Download Swarming motility, secretion of type 3 effectors and biofilm formation

Journal of Medical Microbiology (2010), 59, 511–520
DOI 10.1099/jmm.0.017715-0
Swarming motility, secretion of type 3 effectors and
biofilm formation phenotypes exhibited within a
large cohort of Pseudomonas aeruginosa clinical
isolates
Thomas S. Murray,1 Michel Ledizet2 and Barbara I. Kazmierczak3,4
Correspondence
Barbara I. Kazmierczak
1
[email protected]
2
Department of Pediatrics and Laboratory Medicine (Infectious Diseases and Clinical Microbiology),
Yale University School of Medicine, New Haven, CT 06520, USA
L2 Diagnostics, New Haven, CT 06511, USA
3
Department of Medicine (Infectious Diseases), Yale University School of Medicine, New Haven,
CT 06520, USA
4
Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06520,
USA
Received 19 November 2009
Accepted 19 January 2010
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of acutely
infecting or persistently colonizing susceptible hosts. P. aeruginosa colonizes surfaces in vitro by
either biofilm formation or swarming motility. The choice of behaviour is influenced by the physical
properties of the surface and specific nutrient availability, and subject to regulatory networks that
also govern type 2 and type 3 protein secretion. Biofilm formation by clinical isolates has been
well-studied. However, the swarming behaviour of human isolates has not been extensively
analysed. We collected isolates from 237 hospitalized patients without cystic fibrosis and
analysed motility and secretion phenotypes of each isolate. We found biofilm formation and
swarming to be negatively associated, while swarming was positively associated with the
secretion of both proteases and type 3 exoenzymes. Most isolates were capable of type 3
secretion and biofilm formation, even though these traits are considered to favour distinct modes
of pathogenesis. Our data demonstrate that while clinical isolates display diverse motility, biofilm
and secretion phenotypes, many of the predicted relationships between swarming motility and
other phenotypes observed in laboratory strains also hold true for bacteria isolated from human
patients.
INTRODUCTION
Pseudomonas aeruginosa is a Gram-negative bacterium
associated with both acute and chronic infections of
humans (Lyczak et al., 2000). Many potential P. aeruginosa
virulence factors have been identified using animal models
of infection, including those that allow bacteria to move
over, attach to, and effectively colonize mammalian tissues,
and secreted enzymes and toxins that allow Pseudomonas to
cross tissue barriers and evade host immune defences.
Two surface organelles, flagella and type IV pili (tfp), affect
virulence in acute and chronic models of Pseudomonas
disease. Bacteria lacking flagella caused less inflammation
and death than wild-type counterparts in a murine model
of acute pneumonia (Feldman et al., 1998), possibly a
Abbreviations: CF, cystic fibrosis; EPS, exopolysaccharide; 3-HAA, 3-(3hydroxyalkanoyloxy)alkanoic acid; T3SS, type 3 secretion system; tfp,
type IV pili.
017715 G 2010 SGM
reflection of flagellin’s ability to trigger pro-inflammatory
host responses via Toll-like receptor 5 rather than to a loss
of motility per se (Balloy et al., 2007). Flagella are required
for robust biofilm formation (Klausen et al., 2003), and
likely contribute to persistent colonization. tfp, which
power twitching motility across solid surfaces (Mattick,
2002), mediate attachment to epithelial cells, contribute to
biofilm formation, and are required for full virulence in
corneal infection models (Kang et al., 1997; Mattick, 2002;
O’Toole & Kolter, 1998).
P. aeruginosa uses flagella and tfp to swarm across semisolid surfaces. In vitro, swarming occurs on semi-solid
surfaces (0.5–0.7 % agar) when specific carbon and
nitrogen sources are provided (Kohler et al., 2000; Rashid
& Kornberg, 2000). The role of tfp in swarming may be
strain-dependent, as some P. aeruginosa isolates require tfp
for swarming while others swarm with altered morphology
in the absence of tfp (Kohler et al., 2000; Overhage et al.,
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T. S. Murray, M. Ledizet and B. I. Kazmierczak
2007). Overlapping sets of regulators govern swarming and
biofilm formation in a reciprocal fashion, leading some
investigators to hypothesize that these are alternative
behaviours adopted by P. aeruginosa in response to
environmental cues (Caiazza et al., 2007; Shrout et al.,
2006). For example, strains lacking GacA, required for
exopolysaccharide (EPS) production and biofilm formation, display increased swarming (Parkins et al., 2001),
while P. aeruginosa with increased EPS production swarms
poorly (Caiazza et al., 2007).
supplemented with 0.4 % glucose and 0.05 % glutamate (Kohler
et al., 2000). Swarming plates were incubated overnight at 30 uC, and
placed at room temperature for an additional 24–48 h prior to
measurement of the swarming diameter. Strain PAO1 was included as
a control on each twitching, swimming and swarming plate, and the
swarming or swimming zone of each isolate was expressed as a ratio
relative to PAO1 from the same plate. Any measurable swimming
zone was positive in swimming assays and a swarming zone of .10 %
of that of PAO1 was considered positive in swarm assays. Swarming
assays were repeated in triplicate on swarm-negative strains to
confirm that they were truly negative.
A role for swarming motility during in vivo infection or
colonization has not been established. However, transposon insertions that attenuate P. aeruginosa virulence in a rat
chronic pulmonary infection model map to genes required
for swarming (Potvin et al., 2003). Several cues required for
swarming in vitro, namely rhamnolipids and elevated
glutamate levels, are present in the sputum of cystic fibrosis
(CF) patients (Singh et al., 2000). Lastly, microarray data
show that genes encoding virulence factors, e.g. type 3
secretion system (T3SS) components and extracellular
proteases, are transcribed at higher levels by swarming
bacteria (Overhage et al., 2008).
Surfactant production. Swarm-negative isolates were examined for
Several studies have examined twitching, swimming,
biofilm and T3SS phenotypes of isolates cultured from
specific human infection sites, or from specific subsets of
patients (Feltman et al., 2001; Fonseca et al., 2007; Head &
Yu, 2004). However, no systematic analysis of swarming
motility and its relationship to other putative virulence
factor phenotypes in clinical isolates has been carried out.
We analysed a large set of P. aeruginosa isolates collected
prospectively from 237 unique hospitalized individuals
without CF. Using this extensive dataset, we examined
whether relationships observed in laboratory strains
between swarming and the expression of other proposed
virulence phenotypes held true in isolates obtained from
human patients.
the production of a visible zone of wetting material on swarming
plates, characteristic of 3-(3-hydroxyalkanoyloxy)alkanoic acids
(HAAs) and rhamnolipid secretion. These clinical isolates were also
inoculated to swarming plates with PAO1, and were scored as positive
or negative for their ability to repel PAO1, which is a HAA-dependent
phenomenon (Caiazza et al., 2005). Swarming plates were photographed with a Kodak Easyshare DX6340 3.1 megapixel camera.
Static biofilm formation. Clinical isolates were inoculated to 1.0 ml
LB and grown with shaking at 37 uC overnight. This culture was
diluted 1 : 100 in LB and grown in triplicate in a polystyrene 96-well
plate (Corning) overnight at 37 uC without agitation. PAO1 was
inoculated on each plate as a positive control, while wells without
bacteria served as blanks. The next day, the OD600 of each well was
measured to control for differences in bacterial growth. Plates were
subsequently washed three times in standing water to remove
unattached bacteria. Wells were stained with 1 % (w/v) crystal violet
for 30 min, then washed twice by dipping into standing water baths.
Adherent stain was solubilized in 70 % EtOH and the A592 was
measured (Head & Yu, 2004). Biofilm staining (A592 sample2A592
blank) was divided by the OD600 of the sample (OD600
sample2OD600 blank) to correct for differences in isolate growth
rate. Each value was then normalized to that obtained for PAO1
[(A592 PAO12A592 blank)/(OD600 PAO12OD600 blank)] to allow for
comparisons between assays.
T3SS ELISA. Clinical isolates were inoculated from freshly streaked LB
and adult patients hospitalized at Yale-New Haven Hospital and
identified by the clinical microbiology laboratory were collected
between September 2005 and April 2007. All patients or their legal
guardians provided informed consent, and the study protocol was
approved by the Yale University Human Investigation Committee.
Patients with CF were excluded from this study. Bacterial isolates
were frozen as 15 % glycerol stocks at –80 uC. The first P. aeruginosa
isolate identified from each patient after protocol enrolment was
analysed in this study.
agar plates to 1 ml MinS medium [(106 MinS salts l21534 g KH2PO4,
50.8 g NH4Cl, 297.1 g C4H4O4Na2.6H2O, 25.7 g C6H6NO6-Na3) 16
complete media l215100 ml 106 MinS salts, 100 ml 0.5 M
monosodium glutamate, 50 ml 50 % glycerol, 5 ml 1 M MgSO4,
1 ml 18 mM FeSO4 and 744 ml H2O] and grown for 8 h at 37 uC with
vigorous aeration. Supernatants were collected after centrifuging
cultures at 3000 g for 10 min. The presence of secreted ExoU, ExoS,
ExoT, PcrV and PopD in culture supernatants was detected by ELISA as
previously described (Li et al., 2005). Values were normalized to those
obtained for PAO1 (ExoS) or PA103 (all other secreted proteins),
which were included as positive controls in all assays. An isolate that
secreted .15 % as much PopD as PA103 was considered positive for
type 3 secretion; such isolates also secreted comparable amounts of
PcrV and ExoT (data not shown). The effectors ExoU and ExoS are
variably encoded and secreted by T3SS-positive P. aeruginosa
environmental and clinical isolates and were therefore not used to
determine whether a strain was T3SS-positive or -negative.
Motility assays. Twitching motility was measured by stab-inoculat-
Caseinolytic assay. The caseinolytic activity of secreted LasB
ing 1.5 % agar Luria broth (LB) plates with a single bacterial colony.
Plates were incubated at 37 uC for 18–20 h, and the diameter of the
twitching zone at the plastic–agar interface was noted. Any bacterial
strain that had a detectable twitching zone upon visible inspection
was scored as positive. Swimming motility was measured as the
diameter of zone travelled by bacteria point-inoculated into 0.3 %
agar LB plates and incubated for 18–20 h at 30 uC. Swarming motility
was assayed as previously described on 0.5 % agar M8 plates
(elastase), alkaline protease and protease IV was determined using a
microplate assay. Briefly, 1 ml LB was inoculated with clinical isolates
and grown for 24 h at 37 uC with shaking (250 r.p.m.). Bacterial cells
were removed by centrifugation at 4000 g for 10 min. One microlitre
of the supernatant was diluted fivefold into 0.2 M Tris/HCl (pH 8.0),
then mixed with 25 ml reaction buffer (0.2 M Tris/HCl, pH 8.0;
20 mM CaCl2) and 25 ml FITC-labelled casein (5 mg ml21 in 50 mM
Tris/HCl, pH 7.5; Sigma C3777). After 75 min at 37 uC, 120 ml 5 %
METHODS
Specimen collection. P. aeruginosa isolates cultured from paediatric
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P. aeruginosa swarming, secretion and biofilms
trichloroacetic acid was added to each well. After 20 min further
incubation at 37 uC, insoluble material was removed by centrifugation (500 g for 20 min at 4 uC). Forty microlitres of the supernatant
was diluted into 160 ml reading buffer (0.5 M Tris/HCl, pH 8.8) and
the A492 was measured. Values observed ranged from 20.03 to 0.75
with a median value of 0.34. Repeated measurements of caseinolytic
activity present in PA14 supernatants (positive control) yielded values
of 0.4–0.5.
Statistical analysis. Data were compiled in Excel (Microsoft) and
analysed using Minitab 15 software. The Kruskal–Wallis test was used
as a non-parametric test to compare medians among multiple groups
of clinical strains. The Mann–Whitney test was used to compare
medians between two groups. For binomial variables, chi-square test
analysis was used to assess for significance in comparisons of multiple
groups of isolates while the Fisher’s exact test was used for
comparison of two groups.
RESULTS AND DISCUSSION
allowed us to test whether relationships between swarming
motility and other potential bacterial virulence phenotypes
proposed from studies of laboratory strains were observed
in a large set of patient-derived isolates.
Swarming motility allows P. aeruginosa to move across
semi-solid surfaces in the presence of specific nutritional
cues. The defined medium used in these assays (M8
containing glucose and glutamate as carbon and nitrogen
sources) failed to support the growth of 10 clinical isolates.
Of the remaining 227 isolates, 143 (63 %) displayed
motility of varying morphologies on swarming plates
(Fig. 1). Table 1 reports the distribution of swarm-positive
strains (defined as swarm diameter ¢10 % of that of
PAO1), which did not show significant variation as a
function of the clinical site of culture (P50.23, chi-square).
Swarming motility occurs in the absence of either
twitching or swimming motility
Swarming motility of clinical isolates
We collected P. aeruginosa strains from 237 unique patients
without CF hospitalized at Yale-New Haven Hospital over
a period of 20 months. Bacteria were isolated from blood,
the respiratory tract (sputum and bronchoalveolar lavage),
urine and deep wound cultures (Table 1). Medical record
review documented the presence of signs and symptoms of
clinical infection at these tissue sites in a subset of study
participants. All isolates were assayed for motility,
adherence and protein secretion phenotypes by investigators blinded to the clinical presentation of the patients
from whom isolates were derived. Only the first isolate
collected from each individual at the time of enrolment was
included in our analysis, and no conclusions were drawn
about the acuity or chronicity of the isolate’s association
with any given subject. Host and bacterial variables
potentially associated with clinical signs and symptoms of
infection were analysed using both unadjusted (bivariate)
and multivariate logistic regression models; no association
between any form of motility, including swarming, was
demonstrated by this analysis (M. Ledizet and others,
unpublished). The bacterial phenotype data, however,
Since both flagella and tfp likely influence swarming
behaviours, each isolate was tested for swimming and
twitching motility under defined laboratory conditions. No
significant differences in motility phenotypes were
observed when comparing isolates by the site of recovery
(Table 1).
Swarming motility appears to require motile flagella
(Kohler et al., 2000; Overhage et al., 2007). Within our
series of clinical isolates, we observed that 10 of 20 swimnegative/twitch-positive strains capable of growth on 0.5 %
agar M8 plates displayed swarming motility. However,
swim-negative isolates swarmed less than swim-positive
isolates (Table 2). This difference did not reach statistical
significance (P50.13, Mann–Whitney). Among isolates
that were both swim-positive and swarm-positive, isolates
with the largest swimming diameters tended to form larger
swarming colonies than those with smaller swim diameters
(Fig. 2; P50.008, Kruskal–Wallis). This confirms the
importance of functional flagella for swarming motility.
Many swim-positive isolates (n567) displayed no motility
on swarm plates, consistent with observations that factors
Table 1. Clinical isolate phenotypes as a function of isolate culture site
Site of culture (n)
Respir. (136)
Urine (65)
Wound (22)
Blood (14)
Total (237)
Swim-positive
[n (%)]
Twitch-positive
[n (%)]
Swarm-positive
[n (%)]*
Biofilm [median
(range)]D
121 (89)
54 (83 )
19 (86 )
14 (100)
208 (88 )
100 (74 )
49 (75 )
18 (81 )
13 (93 )
180 (76 )
75 (57 )
41 (63 )
16 (76 )
11 (85 )
143 (63 )
0.98 (0.17–10)
0.96 (0.14–5.4)
0.74 (0.26–1.7)
1.2 (0.4–2.2)
0.96 (0.14–10)
T3SS-positive
[n (%)]d
77
31
9
11
128
(57 )
(48 )
(41 )
(80 )
(54 )
*Isolates were considered positive for swarming if the measured swarming zone diameter was .10 % of that measured for the PAO1 control. Ten
isolates failed to grow on swarming medium so n5227 for this column.
DBiofilm formation on an abiotic surface was measured in a static assay and normalized to a PAO1 control.
dA strain was considered positive for T3SS if it secreted .15 % as much PopD as the PA103 control, as measured by ELISA.
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T. S. Murray, M. Ledizet and B. I. Kazmierczak
(a)
(c)
(b)
Fig. 1. Clinical isolates display variable
swarming phenotypes. Swarming plates
(0.5 % agar M8) were incubated overnight at
30 6C and then at room temperature for
24–48 h. The colony morphology of clinical
isolates varied, even among strains with
identical twitching and swimming phenotypes.
The laboratory strain PAO1 is at the bottom of
each plate. (a) Swim-positive/twitch-negative;
(b) swim-negative/twitch-positive; (c) swimpositive/twitch-positive; (d) swim-negative/
twitch-negative. Enlarged views of colonies
with small swarming diameters are provided in
insets. Bars, 2 cm.
(d)
in addition to flagellar motility are required for swarming
(Deziel et al., 2003). We tested whether these isolates were
capable of producing wetting agents such as HAAs or
rhamnolipids, which appear to be required for swarming
motility in most laboratory strains, by examining whether
they could repel the leading edge of a PAO1 swarming
colony (Fig. 3) (Caiazza et al., 2005; Tremblay et al., 2007).
Fully 49 of the 67 isolates were deficient in this assay, and
also failed to produce a visible ring of wetting material
when grown on swarming plates (Fig. 3). This suggests that
production of HAAs and/or rhamnolipids is a major factor
regulating the expression of swarming motility in clinical
isolates.
The role of tfp in swarming is still subject to debate, as
conflicting data have been published indicating that tfp are
required versus dispensable for swarming in vitro
(Overhage et al., 2007; Shrout et al., 2006). Swarming
colony morphology is affected by the presence or absence
of tfp in some laboratory strains, suggesting that tfp are
expressed and alter the adhesive properties of swarming
bacteria (Murray & Kazmierczak, 2008). We found that
twitching motility did not affect swarming frequency or
swarm diameter among swim-positive isolates (Table 2).
We observed a number of distinct swarming colony
morphologies among our clinical isolates (Fig. 1a–d).
However, morphology was not related to bacterial
swimming or twitching phenotypes in any consistent
fashion, as illustrated by Fig. 1.
Swarming motility occurs when particular amino acids
serve as sole nitrogen sources (Rashid & Kornberg, 2000)
and is not usually observed on rich (undefined) media.
However, two isolates of 237 displayed the unusual
phenotype of swarming on the 0.3 % agar LB plates used
to assay swimming motility (Fig. 4); these isolates also
Table 2. Biofilm formation and swarming motility of isolates with specific swimming and twitching phenotypes
Swim-positive
Swarm-positive
Swarm diameter, median*
Biofilm, median (range)D
Swim-negative
Twitch-positive
Twitch-negative
Total
Twitch-positive
Twitch-negative
Total
106/154 (69 %)
0.52
1.05 (0.4–2.2)
26/40 (65 %)
0.47
0.56 (0.2–3.8)
132/199 (66 %)
0.52
0.96 (0.2–3.8)
10/20 (50 %)
0.34
0.74 (0.1–2.8)
1/8 (13 %)
0.21
0.65 (0.2–2.6)
11/28 (39 %)
0.31
0.68 (0.1–2.8)
*The swarming colony diameter is normalized to the value obtained for PAO1. Swarm-negative isolates are excluded from the calculation of the
median swarm diameter.
DBiofilm formation is normalized to a PAO1 control.
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Fig. 2. Increased swimming motility correlates
with increased swarming motility among
swim-positive/swarm-positive clinical isolates.
Swarming and swimming diameters are
normalized to PAO1. Median normalized
swarm diameters are indicated for each group
and are significantly different (P50.008,
Kruskal–Wallis).
produced a visible zone of surfactant under these
conditions. We grew the two isolates in liquid LB and
examined them by phase-contrast microscopy; both
exhibited characteristic swimming motility (data not
shown), although only one of the two isolates also
generated a swimming zone on 0.3 % agar LB (Fig. 4).
The first isolate (Fig. 4a) failed to swarm on 0.5 % agar LB
but was able to swarm on 0.5 % agar M8 similar to PAO1
(data not shown). Swarming of this isolate on LB required
a lower agar concentration than is routinely needed for this
Fig. 3. Most swarm-negative isolates do not produce wetting
material. Wild-type PAO1 was inoculated in the centre of the
swarming plate. The black arrow shows a swarm-negative clinical
isolate that does not produce wetting material and does not repel
PAO1, while the white arrow shows PAO1 avoiding a swarmnegative clinical isolate due to the production of wetting material.
Bar, 2 cm.
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assay. In contrast, the second isolate (Fig. 4b) was capable
of swarming on both 0.5 % agar LB (Fig. 4d) and 0.5 %
agar M8 (data not shown).
Swarming isolates show diminished biofilm
formation
Biofilm formation provides bacteria with a means of
persistently colonizing either living or inert surfaces within
a human host. Laboratory strains of P. aeruginosa that fail
to twitch or swim form abnormal biofilms (Klausen et al.,
2003; O’Toole & Kolter, 1998). Consistent with this
observation, swim-positive/twitch-positive isolates scored
higher in the in vitro static biofilm assay (median 1.05)
than all other isolates (Table 2; P ,0.001, Kruskal–Wallis).
However, the measured extent of surface attachment varied
among isolates with similar motility phenotypes, suggesting that additional factors influence biofilm formation
(Table 2).
The relationship between swarming and biofilm formation
is debated in the P. aeruginosa literature (Merritt et al.,
2007; Shrout et al., 2006). Both behaviours are nutritionally regulated surface activities that are altered in the
absence of motility organelles. Since we observed that the
absence of either twitching or swimming potentially
altered both biofilm and swarming behaviours, we
restricted our subsequent analysis to isolates that were
positive for both swimming and twitching. Based upon
published observations of laboratory strains (Merritt et al.,
2007; Yeung et al., 2009), we hypothesized that strains that
did not swarm would score higher than swarming isolates
in our biofilm assay. We did indeed observe an inverse
relationship between swarming motility and biofilm
formation among the 154 swim-positive/twitch-positive
isolates that grew on M8 swarming plates, as swarmnegative isolates scored significantly higher than swarmpositive isolates in the biofilm assay (Table 3; P50.009,
Kruskal–Wallis).
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T. S. Murray, M. Ledizet and B. I. Kazmierczak
(a)
(b)
(c)
(d)
Fig. 4. Swarming motility on LB plates. Two
clinical isolates displayed colony spread consistent with swarming when assayed on 0.3 %
agar LB plates. The first isolate (a) displayed
no swimming, while the second (b) displayed
both a swimming zone and a swarming
morphology on the agar surface. Bars, 1 cm.
(c) PAO1 does not swarm on 0.5 % agar LB
(48 h post-inoculation), while the clinical
isolate pictured in (b) shows extensive swarming on 0.5 % agar LB at this time (d).
Swarm-positive isolates secrete increased levels
of proteases
The transcription of genes encoding the extracellular
proteases LasB, alkaline protease and protease IV is
upregulated in swarming P. aeruginosa (Overhage et al.,
2008). We measured casein hydrolytic activity present in
bacterial culture supernatants after 24 h of planktonic
growth, and asked whether secreted protease activity was
correlated with bacterial swarming. Isolates that displayed
large swarm diameters exhibited increased secreted caseinolytic activity relative to isolates that displayed reduced
or absent swarming (Fig. 5a; P ,0.001, Kruskal–Wallis).
Because protease secretion was assayed under conditions
that do not promote swarming, this result indicates that
protease secretion is inherently greater in strains capable of
swarming and not simply upregulated during swarming.
To determine whether protease activity is required for
swarming, we tested a number of transposon insertions in
protease genes [lasA (PA1871), lasB (PA3724), aprA
(PA1249) and protease IV (PA4175)] from the PAO1
transposon library for their ability to swarm. No single
protease mutant was defective for swarming (data not
shown).
Swarming isolates are more likely to be T3SSpositive
T3SS gene expression was induced in actively swarming
bacteria (Overhage et al., 2008). We therefore examined
whether swim-positive/twitch-positive isolates were more
Table 3. Phenotype associated with swarming motility among twitch-positive/swim-positive isolates
Swarming diameterD
Biofilm formationd
PopD secretion, median§
T3SS-positive||
Quartile 1 (n548)
Quartile 2 (n535)
Quartile 3 (n530)
Quartile 4* (n541)
0–0.09
1.41
16
25 (52 %)
0.1–0.3
1.02
19
18 (53 %)
0.31–0.69
0.98
12
15 (50 %)
.0.7
0.88
51
35 (85 %)
*T3SS activity was increased in quartile 4 (P50.001, Kruskal–Wallis).
DSwarming diameter is normalized relative to PAO1.
dBiofilm formation is normalized relative to PAO1. Biofilm formation differs across quartiles (P50.009, Kruskal–Wallis).
§Median expressed as percentage of PA103 control.
||Positive defined as ¢15 % of PA103 control.
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Fig. 5. Relationship between swarming, protease activity and type 3 secretion. (a)
Swarming is positively correlated with in vitro
protease activity (P ,0.001, Kruskal–Wallis).
(b) Isolates with the largest swarming zones
secrete more PopD than isolates with
decreased or absent swarming in an in vitro
assay for T3SS (P ,0.001, Kruskal–Wallis).
(c) Isolates that secrete the highest amount of
PopD display more active protease activity
(P50.06, Kruskal–Wallis). The median is
shown for each group of isolates; values are
expressed as percentages of the PA103
control.
likely to be T3SS-positive in vitro if they were swarmpositive. Eighty-five per cent (35/41) of swim-positive/
twitch-positive bacteria that showed the largest swarming
colony diameters (.0.7 normalized to PAO1) were T3SSpositive compared with bacteria that showed either no
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(25/48 T3SS-positive, 52 %) or reduced (18/35 T3SSpositive, 53 %) swarming diameters (Table 3; P50.001,
Kruskal–Wallis). The median amount of PopD secreted by
bacteria with the greatest swarming colony diameters (51 %
of PA103 control) was also significantly greater than that
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T. S. Murray, M. Ledizet and B. I. Kazmierczak
secreted by bacteria that demonstrated no (16 % of PA103
control) or little (19 % of PA103 control) swarming (Fig.
5b; P ,0.001, Kruskal–Wallis). Since bacteria that scored
the highest in swarming motility were more likely to secrete
both proteases and T3SS effectors, we also examined
whether there was a direct correlation between protease
secretion and T3SS activity in vitro. Indeed, isolates with
higher in vitro T3SS activity showed greater secreted
caseinolytic activity than T3SS-negative bacteria (Fig. 5c;
P50.06, Kruskal–Wallis).
Since swarming was inversely correlated with biofilm
formation and positively associated with T3SS, we
hypothesized there would be an inverse relationship
between T3SS and biofilm formation, as reported for
laboratory strains harbouring mutations in the sensor
kinases GacS, RetS or LadS (Goodman et al., 2004; Kuchma
et al., 2005; Laskowski & Kazmierczak, 2006; Ventre et al.,
2006). However, most clinical isolates were capable of both
biofilm formation and T3SS activity when assayed in vitro
(Table 4), and we observed no inverse relationship between
biofilm formation and the frequency of T3SS-positive
isolates (P50.3, chi-square). Most clinical isolates, therefore, retain the ability to respond to the environmental cues
that elicit one or the other of these behaviours in vitro.
Several authors have described phenotypes that suggest coordinated regulation between the T3SS and tfp (Beatson et
al., 2002; Whitchurch et al., 2005) or between T3SS and
flagella (Soscia et al., 2007). We examined whether T3SS
was associated with tfp-dependent twitching and flagelladependent swimming motility in our clinical isolates.
There was no apparent relationship between T3SS and
flagellar motility in our isolates, as 48 % (16/33) of swimnegative isolates were T3SS-positive compared with 55 %
(113/204) of swim-positive isolates (P50.46, chi-square
test). However, twitch-positive isolates were significantly
more likely to be T3SS-positive, 60 % (108/180), compared
with twitch-negative isolates, 38 % (21/56) (P50.003,
chi-square test).
Relationship of swarming motility to virulence
factors of P. aeruginosa
We have collected and examined P. aeruginosa isolates
from 237 unique individuals hospitalized at a tertiary care
hospital. The isolates were obtained from patients whose
clinical presentations ranged from no signs of acute
infection to life-threatening acute infection and sepsis.
The isolates displayed diverse motility, biofilm and protein
secretion phenotypes. A primary goal of our work was to
analyse swarming motility in a large sample of clinical
isolates, as the role of swarming in pathogenesis is poorly
understood. Additionally, we examined whether relationships between swarming motility and other phenotypes
associated with virulence initially proposed from studies of
laboratory strains could also be observed in a large set of
clinical isolates. The results of this analysis support several
important conclusions.
The role of swarming motility in human colonization and
infection has not been previously addressed. In our study,
P. aeruginosa swarming motility was associated with
neither increased nor decreased risk of infection (M.
Ledizet and others, unpublished). However, a majority of
clinical isolates were capable of swarming motility in vitro.
The isolates displayed varied patterns of tendril formation
and a range of colony sizes as illustrated in Fig. 1.
Swarming motility is promoted by the production of
bacterial wetting agents, such as HAAs and mono- and dirhamnolipids (Caiazza et al., 2005; Deziel et al., 2003;
Tremblay et al., 2007). In our series, the majority of swimpositive isolates that failed to swarm did not produce
detectable wetting agents, which may have contributed to
this motility defect.
Biofilm formation is a behaviour of sessile bacteria;
nonetheless, it shares many features with swarming
motility. Both are community behaviours that bacteria
exhibit on surfaces. Common sets of bacterial surface
structures (flagella, tfp, EPS) and secreted products
(rhamnolipids) influence both biofilm and swarming
behaviours, and common nutritional cues affect the extent
and structure of both swarming colonies and biofilms
(Shrout et al., 2006). However, it is not clear whether
swarming and biofilm formation represent bacterial
commitment to more or less exclusive modes of behaviour,
as is suggested by some experimental observations.
Phenotypes such as increased EPS formation suppress
swarming and increase biofilm formation among laboratory isolates (Caiazza et al., 2007). Furthermore, swarming-deficient mutants identified in the PA14 transposon
Table 4. Biofilm formation does not predict T3SS activity among swim-positive/twitch-positive isolates
Biofilm score (n)*
PopD secretion, medianD
T3SS-positive (n594)d
Quartile 1
Quartile 2
Quartile 3
Quartile 4
,0.75 (40)
32
26/40 (65 %)
0.75–1.04 (40)
14.5
20/40 (50 %)
1.05–1.59 (40)
40
26/40 (65 %)
¢1.6 (39)
20
23/39 (59 %)
*Biofilm formation is normalized to PAO1. The number of isolates in each quartile is indicated.
DMedian is expressed as a percentage of the PA103 control.
dPositive defined as ¢15 % of PA103 control; percentage of T3SS-positive strains across quartiles not significantly different (P50.3, chi-square).
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Journal of Medical Microbiology 59
P. aeruginosa swarming, secretion and biofilms
insertion mutant library illustrate an inverse relationship
between swarming motility and biofilm formation even in
the absence of obvious swimming or twitching motility
defects (Yeung et al., 2009).
Although we observed a range of behaviours in our clinical
isolates, in general non-swarming isolates scored higher in
the biofilm assay than isolates with the largest swarming
diameters (Table 3). This held true after we controlled for
both twitching and swimming phenotypes, which can
affect biofilm formation and swarming (Klausen et al.,
2003; O’Toole & Kolter, 1998). Many of the swarmnegative strains did not produce detectable wetting agents
such as rhamnolipids in vitro. As rhamnolipids are
associated with biofilm dispersion, their absence could
contribute to the increased biofilm formation observed for
these swarm-negative isolates (Boles et al., 2005; Irie et al.,
2005). Only seven isolates (0.3 %) scored higher than the
reference strain PAO1 in assays for both biofilm formation
and swarming motility. These data are consistent with the
hypothesis that swarming and biofilm formation represent
distinct and largely non-overlapping mechanisms for
surface colonization, rather than a continuum of behaviours, and suggest that clinical isolates exhibit a bias for
one or the other behaviour that persists when they are
analysed ex vivo.
Laboratory strains secrete type 3 exotoxins on swarming
plates, suggesting that the environmental cues that trigger
swarming behaviour may also activate the T3SS (Overhage
et al., 2008). While our data do not explicitly test this
hypothesis, isolates with the largest swarming zones were
significantly more likely to be T3SS-positive than all other
isolates. Likewise, swarm-positive isolates were also found
to secrete significantly more proteases than swarm-negative
isolates. These data are consistent with the hypothesis that
a common set of environmental signals may trigger
swarming, T3SS and protease secretion. Specific isolates
may respond to these cues more robustly than other
isolates.
We observed that the majority of clinical isolates could
both form static biofilms and secrete T3SS effectors in vitro.
These two behaviours are associated with distinct gene
expression profiles (Goodman et al., 2004). Laboratory
strains in which the sensor kinases LadS, GacS or RetS or
the response regulator GacA have been mutated can be
locked into one mode of behaviour or the other (Goodman
et al., 2004; Kuchma et al., 2005; Laskowski & Kazmierczak,
2006; Ventre et al., 2006). However, it is not surprising that
isolates in which these proteins are presumably functional
demonstrate both biofilm formation and T3SS activity in
response to the appropriate environmental cues. This is
distinct from the situation described for chronically
colonized patients with CF, where the ability to secrete
T3SS effectors is likely selected against during bacterial
adaptation to the CF airway (Smith et al., 2006). Most
isolates from the current study retain the ability to carry
out behaviours typically associated with acute infection and
http://jmm.sgmjournals.org
persistence, suggesting that the ability to respond to a range
of environments – rather than specialization for a
particular type of behaviour – is a general characteristic
of clinical P. aeruginosa isolates from non-CF patients.
ACKNOWLEDGEMENTS
We thank Maria Lebron and Xiao Bai for technical assistance, and
Roberta Wilkerson for assistance with patient enrolment. This
publication was made possible by CTSA grant KL2 RR024138 from
the National Center for Research Resources (NCRR), a component of
the National Institutes of Health (NIH), and NIH roadmap for
research and Yale Child Health Research Center grant K12 HD001401
(T. S. M.), the Patrick and Catherine Weldon Donaghue Medical
Research Foundation (B. I. K.) and NIH grant R42 AI058659 (B. I. K.).
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institute of
Allergy and Infectious Diseases, the NCRR or NIH.
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