The role of RND-type efflux pumps in multidrug-resistant mutants of Klebsiella pneumoniae

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10(-7)-10(-9). Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites

Characterization of MATE-Type Multidrug Efflux Pumps from <i>Klebsiella pneumoniae</i> MGH78578

<div><p>We previously described the cloning of genes related to drug resistance from <i>Klebsiella pneumoniae</i> MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it <i>ketM</i>. <i>Escherichia coli</i> KAM32 possessing <i>ketM</i> on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in <i>E</i>. <i>coli</i> KAM32 possessing <i>ketM</i> on a plasmid. The expression of mRNA for <i>ketM</i> was observed in <i>K</i>. <i>pneumoniae</i> cells, and we subsequently disrupted <i>ketM</i> in <i>K</i>. <i>pneumoniae</i> ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and <i>ketM</i> disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in <i>K</i>. <i>pneumoniae</i>. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces.</p></div

Phylogeny of KetM and MATE-type proteins in cluster 1.

<p>MATE-type proteins were analyzed with CLUSTAL W (the program used was ClustalW2.1). Na⁺-coupling MATE proteins were shown in red and H⁺-coupling MATE proteins were shown in blue. NorM_<i>Vc</i>, which is considered to utilize both Na⁺ and H⁺, was shown in pink. KetM and YdhE, the coupling cations of which have not yet been identified, were shown in black. The criteria to determine cation utilization by each MATE-type transporter was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121619#pone.0121619.s002" target="_blank">S1 Table</a>. The accession number of each protein was follows; ABR77432.1 (KetM (<i>K</i>. <i>pneumoniae</i>)), AAB47941.1(YdhE (<i>E</i>. <i>coli</i>)), BAC59742.1 (NorM_<i>Vp</i> (<i>V</i>. <i>parahaemolyticus</i>)), Q9KRU4.2 (VcmA (<i>V</i>. <i>cholerae</i> non-O1)), EKY33397.1 (NorM_<i>Vc</i> (<i>V</i>. <i>cholerae</i> non-O1)), AB010463.1 (PdrM (<i>S</i>. <i>pneumoniae</i>)), WP_011011952.1 (PfMATE (<i>Pyrococcus furiosus</i>)), BAB70470.1 (VcrM (<i>V</i>. <i>cholerae</i> non-O1)), NP_798828.1 (VmrA (<i>V</i>. <i>parahaemolyticus</i>)), BAD98611.1(VcmB (<i>V</i>. <i>cholerae</i> non-O1)), BAD98614.1(VcmN (<i>V</i>. <i>cholerae</i> non-O1)), ADF62863.1 (ECL_03329 (<i>Enterobacter cloacae</i>)), BAD98612.1 (VcmD (<i>V</i>. <i>cholerae</i> non-O1)), BAD98613.1 (VcmH (<i>V</i>. <i>cholerae</i> non-O1)), AAW89139.1 (NorM_<i>Ng</i> (<i>Neisseria gonorrhoeae</i>), BAD89844.2 (AbeM (<i>A</i>. <i>baumannii</i>)), AAG04750.1 (PmpM (<i>P</i>. <i>aeruginosa</i>)), and P45272.1 (HmrM (<i>H</i>. <i>influenzae</i>)).</p

mRNA expression of deduced MATE-type transporter genes in <i>K</i>. <i>pneumoniae</i>.

<p>Gene expression was investigated by RT-PCR. Panel A: <i>ketM</i>, Panel B: <i>dinF</i>, Panel C: <i>yeeO</i>, and Panel D: <i>uncB</i>. The amplification of <i>uncB</i> was used as a standard control. Amplification was performed without a reverse-transcriptase reaction for samples in the RT (-) lanes. The experiment was repeated more than five times and the most reproducible result was shown. Lane 1: MGH78578, Lane 2: ATCC10031, Lane 3: NCTC9632, Lane 4: SKYM</p

Alignment of NorM from <i>V</i>. <i>parahaemolyticus</i>, NorM from <i>V</i>. <i>cholerae</i>, and KetM from <i>K</i>. <i>pneumoniae</i>.

<p>NorM from <i>V</i>. <i>parahaemolyticus</i> (NorMVp, Accession: BAC59742.1), NorM from <i>V</i>. <i>cholerae</i> (NorMVc, Accession: EKY33397.1), and KetM from <i>K</i>. <i>pneumoniae</i> (KetMKp, Accession: ABR77432.1) were aligned (Clustal W version 2.1 in DDBJ). The transmembrane (TM) domains deduced from the 3D structure of NorM from <i>V</i>. <i>cholerae</i> are shown in pink. Amino acid residues that were common between NorM from <i>V</i>. <i>cholerae</i> (NorMVc) and KetM from <i>K</i>. <i>pneumoniae</i> (KetMKp) only, and deduced to be structurally different from NorM from <i>V</i>. <i>parahaemolyticus</i> (NorMVp) were marked in blue (NorMVc and KetMKp). As a reference, the corresponding amino acid residues in NorM from <i>V</i>. <i>parahaemolyticus</i> were marked in red. Asp367 in NorM from <i>V</i>. <i>parahaemolyticus</i>, Asp371 in NorM from <i>V</i>. <i>parahaemolyticus</i>, and Asp368 in KetM from <i>K</i>. <i>pneumoniae</i> were marked in green. Asterisks indicate amino acids that were common to all sequences at a particular position. Colons and dots indicate structurally similar amino acids as calculated by the Clustal W program.</p

Primers used in this study.

<p>Primers used in this study.</p

Minimum inhibitory concentrations of various chemicals in <i>Klebsiella</i> strains.

<p>DAPI, 4',6-diamidino-2-phenyl indole</p><p>Minimum inhibitory concentrations of various chemicals in <i>Klebsiella</i> strains.</p

DAPI efflux was accelerated by cations.

<p>Lactate-TMAH (pH7.0) was added at a final concentration of 20 mM at the arrow point of lactate. A total of 20 mM Na₂SO₄ was added at the arrow point of the salt in curve a while 20 mM K₂SO₄ was added in curve b. The cells used for the assay were <i>E</i>. <i>coli</i> KAM32/pDSH8. The experiment was repeated twice and a representative of typical data was shown here.</p

DAPI efflux activity when the salt was added prior to lactate.

<p>A total of 20 mM NaCl (a) or 20 mM KCl (b) was added prior to lactate-TMAH at the arrow point of the salt. The same volume of H₂O was added as a control (c). Lactate-TMAH (pH7.0) was added at a final concentration of 20 mM at the arrow point of lactate. The experiment was repeated twice and a representative of typical data was shown here.</p

DAPI efflux activity was enhanced by NaCl and KCl.

<p>Lactate-TMAH (pH7.0) was added at a final concentration of 20 mM at the arrow point of lac. Panel A shows the results obtained with <i>E</i>. <i>coli</i> KAM32/pDSH8 and panel B shows those with <i>E</i>. <i>coli</i> KAM32/pBluescript II SK (-) as a control. A total of 20 mM NaCl (a) or 20 mM KCl (b) was added at the arrow point of the salt. Experiments were repeated four times and a representative of typical data was shown here.</p