ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2010, p. 2720–2723
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 6
ramR Mutations in Clinical Isolates of Klebsiella pneumoniae with
Reduced Susceptibility to Tigecycline?
M. Hentschke,* M. Wolters, I. Sobottka, H. Rohde, and M. Aepfelbacher
Institute of Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf,
Martinistrasse 52, 20246 Hamburg, Germany
Received 20 January 2010/Returned for modification 7 February 2010/Accepted 21 March 2010
Five Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline (MIC, 2 ?g/ml) were analyzed.
A gene homologous to ramR of Salmonella enterica was identified in Klebsiella pneumoniae. Sequencing of ramR
in the nonsusceptible Klebsiella strains revealed deletions, insertions, and point mutations. Transformation of
mutants with wild-type ramR genes, but not with mutant ramR genes, restored susceptibility to tigecycline and
repressed overexpression of ramA and acrB. Thus, this study reveals a molecular mechanism for tigecycline
resistance in Klebsiella pneumoniae.
Klebsiella pneumoniae is an important pathogen of nosoco-
mial infections, including urinary tract infections, pneumonia,
wound infections, and sepsis (22). Klebsiella pneumoniae rap-
idly acquires resistance to most commonly used beta-lactam
antibiotics by different mechanisms, including expression of
extended-spectrum beta-lactamases (ESBLs), plasmid-medi-
ated AmpC beta-lactamases, and recently also carbapen-
emases (29). Isolates are also frequently nonsusceptible to
fluoroquinolones and aminoglycosides, leaving only few if any
treatment options, and even infections with untreatable, pan-
resistant strains have been reported (6). Infections with such
multidrug-resistant (MDR) pathogens represent an important
field of application for treatment with the recently introduced
antibiotic tigecycline (8, 14, 20, 30), the first member of the
novel class of glycylcyclines (19). It has an extraordinarily
broad spectrum of antibacterial activity, covering most Gram-
positive, Gram-negative, and anaerobic pathogens, including
vancomycin-resistant enterococci (VRE), methicillin-resistant
Staphylococcus aureus (MRSA), and ESBL- and carbapen-
emase-producing strains (8). Unfortunately, the emergence of
resistance to tigecycline in Klebsiella pneumoniae isolates has
already been reported (25, 28).
Overexpression of RamA, which is a positive regulator of
the AcrAB efflux system, has been observed in tigecycline-
resistant Klebsiella pneumoniae strains (2, 25, 28) and also in
tigecycline-resistant Enterobacter cloacae isolates (11). Fur-
thermore, AcrAB and related efflux pumps which confer resis-
tance to multiple antibiotics, including tetracyclines, fluoro-
quinolones, chloramphenicol, and others (21, 23), have been
implicated in resistance to tigecycline in several other species
(4, 5, 9–12, 16, 26, 27, 32). The overexpression of ramA seemed
to be causative for overexpression of AcrAB in Klebsiella pneu-
moniae and Enterobacter cloacae, but the molecular basis of
ramA upregulation could not be defined in these species. We
were recently able to show that upregulation of ramA and
consecutively AcrAB in a tigecycline-resistant Salmonella en-
terica isolate was due to an inactivating mutation in ramR, a
repressor of ramA (1, 13, 17, 24) in Salmonella (9). How ramA
is regulated in bacteria other than Salmonella is currently un-
We collected five independent Klebsiella pneumoniae iso-
lates from our diagnostic service, and they exhibited suspi-
ciously small disk diffusion zone diameters (?19 mm), and
further analyzed these strains. For tigecycline, MICs were de-
termined by broth microdilution with a commercially available
tigecycline panel (Merlin Diagnostika GmbH, Bornheim-Her-
sel, Germany) using freshly prepared (?12 h old) Mueller-
Hinton II broth (BBL, BD Bioscience, Sparks, MD). For cip-
rofloxacin and chloramphenicol, MICs were determined by
Etest (AB Biodisk, Solna, Sweden). MICs were interpreted
according to the European Committee on Antimicrobial Sus-
ceptibility Testing (EUCAST) clinical breakpoints (for tigecy-
cline, ?1.0 ?g/ml is susceptible, 2.0 ?g/ml is intermediate, and
?2.0 ?g/ml is resistant; for ciprofloxacin, ?0.5 ?g/ml is sus-
ceptible, 1.0 ?g/ml is intermediate, and ?1.0 ?g/ml is resistant;
for chloramphenicol, ?8.0 ?g/ml is susceptible and ?8.0 ?g/ml
is resistant). All five isolates exhibited MICs of 2 ?g/ml, which
was interpreted as intermediate. Testing of 12 randomly col-
lected Klebsiella pneumoniae patient isolates with disk diffusion
zone diameters of ?19 mm uniformly revealed MICs of 0.25
?g/ml. Resistance to tigecycline in Klebsiella pneumoniae has
previously been linked to overexpression of ramA (28). Be-
cause we recently found in a tigecycline-resistant Salmonella
isolate that ramA overexpression was due to a mutation in
ramR, a known negative regulator of ramA (9), we asked
whether a similar mechanism is instrumental in Klebsiella. A
BLAST search identified a predicted Klebsiella pneumoniae
protein (accession number YP_001334235) with 63% identity
to Salmonella RamR (NP_459572.1). Strikingly, the gene for
this protein is located directly upstream of the Klebsiella pneu-
moniae ramA gene (YP_001334236.1) in a head-to-head ar-
rangement (Fig. 1), a genomic organization reminiscent of the
respective situation in Salmonella. The intergenic region be-
tween ramR and ramA additionally harbors a predicted gene,
romA, with homology to beta-lactamase genes. It was previ-
* Corresponding author. Mailing address: Institut fu ¨r Mediz-
inische Mikrobiologie, Virologie und Hygiene, Universita ¨tsklini-
kum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Ger-
many. Phone: 49 40 74105 8184. Fax: 49 40 74105 3250. E-mail:
?Published ahead of print on 29 March 2010.
ously shown not to be involved in the ramA-mediated MDR
phenotype (7), but if expressed, it may be coregulated by
RamR due to its genomic localization, albeit with unknown
significance. Interestingly, a putative palindromic binding ele-
ment for RamR mutated in some fluoroquinolone-resistant
Salmonella isolates (1, 13) is highly conserved in Klebsiella
pneumoniae and located in the intergenic region between ramR
and ramA (nucleotides 622742 to 622762). These similarities
strongly suggest that the identified gene represents the Kleb-
siella pneumoniae homologue of Salmonella ramR.
We amplified the ramR gene and the surrounding genomic
region from the tigecycline-resistant strains and the 12 randomly
collected strains with MICs of 0.25 ?g/ml and performed se-
quence analysis (forward [5?-CTGCAG-TGCCCGGTGAACCC
TGGCGT] and reverse [5?-CTGCAG-ATTTGCTGATTCAGC
AGCGAC] primers). In all five non-tigecycline-susceptible
strains, mutations in ramR relative to the reference se-
quence Klebsiella pneumoniae subsp. pneumoniae MGH
78578 (CP000647), as depicted in Fig. 1, were detected. Four
strains (UR11100, VA14419, VA14743, and VA21266) har-
bored deletions, insertions, or point mutations leading to a
premature stop codon, which result in predicted truncated
RamR proteins highly likely to be nonfunctional. VA6048 har-
bored two mutations leading to amino acid exchanges in the
coding region of ramR. None of these mutations were found in
the 12 tigecycline-susceptible strains. Instead, two different
silent polymorphisms (594G3T and 150G3A [gene]) were
detected and two strains harbored polymorphisms in the ramR
gene, which resulted in amino acid exchanges (VA21490 har-
bored two exchanges, 437A3G [gene]/146I3T [protein] and
454A3T [gene]/152Y3N [protein], and VA21488 harbored
one exchange identical to the first in VA24190, 437A3G
[gene]/146I3T [protein]). We cloned the ramR genes of all the
mutants, of a Klebsiella pneumoniae strain with a wild-type
(WT) MIC to tigecycline, a wild-type sequence of ramR (from
VA12262), and ramR sequences of the two strains (VA21488
and VA21490) harboring the coding polymorphisms together
with the surrounding genomic regions into the PstI site of the
pACYC177 vector using PCR products with the forward and
reverse primers (see above). All constructs were verified by
sequencing. Two of the mutant strains, VA6048 and VA14743,
were amenable for transformation. Transformation of VA6048
and of VA14743 with wild-type ramRVA12262[Klebsiella pneu-
moniae VA6048 (ramRVA12262-WT) and Klebsiella pneumoniae
VA14743 (ramRVA12262-WT), respectively] lowered the MIC
for tigecycline in both strains from 2 ?g/ml to 0.25 ?g/ml, as
shown in Table 1. Both ramRVA21488and ramRVA21490lowered
the MICs for tigecycline of VA6048 and VA14743 to the same
extent as ramRVA12262-WT(data not shown), indicating that the
amino acid exchanges represent nonfunctional polymorphisms.
In contrast, introduction of any of the mutated ramR genes
FIG. 1. Schematic diagram of the genomic region comprising ramR
and ramA of Klebsiella pneumoniae subsp. pneumoniae MGH 78578
(CP000647). The mutations identified in the ramR genes of the non-
susceptible Klebsiella strains are indicated. g., gene (nucleotide posi-
tion); p., protein (amino acid position); del, deletion; ins, insertion.
TABLE 1. MICs and relative expressions of ramA and acrB of strains used in this study
Klebsiella pneumoniae isolate
MIC (?g/ml) and statusa
28.9 ? 8.1
25.1 ? 5.9
40.5 ? 11.4
48.9 ? 10.5
38.9 ? 10.3
0.9 ? 0.6
32.4 ? 7.2
46.5 ? 16.4
25.0 ? 11.6
21.2 ? 4.2
29.9 ? 10.7
0.8 ? 0.1
50.4 ? 8.9
45.6 ? 5.4
43.8 ? 7.0
57.6 ? 15.3
66.7 ? 30.1
11.6 ? 1.3
10.9 ? 0.4
14.9 ? 1.6
7.8 ? 1.0
15.8 ? 2.8
1.5 ? 0.2
10.9 ? 1.8
18.9 ? 6.6
5.8 ? 0.9
8.4 ? 3.0
14.5 ? 3.3
1.5 ? 0.1
17.8 ? 9.3
5.1 ? 0.5
11.0 ? 2.3
22.0 ? 10.2
10.1 ? 4.7
aS, susceptible; I, intermediate; R, resistant. Status determinations are according to EUCAST clinical breakpoints (www.eucast.org/).
bTested by broth microdilution.
cTested by Etest.
dMeasured by quantitative RT-PCR, shown as x-fold expression of VA12262 (expression ? 1). Results are means of 3 (ramA) or 2 (acrB) runs ? standard deviations.
eAll isolates were obtained as a result of this study.
VOL. 54, 2010TIGECYCLINE-RESISTANT KLEBSIELLA PNEUMONIAE ISOLATES 2721
(ramRVA6048, ramRUR11100, ramRVA14419, ramRVA14743, and
ramRVA21266) or the empty pACYC177 vector did not affect
the MIC for tigecycline in VA6048 or VA14743. These findings
suggest that the identified ramR homologue is involved in
resistance to tigecycline and that the identified mutations in
the nonsusceptible strains are functionally relevant. The Ac-
rAB system is involved in resistance to antibiotics from multi-
ple classes. Thus, we also tested MICs for ciprofloxacin and
chloramphenicol in our strains, as both are known to be
substrates of AcrAB. Consistent with the findings for tige-
cycline, MICs for ciprofloxacin and for chloramphenicol of
VA6048 and VA14743 were lowered when wild-type ramR
was introduced but remained unchanged when mutated
ramR or empty pACYC177 vector was transformed (Table
1). These results strongly imply that ramR in Klebsiella pneu-
moniae is involved in the regulation of AcrAB in a manner
similar to that in Salmonella.
Next, we directly analyzed the influence of Klebsiella pneu-
moniae ramR on the transcriptional expression level of ramA
by Northern blot hybridization (hybridization probes for ramA
were generated with primers 5?-ATGACGATTTCCGCTCAG
GTGA and 5?-CAGTGGGCGCGACTGTGGTTC, and those
for 16S rRNA [rrsE] were generated with primers 5?-TTGAC
GTTACCCGCAGAAGAA and 5?-TCTACAAGACTCTAG
CCTGCCA; these were labeled with [?-32P]dCTP [Hartmann-
Analytic, Braunschweig, Germany] by using the Megaprime
DNA labeling system from GE Healthcare). We also used
SYBR green quantitative reverse transcription-PCR (qRT-
PCR) using the qPCR Core SYBR green I kit from Eurogen-
tec, Seraing, Belgium (primers used for qRT-PCR are the
same as for the generation of the Northern blot hybridization
probes except ramA-rev-qPCR [5?-CAGCCGTTGCAGATG
CCATTTC]). RNA was isolated with the RNeasy kit (Qiagen,
Hilden, Germany). First, expression levels of ramA in the 12
randomly selected Klebsiella pneumoniae strains with MICs of
0.25 ?g/ml were compared to those in the five nonsusceptible
strains by Northern blot hybridization. Three micrograms of
the isolated total RNA was separated by electrophoresis in a
gel containing 1% agarose and 1.2% formaldehyde and was
subsequently transferred to a nylon membrane (Macherey-
Nagel, Dueren, Germany) by neutral capillary elution in 20?
SSC (3 M NaCl, 0.3 M trisodium-citrate dehydrate). Hybrid-
ization was carried out at 65°C in 10 ml hybmix (7% SDS, 10%
PEG 20000, 0.22 M NaCl, 1.5 mM EDTA, 15 mM sodium
phosphate, 5 ?g/ml sonicated salmon sperm DNA, 500,000
cpm specific probe) overnight, washed three times at 65°C with
2? SSC–0.1% SDS, and then exposed to Kodak MS autora-
diograph films. While expression of ramA was uniformly low in
strains with wild-type MICs to tigecycline, ramA expression
was very prominent in the nonsusceptible strains (VA6048,
UR11100, VA14419, VA14743, and VA21266), suggesting
massive upregulation (Fig. 2A).
For qRT-PCR, RNA was pretreated with DNase I (Roche,
Mannheim, Germany) and then reverse transcribed with the
SuperScript kit (Invitrogen, Karlsruhe, Germany). qRT-PCRs
were run on a Rotor Gene Q cycler (Qiagen, Hilden, Ger-
many) with 45 cycles of 20 s at 95°C, 20 s at 60°C, and 30 s at
72°C. Data were analyzed by using the 2???CTmethod (15).
Quantification by qRT-PCR demonstrated 25-fold to nearly
50-fold upregulation in the nonsusceptible strains compared to
VA12262-WT, which served as the reference strain (Table 1).
Furthermore, qRT-PCR of acrB (forward primer, 5?-TTAAT
ACCCAGACCGGATGC; reverse primer, 5?-TGGCCGCGG
GCCAGTTAGGCGGTA), a target gene of RamA, revealed
concomitant 8-fold-to-15-fold upregulation in the mutants
compared to the level for the wild-type strain VA12262 (Table
1). Transformation of wild-type ramR (from VA12262-WT) into
VA6048 [Klebsiella pneumoniae VA6048 (ramRVA12262-WT)] and
into VA14743 [Klebsiella pneumoniae VA14743 (ramRVA12262-WT)]
resulted in strongly repressed ramA expression, while no
change in ramA expression was noted in strains transformed
with any of the mutated ramR genes from the nonsusceptible
strains or the empty pACYC177 vector as analyzed by Northen
blot hybridization (Fig. 2B) and by qRT-PCR (Table 1).
Again, changes in the expression levels of acrB accompanied
those observed for ramA (Table 1). All strains harboring
mutated ramR overexpressed acrB in comparison to the
wild-type strain VA12262 or the mutants VA6048 and
VA14743 complemented with wild-type ramR [Klebsiella
pneumoniae VA6048 (ramRVA12262-WT) and Klebsiella pneu-
moniae VA14743 (ramRVA12262-WT), respectively]. These
FIG. 2. RamR represses expression of ramA in Klebsiella pneu-
moniae. Expression levels of ramA were analyzed by Northern blot
hybridization. Total RNAs of the strains indicated were isolated from
mid-log-phase cultures. Three micrograms of total RNA was loaded
into each lane, and the filters were hybridized with [32P]dCTP-labeled
probes of ramA and subsequently 16S rRNA (rrsE) as a loading con-
trol. Values on the left of the panels are band sizes in kbp. (A) Com-
parison of ramA expression levels in non-tigecyline-susceptible strains
and susceptible strains. VA14743 is included in the Northern blot on
the right side as a positive control. (B) ramA expression in nonsuscep-
tible strains transformed with mutated or wild-type ramR.
2722HENTSCHKE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
experiments establish ramR in Klebsiella pneumoniae as a
repressor of ramA.
In summary, we identified a gene in Klebsiella pneumoniae
with homology to ramR, a repressor of ramA in Salmonella
enterica, which is mutated in strains resistant to tigecycline (9)
and to ciprofloxacin (1, 13, 17, 24). Our results imply that ramA
in Klebsiella pneumoniae is regulated in a manner similar to its
regulation in Salmonella and provide a molecular mechanism
for tigecycline resistance in Klebsiella pneumoniae. All of our
non-tigecycline-susceptible Klebsiella pneumoniae strains har-
bored mutations in the ramR gene, suggesting that this is a
major molecular mechanism for tigecycline resistance in Kleb-
siella pneumoniae. Though the susceptibilities of all of our
strains were clearly reduced compared to those of wild-type
strains, none of our strains exhibited full resistance to tigecy-
line by definition (MIC ? 2 ?g/ml). However, fully resistant
Klebsiella strains have been described previously (6, 28), sug-
gesting that several mechanisms might contribute to tigecycline
resistance. Acquisition of mutations in ramR may represent
one step in the development of full resistance; however, in
certain body compartments like the bloodstream, where only
low concentrations of tigecycline can be achieved, an interme-
diate phenotype may be sufficient to result in therapeutic fail-
ure of tigecycline (3, 18). Other resistance mechanisms, like
Tn1721-associated tet(A) (9, 31), may be additive and upon
acquisition successively result in full resistance. It is particu-
larly worrisome that AcrAB-mediated multidrug resistance can
be induced by prior treatment with a multitude of antibiotics.
Furthermore, it cannot be unambiguously inferred from the
resistance phenotype exhibited by an individual isolate in vitro
during routine diagnostic resistance testing. Thus, susceptibil-
ity testing to tigecycline of all relevant isolates would be ben-
eficial when tigecyline treatment is an option.
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