AAC Accepted Manuscript Posted Online 20 April 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00092-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Anti-biofilm peptides increase the susceptibility of carbapenemase-
2
producing Klebsiella pneumoniae clinical isolates to β-lactam
3
antibiotics
4
Suzana Meira Ribeiroa, César de la Fuente-Núñezb,BeverlieBaquirb, Celio Faria-Juniorc, Octávio
5
L. Francoa#andRobert. E.W.Hancockb#
6 7 8 9 10
a
Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises
Proteômicas e Bioquímicas, Universidade Católica de Brasília, Brazil. b
Centre for Microbial Diseases and Immunity Research, Department of Microbiology and
Immunology, University of British Columbia, Vancouver, British Columbia, Canada. c
LACEN, Laboratório Central de Saúde Pública do Distrito Federal, Brasília, DF, Brazil
11
Running Head: Effect of anti-biofilm peptides on K. pneumoniae biofilms
12
#Address correspondence to Robert E. W. Hancock, [email protected] or Octávio L. Franco,
13
[email protected]
14
Phone number:+1 (604) 822-2682; Fax: 604-827-5566
15 16 17 18 19 20
1
21
ABSTRACT
22
Multi-drug resistant carbapenemase-producing Klebsiella pneumoniae (KpC)are becoming
23
acommon cause of infections in health care centers.Furthermore,Klebsiellacan develop
24
multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe
25
the antimicrobial and anti-biofilm activities of synthetic peptides DJK-5, DJK-6 and 1018
26
against five KpC isolates.Using static microplate assays,it was observed that the concentration
27
required to prevent biofilm formation by these clinical isolates was below theminimum
28
inhibitory concentration (MIC) for planktonic cells. More sophisticated flow cell experiments
29
confirmed the anti-biofilm activity of the peptides against 2 day-old biofilms of different
30
KpCisolates and, in some cases, the peptides inducedsignificant biofilm cell death.Clinically
31
relevant combinations of DJK-6 togetherwith β-lactam antibiotics, including the carbapenem
32
meropenem, alsoprevented planktonic growth and biofilm formation of KpC strain1825971.
33
Interestingly, peptide DJK-6 was able to enhance,by at least 16-fold, the ability of meropenem to
34
eradicate pre-formedbiofilms formed by this strain. Using peptide DJK-6 to potentiate the
35
activity
36
treatinfectionscaused by KpCisolates.
37
Keywords: small cationic peptides, biofilms, carbapenemase-producing K. pneumoniae.
38
INTRODUCTION
of
β-lactams
including
meropenem
represents
a
promising
strategy
to
39
Klebsiella pneumoniae is a Gram-negative, encapsulated, opportunisticpathogen of
40
theFamilyEnterobacteriaciae(1). It has become a significant cause of infections,especially
41
pneumonia, but also meningitis,sepsis, urinary tract,device-associated and surgical wound-
42
siteinfections(2–7).The most severe clinical cases of such infections are often associated with 2
43
K.pneumoniae that produce a plasmid-borne carbapenemase(KpC).
44
The rate of fatalityfrom KpC-producing K. pneumoniaeis high (>50%) among patients with
45
bloodstream infection(2, 8, 9).The treatment of these infections is difficult since KpCstrains are
46
resistant to all β-lactam antibiotics, including carbapenemsthat are typically used as a last resort
47
(10). In addition, bacteria harboringcarbapenemase genes are often resistant to other antibiotics.
48
As a consequence of this multi-drug resistance, failure in treatment contributes to increasedrates
49
of morbidity and mortality (9, 11, 12). Tigecycline, aminoglycosides and the polymyxins
50
(polymyxin B and colistin) are currently the best treatment options for carbapenemase-producing
51
strains(13). However, these strains also have the potential to develop resistance to these
52
antibiotics(14–16).
53
The treatment of infections caused by multi-drug resistant bacteria is a challengeand
54
theability of bacterial pathogens to form biofilms can further complicate this scenario. Biofilms,
55
a distinct microbial lifestyle of bacteria,aresurface-associated multicellular communities of
56
bacteriaencased in a protective polymeric matrix that can include polysaccharides, proteins and
57
DNA(17). They represent a substantial problem in the clinic, as theycan lead to the development
58
of chronic long-lived infections on body surfaces including the lung, skin, heart and bones (18–
59
21), and can also be formedon medical devices (central venous catheters, urinary tract catheters,
60
mechanical heart valve, contact lenses, endotracheal tube and implants)(18, 22–25).
61
Biofilms arehighly (adaptively) resistant to antibiotics as well as removal by host immune
62
defense mechanisms and thus are very difficult to eradicatewhen compared to their planktonic
63
(free-swimming) counterparts, leading to increased health care costs(26). Although biofilms have
64
a significant impact in public health, most research to date has focused on developing natural or
65
synthetic antimicrobial agentsthat inhibit planktonic bacteria. Cationic amphipathicantimicrobial
3
66
peptideshave emerged in recent years as an alternative strategy for the treatment of antibiotic-
67
resistant bacteria(27). Other so-called host defence peptidesfunction as immunomodulatory
68
agentswithanti-infective, anti-inflammatory and wound healing properties, independent of direct
69
antimicrobial activity(27).
70
Recently, it was shown that some cationic amphipathic peptidescould prevent and/or
71
eradicate bacterial biofilms in a manner that was independent of direct antimicrobial activity
72
against free swimming bacteria(28–30). Others studiesdemonstrated thattheanti-biofilmpeptides
73
1018 (a synthetic variant of host defence peptides), DJK-5 and DJK-6(both, designed D-
74
enantiomeric protease resistant peptides) had the ability to work synergistically with several
75
conventional antibiotics, thus uncovering a novel approach to potentiating antibiotic action
76
against bacterial biofilms(31, 32). Based on these studies, we hypothesizedthat the small cationic
77
anti-biofilm peptides 1018, DJK-5 and DJK-6could both prevent biofilm formation and target
78
pre-formed biofilmsformed by carbapenemase-producingK.pneumoniae. The objective of this
79
study was to investigate the biofilm-forming ability of KpC clinical strains isolated from patients
80
atBrasilia Hospital, and to test the activity of anti-biofilm peptides against these isolates.
81 82
MATERIALS AND METHODS
83
Bacterial strains.
84
Clinical isolates of K. pneumoniawere obtainedfrom hospitalized patients in Brasília, Brazil,
85
between 2010 and 2012. The isolate 1825971 was obtainedfromendotracheal aspirate and the
86
isolates 1789769, 2144392, 2210477 and 1450421 wereobtained from blood. The isolates were
87
suspended in Mueller Hinton broth and incubated for 18-24 h at 36ºC. They were confirmed as
88
Klebsiella pneumoniae strains using the MicroScanWalkAwayautomated system (Siemens 4
89
Healthcare Diagnostics), according to the manufacturer´s instructions. Standard biochemical
90
tests were also used to confirm the species(33). Isolates were preservedat -70ºC with the addition
91
of 8% dimethyl sulphoxide.
92
Peptide Synthesis.
93
DJK-5 (VQWRAIRVRVIR-NH2) and DJK-6 (VQWRRIRVWVIR-NH2), both D-
94
enantiomeric peptides, and the peptide 1018 (VRLIVAVRIWRR-NH2) were synthesized by CPC
95
Scientific using solid-phase 9-fluorenylmethoxy carbonyl (Fmoc) chemistry and purified to 95%
96
using reverse-phase high-performance liquid chromatography (HPLC). Peptide mass was
97
confirmed by mass spectrometry.
98 99 100 101
Antibiotic susceptibility of K.pneumoniae clinical isolates, screening for carbapenemases and detection of blaKPC genes. Susceptibility
testing
was
performed
susceptibility breakpoints
using
the
MicroScanWalkAwayautomated
102
method. The
103
protocols(34).Resistance to carbapenem antibiotics was due tothe production of a
104
carbapenemase, which was detected using the modified Hodge test(35). K. pneumoniae ATCC
105
700603 was used as negative control andK. pneumoniaeIOC (carbapenemase-producing strain
106
from the culture collectionof the Oswaldo Cruz Institute, Rio De Janeiro, Brazil) was used as a
107
positive control. Specific primers were used under standard polymerase chain reaction (PCR)
108
conditions to detect and confirm the presence ofgenes encoding K. pneumoniae
109
carbapenemase,blaKPC
110
CTCAGTGCTCTACAGAAAACC)(36). The strains used as controls for the modified Hodge
111
test were also used as negative and positive controls for PCR.
genes,
were interpreted in accordance to the
(F,5’-TGTCACTGTATCGCCGTC
andR,
CLSI
5’-
5
112 113
Biofilm formation and minimal inhibitory concentration of planktonic cells and biofilms.
114
K. pneumoniae biofilm formation was assessed in both rich mediaLuria-Bertani (LB) broth
115
(BD Difco) and Todd Hewitt Broth (BD Difco) and in BM2 minimal medium (62 mM potassium
116
phosphate buffer, pH 7.0, 7 mM (NH4)2SO4, 2 mM MgSO4, 10 μM FeSO4, 0.5% glucose).
117
Overnight cultures of K. pneumoniae strains grown in LB broth were diluted 1 in 100 in each of
118
these mediaand 100 μL per well added into 96-well round bottom microplates. Planktonic cell
119
growth was assessed by measuring absorbance at 600 nm.Non-adherent planktonic cells were
120
removed from microtitre wells and washed twice with deionized water. Adherent cells were
121
stained with 0.01% crystal violet for 20 min. The microplate wells were washed twice with
122
deionized water, air-dried and solubilized with 110 μlof 70% ethanol. Biofilm formation was
123
assessedby measuring the absorbance at 595 nm using a microplate reader (Bio-Tek Instruments,
124
Inc., United States). Three biological replicates were performed for eachexperimentinvolving
125
eighttechnicalreplicates.Afterestablishing the bestconditions for biofilm growth, antimicrobial
126
peptideswere
127
methodforcationic peptides(37)was used to determine theMinimal Inhibitory Concentration
128
(MIC)for planktonic cellsand the minimal biofilminhibitory concentration(MBIC) to inhibit
129
biofilm formation by50% (MBIC50) or 100% (MBIC100).
evaluated
fortheir
ability
toinhibit
biofilm
formation.Themicrodilution
130 131
Checkerboard assay.
132
Synergy between DJK-6 and various antibiotics was assessedbycheckerboard titration in 96-
133
well microplates, wherein the peptide was diluted along the rows of a microtiter tray and the
134
antibiotics were diluted along the columns. Planktonic growth and biofilm formationwas 6
135
accessed as described above. The resultswere expressed as the fractional inhibitory concentration
136
index(FIC), which is determined as follows: FIC = [A]/MICA + [B]/MICB, where MICA and
137
MICB are the MICs of antibiotic A and peptide B alone and [A] and [B] are the MICs of A and B
138
when in combination. The FIC index was interpreted as follows: FIC16/8
S R R
16 >16/8
S R R
32 >16 >16/8
I R R
16 >16/8
S R R
32 >16 >16/8
I R R
>16 >16 >32 >32 >16 >32 >16 >16 >2 >4 >8 >8 >4 >8 >64 >64 >64
R R R S R R R R R R R R R R R R R
>16 >16 >32 >32 >16 >32 >16 >16 >2 >4 >8 >8 8 >64 >64 >64
R R R R R R R R R R R R S R R R R
>16 >16 >32 >32 16 >32 >16 >16 >2 >4 >8 >8 4 >8 64 >64 >64
R R R R R R R R R R R R I R I R R
>16 >16 >32 32 8 >32 >16 >16 >2 >4 >8 >8 >4 8 >64 >64 >64
R R R I I R R R R R R R R R R R R
>16 >16 >32 32 >16 >32 >16 >16 >2 >4 >8 >8 >4 >8 >64 >64 >64
R R R R R R R R R R R R R R R R R
>8 2/38
R S I R
8 8 2/38
I S R R
2/38
R S R R
447 448 449 450 21
451
Table 2. Assessment of biofilm formation of KPC-producing K. pneumoniae isolates in different
452
media. The assay examined adherence to the surfaces of the wells of 96-well microtiter plates.
453
After incubation for 24 h at 37°C, the growth of planktonic cells was measured as the absorbance
454
at 600 nm (A600) while biofilm growth was assessed at 595 nm (A595) after crystal violet staining
455
and ethanol extraction. KpC isolates 1450421 1789769 1825971 2144392 2210477
Luria Broth Planktonic Biofilm
Growth Todd Hewitt Broth Planktonic Biofilm
BM2 glucose Planktonic Biofilm
1.29 ± 0.04 1.18 ± 0.05 1.30 ± 0.06 1.20 ± 0.05 1.25 ± 0.07
1.48 ± 0.04 1.53± 0.03 1.51 ± 0.04 1.52 ± 0.03 1.46 ± 0.05
1.17 ± 0.06 1.22 ± 0.06 1.15 ± 0.04 1.16 ± 0.06 1.13 ± 0.01
0.20 ± 0.05 0.05 ± 0.03 0.27 ± 0.06 0.02± 0.01 0.15± 0.03
0.17 ± 0.03 0.11 ± 0.02 0.12 ± 0.02 0.04 ± 0.02 0.14± 0.06
1.28 ± 0.11 0.80 ± 0.07 0.79 ± 0.02 0.62 ± 0.04 1.18 ± 0.04
456 457
Table 3. Evaluation of the antimicrobial activity of peptides against planktonic and biofilm cells
458
of KPC-producing K. pneumoniae isolates using microtiter assays. Shown is the MIC for
459
planktonic cells and MBIC50 and MBIC100 for preventing 50% or 100% of biofilm formation.
460 KpC isolates 1450421 1789769 1825971 2144392 2210477
IDR 1018 (µg.mL-1) MIC MBIC50 MBIC100
MIC
32 32 >64 32 32
8 16 >64 32 16
16 4 2 1 8
32 32 64 4 32
DJK-5 (µg.mL-1) MBIC50 MBIC100 4 4 32 1 4
8 8 >64 4 8
MIC 16 8 64 8 >64
DJK-6 (µg.mL-1) MBIC50 MBIC100 8 2 4 1 8
16 4 32 2 >64
461 462 463 464 465 466 467 22
468 469
Table 4. Evaluation of the antimicrobial activity of conventional antibiotics in preventing
470
planktonic (MIC) and biofilm (MBIC) growth of K. pneumoniaeKpCstrain 1825971 in microtiter
471
assays, and effect of the combination of peptide DJK-6 and the conventional antibiotics by
472
checkerboard titration assessing the fractional inhibitory concentration index (FIC). Syn,
473
synergy; Ind, indifferent. All FIC calculations were determined taking the next lowest MBIC100
474
value (e.g., for >64 μg.mL-1 we used 128 μg.mL-1).
475 Antibiotics
MIC (µg.mL-1)
MBIC50 (µg.mL-1)
MBIC100 (µg.mL-1)
MBIC of antibiotic in combination with DJK-6 (µg.mL-1)
MBIC of DJK-6 in combination with antibiotic (µg.mL-1)
FIC with peptide
Interpretation
Fold reduction in antibiotic concentration at the FIC
Meropenem Imipenem Cefepime Cefotaxime Colistin Polymyxin
>64 >64 >64 >64 >64 >64
>64 >64 >64 >64 >64 >64
>64 >64 >64 >64 >64 >64
4 8 8 64 64 64
12 12 12 32 48 48
0.4 0.4 0.4 1.5 2.0 2.0
Syn Syn Syn Ind Ind Ind
32 16 16 2 2 2
476 477 478
23
Figure 1. Effect of peptides 1018 and DJK-6 against pre-formed biofilms of KpC isolates. Biofilms were grown using the flow cell system. Treatments were applied on 2 day-old biofilms for 24 h. After this time, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).
Figure 2. Effect of the combination between peptide DJK-6 and meropenem on pre-formed biofilms of KpC 1825971. Biofilms were grown in the flow cell system. Treatments were applied to 2 day-old biofilms for 24 h. After this time, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).
Figure 3. Effect of the combination between peptide DJK-6 and meropenem on pre-formed biofilms of KpC strains 1450421 and 2144392. Biofilms were grown in flow cell system. Treatments were applied to a 2 days-old biofilm, for 24 h. After this time, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).
1
Anti-biofilm peptides increase the susceptibility of carbapenemase-
2
producing Klebsiella pneumoniae clinical isolates to β-lactam
3
antibiotics
4
Suzana Meira Ribeiroa, César de la Fuente-Núñezb,BeverlieBaquirb, Celio Faria-Juniorc, Octávio
5
L. Francoa#andRobert. E.W.Hancockb#
6 7 8 9 10
a
Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises
Proteômicas e Bioquímicas, Universidade Católica de Brasília, Brazil. b
Centre for Microbial Diseases and Immunity Research, Department of Microbiology and
Immunology, University of British Columbia, Vancouver, British Columbia, Canada. c
LACEN, Laboratório Central de Saúde Pública do Distrito Federal, Brasília, DF, Brazil
11
Running Head: Effect of anti-biofilm peptides on K. pneumoniae biofilms
12
#Address correspondence to Robert E. W. Hancock, [email protected] or Octávio L. Franco,
13
[email protected]
14
Phone number:+1 (604) 822-2682; Fax: 604-827-5566
15 16 17 18 19 20
1
21
ABSTRACT
22
Multi-drug resistant carbapenemase-producing Klebsiella pneumoniae (KpC)are becoming
23
acommon cause of infections in health care centers.Furthermore,Klebsiellacan develop
24
multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, we describe
25
the antimicrobial and anti-biofilm activities of synthetic peptides DJK-5, DJK-6 and 1018
26
against five KpC isolates.Using static microplate assays,it was observed that the concentration
27
required to prevent biofilm formation by these clinical isolates was below theminimum
28
inhibitory concentration (MIC) for planktonic cells. More sophisticated flow cell experiments
29
confirmed the anti-biofilm activity of the peptides against 2 day-old biofilms of different
30
KpCisolates and, in some cases, the peptides inducedsignificant biofilm cell death.Clinically
31
relevant combinations of DJK-6 togetherwith β-lactam antibiotics, including the carbapenem
32
meropenem, alsoprevented planktonic growth and biofilm formation of KpC strain1825971.
33
Interestingly, peptide DJK-6 was able to enhance,by at least 16-fold, the ability of meropenem to
34
eradicate pre-formedbiofilms formed by this strain. Using peptide DJK-6 to potentiate the
35
activity
36
treatinfectionscaused by KpCisolates.
37
Keywords: small cationic peptides, biofilms, carbapenemase-producing K. pneumoniae.
38
INTRODUCTION
of
β-lactams
including
meropenem
represents
a
promising
strategy
to
39
Klebsiella pneumoniae is a Gram-negative, encapsulated, opportunisticpathogen of
40
theFamilyEnterobacteriaciae(1). It has become a significant cause of infections,especially
41
pneumonia, but also meningitis,sepsis, urinary tract,device-associated and surgical wound-
42
siteinfections(2–7).The most severe clinical cases of such infections are often associated with 2
43
K.pneumoniae that produce a plasmid-borne carbapenemase(KpC).
44
The rate of fatalityfrom KpC-producing K. pneumoniaeis high (>50%) among patients with
45
bloodstream infection(2, 8, 9).The treatment of these infections is difficult since KpCstrains are
46
resistant to all β-lactam antibiotics, including carbapenemsthat are typically used as a last resort
47
(10). In addition, bacteria harboringcarbapenemase genes are often resistant to other antibiotics.
48
As a consequence of this multi-drug resistance, failure in treatment contributes to increasedrates
49
of morbidity and mortality (9, 11, 12). Tigecycline, aminoglycosides and the polymyxins
50
(polymyxin B and colistin) are currently the best treatment options for carbapenemase-producing
51
strains(13). However, these strains also have the potential to develop resistance to these
52
antibiotics(14–16).
53
The treatment of infections caused by multi-drug resistant bacteria is a challengeand
54
theability of bacterial pathogens to form biofilms can further complicate this scenario. Biofilms,
55
a distinct microbial lifestyle of bacteria,aresurface-associated multicellular communities of
56
bacteriaencased in a protective polymeric matrix that can include polysaccharides, proteins and
57
DNA(17). They represent a substantial problem in the clinic, as theycan lead to the development
58
of chronic long-lived infections on body surfaces including the lung, skin, heart and bones (18–
59
21), and can also be formedon medical devices (central venous catheters, urinary tract catheters,
60
mechanical heart valve, contact lenses, endotracheal tube and implants)(18, 22–25).
61
Biofilms arehighly (adaptively) resistant to antibiotics as well as removal by host immune
62
defense mechanisms and thus are very difficult to eradicatewhen compared to their planktonic
63
(free-swimming) counterparts, leading to increased health care costs(26). Although biofilms have
64
a significant impact in public health, most research to date has focused on developing natural or
65
synthetic antimicrobial agentsthat inhibit planktonic bacteria. Cationic amphipathicantimicrobial
3
66
peptideshave emerged in recent years as an alternative strategy for the treatment of antibiotic-
67
resistant bacteria(27). Other so-called host defence peptidesfunction as immunomodulatory
68
agentswithanti-infective, anti-inflammatory and wound healing properties, independent of direct
69
antimicrobial activity(27).
70
Recently, it was shown that some cationic amphipathic peptidescould prevent and/or
71
eradicate bacterial biofilms in a manner that was independent of direct antimicrobial activity
72
against free swimming bacteria(28–30). Others studiesdemonstrated thattheanti-biofilmpeptides
73
1018 (a synthetic variant of host defence peptides), DJK-5 and DJK-6(both, designed D-
74
enantiomeric protease resistant peptides) had the ability to work synergistically with several
75
conventional antibiotics, thus uncovering a novel approach to potentiating antibiotic action
76
against bacterial biofilms(31, 32). Based on these studies, we hypothesizedthat the small cationic
77
anti-biofilm peptides 1018, DJK-5 and DJK-6could both prevent biofilm formation and target
78
pre-formed biofilmsformed by carbapenemase-producingK.pneumoniae. The objective of this
79
study was to investigate the biofilm-forming ability of KpC clinical strains isolated from patients
80
atBrasilia Hospital, and to test the activity of anti-biofilm peptides against these isolates.
81 82
MATERIALS AND METHODS
83
Bacterial strains.
84
Clinical isolates of K. pneumoniawere obtainedfrom hospitalized patients in Brasília, Brazil,
85
between 2010 and 2012. The isolate 1825971 was obtainedfromendotracheal aspirate and the
86
isolates 1789769, 2144392, 2210477 and 1450421 wereobtained from blood. The isolates were
87
suspended in Mueller Hinton broth and incubated for 18-24 h at 36ºC. They were confirmed as
88
Klebsiella pneumoniae strains using the MicroScanWalkAwayautomated system (Siemens 4
89
Healthcare Diagnostics), according to the manufacturer´s instructions. Standard biochemical
90
tests were also used to confirm the species(33). Isolates were preservedat -70ºC with the addition
91
of 8% dimethyl sulphoxide.
92
Peptide Synthesis.
93
DJK-5 (VQWRAIRVRVIR-NH2) and DJK-6 (VQWRRIRVWVIR-NH2), both D-
94
enantiomeric peptides, and the peptide 1018 (VRLIVAVRIWRR-NH2) were synthesized by CPC
95
Scientific using solid-phase 9-fluorenylmethoxy carbonyl (Fmoc) chemistry and purified to 95%
96
using reverse-phase high-performance liquid chromatography (HPLC). Peptide mass was
97
confirmed by mass spectrometry.
98 99 100 101
Antibiotic susceptibility of K.pneumoniae clinical isolates, screening for carbapenemases and detection of blaKPC genes. Susceptibility
testing
was
performed
susceptibility breakpoints
using
the
MicroScanWalkAwayautomated
102
method. The
103
protocols(34).Resistance to carbapenem antibiotics was due tothe production of a
104
carbapenemase, which was detected using the modified Hodge test(35). K. pneumoniae ATCC
105
700603 was used as negative control andK. pneumoniaeIOC (carbapenemase-producing strain
106
from the culture collectionof the Oswaldo Cruz Institute, Rio De Janeiro, Brazil) was used as a
107
positive control. Specific primers were used under standard polymerase chain reaction (PCR)
108
conditions to detect and confirm the presence ofgenes encoding K. pneumoniae
109
carbapenemase,blaKPC
110
CTCAGTGCTCTACAGAAAACC)(36). The strains used as controls for the modified Hodge
111
test were also used as negative and positive controls for PCR.
genes,
were interpreted in accordance to the
(F,5’-TGTCACTGTATCGCCGTC
andR,
CLSI
5’-
5
112 113
Biofilm formation and minimal inhibitory concentration of planktonic cells and biofilms.
114
K. pneumoniae biofilm formation was assessed in both rich mediaLuria-Bertani (LB) broth
115
(BD Difco) and Todd Hewitt Broth (BD Difco) and in BM2 minimal medium (62 mM potassium
116
phosphate buffer, pH 7.0, 7 mM (NH4)2SO4, 2 mM MgSO4, 10 μM FeSO4, 0.5% glucose).
117
Overnight cultures of K. pneumoniae strains grown in LB broth were diluted 1 in 100 in each of
118
these mediaand 100 μL per well added into 96-well round bottom microplates. Planktonic cell
119
growth was assessed by measuring absorbance at 600 nm.Non-adherent planktonic cells were
120
removed from microtitre wells and washed twice with deionized water. Adherent cells were
121
stained with 0.01% crystal violet for 20 min. The microplate wells were washed twice with
122
deionized water, air-dried and solubilized with 110 μlof 70% ethanol. Biofilm formation was
123
assessedby measuring the absorbance at 595 nm using a microplate reader (Bio-Tek Instruments,
124
Inc., United States). Three biological replicates were performed for eachexperimentinvolving
125
eighttechnicalreplicates.Afterestablishing the bestconditions for biofilm growth, antimicrobial
126
peptideswere
127
methodforcationic peptides(37)was used to determine theMinimal Inhibitory Concentration
128
(MIC)for planktonic cellsand the minimal biofilminhibitory concentration(MBIC) to inhibit
129
biofilm formation by50% (MBIC50) or 100% (MBIC100).
evaluated
fortheir
ability
toinhibit
biofilm
formation.Themicrodilution
130 131
Checkerboard assay.
132
Synergy between DJK-6 and various antibiotics was assessedbycheckerboard titration in 96-
133
well microplates, wherein the peptide was diluted along the rows of a microtiter tray and the
134
antibiotics were diluted along the columns. Planktonic growth and biofilm formationwas 6
135
accessed as described above. The resultswere expressed as the fractional inhibitory concentration
136
index(FIC), which is determined as follows: FIC = [A]/MICA + [B]/MICB, where MICA and
137
MICB are the MICs of antibiotic A and peptide B alone and [A] and [B] are the MICs of A and B
138
when in combination. The FIC index was interpreted as follows: FIC16/8
S R R
16 >16/8
S R R
32 >16 >16/8
I R R
16 >16/8
S R R
32 >16 >16/8
I R R
>16 >16 >32 >32 >16 >32 >16 >16 >2 >4 >8 >8 >4 >8 >64 >64 >64
R R R S R R R R R R R R R R R R R
>16 >16 >32 >32 >16 >32 >16 >16 >2 >4 >8 >8 8 >64 >64 >64
R R R R R R R R R R R R S R R R R
>16 >16 >32 >32 16 >32 >16 >16 >2 >4 >8 >8 4 >8 64 >64 >64
R R R R R R R R R R R R I R I R R
>16 >16 >32 32 8 >32 >16 >16 >2 >4 >8 >8 >4 8 >64 >64 >64
R R R I I R R R R R R R R R R R R
>16 >16 >32 32 >16 >32 >16 >16 >2 >4 >8 >8 >4 >8 >64 >64 >64
R R R R R R R R R R R R R R R R R
>8 2/38
R S I R
8 8 2/38
I S R R
2/38
R S R R
447 448 449 450 21
451
Table 2. Assessment of biofilm formation of KPC-producing K. pneumoniae isolates in different
452
media. The assay examined adherence to the surfaces of the wells of 96-well microtiter plates.
453
After incubation for 24 h at 37°C, the growth of planktonic cells was measured as the absorbance
454
at 600 nm (A600) while biofilm growth was assessed at 595 nm (A595) after crystal violet staining
455
and ethanol extraction. KpC isolates 1450421 1789769 1825971 2144392 2210477
Luria Broth Planktonic Biofilm
Growth Todd Hewitt Broth Planktonic Biofilm
BM2 glucose Planktonic Biofilm
1.29 ± 0.04 1.18 ± 0.05 1.30 ± 0.06 1.20 ± 0.05 1.25 ± 0.07
1.48 ± 0.04 1.53± 0.03 1.51 ± 0.04 1.52 ± 0.03 1.46 ± 0.05
1.17 ± 0.06 1.22 ± 0.06 1.15 ± 0.04 1.16 ± 0.06 1.13 ± 0.01
0.20 ± 0.05 0.05 ± 0.03 0.27 ± 0.06 0.02± 0.01 0.15± 0.03
0.17 ± 0.03 0.11 ± 0.02 0.12 ± 0.02 0.04 ± 0.02 0.14± 0.06
1.28 ± 0.11 0.80 ± 0.07 0.79 ± 0.02 0.62 ± 0.04 1.18 ± 0.04
456 457
Table 3. Evaluation of the antimicrobial activity of peptides against planktonic and biofilm cells
458
of KPC-producing K. pneumoniae isolates using microtiter assays. Shown is the MIC for
459
planktonic cells and MBIC50 and MBIC100 for preventing 50% or 100% of biofilm formation.
460 KpC isolates 1450421 1789769 1825971 2144392 2210477
IDR 1018 (µg.mL-1) MIC MBIC50 MBIC100
MIC
32 32 >64 32 32
8 16 >64 32 16
16 4 2 1 8
32 32 64 4 32
DJK-5 (µg.mL-1) MBIC50 MBIC100 4 4 32 1 4
8 8 >64 4 8
MIC 16 8 64 8 >64
DJK-6 (µg.mL-1) MBIC50 MBIC100 8 2 4 1 8
16 4 32 2 >64
461 462 463 464 465 466 467 22
468 469
Table 4. Evaluation of the antimicrobial activity of conventional antibiotics in preventing
470
planktonic (MIC) and biofilm (MBIC) growth of K. pneumoniaeKpCstrain 1825971 in microtiter
471
assays, and effect of the combination of peptide DJK-6 and the conventional antibiotics by
472
checkerboard titration assessing the fractional inhibitory concentration index (FIC). Syn,
473
synergy; Ind, indifferent. All FIC calculations were determined taking the next lowest MBIC100
474
value (e.g., for >64 μg.mL-1 we used 128 μg.mL-1).
475 Antibiotics
MIC (µg.mL-1)
MBIC50 (µg.mL-1)
MBIC100 (µg.mL-1)
MBIC of antibiotic in combination with DJK-6 (µg.mL-1)
MBIC of DJK-6 in combination with antibiotic (µg.mL-1)
FIC with peptide
Interpretation
Fold reduction in antibiotic concentration at the FIC
Meropenem Imipenem Cefepime Cefotaxime Colistin Polymyxin
>64 >64 >64 >64 >64 >64
>64 >64 >64 >64 >64 >64
>64 >64 >64 >64 >64 >64
4 8 8 64 64 64
12 12 12 32 48 48
0.4 0.4 0.4 1.5 2.0 2.0
Syn Syn Syn Ind Ind Ind
32 16 16 2 2 2
476 477 478
23
Figure 1. Effect of peptides 1018 and DJK-6 against pre-formed biofilms of KpC isolates. Biofilms were grown using the flow cell system. Treatments were applied on 2 day-old biofilms for 24 h. After this time, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).
Figure 2. Effect of the combination between peptide DJK-6 and meropenem on pre-formed biofilms of KpC 1825971. Biofilms were grown in the flow cell system. Treatments were applied to 2 day-old biofilms for 24 h. After this time, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).
Figure 3. Effect of the combination between peptide DJK-6 and meropenem on pre-formed biofilms of KpC strains 1450421 and 2144392. Biofilms were grown in flow cell system. Treatments were applied to a 2 days-old biofilm, for 24 h. After this time, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).
