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Mechanisms and physiological effects of protamine resistance

in Salmonella enterica serovar Typhimurium LT2


Maria Pra nting and Dan I. Andersson*
Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23 Uppsala, Sweden
*Corresponding author. Tel: 46-18-4714175; Fax: 46-18-4714673; E-mail: Dan.Andersson@imbim.uu.se
Received 6 November 2009; returned 5 January 2010; revised 30 January 2010; accepted 6 February 2010
Objectives: Protamines are cationic peptides that exert antimicrobial activity. We have examined the evolution
of bacterial resistance to protamine sulphate and the resulting effects on tness and physiology, with the
objective of increasing knowledge about mechanisms of bacterial resistance to antimicrobial peptides.
Methods: Spontaneous, protamine-resistant Salmonella enterica serovar Typhimurium (i.e. Salmonella
Typhimurium) LT2 mutants were isolated on agarose plates containing protamine sulphate. Resistance
mutations were identied using transposon insertions and DNA sequencing. Peptide susceptibility was
determined by broth dilution tests and antibiotic susceptibility using Etests. Fitness was determined as
log-phase growth rates. Growth-compensated strains were isolated by serial passage through population
bottlenecks followed by visual screening for large colonies.
Results: Protamine-resistant mutants appeared at a rate of 2.310
27
/cell/generation. These mutants were
220 times more resistant to protamine than the parental strain and less susceptible to several other antimi-
crobials, including colistin, gentamicin, lactoferricin and human defensin HNP-1. The resistance mutations were
mapped to genes involved in haem biosynthesis and respiration, and were associated with a reduction in bac-
terial tness. Some mutants could, in the absence of protamine, be evolved to higher tness by acquiring
second-site compensatory mutations.
Conclusions: Spontaneous mutants resistant to protamine sulphate were readily selected in Salmonella
Typhimurium LT2. These mutants were less susceptible to several other peptides and antibiotics, and had
the characteristics of small colony variants, a phenotype often associated with persistent and recurrent infec-
tions that are difcult to treat and which could be a strategy for bacteria to escape the killing effects of anti-
microbial peptides.
Keywords: small colony variants, antimicrobial peptides, tness costs, haem biosynthesis
Introduction
Antimicrobial peptides (AMPs) are molecules involved in innate
immunity and are found in organisms ranging from bacteria to
man. These peptides have killing activity against many microbes,
but are also involved in several additional immune reactions
including wound healing, recruitment of leucocytes and modu-
lation of the inammatory response.
1,2
As antibiotic-resistant
bacteria have increased in prevalence the need for alternative
treatments has become more important, making AMPs potential
candidates as antibacterial drugs. However, little is known about
the consequences of clinical AMP usage with regard to resistance
development. For example, concerns have been raised that wide-
spread pharmaceutical use of AMPs could select for microbes
that are not only resistant to the peptide in question, but that
can also evade the normal repertoire of AMPs in the human
body.
3
AMPs are generally sensitive to environmental conditions (e.g.
agar, salt and cation concentrations) and are therefore often
inactivated in standard laboratory media, making it difcult
and expensive to perform agar plate-based genetic studies of
resistance.
46
In contrast, protamine from salmon sperm cells
is a highly cationic and basic peptide that has been used as a
cheap and active model peptide in several studies substituting
for more expensive or easily inactivated AMPs. Although lacking
the typical amphiphilic amino acid composition of many AMPs,
protamine is still capable of killing a variety of different microor-
ganisms.
711
Groisman et al.
12
screened a library of transposon
insertions in Salmonella enterica serovar Typhimurium (i.e.
Salmonella Typhimurium) 14028s for hypersusceptibility to
# The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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J Antimicrob Chemother
doi:10.1093/jac/dkq059
1 of 12
Journal of Antimicrobial Chemotherapy Advance Access published March 16, 2010

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protamine, and identied several genes important for bacterial
defence against AMPs. Dhawan et al.
6
used protamine sulphate
resistance as a surrogate marker when selecting bacterial
mutants with increased resistance to thrombin-induced platelet
microbicidal protein (tPMP-1), and protamine has subsequently
been used in several studies of tPMP-1.
1315
Sadowska et al.
16
used protamine as a model to study the cationic peptides
encountered by Staphylococcus aureus in the human body.
They observed that subinhibitory concentrations of protamine
selected for small colony variants (SCVs) of S. aureus.
SCVs are mutant subpopulations of bacteria that differ in
many aspects from their parental counterparts. Typical charac-
teristics include slow growth, aminoglycoside resistance, lack of
pigmentation and inability to use many carbon sources.
17
Several bacterial species are known to form SCVs, and these
cells are often involved in recurrent and persistent infections
and are very resilient to antibiotic treatment.
1719
It has been
suggested that the ability to form SCVs is important for the
disease progression of intracellular pathogens such as
Salmonella and that the SCV phenotype may be selected by cat-
ionic peptides present in the body.
16,17,20
The SCV phenotype
could be a way for bacteria to improve intracellular persistence
and avoid killing by host defences.
In this study, we examined the mechanism and rate of for-
mation of mutations conferring decreased susceptibility to pro-
tamine sulphate in Salmonella Typhimurium LT2, with the
objective of increasing knowledge about different bacterial
resistance mechanisms towards AMPs. We report that spon-
taneous, protamine-resistant mutants arise at a comparatively
high rate and that most of these mutants had the typical charac-
teristics of SCVs, such as slow growth and aminoglycoside resist-
ance. A variety of resistance-associated mutations were
identied in genes whose products are involved in electron trans-
port and respiration. When the selective pressure was removed,
some of the mutants quickly re-established fast growth and
lost the resistance phenotype, while others were stable for
several hundred generations of growth.
Materials and methods
Bacterial strains, media and growth conditions
The strains used in this study are derivatives of Salmonella Typhimurium
LT2, and are listed in Table 1. Unless indicated, bacteria were grown at
378C in Iso-Sensitest broth (Oxoid Ltd, Hampshire, UK) supplemented
with 0.2% glucose. For solid media, LE SeaKem agarose (Lonza, Rockland,
ME, USA) was added to a nal concentration of 1%. LuriaBertani broth
(LB) and agar (LA) were used when indicated. Protamine sulphate, colis-
tin, chloramphenicol, streptomycin and tetracycline were added to the
growth medium when appropriate (all from SigmaAldrich, St Louis,
MO, USA). Martin Malmsten (Uppsala University, Uppsala, Sweden),
Mikael Rhen, Birgitta Agerberth and Katrin Pu tsep (all at Karolinska Insti-
tutet, Stockholm, Sweden) kindly provided CNY100 H-L, bleomycin,
mCRAMP/rCRAMP and HNP-1, respectively. The peptides LL-37 and
PR-39 were purchased from Innovagen (Lund, Sweden) and lactoferricin
B (fragment 414) from SigmaAldrich.
PCR, sequencing and DNA isolation
Genes of interest were PCR amplied with AmpliTaq Gold polymerase in a
GeneAmp PCR System 9700 (Applied Biosystems) using genomic DNA,
bacterial colonies or 1 mL of an overnight culture as template. Genomic
DNA was isolated using a Wizard Genomic DNA purication kit (Promega,
Madison, WI, USA) or a Qiagen Genomic tip 100/G (Hilden, Germany).
The PCR program used was as follows: 5 min of denaturation at 958C;
and 31 cycles of 30 s at 948C, 30 s at the appropriate annealing tempera-
ture for each primer pair and elongation at 728C (1 min/kb). An additional
7 min elongation step was included after the nal cycle. PCR products
were puried from solution or 1% agarose gels using the Illustra GFX
PCR DNA and gel band purication kit (GE Healthcare, Buckinghamshire,
UK), lyophilized together with a sequencing primer and sent to Eurons
MWG Operon, Ebersberg, Germany, for sequencing. Primers used for
amplication and sequencing can be found in Table S1 [available as Sup-
plementary data at JAC Online (http://jac.oxfordjournals.org/)].
Isolation of mutants
Drops of 10 mL containing 410
6
bacteria from independent cultures
were placed on medium containing 300 mg/L protamine sulphate. The
plates were incubated at 378C and examined over several days. Potential
mutants (i.e. colonies appearing in the drop) were puried on LA plates.
These clones were conrmed to have lower susceptibility by repeating the
selection procedure. Only clones that survived the second exposure were
included for further study. In total, 23 independent mutants were
isolated.
Determination of the in vitro mutation rate to
protamine resistance
To determine the mutation rate to protamine resistance, 410
6
cfu
from each of 75 independent Salmonella Typhimurium LT2 overnight cul-
tures were spotted on plates supplemented with protamine sulphate at
300 mg/L. The number of colonies in each spot was scored over the
next couple of days. The mutation rate was calculated by the p
0
method: 2[ln(p
0
/p
tot
)]/N, where p
0
is the number of cultures (i.e. spots)
with no mutants, p
tot
is the total number of cultures and N is the
number of bacteria applied in each spot.
21
Growth rate measurements
An overnight culture of each strain to be tested was diluted to
10
5
10
6
bacteria/mL and transferred in quadruplicate to a bioscreen
plate (400 mL/well). Each strain was assayed in at least three separate
experiments in a Bioscreen C Analyzer (Oy Growth Curves Ab Ltd, Helsinki,
Finland) at 378C. Absorbance was measured at 600 nm every 4 min. The
growth rate of the wild-type strain was set to 1 and the relative growth
rate was calculated as t
gen
(wild-type)/t
gen
(mutant). The calculations
were based on OD
600
values from 0.05 to 0.1 (protamine-resistant
mutants) or 0.04 to 0.1 (compensated mutants).
Protamine susceptibility assay
MIC assays were performed in 10 mL glass tubes. Bacteria were grown to
mid-exponential phase and diluted to 510
5
cfu/mL. Bacterial suspen-
sion (800 mL) was mixed with 200 mL of protamine sulphate solution for
nal protamine concentrations of 2000, 1000, 500, 250, 125, 62.5 and
31.25 mg/L. The tubes were incubated at 378C without shaking for 18 h
or 2430 h for the wild-type and protamine-resistant mutants, respect-
ively (due to the slow growth of the mutants), and the MIC was set to the
lowest concentration of protamine sulphate yielding no visible growth. All
experiments were done with duplicate samples on at least two separate
occasions, and the average MIC was calculated. To further verify the
resistance phenotype of the mutants, protamine susceptibility was also
investigated by plate assays. Stationary-phase bacteria (varying
Pranting and Andersson
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between 100 and 10
7
cfu) were spotted and/or streaked on media con-
taining different concentrations of protamine.
Genetic mapping of resistance mutations
Isolation of mini-Tn10 insertions
P22 phage was grown on a pool of mini-Tn10s (D16D17, tetracycline
resistant, TetR) inserted randomly in a wild-type genetic background.
The resulting phage lysate was used to infect the slow-growing
protamine-resistant strain of interest. Transductants were selected on
LA plates supplemented with tetracycline (15 mg/L), and transposon
insertions that restored the normal colony size phenotype were puried
and saved. Linkage between the protamine resistance mutation (also
assumed to be responsible for the slow growth phenotype) and the
tetracycline marker was established by backcrosses into the original
mutant. An isogenic pair of a tetracycline-resistant, fast-growing
(protamine-susceptible) strain and a tetracycline-resistant, slow-growing
(protamine-resistant) strain was saved at 2808C. The transposon
insertions were also used in genetic linkage experiments to examine if
any of the unmapped protamine-resistant mutants carried mutations
in the same area.
Identication of insertion points
Arbitrarily primed PCRs were carried out to locate the transposon inser-
tion points. In a rst reaction, one transposon-specic primer was
mixed with an array of arbitrary primers each comprising one variable
part (15 bp) and one dened part (20 bp). The following PCR program
was used: 5 min of denaturation at 958C; 5 cycles of 30 s at 948C, 30 s
at 308C and 1 min at 728C; 30 cycles of 30 s at 948C, 30 s at 558C and
1 min at 728C; and a last elongation step of 5 min. The products from
the rst reaction then functioned as template in a second, nested PCR:
5 min of denaturation at 958C; and 29 cycles of 30 s at 948C, 30 s at
548C and 1 min at 728C, with the last elongation extended to 5 min.
The PCR products were puried and sequenced using primers with
homology to the transposon. Genes in the vicinity of the transposon
were then PCR amplied and sequenced to identify the resistance
mutations.
Table 1. Genotypes, protamine susceptibility (MICs) and relative growth rates of strains used in this study
Strain Genotype and amino acid change (one-letter code) MIC (mg/L) Growth rate Reference
DA6192 wild-type 95 1 laboratory collection
DA10886 hemL acc!ccc, T297P 250 0.53 this study
DA10887 hemC acc!ccc, T241P .2000 0.27 this study
DA10888 hemA gtc!ggc, V45G 1000 0.29 this study
DA10889 hemA gtc!ggc, V45G 1000 0.31 this study
DA10890 hemA gca!cca, A318P 1000 0.30 this study
DA10891 del (prpE, hemB, yaiU) 1765 bp [bp 423571425335] 1500 0.30 this study
DA10892 hemA 11 bp del [bp 18756151 875625] !FS 1000 0.29 this study
DA10893 hemA gtc!ggc, V45G 1000 0.32 this study
DA10894 hemA 36 bp del [bp 1 8746911874726], aa 329340 1000 0.30 this study
DA10895 del (prpE, hemB) 777 bp [bp 423405424181] 1500 0.29 this study
DA11799 hemL tgg!cgg, W59R 250 0.60 this study
DA11800 hemA gtc!ggc, V45G 1000 0.29 this study
DA11801 hemA 36 bp del [bp 18746911874726], aa 329340 1000 0.32 this study
DA11802 hemL acc!ccc, T297P 250 0.56 this study
DA11803 hemC cag!ccg, Q287P 500 0.33 this study
DA11805 hemC gtg!gag, V249E 500 0.39 this study
DA11806 hemL 1 bp ins [237886 C] !FS 250 0.60 this study
DA11807 cydC gag!tag, E235Stop 250 0.77 this study
DA11808 hemL 1 bp ins [238057 T] !FS 250 0.57 this study
DA11809 cydC gag!tag, E235Stop 250 0.85 this study
DA11810 mutation linked to Tn10dTet inserted at bp 4185115 125 0.25 this study
DA11811 cydC gag!tag, E235Stop 250 0.84 this study
DA11812 hemA tcg!tag, S232Stop 1000 0.28 this study
DA15245 hemB484::MudJ 700 ND this study
DA15248 hemG614::MudJ 750 0.27 this study
DA15082 env-53, hemA

linked to narK::Tn10dTet 62.5 ND this study


DA15240 env-53, hemAV45G linked to narK::Tn10dTet 250 ND this study
DA15260 cysG1510::Tn10 (TetR) 62.5 ND this study
DA15055 cysG1510::Tn10 (TetR), hemAV45G 375 ND this study
DA8326 fmt 5 bp del 62.5 0.18 Nilsson et al.
51
DA14505 rplQ from Staphylococcus aureus 62.5 0.18 laboratory collection
FS, frameshift mutation; aa, amino acid; ND, not determined.
Protamine resistance in Salmonella Typhimurium LT2
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JAC

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Construction of haemin-permeable strains and a
hemA/cysG double mutant
P22 phage was grown on a strain with a hemA (V45G) mutation linked to
a mini-Tn10 (identied from the mapping experiments above). The result-
ing lysate was used to transduce a haemin-permeable strain, carrying an
env-53 mutation.
22
Transductants were selected on tetracycline and con-
rmed by colony PCR and sequencing. An isogenic pair of one haemin-
permeable, protamine-susceptible (hem

, fast-growing) strain and one


haemin-permeable, protamine-resistant (hem
2
, slow-growing) strain
was saved. A hemA (V45G)/cysG double mutant was constructed by
moving a cysG1510::Tn10 (TetR) insertion into the hemA
2
background
by transduction.
Aminolevulinic acid (ALA) and haemin complementation
studies
ALA and haemin (both from SigmaAldrich) were dissolved in water and
0.1 M NaOH and used at a nal concentration of 50 and 10 mg/L,
respectively. MIC assays with protamine in the presence of ALA or
haemin were performed for the wild-type parental strain and a
subset of the mutants as described above. Control experiments without
ALA/haemin supplementation (but with identical protamine and NaOH
concentrations) were run in parallel.
Cross-resistance studies
Susceptibility to antibiotics was determined using Etests (AB bioMerieux,
Solna, Sweden). Bacteria, diluted to 10
7
cfu/mL, were spread with a
cotton swab on Iso-Sensitest agar, after which the Etest was applied.
Results were read after 16 h for the wild-type strain and after 2440 h
for the mutants. In addition, bacterial susceptibility was determined in
timekill assays with four antibiotics: tetracycline (3 mg/L); chloramphe-
nicol (6 mg/L); streptomycin (8 mg/L); and colistin (1 mg/L). Bacteria were
grown to mid-log phase and diluted to 510
5
cfu/mL. A 5 mL aliquot of
bacterial solution was transferred to 50 mL E-asks after which antibiotic
was added. One ask per strain was left untreated as a control. Samples
were diluted and spread on LA plates to determine the viable count
before addition of antibiotic and after 0.5, 1, 4 and 24 h. The experiment
was performed three times for each strain and antibiotic, and survival
was calculated as the number of viable cells at each timepoint divided
by the number of cells at the start of the experiment.
Bleomycin is a glycopeptide antibiotic used in cancer therapy and
CNY100 H-L is a synthetic peptide based on a fragment of C3a from
the human complement system.
23,24
LL-37, mCRAMP and rCRAMP are
cathelicidin peptides that form pores in the bacterial membrane and
are found in humans, mice and rats, respectively, while PR-39 is a
porcine peptide that enters the bacterial cell and interferes with DNA
and protein synthesis.
2528
Lactoferricin B is a bovine broad-spectrum
AMP, while HNP-1 is a human a-defensin with in vitro activity against bac-
teria, fungi and viruses.
29,30
The MICs of bleomycin and the AMPs CNY100 H-L, mCRAMP, rCRAMP,
LL-37 and PR-39 were determined in MuellerHinton broth (Becton
Dickinson and Company, Sparks, MD, USA) with 0.2% glucose in 96-well
microtitre plates. Log-phase bacteria were diluted to 510
5
cfu/mL
and 45 or 90 mL was added to wells containing 5 or 10 mL of peptide sol-
ution, respectively. The MIC was set to the lowest concentration giving no
visible growth after 18 h (wild-type) or 2440 h (protamine-resistant
mutants) of incubation at 378C. Bacterial susceptibility to lactoferricin B
(414) and HNP-1 was determined in 10 mM sodium phosphate buffer
supplemented with 0.03% trypticase soy broth (TSB; Becton Dickinson
and Company). Log-phase bacteria were washed and diluted to
310
6
cfu/mL, and 90 mL of bacterial solution was mixed with 10 mL
of peptide solution in microcentrifuge tubes to give nal concentrations
of 0, 2.5, 5 or 10 mg/L (lactoferricin) and 0 or 100 mg/L (HNP-1).
Samples (10 mL) were diluted and spread on LA after 0, 0.33, 1 and 3 h
for viable counts (a 5 h timepoint was added for HNP-1).
Compensatory evolution
Compensatory evolution was carried out in LB supplemented with 0.2%
glucose. Ten independent lineages were started for each strain and
grown overnight. Every 24 h, the lineages were serially transferred with a
population bottleneck of 10
6
bacteria into fresh medium. Each cycle cor-
responded to 710 generations of growth, depending on the extent of
growth of the mutant. Samples from all cultures were spread on LA
plates every 13 days and the plates were visually examined for growth-
compensated strains (i.e. clones forming larger colonies than the ancestral
mutant). When .50% of the colonies were large, one single fast-growing
colony was picked and saved at 2808C. For a subset of the compensated
mutants, exponential growth rates were measured and the genes carrying
the original mutation were PCR amplied and sequenced.
Results
Isolation of mutants and determination of mutation rate
Twenty-three spontaneous, protamine-resistant Salmonella
Typhimurium LT2 mutants were isolated and the mutation rate
to protamine resistance was determined using the p
0
method.
On average, 3.810
6
cfu from 75 independent cultures were
spotted on medium containing protamine sulphate. In 31
spots, no colonies appeared and the mutation rate was calcu-
lated to be 2.310
27
/cell/generation.
Identication of resistance mutations
Mini-Tn10dTet transposon insertions were used to identify
mutations responsible for protamine resistance in six mutants
with different growth rates all slower than that of the parental
strain. The six selected strains all carried the TetR marker at differ-
ent positions on the chromosome. The remaining mutants were
linked to one of these six TetR markers, indicating that all the 23
mutations were locatedinanyone of six areas of thechromosome.
By performing an arbitrarily primed PCR out from the transposon
followed by DNA sequencing of the amplied area, the transposon
insertion points were identied in the genes yaiU, narK, stfG,
STM3940 and ybjE and between yigC and fre [Figure S1a, available
as Supplementarydataat JACOnline(http://jac.oxfordjournals.org/
)]. Thesegenes areall locatedincloseproximitytogenes involvedin
haembiosynthesis or respiration(FigureS1a). Mutations inrespirat-
ory genes are common in SCVs and could explain many of the phe-
notypic traits seen in the protamine-resistant mutants. We
therefore PCR amplied and sequenced genes with predicted func-
tions in respiration and maintenance of the electrochemical gradi-
ent and could in this way identify the mutations in 22 of the
mutants (Table 1). Nine mutants carried mutations in hemA, of
which three were deletions, one was a nonsense mutation and
ve were missense mutations. Five mutants had mutations in
hemL; three strains carried missense mutations and two strains a
single base pair insertion. Three strains were mutated in hemC
(all missense mutations) and two strains harboured deletions of
hemB and the upstream and downstream genes prpE and yaiU.
All of the above genes are directly involved in the biosynthesis of
haem [Figure S1b, available as Supplementary data at JAC Online
Pranting and Andersson
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(http://jac.oxfordjournals.org/)].
31
Of the remaining mutants, three
strains carried nonsense mutations in cydC, which encodes the
membrane/ATP-binding component of a cysteine/glutathione
ABC transporter, and one mapped close to the transposon
between yigC and fre (mutation not identied).
32,33
Characterization of mutants
The MIC of protamine needed to inhibit growth varied between
the mutants and ranged from 125 to .2000 mg/L, compared
with 95 mg/L for the wild-type strain (Table 1). The hemL and
cydC mutants were less resistant than those carrying mutations
in hemA, hemC and hemB (MIC 250 mg/L compared with 500 to
.2000 mg/L). One mutant in particular, DA10887 (hemC T241P),
was highly resistant to protamine and survived at all concen-
trations tested. Two independently constructed mutants with
MudJ insertions in hemB and hemG, respectively (DA15245 and
DA15248), were also more resistant to protamine than the wild-
type strain (Table 1). When spotted or streaked on agarose plates
supplemented with protamine, all mutants could also grow and
form colonies on higher protamine concentrations than the
wild-type strain (data not shown). The cysG gene product is
involved in several catalytic steps in the synthesis of sirohaem
and cobalamin from uroporphyrinogen III, an intermediate in
the haem biosynthetic pathway.
34
A constructed cysG mutant
(cysG1510::Tn10, DA15260) showed no growth defects on agar
plates and was not resistant to protamine (Table 1), indicating
that defects in this branch of the pathway are not involved in
the observed resistance (Figure S1b).
For the mutants isolated in this study, there was a correlation
between growth rate and level of protamine resistance
[Figure S2, available as Supplementary data at JAC Online (http://
jac.oxfordjournals.org/)]. Generally, the slower the growth rate,
the higher the resistance. However, protamine susceptibility was
also determined for two slow-growing strains with mutations in
fmt and rplQ, two genes with no known function in respiration
(DA8326 and DA14505, respectively). These strains were as sus-
ceptible to protamine as the wild-type strain, indicating that
slow growth alone is not sufcient to cause protamine resistance
(Table 1).
Several previous studies have reported difculties when
measuring the MIC of protamine in liquid broth because the posi-
tively charged protamine is prone to interact with components of
the broth and cause precipitation.
7,8,11
To assess these concerns
we compared the precipitation of protamine in LB, TSB and Iso-
Sensitest broth. With Iso-Sensitest broth, a faint turbidity was
observed in the tubes with the highest concentrations of prota-
mine. This was much less pronounced than that seen in LB or
TSB (data not shown). It is still possible that the MIC is slightly
overestimated due to precipitation of protamine; however, this
should affect all strains in the same way, and all mutants were
signicantly more resistant than the wild-type strain. It has
also been observed that protamine causes clumping of cells,
but in our experiments this was not observed when examining
protamine-treated cells under the microscope.
8,9
ALA and haemin complementation studies
The hemA and hemL gene products are involved in the early
steps of haem biosynthesis where glutamate is converted into
ALA.
35,36
Supplementation with extracellular ALA is expected to
compensate for the defects caused by these mutations and
restore protamine susceptibility in the mutants. Therefore, com-
plementation studies with ALA were performed for representa-
tive strains with mutations in the following genes: hemL
(DA10886); hemA (DA10888); cysG (DA15260); hemA and cysG
(DA15055); hemC (DA10887); and hemG (DA15248).
When supplemented with ALA, growth of the examined hemL
and hemA mutants was improved, with a concomitant loss of
the protamine resistance phenotype (i.e. susceptibility was
restored to that of the wild-type strain). The results are presented
in Table 2. The protamine susceptibility of the wild-type strain
(MIC 125 mg/L in these experiments, compared with 95 mg/L
when averaging all MIC assays) and the cysG mutant was
similar in medium with and without ALA. The hemC and hemG
mutants (involved in biosynthetic steps downstream of ALA
completion) were not complemented by ALA and retained the
protamine resistance phenotype, as expected (Table 2).
Given that mutations in several genes involved in haem bio-
synthesis resulted in protamine resistance and a slow growth
Table 2. MIC measurements in the presence of ALA or haemin
MIC (mg/L)
Strain Genotype and amino acid change (one-letter code) 2ALA ALA (50 mg/L)
DA6192 wild-type 125 125
DA10886 hemL (T297P) 250 125
DA10888 hemA (V45G) 1000 125
DA15260 cysG1510::Tn10 62.5 62.5
DA15055 cysG1510::Tn10, hemA (V45G) 375 125
DA10887 hemC (T241P) .2000 .2000
DA15248 hemG614::MudJ 750 750
2haemin haemin (10 mg/L)
DA15082 env-53, hemA

linked to narK::Tn10dTet 62.5 62.5


DA15240 env-53, hemA (V45G) linked to narK::Tn10dTet 250 62.5
Protamine resistance in Salmonella Typhimurium LT2
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phenotype, it seemed likely that the observed protamine resist-
ance was caused by lack of the end product haem rather than
by the lack of intermediates of the haem biosynthetic
pathway. To conrm this idea we determined the growth and
resistance pattern of a hemA mutant (V45G) in the presence of
haemin. Since Salmonella Typhimurium LT2 is not naturally per-
meable to haemin, this hem mutation was moved into a
haemin-permeable background by transduction. The resulting
strain was more susceptible to protamine than the original
mutant, but was still more resistant than the isogenic, haemin-
permeable control. The strain was able to grow better than the
parental mutant when supplemented with haemin in a disc on
LA, but still had a growth defect compared with the wild-type
strain (data not shown). As can be seen in Table 2, growth in
the presence of haemin restored protamine susceptibility in the
hem mutant to the level seen in the control, thus indicating
that haem deciency was responsible for the observed prota-
mine resistance in the strains carrying hem mutations.
Fitness of protamine-resistant mutants
All of the protamine-resistant mutants showed a drastic
reduction in growth, forming tiny colonies on LA plates after 24
or 48 h incubation. As a measure of tness, the exponential
growth rate relative to the wild-type strain was determined for
the mutants. All of the examined mutants were signicantly
impaired in growth, with relative growth rates ranging from
0.25 to 0.85 as compared with that of the susceptible wild-type
strain that was set to 1 (Table 1). The hemL and cydC mutants
were more t (relative growth rates 0.530.85) than mutants
defective in other hem genes (relative growth rates 0.270.39).
Apart from a reduction in the exponential growth rate, most of
the examined mutants also had a substantially longer lag
phase than the wild-type strain (data not shown).
Antibiotic susceptibility of protamine-resistant mutants
SCVs have been associated with infections that are persistent and
difcult to treat.
1719
Although the parental strain is susceptible to
a certain antibiotic, the SCVs often have a different resistance
pattern.
17,37,38
In order to characterize the protamine-resistant
mutants isolated in the present study, the susceptibility of a
subset of the strains to commonly used antibiotics and the lipo-
peptide colistin was investigated using Etests. The resulting MICs
are presented in Table 3 together with the MICs determined for
the wild-type parental strain. All the examined mutant strains
were resistant to aminoglycosides, in accordance with what has
been observed in previous studies with SCVs.
17,38,39
The MICs of
gentamicin and streptomycin for the mutants were increased
32-fold and 4- to 24-fold relative to the wild-type, respectively.
The mutants were also less susceptible to colistin (3- to 4-fold
increase in the MIC), with the exception of the hemL mutant
(the cydC mutant was not tested). On the other hand, the exam-
ined mutants were generally more susceptible to chloramphenicol
and nitrofurantoin, andall except the cydC mutant were more sus-
ceptible to ampicillin and tetracycline. Ciprooxacin was equally
active against all strains.
To further dene the resistance phenotype, we investigated
the timekill kinetics of colistin, streptomycin, chloramphenicol
and tetracycline for DA10887 (hemC T241P), DA10888 (hemA
V45G) and DA6192 (wild-type). In Figure 1, cell viability (as a per-
centage of the starting inocula) is plotted over time. As was seen
in the Etest assay, both hem mutants were less susceptible to
colistin (1 mg/L) and streptomycin (8 mg/L) than the wild-type
strain. The growth curves of the mutants were similar both
with and without antibiotics. In experiments with colistin and
streptomycin both treated wild-type and mutant cells grew to
10
8
10
9
cfu/mL in 24 h (data not shown). Chloramphenicol
(6 mg/L) and tetracycline (3 mg/L) had a bacteriostatic effect
on the hem mutants, while the wild-type strain, although
affected, grew quite well in the presence of these antibiotics.
AMP susceptibility of protamine-resistant mutants
Given the low protamine susceptibility of several of the mutants,
we wanted to examine if this resistance could protect from the
killing effects of other types of AMPs. One hem mutant with
high protamine resistance, DA10887 (hemC T241P), was
chosen and subjected to various AMPs of different origin and
mode of action. The MICs of each peptide are presented in
Table 4. In the MIC experiments, no cross-resistance to the
tested peptides was observed for the protamine-resistant
Table 3. Antibiotic susceptibility of protamine-resistant mutants as determined by Etests
MIC (mg/L)
wild-type
DA6192
hemL T297P
DA10886
hemC T241P
DA10887
hemAV45G
DA10888
hemB del
DA10891
cydC E235Stop
DA11807
Ampicillin 0.75 0.38 0.25 0.25 0.25 0.75
Chloramphenicol 4 4 1 1 1 1.5
Ciprooxacin 0.012 0.012 0.012 0.012 0.012 0.012
Colistin 0.25 0.25 0.75 1 1 ND
Gentamicin 0.125 4 4 4 4 4
Nitrofurantoin 12 2 1.5 2 1.5 8
Streptomycin 4 64 96 96 96 16
Tetracycline 1.0 0.5 0.5 0.25 0.19 1.0
ND, not determined.
Pranting and Andersson
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hemC mutant. In fact, the mutant was more susceptible than
the wild-type strain to several peptides. The largest difference
in resistance was for bleomycin, for which the mutant was
three times more susceptible than the wild-type. However, the
hemC mutant was killed at a slower rate and to a lesser extent
upon both lactoferricin and HNP-1 treatment compared with
the wild-type strain (Figure 2a and b, respectively). After 3 h of
lactoferricin treatment, the percentage of viable cells remaining
of the initial inocula was 100%, 15% and 8% for the mutant in
2.5, 5 and 10 mg/L lactoferricin, respectively. During the same
time, the wild-type decreased to 7%, 0.5% and 0.005% of the
initial inocula. Approximately 60% of the mutant cells were
viable after a 5 h treatment with 100 mg/L HNP-1, compared
with only 0.1% of the wild-type cells.
Instability and compensatory evolution in protamine-
resistant mutants
The slow growth phenotype of some of the mutants was not
stable. Occasionally during overnight growth on LA, one or
a few large colonies would appear among the slow-growing
mutants. This was observed in particular for hemC mutants
Time (h)
1
0 5 10 15 20 25
10
10
2
10
3
10
4
10
5
10
6
V
i
a
b
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e

b
a
c
t
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i
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(
%
)
V
i
a
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b
a
c
t
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(
%
)
Streptomycin (8 mg/L)
0 1 2 3 4
10
1
10
2
10
3
10
4
10
5
10
6
10
7
Time (h)
1
0 5 10 15 20 25
10
10
2
10
3
10
4
10
5
10
6
V
i
a
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e

b
a
c
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a

(
%
)
10
7
V
i
a
b
l
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b
a
c
t
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a

(
%
)
Colistin (1 mg/L)
Time (h)
Chloramphenicol (6mg/L) Tetracycline (3mg/L)
0 1 2 3 4
10
1
10
2
10
3
10
4
10
5
10
6
Time (h)
DA6192 (wild-type) ab DA6192 (wild-type) +ab DA10887 (hemC T241P) ab
DA10887 (hemC T241P) +ab DA10888 (hemA V45G) ab DA10888 (hemA V45G) +ab
Figure 1. Killing curves of hem mutants (SCVs) subjected to antibiotics. The curves represent the change in the percentage of viable cells over time
relative to that at the start of the experiment. 2ab, control without antibiotic; ab, antibiotic-treated bacteria.
Table 4. Susceptibility of a hemC mutant to various antimicrobial
peptides
MIC (mg/L)
Peptide/antibiotic wild-type DA6192 hemC T241P DA10887
Bleomycin 0.47 0.14
CNY100 H-L 31.5 31.5
mCRAMP 48 24
rCRAMP 32 32
LL-37 90 59
PR-39 7 5.5
Protamine resistance in Salmonella Typhimurium LT2
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and certain hemA mutants. To examine the phenotypic stability
in detail, four low-tness mutants with different mutations
(DA10886, hemL T297P; DA10887, hemC T241P; DA10888,
hemA V45G; DA10891, hemB deletion) were subjected to com-
pensatory evolution by serial passage through population bottle-
necks. Fitness was measured, relative to the wild-type strain, for
all compensated lineages (Figure 3).
For strains DA10886 and DA10888 only 4/10 lineages com-
pensated within 370 generations of growth and no compen-
sation was observed for strain DA10891 (Table 5). Interestingly,
all 10 lineages of strain DA10887 had compensated within 70
generations of growth and most compensated within 30 gener-
ations of growth (four passages, Table 5). The majority of these
lineages still grew signicantly more slowly than the wild-type
strain (Figure 3). The hemL, hemA and hemC genes from a set
of the respective compensated mutants were sequenced to
identify intragenic compensatory mutations (Table 5). In all
hemL lineages, the proline at position 297 of the protein in the
resistant strain had been replaced by a serine. Three of the com-
pensated hemA lineages were true genetic revertants. The com-
pensatory mutation was not identied for the fourth lineage. In
one of the compensated hemC lineages, the proline at position
241 of the protein had been replaced by leucine and in
another by asparagine. One hemC lineage compensated in two
steps; rst to a growth rate of 0.54, then to 0.85 in 14 and 41
generations, respectively. The intermediately t strain had no
secondary mutations in hemC, while the fast-growing strain
carried a histidine at amino acid position 241. In two strains,
there was double sequence at the position of the compensatory
mutation, indicating the presence of a duplication that included
the hemC gene.
To address whether hemC mutants were generally unstable,
two additional strains with mutations in hemC (DA11803,
Q287P; DA11805, V249E) were evolved in a subsequent cycling
experiment. These strains compensated even more quickly and
all lineages formed normal or near to normal sized colonies
within only 30 generations of growth (data not shown). In
general, compensation of growth defects led to a loss of prota-
mine resistance, as shown by MIC determinations (Table 5).
The lineage of strain DA10887 that increased the least in
tness partly retained the resistance phenotype and could
survive a protamine concentration of 375 mg/L.
Discussion
Protamines are cationic peptides with antimicrobial activity that
have been used as model peptides in studies of more easily inac-
tivated or expensive AMPs. The mechanism of bacterial killing by
protamine is not fully understood, but is thought to involve initial
contact through electrostatic interactions between the positively
charged peptide and the negatively charged bacterial envelope,
followed by a general disruption of the membrane, leakage
of K

, ATP and other cellular components, and inhibition of


metabolic processes, including protein synthesis.
4042
In this
study, spontaneous, protamine-resistant mutants of Salmonella
Typhimurium LT2 were isolated at a rate of 2.310
27
/cell/gener-
ation. In general, the strains with the slowest growth rates were
more resistant to protamine. It should, however, be noted that
slow growth per se did not cause protamine resistance, since
slow-growing strains with mutations in genes with no known
0.0
0.2
0.4
0.6
0.8
1.0
1.2
DA10886
hemL T297P
DA10887
hemC T241P
DA10888
hemA V45G
DA10891
hemB del
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Slow growing parental strains (SCVs)
Compensated strains
Figure 3. Fitness of compensated mutants and the respective parental
mutant (strain and mutation indicated below the chart) relative to the
wild-type strain. The tness of the wild-type strain was set to 1.
10
3
0 1 2 3
10
2
10
1
1
10
10
2
10
3
10
4
Time (h)
0 1 3 2 4 5
Time (h)
V
i
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b
a
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(
%
)
10
3
10
2
10
1
1
10
10
2
10
3
10
4
V
i
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b
a
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(
%
)
Lactoferricin B (mg/L)
0
2.5
5
10
0
2.5
5
10
HNP-1 (mg/L)
DA6192
(wild-type)
DA10887
(hemC T241P)
100
0
100
0
(a)
(b)
DA10887
(hemC T241P)
DA6192
(wild-type)
Figure 2. Killing effect of (a) lactoferricin B (414) and (b) HNP-1 on the
protamine-resistant hemC mutant DA10887 (an SCV) and the susceptible
parental strain DA6192. The curves represent the change in the
percentage of viable bacteria over time relative to that at the start of
the experiment.
Pranting and Andersson
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involvement in respiration (e.g. fmt and rplQ mutants) were not
resistant to protamine.
The mutations responsible for protamine resistance were
identied in 22 of the 23 mutants. Nineteen mutants carried
mutations in genes involved in four different steps of haem bio-
synthesis. These mutations were found at 14 different locations
in the genes. The variety of different mutations conrms the
presence of a large mutational target and explains the relatively
high mutation rate. Furthermore, it indicates that it is the lack of
the end product of the pathway, haem, that causes resistance
and not the individual proteins or metabolic intermediates of
the pathway. In bacteria, haem is involved in multiple cellular
functions including respiration and regulation of gene expression.
Haem serves as cofactor for many cytochromes and catalases,
and sirohaem and cobalamin production is dependent on inter-
mediates of the haem biosynthetic pathway.
31,34,35,43
Mutations
in the different hem genes lead to defects in electron transport
and respiration. This in turn causes slow growth by reducing
the amount of ATP available for metabolism and leads to a
decreased transmembrane potential, reducing the afnity for
and uptake of cationic compounds, explaining the observed pro-
tamine resistance.
17
Three of the protamine-resistant mutants carried a nonsense
mutation at the same position of cydC. These mutants had a
growth rate that was 15% lower than that of the wild-type
strain and were among the least resistant mutants. In
Escherichia coli, cydC encodes a subunit of an ATP-binding cas-
sette transporter (CydDC).
32,33
The sequence similarity of the
E. coli and Salmonella Typhimurium LT2 cydC genes is 75% at
the nucleotide level, and the protein probably has a similar func-
tion in the two strains. Goldman et al.
44
found the periplasm of
an E. coli cydC mutant to be more oxidized than that of the wild-
type strain and it was suggested that the CydDC transporter
maintains redox balance by exporting reducing molecules to
the periplasm. It was later demonstrated that cysteine and the
antioxidant glutathione are substrates for the transporter.
32,33
The lowered reducing power in the cydC mutants is thought to
affect several important functions, including assembly of c-type
cytochromes and functioning periplasmic cytochrome b.
33
These defects will in turn affect several cellular functions includ-
ing electron transport, which would explain the lower protamine
susceptibility. The less severe phenotype changes in these
mutants might reect redundancy in the regulation of redox
homeostasis.
The hemA and hemL gene products are involved in production
of 5-ALA, an intermediate in the haem biosynthetic pathway.
35
Protamine resistance and growth defects of the examined
hemL and hemA mutants could be reversed by addition of ALA
to the growth medium, establishing the connection between
defects in ALA production and the resistance phenotype of
these mutants. It has been observed that ALA can be produced
non-enzymatically in a hemL-independent fashion, which may
explain the somewhat higher tness observed for the hemL
mutants compared with other hem mutants.
36,45
An alternative
explanation would be that the defective enzyme retains residual
activity, allowing a low level of ALA production. Furthermore,
hemA is the rst gene in an operon and expression of the down-
stream genes might, due to polarity effects, be altered in the
Table 5. Characterization of compensatory mutations in independently passaged lineages of protamine-resistant mutants
Parental strain, resistance
mutation and amino acid change
Growth-compensating mutation
and amino acid changes Generations of growth MIC (mg/L)
DA6192 (wild-type) NA not passaged 95
DA10886 hemL (ccc!tcc, P297S) 140 95
hemL (acc!ccc, T297P) hemL (ccc!tcc, P297S) 150 ND
hemL (ccc!tcc, P297S) 170 ND
hemL (ccc!tcc, P297S) 360 ND
no compensation, 6 lineages 370 NA
DA10887 hemC (ccc!aac, P241N) 21 ND
hemC (acc!ccc, T241P) extragenic 14 375
hemC (ccc!cac, P241H) 41 95
hemC (possible amplication) 28 95
hemC (possible amplication) 56 62.5
hemC (ccc!ctc, P241L) 7 ND
not identied, 5 lineages 21/21/35/63/70 ND
DA10888 reversion 14 95
hemA (gtc!ggc, V45G) reversion 84 ND
reversion 224 ND
not identied 30 95
no compensation, 6 lineages 260 NA
DA10891 del prpE-hemB-yaiU no compensation 260 NA
ND, not determined; NA, not applicable.
Protamine resistance in Salmonella Typhimurium LT2
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hemA mutants, resulting in tness costs unrelated to the pro-
duction of ALA.
As expected, ALA supplementation had no effect on hemC
and hemG mutants impaired in haem biosynthetic reactions sub-
sequent to completion of ALA synthesis. In addition, a mutant
defective in the rst steps of sirohaem and cobalamin synthesis
was not resistant to protamine in either the presence or absence
of ALA, enabling us to rule out involvement of these two pro-
ducts in protamine resistance. Protamine susceptibility of a
haemin-permeable hemA mutant could be reversed by exogen-
ous haemin, further indicating that haem deciency caused
the resistance phenotype.
Mutants decient in respiratory genes have been isolated
from clinical specimens from various sites of the body and are
being increasingly recognized as a major contributor to persist-
ent and recurrent diseases such as osteomyelitis, cystic brosis
and foreign body-related infections.
17,19
These SCVs have an
improved capacity to survive within cells and are more resistant
to many antimicrobials, including aminoglycosides.
20,38,46,47
Similar to clinical SCVs, all examined protamine-resistant
mutants were less susceptible to aminoglycosides than the par-
ental strain, irrespective of whether they carried hem or cydC
mutations. Aminoglycoside transport over the bacterial mem-
brane is dependent on a charge differential across the mem-
brane, formed during electron transport and respiration.
19,39
Thus, the respiratory defects of the mutants interrupt uptake
and function of the antibiotic. The mutants were also less sus-
ceptible to the lipopeptide antibiotic colistin. Colistin activity is
dependent on electrostatic interactions between the antibiotic
and the negatively charged bacterial membrane, and, following
the same reasoning as above, the respiratory defects of the
mutants probably produce changes in the bacteria that reduce
this interaction.
48
It has been previously observed that SCVs of
S. aureus with different auxotrophisms are resistant to the AMP
lactoferricin B.
49
Similarly, we found a Salmonella Typhimurium
LT2 SCV (hemC T241P) to be less susceptible to this peptide.
Samuelsen et al.
49
suggested that lactoferricin resistance was
caused by a reduced metabolic activity rather than by decreased
membrane interaction and uptake, based on the observation
that labelled peptide was efciently internalized by the SCV.
The examined hemC mutant was also less susceptible to the
human defensin HNP-1. Together with HNP-2 and 3, this
peptide accounts for 5% of the human neutrophil proteins.
2,30
Other immune cells, e.g. T cells, also express HNP-1, and it seems
likely that resistance could facilitate bacterial survival in the
human body.
2
That is, respiratory defects of SCVs can increase
resistance to several antimicrobial agents, including peptides
present in the human body, and probably does this through
several different mechanisms.
Clinically isolated SCVs are often described as unstable, fre-
quently reverting to a normal colony phenotype. Generally, pro-
tamine resistance conferred a high tness cost displayed as a
reduced growth rate. The majority of the mutants appeared to
be phenotypically stable, but occasionally certain mutants
re-established fast growth. For some mutant strains it was poss-
ible to select variants with increased tness and second-site
mutations by serial passage of the mutants in the absence of
selection. For the examined hemA and hemL mutants only a
few of the individual lineages compensated and a substantial
fraction of these were genetic revertants. No compensation
was observed for a strain with a deletion of hemB, whereas, in
contrast, all lineages of strain DA10887 (hemC, T241P) compen-
sated rapidly. The growth rates of these lineages were signi-
cantly lower than that of the wild-type strain, indicating that
they did not revert back to the parental genotype. When the
hemC gene of these mutants was sequenced, some strains
were found to have a double DNA sequence at specic positions
in the gene, indicating the presence of a duplication and sub-
sequent mutational divergence. This in turn provides a possible
explanation for the fast compensation of all DA10887 lineages.
The hemC gene is located between two rRNA operons in an
area of the chromosome known to undergo frequent dupli-
cations (sequence repeats provide substrate for duplications),
which may play a role in the fast compensation in these
strains.
50
If so, this would provide a novel mechanism for the
reported phenotypic instability of many SCVs. The potential
occurrence of gene duplications in these strains is currently
under investigation.
In the present study, all isolated protamine-resistant mutants
have the typical characteristics of SCVs. They grow slowly and
carry resistance mutations in genes associated with redox reac-
tions and respiration, are resistant to aminoglycosides, other
antibiotics and peptides, and some are also genetically unstable.
A large tness cost, as observed for the mutants in this study,
would normally imply a competitive disadvantage in the
absence of selective pressure, and it is expected that such
strains would be outcompeted by faster growing strains.
However, SCVs are frequently recovered from patients and have
been suggested to be an important part of the disease pro-
gression and survival in the host for intracellular pathogens.
Thus, the low tness observed might actually provide a survival
advantage in this case. Spontaneously formed SCVs of
Salmonella have only been isolated and characterized in a few
studies, but, as emphasized by Proctor and co-workers in their
studies of SCVs in S. aureus, certain AMPs in the body might
select for or stabilize the SCV phenotype.
17,18,20
Formation of
SCVs could be a way for the bacteria to avoid killing by certain
AMPs, and could be a potential problem for the use of pharma-
ceuticals based on AMP structures whose mode of action is
based on an energized bacterial membrane or actively growing
bacteria.
Acknowledgements
We thank Diarmaid Hughes and Annika Nilsson for comments.
Funding
This work was supported by grants from the Swedish Research Council
(2009-3486), 6th EU Framework Program (2005-318152) and Vinnova
(2009-00169) to D. I. A.
Transparency declarations
None to declare.
Pranting and Andersson
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Supplementary data
Table S1, Figure S1 and Figure S2 are available as Supplementary data at
JAC Online (http://jac.oxfordjournals.org/).
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