FEMS Microbiology Letters 83 (1991) 291-298
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
ADONIS 037810979100451K
291
FEMSLE 04644
Isolation and characterization of bacteriophage FC3-10
from Klebsiella spp.
Silvia C a m p r u b ~ 1, S u s a n a M e r i n o l, V i c e n t e - J a v i e r Bened~ 2 a n d J u a n M. Tom(is
1 Departamento de Microbiolog[a, Universidad de Barcelona, Barcelona, and 2 Laboratorio de Microbiolog{a,
Universidad de las Islas Baleares, Palma de Mallorca, Spain
Received 29 May 1991
Revision received 29 July 1991
Accepted 31 July 1991
Key words: Klebsiella spp.; Bacteriophage FC3-10; Lipopolysaccharide
1. S U M M A R Y
2. I N T R O D U C T I O N
FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707,
chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be
the low-molecular mass lipopolysaccharide (LPS)
fraction (LPS-core oligosaccharides), specifically
the heptose content of the LPS inner-core. This
is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was
unable to plate on Salmonella typhimurium LPS
mutants with chemotypes Rd 2 or Re showing
incomplete or no heptose content on their LPScore, respectively. Spontaneous phage-resistant
mutants from different Klebsiella strains were
deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.
Klebsiella pneumoniae is a naturally encapsulated bacterium and an important opportunistic
pathogen [1]. The presence of a complete
lipopolysaccharide (LPS) in its cell wall provides
a good model to study the contribution of LPS
and capsular polysaccharide to bacterial virulence. We previously isolated several K. pneumoniae bacteriophage whose receptor was the O-antigen repeating units of the LPS [2,3]. Using these
bacteriophage we were able to obtain LPS mutants by selecting phage-resistant mutants, which
were encapsulated or unencapsulated depending
of the strain used for the selection (wild-type or
isogenic unencapsulated mutant). With all these
mutants we were able to examine the role of
capsular polysaccharide and LPS in different aspects of K. pneumoniae pathogenesis [4,5]. However, none of the Klebsiella bacteriophage previously isolated from the wild-type or isogenic unencapsulated mutants (always with a complete
LPS) used the LPS-core oligosaccharides as their
bacterial surface receptor.
Correspondence to: J.M. Tom~s, Departamento de Microbiolog~a, Universidad de Barcelona, Diagonal 645, 08071
Barcelona, Spain.
292
In this report we isolated bacteriophage FC310 on a LPS mutant from K. pneumoniae previously described [3], a bacteriophage that used the
LPS-inner-core as their bacterial receptor (more
precisely, the heptose content of the LPS-core).
With these bacteriophage we were able to obtain
very deep rough LPS mutants and encapsulated
revertants from unencapsulated strains.
3. M A T E R I A L S A N D M E T H O D S
( w / v ) (LB-agar) and LB soft-agar with 0.6% agar.
For titration and inactivation assays, phage suspensions were diluted in phage buffer [9]. Spontaneous mutants of K. pneumoniae resistant to bacteriophage FC3-10 were isolated by spreading a
mixture containing ca. 10 s bacteria and 1() '~ phage
PFU on LB-agar. After 36 h at 37°C, colonies of
phage-resistant mutants were picked and purified
by streaking and were cross-streaked against
FC3-10 to confirm resistance. Bacteriophage sensitivity was assayed by spot test.
3.1. Bacteria, bacteriophage and media
3.2. Bacteriophage isolation
The strains, their relevant properties and origins, as well as their phage sensitivity are listed in
Table 1. Bacteriophage FC3-1, FC3-9, and 4,1
were previously described [2,6,7]. Luria broth (LB)
[8] was used for bacterial growth and phage propagation. LB was supplemented with 1.5% agar
Freshwater samples were centrifuged and the
supernatant was incubated at 37°C with an exponential growth-phase culture of K. pneumoniae
KT707. After overnight incubation, the bacteria
were removed by centrifugation and filtration,
and the supernatant was plated on the same
Table 1
K. pneumoniae strains used, their relevant characteristics, p h a g e sensitivity and origin
Strain
R e l e v a n t characteristics
Sensitivity to
FC3-1
C3
O1 : K66
KT791
O1 : K
KT856
O1 ; K 6 6 , F C 3 - 1 0 resistant
KT857
O
KT707
O
:K66, chemotype Rd
KT858
O
:K
m u t a n t derived from K T 7 0 7
-
KT794
O
-
KT831
O
: K , F C 3 - 1 0 resistant
m u t a n t derived from K T 7 9 4
, derived from C 3
m u t a n t derived from K T 7 9 1
: K , F C 3 - 1 0 resistant
m u t a n t derived from K T 7 9 1
Source
FC3-9
FC3-10
d) 1
+
+
-
N T ;'
[2]
+
-
+
NT
[20]
+
+
NT
o u r lab
-
-
NT
our lab
+
NT
[3]
NT
our lab
+
NT
[20]
NT
our lab
, F C 3 - 1 0 resistant
:K--, c h e m o t y p e R b or Rc
DL1
O1 : K1
KT836
OI:K
KT952
O1 : K I , F C 3 - 1 0 resistant
m u t a n t derived from K T 8 3 6
O
: K , F C 3 - 1 0 resistant
m u t a n t derived from K T 8 3 6
KT950
O
KT954
O
NT
-
+
NT
+
-
NT
-
-
NT
-
-
NT
: K , F C 3 - 1 0 resistant
m u t a n t derived from K T 9 5 0
KT951
O-
KT955
O
KT953
:K
:K
, derived from D L 1
, chemotype Ra
, chemotype Rb or Rc
: K , F C 3 - 1 0 resistant
m u t a n t derived from K T 9 5 1
:' N T , not tested.
+
[20]
[20]
+
our lab
+
+
our lab
our lab
NT
-
-
NT
+
-
NT
our lab
our lab
our lab
293
strain by using the double agar layered method of
Adams [10]. Plaques which formed on the plates
were stabbed with a needle and eluted with a
small volume of phage buffer. Each phage suspension was serially propagated twice on the same
strain.
3.3. General phage techniques and production of
phage lysates
The methods of Adams [10] were used. The
incubation temperature was 37°C, and the plates
were incubated for 24 h. The bacteriophage host
range was assayed by spot test. One-step growth
experiments were done using K. pneumoniae
KT707 as host at a multiplicity of infection of 1.
Solvent inactivation (ether and chloroform),
phage particle purification, determination of the
buoyant density and nucleic acid type as well as
the polypeptide analyses were performed as previously described [11].
lyzed against distilled water, first at room temperature and then at 4°C, and analyzed by SDSP A G E and silver stained by the method of Tsai
and Frasch [15]. For chemical analyses, purified
LPS or LPS fractions were hydrolyzed with 1 N
HC1 for 2 h at 100°C. Colorimetric analyses of the
2-keto-3-deoxy-octulosonic acid (KDO) content
of LPS were performed by the method of
Karkhanis et al. [16]. Monosaccharides were also
analyzed as previously described by us [2]. Purified LPS was further analyzed by SDS-PAGE and
silver stained by the method of Tsai and Frasch
[151.
3. 6. Antisera
Anti-LPS serum was obtained as previously
described, as well as anti-K66 or anti-K1 specific
immune sera [4].
4. R E S U L T S AND DISCUSSION
3.4. Bacteriophage inactiL,ation experiments
Bacteriophage (103 PFU) were incubated for
20 rain at 37°C with one of the following: 10 7
bacterial cells, 200 txg of 1% deoxycholate
(DOC)-solubilized outer membrane (OM) proteins with or without treatment with 10 /~g of
proteinase K (2), 100 /xg of purified LPS (unless
otherwise indicated) or 50 ~zg of high-molecular
mass LPS (HMW-LPS) (O-antigen enriched fraction) or 50 txg of low-molecular mass LPS (LMWLPS) (LPS core and lipid A enriched fraction).
Chloroform (2-3 drops) was added, mixed for 1
min, and the mixture was centrifuged at 12000 × g
for 10 min at 4°C. The supernatants were assayed
directly on K. pneumoniae KT707.
3.5. Cell surface isolation and analyses
Cell envelopes were prepared as previously
described [2]. Membrane proteins were analyzed
by sodium-dodecyl sulphate-polyaerylamide gel
electrophoresis (SDS-PAGE) as previously described.
LPS was purified by the method of Westphal
and Jann [12] as modified by Osborn [13]. Subfractionation of purified LPS by column chromatography was performed as previously reported by us [14]. Fractions were extensively dia-
4.1. Bacteriophage isolation and characterization
We isolated bacteriophage FC3-10 from one of
three different freshwater samples. Phage FC3-10
gave clear plaques of 4 mm with round edges on
K. pneumoniae KT707 (rough mutant). Phage
FC3-10 incorporated 3H-thymidine in their nucleic acid and therefore is D N A based. Furthermore, Bradley's method [17] confirmed that phage
FC3-10 contained double-stranded DNA. Restriction enzymes EcoRI EcoRV, BamHI, Pr,uI,
XbaI, and Pt~ulI were able to cleave bacteriophage FC3-10 DNA, while BglII, Pstl, Sall and
HindlII were without effect. Sixteen polypeptides
were observed on SDS-PAGE with silver stain:
the three major ones of 68.0, 46.0, and 22.6 kDa.
Neither chloroform nor. ether caused any loss of
infectivity. Bacteriophage FC3-10 had a buoyant
density of 1.51 g / c m 3, a latent period of 25 min,
a rise period of 10 min and a burst size of 84
PFU. These phage were polyhedric contractile
tail and a base plate with probably six spikes but
no collar (Fig. 1).
The host range of bacteriophage FC3-10 was
found to be wide, producing clear plaques on (K.
pneumon&e cells lacking the O-antigen or the
K-antigen (capsular polysaccharide), or both anti-
294
FC3-10 is a bacteriophage that can be classified in the Myouiridae family by Matthews and in
a group A2 by Ackermann [18] according to their
morphological, physico-chemical and biological
characteristics.
4.2. FC3-10 bacteriophage surface receptor
Mutants resistant to bacteriophage FC3-10
from strains O : K + or O : K - occurred at a
frequency of 3 x 10 -~ and fell into a single class
based on their phage sensitivity pattern and LPS
profile. All of them (more than 10 tested for each
strain) were resistant to bacteriophages FC3-1,
FC3-9, and rbl; and all of them showed a LPS
profile with alterations in the relative electrophoretic moiety of their LPS-core (Fig. 2). The
FC3-10 resistant mutants obtained from strains
O - : K + like (KT707 and KT950) were unencapsulated when we tested with specific anti-K sera.
However, mutants resistant to bacteriophage
FC3-10 from unencapsulated strains (O + : K - )
fell into two different classes by the criteria mentioned above. Some of them (about 50%) showed
identical characteristics as the FC3-10 resistant
Fig. 1. Electron micrography of purified bacteriophage FC3-10
negatively stained with 1% phosphotungstic acid (bar = 100
nm).
gens; while wild-type strains (O + : K +) were resistant or partially sensitive to this bacteriophage
(the plaques were extremely turbid and there was
a hundred-fold decrease in the number of PFU).
For instance, strains belonging to serotype K1
were completely resistant to the bacteriophage
while strains belonging to serotype K66 were partially sensitive. All the unencapsulated Klebsiella
strains that we tested (more than 50 obtained like
isogenic mutants from more than 20 different K
serotypes) were sensitive to this bacteriophage,
belonging to all species of this genera (pneumoniae, terrigena, oxytoca, ozanae, planticola, and
rhinosclerosmatis). Also, some strains of Proteus
rettgeri and Proteus uulgaris and LPS mutants
from S. typhimurium and E. coli with chemotype
Rb were sensitive to this phage. The other enterobacteria tested were resistant to this phage.
1 234
5 6 78
910
Fig. 2. SDS-PAGE of purified LPS from K. pneumoniae
strains. Purified LPS was assayed by the method of Tsai and
Frasch [15]. Lanes: 1, strain C3; 2, strain KT791; 3, strain
DL1; 4, strain KT836; 5, strain KT857; 6, strain KT858; 7,
strain KT856; 8, strain KT952; 9, strain KT953; and 10, strain
KT954.
295
m u t a n t s o b t a i n e d from strains O - : K + or
O - : K - like (KT857 a n d KT953); while the o t h e r
50% of FC3-10 resistant m u t a n t s o b t a i n e d from
u n e n c a p s u l a t e d strains (KT856 a n d KT952) reg a i n e d the same characteristics, according to their
phage sensitivity p a t t e r n s a n d LPS profile o n
S D S - P A G E (Fig. 2), as their c o r r e s p o n d e n t wild
types (O + : K + ) . F o r i n s t a n c e they recovered the
sensitivity to specific b a c t e r i o p h a g e s for the K - a n tigen; like ~bl for serotype K1, or FC3-9 for
serotype K66. Also, these FC3-10 resistant m u tants, like KT856 a n d KT952, were c a p s u l a t e d
w h e n we tested with specific a n t i s e r a for their K antigen.
P h a g e FC3-10 a d s o r b e d readily to K. pneumoniae O + : K - , O - : K +, a n d O - : K - b u t n o t to
the resistant m u t a n t s ( T a b l e 2). D O C - s o l u b i l i z e d
O M from strains O - : K +, O - : K - , or their FC310 resistant m u t a n t s showed similar p h a g e adsorption as did the c o r r e s p o n d i n g whole cells
(Table 2). D O C - s o l u b i l i z e d O M from strains
O + : K - also showed similar p h a g e a d s o r p t i o n as
did the c o r r e s p o n d i n g whole cells, while D O C solubilized O M from strains O + : K + (wild-types,
or FC3-10 resistant m u t a n t s from O + : K - - l i k e
KT856 a n d KT952) showed FC3-10 bacteriophage inactivation, a n d the whole cells did not
(Table 2). P r o t e i n a s e K digestion did not alter the
ability of D O C - s o l u b i l i z e d O M from the strains
O + : K - , O : K +, or O - : K - to inactivate bacteriophage FC3-10, t h e n excluding O M p r o t e i n s as
the b a c t e r i o p h a g e receptor.
Purified LPS from K. pneumoniae strains sensitive to b a c t e r i o p h a g e FC3-10 was fully able to
inactivate this b a c t e r i o p h a g e , as well as purified
LPS from strains O + : K + b e i n g able to do it
besides despite the fact that the whole cells were
resistant to this b a c t e r i o p h a g e . Purified LPS from
the FC3-10 resistant m u t a n t s (KT857, KT858,
KT831, KT953, KT954 a n d KT955) was u n a b l e to
inactivate b a c t e r i o p h a g e FC3-10 (Table 2). Also,
LPS fractions c o n t a i n i n g h i g h - m o l e c u l a r mass
Table 2
Inactivation of bacteriophage FC3-10 by K. pneumoniae whole cells and OM components
Strain
% of FC3-10 inactivation by:
DOC-solubilized OM a
LPS b
Whole
cells
Without c
proteinase K
With c
proteinase K
Complete
HMW-LPSa
LMW-LPSd
C3
KT791
KT856
KT857
KT707
KT758
KT794
KT831
< 1.0
91
< 1.0
< 1.0
93
< 1.0
94
< 1.0
78
82
77
< 1.0
86
< 1.0
87
< 1.0
77
83
78
< 1.0
87
< 1.0
89
< 1.0
76
78
77
< 1.0
81
< 1.0
79
< 1.0
< 1.0
< 1.0
< 1.0
NT °
NT
NT
NT
NT
75
76
75
NT
NT
NT
NT
NT
DL1
KT836
KT952
KT953
KT950
KT954
KT951
KT955
< 1.0
90
< 1.0
< 1.0
92
< 1.0
93
< 1.0
73
83
74
< 1.0
85
< 1.0
86
< 1.0
74
82
75
< 1.0
86
< 1.0
85
< 1.0
75
76
75
< 1.0
77
< 1.0
76
< 1.0
< 1.0
< 1.0
< 1.0
NT
NT
NT
NT
NT
73
74
73
NT
NT
NT
NT
NT
a OM solubilized with 1% DOC and 2 mM EDTA [2].
b Purified LPS (100/xg).
c Treatment with proteinase K (10/zg/ml) for 2 h at 45°C.
d Pooled high-molecular mass LPS fractions and pooled low-molecular mass LPS fractions (50 p~g).
e
NT, not tested.
296
Table 3
Chemical composition of purified LPS from different K. pneumoniae strains
Strain
Amount ( / * m o l / m g of LPS) of:
KDO ~
Heptose ~'
Hexoses b
Hexosamines b
Ribose b
Arabinose h
C3
KT791
KT856
KT857
KT707
KT858
KT794
KT831
0.035
0.036
0.035
0.160
0.112
0.162
0.086
0.159
0.28
0.27
0.27
0.52
0.79
0.51
0.63
0.53
0.10
0.10
0.11
0
0
0
0.06
0
0.12
0.13
0.13
0
0
0
0
0
0.14
0.13
0.13
0
0
0
0
0
0.13
0.12
0.13
0
0
0
0
0
~ Assayed by colorimetric method [17].
b Assayed by gas liquid chromatography.
LPS (O-antigen repeating units) were not able to
inactivate bacteriophage FC3-10 (phage inactivation in all cases < 1%), while LPS fractions containing low-molecular mass LPS (LPS core defficient in O-antigen) were able to inactivate bacteriophage FC3-10 by about 75%.
Purified LPS from the wild-type strains and
their isogenic phage-resistant mutants were studied on S D S - P A G E by the method of Tsai and
Frasch [15]. As can be observed in Fig. 2 the
strains O + showed high-molecular mass LPS,
while the strains O
lack the high-molecular
weight LPS.
We also examined the protein composition of
the O M of K. pneumoniae strains. No differences
could be observed between the phage-resistant
mutants and their corresponding wild-type strains
(data not shown).
The chemical analysis of purified LPS from K.
pneumoniae KT791, KT707, KT794, and their
corresponding phage-resistant mutants is shown
in Table 3. The FC3-10 resistant mutants obtained from strains O - : K + or O - : K - showed
an increase in the K D O content and a decrease
in the heptose content of their LPS (values of
Table 3 are per mg of LPS). Similar results were
obtained for the LPS of strain KT857 (FC3-10
resistant mutant from KT791 lacking the O-antigen repeating units), while the other kind of
FC3-10 resistant mutants obtained from KT791
(like KT856) showed identical LPS composition
as the wild-type strain (Table 3).
Thus it is apparent that the low-molecular
mass LPS (LPS core oligosaccahrides) alone is
the true receptor for bacteriophage FC3-10, and
furthermore a complete heptose content on the
LPS-core is essential for the FC3-10 bacteriophage adsorption. Also, LPS mutants from S.
typhimurium or E. coli with chemotype Rb were
partially sensitive to phage FC3-10, while none of
the LPS mutants from the same strains with
chemotype Re (without heptose content on their
LPS-core) or chemotype Rd 2 (incomplete heptose content on their LPS-core) were sensitive to
phage FC3-10. It is also interesting that none of
the LPS-specific bacteriophage for the LPS-core
of S. typhimurium, like Br60, Ffm, Br2 or C21
[19], were able to plate on FC3-10-resistant mutants (deep-rough)-like on S. typhimuriurn LPS
mutants with chemotype Re. Another point to
note, by using this bacteriophage we obtained
encapsulated revertants (like KT856 or KT952)
from unencapsulated strains, this fact being important in the study of Klebsiella pathogenesis.
Finally, it is interesting to point out that phageresistant mutants obtained from O - : K + are always K - , suggesting the possibility that this LPScore region (the heptose content) could be the
determinant of the capsular polysaccharide linkage to the cell.
297
ACKNOWLEDGEMENTS
T h i s i n v e s t i g a t i o n was s u p p o r t e d by C I C Y T
grant PM88-0076, and fellowship from CIRIT
( G e n e r a l i t a t de C a t a l u n y a ) to S.C.
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