38
upregulating the endogenous LPS modification systems (10-14), or by the acquisition of
39
exogenous genes encoding LPS modification systems (15). Mutations causing loss of function
40
of the MgrB protein, a negative feedback regulator of the PhoP-PhoQ signal transduction
41
system that controls several biochemical pathways, including those involved in LPS
42
modification, are a frequent mechanism of acquired colistin resistance among KPC-KP
43
circulating in the clinical setting (14,16,17).
44
We previously showed that colistin resistance associated with inactivation of the mgrB gene is
45
not associated with a significant biological cost and is stably maintained in the absence of
46
selective pressure (18), a finding consistent with the rapid and efficient dissemination of
47
colistin-resistant (COL-R) KPC-KP carrying this resistance mechanism observed in the clinical
48
setting (7, 9).
49
In this work we investigated the impact of colistin resistance, mediated by mgrB inactivation,
50
on the virulence of KPC-KP strains representative of the two clades of the pandemic ST258
51
clonal lineage (19) using the Galleria mellonella infection model, which is considered a
52
validated model for testing virulence of K. pneumoniae (20, 21).
53
The KPC-KP strains used in the experiments are described in table 1. KK207-1 and KKBO-1
54
are two previously described strains, representative of the two clades of ST258 (clade 1 and
3
55
clade 2, respectively) (22). The mgrB insertional mutant of KKBO-1 (named mutKKBO-1) was
56
previously described (18), while the mgrB mutant of KK207-1 (named mutKK207-1) was
57
obtained with the same approach as mutKKBO-1 and carried an ISKpn18 insertion disrupting
58
the mgrB gene. Colistin MICs of the strains were determined by reference broth microdilution
59
(23).
60
For experiments in G. mellonella, bacteria were grown in LB broth aerobically at 37°C,
61
harvested during exponential phase (OD600≈0.7), and washed once with 10 mM phosphate-
62
buffered saline (PBS, pH 6.5). Bacteria were then suspended in PBS to an OD600 of 1.5,
63
corresponding approximately to 1 x 109 CFU/ml. Larvae weighing 450 to 600 mg were used
64
for the experiments. For comparative evaluation of virulence, groups of 10 larvae were
65
injected with 5 x 105 CFU of each strain and with sterile PBS as control. Larvae were kept at
66
37 °C in the dark, in humidified atmosphere, with food, and daily examined for pigmentation
67
and mobility. Time of death was recorded at 24, 48 and 72 hours. Three independent
68
experiments were performed. The 50 % lethal dose (LD50) was determined inoculating larvae
69
with 10-fold serial dilutions of each strain containing 5 x 104 to 5 x 107 CFU. Ten larvae were
70
injected with each dilution. For each strain, data from three independent experiments were
71
combined. LD50s were calculated using the GraphPad Prism 6.0 software (GraphPad Software
72
Inc., La Jolla, CA). LD50 values were compared using the two-tailed t test. P values of
1
Colistin resistance caused by inactivation of the MgrB regulator is not associated with
2
decreased virulence of ST258 KPC carbapenemase-producing Klebsiella pneumoniae
3 4
Fabio Arena,a Lucia Henrici De Angelis,a Antonio Cannatelli,a Vincenzo Di Pilato,b Marina
5
Amorese,a Marco Maria D’Andrea,a Tommaso Giani a and Gian Maria Rossolini a,c,d,e*
6 7
Department of Medical Biotechnologies, University of Siena, Siena, Italya; Department of
8
Surgery and Translational Medicine, University of Florence, Florence, Italyb; Department of
9
Experimental and Clinical Medicine, University of Florence, Florence, Italyc; Clinical
10
Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italyd; Don
11
Carlo Gnocchi Foundation, Florence, Italye
12 13
Running Head: Virulence of colistin-resistant ST258 KPC K. pneumoniae
14 15 16
* Corresponding author: Gian Maria Rossolini, Dipartimento di Medicina Sperimentale e
17
Clinica, Università di Firenze, S.O.D. Microbiologia e Virologia, Azienda Ospedaliera-
18
Universitaria
19
[email protected]
Careggi,
Via
San
Damiano,
50134
Florence,
Italy.
E-mail:
20 21 22
1
23
Abstract
24
Using the Galleria mellonella animal model, we compared the virulence of two ST258 KPC-
25
producing K. pneumoniae strains, representative of the two clades of this clonal lineage, with
26
that of isogenic colistin-resistant mgrB mutants. With both strains, the mgrB mutants did not
27
exhibit modification in virulence. In the G. mellonella model, the clade 1 strain (capsular type
28
cps-1 [wzi29, producing KPC-2]) was significantly more virulent than the clade 2 strain
29
(capsular type cps-2 [wzi154, producing KPC-3]).
2
30
Colistin is a backbone component of combination antimicrobial regimens for serious
31
infections caused by carbapenemase-producing strains of Klebsiella pneumoniae (CP-KP) (1,
32
2), which are among the most challenging antibiotic-resistant pathogens spreading globally
33
(3, 4). However, in settings of CP-KP endemicity, the emergence and rapid dissemination of
34
colistin resistance has been reported, especially among KPC-type carbapenemase-producing
35
K. pneumoniae (KPC-KP), including those of the pandemic ST258 clonal lineage (5-9).
36
Colistin resistance is mostly due to modification of the antibiotic target (the lipid A moiety of
37
the bacterial lipopolysaccharide (LPS), which can be mediated by different mutational events
38
upregulating the endogenous LPS modification systems (10-14), or by the acquisition of
39
exogenous genes encoding LPS modification systems (15). Mutations causing loss of function
40
of the MgrB protein, a negative feedback regulator of the PhoP-PhoQ signal transduction
41
system that controls several biochemical pathways, including those involved in LPS
42
modification, are a frequent mechanism of acquired colistin resistance among KPC-KP
43
circulating in the clinical setting (14,16,17).
44
We previously showed that colistin resistance associated with inactivation of the mgrB gene is
45
not associated with a significant biological cost and is stably maintained in the absence of
46
selective pressure (18), a finding consistent with the rapid and efficient dissemination of
47
colistin-resistant (COL-R) KPC-KP carrying this resistance mechanism observed in the clinical
48
setting (7, 9).
49
In this work we investigated the impact of colistin resistance, mediated by mgrB inactivation,
50
on the virulence of KPC-KP strains representative of the two clades of the pandemic ST258
51
clonal lineage (19) using the Galleria mellonella infection model, which is considered a
52
validated model for testing virulence of K. pneumoniae (20, 21).
53
The KPC-KP strains used in the experiments are described in table 1. KK207-1 and KKBO-1
54
are two previously described strains, representative of the two clades of ST258 (clade 1 and
3
55
clade 2, respectively) (22). The mgrB insertional mutant of KKBO-1 (named mutKKBO-1) was
56
previously described (18), while the mgrB mutant of KK207-1 (named mutKK207-1) was
57
obtained with the same approach as mutKKBO-1 and carried an ISKpn18 insertion disrupting
58
the mgrB gene. Colistin MICs of the strains were determined by reference broth microdilution
59
(23).
60
For experiments in G. mellonella, bacteria were grown in LB broth aerobically at 37°C,
61
harvested during exponential phase (OD600≈0.7), and washed once with 10 mM phosphate-
62
buffered saline (PBS, pH 6.5). Bacteria were then suspended in PBS to an OD600 of 1.5,
63
corresponding approximately to 1 x 109 CFU/ml. Larvae weighing 450 to 600 mg were used
64
for the experiments. For comparative evaluation of virulence, groups of 10 larvae were
65
injected with 5 x 105 CFU of each strain and with sterile PBS as control. Larvae were kept at
66
37 °C in the dark, in humidified atmosphere, with food, and daily examined for pigmentation
67
and mobility. Time of death was recorded at 24, 48 and 72 hours. Three independent
68
experiments were performed. The 50 % lethal dose (LD50) was determined inoculating larvae
69
with 10-fold serial dilutions of each strain containing 5 x 104 to 5 x 107 CFU. Ten larvae were
70
injected with each dilution. For each strain, data from three independent experiments were
71
combined. LD50s were calculated using the GraphPad Prism 6.0 software (GraphPad Software
72
Inc., La Jolla, CA). LD50 values were compared using the two-tailed t test. P values of
