Natural being Klebsiella pneumoniae (NB-K.p) bacteria were purchased from American Type Culture Collection (Klebsiella pneumoniae subsp. Pneumoniae; ATCC^® 43816™). Klebsiella pneumoniae bacteria from patients with pneumonia (PD-K.p) were isolated from a 56-year male patient with pneumonia who suffered from the disease for approximately 30 years. Cells of Klebsiella pneumoniae were grown in LBmedium at 37˚C for 24 h.

The Klebsiella pneumoniae bacteria were cultured in 10 mg/ml penicillin medium with or without penicillin-binding proteins (PBPs, 0.67 µg/ml, Sigma-Aldrich; Merck KGaA) for 24 h. The number of Klebsiella pneumoniae cells was calculated in the agar plating. The detailed procedures were conducted according to a previous study (24).

To investigate the site of the zinc finger nuclease, a recombinant plasmid expressing GFP and ZFN (rpGFP-ZFN) was constructed. All plasmids were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). A full-length ZFN fragment was amplified and subcloned into rpGFP-pET27b. The recombinant plasmid was named rpGFP-ZFN. All expression plasmids were confirmed by sequencing. Cells were transfected with rpGFP-ZFN or pET27b by using electrotransfection according to the manufacturer's instructions. After a 48-h transfection, the cells were captured using a Leica DM5000 microscope equipped with Q-Imaging Retiga 4000RV camera (Teledyne QImaging).

Antimicrobial susceptibility tests of Klebsiella pneumoniae were performed by the disk diffusion method, according to Clinical and Laboratory Standards Institute (CLSI) recommendations (25). The final results were performed according to the respective standards for antimicrobial susceptibility testing.

This study analyzed the affinity of PBP with penicillin by using ELISA Kit (cat. no. B21210; R&D Systems). The ELISA assays were performed according to the manufacturer's protocols. The results were measured at 450 nm in an ELISA reader.

Total RNA was obtained from Klebsiella pneumoniae by using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The cDNA was synthesized with ReverTraAce (Toyobo Corp.). All forward and reverse primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and are listed in Table I. PCR amplification was followed by preliminary denaturation at 94˚C for 2 min, followed by 45 cycles of 95˚C for 30 sec; annealing temperature was reduced to 57˚C for 30 sec, and 72˚C for 30 sec. A volume of 20 µl containing 50 ng of genomic DNA, 200 µM dNTP, 2.5 units of Taq DNA polymerase, and 200 µM primers was used. Relative mRNA expression level changes were calculated using the 2^-ΔΔCq method (25). The results are expressed as n-fold compared with the β-actin control.

Klebsiella pneumonia cells were homogenized using RIPA buffer (M-PER reagent for the cells and T-PER reagent for the tissues; Thermo Fisher Scientific, Inc.) and centrifuged at 6,000 x g at 4˚C for 15 min. The protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 30 µg protein extracts was electrophoresed on 12% polyacrylamide gradient gels and then transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were incubated in 5% BSA (Sigma-Aldrich; Merck KGaA) for 2 h at 37˚C and then incubated with primary antibodies (dilution 1:1,000, cat. no. ab11251; Abcam) or β-actin (dilution 1:1,000, cat. no. ab8226; Abcam) for 2 h at 37˚C. After washing with PBS three times, the membranes were then incubating with secondary antibodies (dilution 1:1,000, cat. no. SAB11045182; Sigma-Aldrich; Merck KGaA) for 12 h at 4˚C. After three washings in PBST, the membranes were developed using a chemiluminescence assay system (Roche) and exposed to Kodak exposure film. Densitometric quantification of the immunoblot data was performed by using Quantity-One software (version 1.2; Bio-Rad Laboratories, Inc.).

Klebsiella pneumoniae cells were prepared as standard operation. Klebsiella pneumoniae cells were fixed with formaldehyde solution (10%). Rehydrated slides or cells were incubated with primary antibodies: Anti-PBP (dilution 1:500, cat. no. ab226275, Abcam) or anti-zinc finger nuclease (dilution 1:1,000, cat. no, WH0284312M2-100UG; Sigma-Aldrich; Merck KGaA) for 2 h at 37˚C. Samples were washed with PBS three times and then incubated with anti-IgG-FITC (dilution 1:1,000, cat. no. ab6785; Abcam). Samples were washed and imaged using a fluorescence microscopy (Nikon; magnification, x100).

The mass spectrometry was recorded using a quadrupole time-of-flight mass filter (Micromass, UK). Spectrometric analysis was scanned over a range 100-2,000 units of ratio m/z of mass to charge, with scan step 2 sec and interscan 0.1 sec/step. The quadrupole scan mode was performed under an electrospray voltage 3 kV at the tip of a stainless-steel capillary needle. The elution conditions were acetonitrile (50%) containing formic acid (0.1%) at rate 2 µl/min.

A total of 1x10⁶ cells in 100 µl H₂O were transfected with control siRNA negative control (control, 30 nM, 5'-UCACAACCUCCUAGAAAGAGUAGA-3') or siRNA targeting PBPs (PBP, 30 nM, 5'-CUGAAGUGAUGUGUAACUGAUCAG-3') using Hiperfect (Qiagen) transfection reagent. After 48 h of transfection, the transfected cells were treated with zinc finger nuclease (0.5 µg/ml, Sigma-Aldrich; Merck KGaA) for 12 h at 37˚C.

All presented data are reported as means ± SEM. Unpaired data were analyzed by Student's t-test. Comparisons of data between multiple groups were analyzed by analysis of variance (ANOVA) followed by Tukey post hoc test. ^*P<0.05 and ^**P<0.01 were assigned to indicate statistical significance and are denoted with the relevant asterisks in the figures.

In order to analysis the antimicrobial drug resistance of Klebsiella pneumoniae for β-lactam, we first determined ESBL gene expression and evaluated β-lactamase activity. The results in Fig. 1A demonstrate that both the mRNA and protein expression levels of ESBL were higher in Klebsiella pneumoniae isolated from patients with pneumonia (PD-K.p) than natural being Klebsiella pneumonia (NB-K.p). A band at 42 kDa protein was identified in the cell extract of Klebsiella pneumoniae. To determine the role of ESBL in the antimicrobial resistance of Klebsiella pneumoniae, the growth of Klebsiella pneumoniae was assessed. Our data showed that β-lactamase could hydrolyze the β-lactam ring, as determined by electrospray ionization-mass spectrometric analysis (Fig. 1B). PD-K.p presented a higher survival than NB-K.p in an environment with penicillin-based antibiotics (Fig. 1C). This may be due to the higher production of β-lactamase in PD-K.p than NB-K.p To determine the β-lactamase activity in vivo, ampicillin was utilized as a substrate to confirm the activity of β-lactamase. The data in Fig. 1D illustrate that new signals with m/z 368 units M-H⁺ and 390 units M-Na⁺ were observed in NB-K.p after incubation with β-lactamase protein at 25˚C for 30 min (m/z of ampicillin is 150 units M-H⁺ and 172 units M-Na⁺ of PD-K.p). These data suggest that β-lactamase hydrolyzed the amide bond of the β-lactam ring and promoted antimicrobial drug resistance of PD-K.p.

Penicillin-binding proteins (PBPs), bacterial peripheral membrane enzymes, play an essential role in catalysis in the final steps for the biosynthesis of the essential bacterial cell wall peptidoglycan. PBPs are involved in antimicrobial drug resistance In this study, we aimed to ascertain whether PBPs (25 mg/ml) could attenuate antimicrobial drug resistance of Klebsiella pneumoniae for β-lactam antibiotics. Our results showed that PBPs could bind with β-lactam antibiotic (penicillin) in vitro (Fig. 2A). It was also found that PBPs inhibited Klebsiella pneumoniae growth (PD-K.p and NB-K.p) compared to the control after incubation with penicillin (Fig. 2B). This revealed that PBPs significantly inhibited the growth of PD-K.p compared to NB-K.p (P<0.01 vs. NB-K.p). In addition, our data in Fig. 2C showed that penicillin could bind the FITC-labeled PBP-specific antibody, and these results suggested that PBPs were expressed and presented at the cell surface of Klebsiella pneumonia, as determined by confocal fluorescence microscope. Furthermore, PBPs downregulated expression of β-lactamase (Fig. 2D). These results suggest that the binding of PBPs to β-lactam may interfere with the synthesis of β-lactamases.

A previous study indicates that zinc finger nuclease technology is able to target and disrupt gene-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of antibiotic resistant bacteria and antibiotic resistance genes (23). Therefore, we analyzed the function of zinc finger nuclease in Klebsiella pneumoniae. Fig. 3A shows the antimicrobial susceptibility profile of Klebsiella pneumoniae. The results revealed that Klebsiella pneumoniae bacteria exhibit varying degrees of multi-drug resistance (CD, clindamycin; E, erythromycin; LZD, linezolid; PG, penicillin G; RP, rifampin; OX, oxacillin; concentrations of all the antibiotics were 1 mg/ml). However, zinc finger nuclease treatment decreased drug resistance of Klebsiella pneumoniae compared to control. Fig. 3B indicates that the growth of Klebsiella pneumoniae was inhibited after transduction of plasmids containing zinc finger nuclease. The optical density experiment demonstrated a marked decrease in Klebsiella pneumoniae growth after treatment with ampicillin compared with the control groups (P<0.01) at the indicated temperature, which was consistent with the results in Fig. 3A (Fig. 3C). In addition, the kinetics of zinc finger nuclease in Klebsiella pneumoniae was analyzed. The data showed that zinc finger nuclease markedly increased the binding potential of PD-K.p to penicillin compared with PBS (control) (Fig. 3D). The data suggest that zinc finger nuclease inhibited Klebsiella pneumoniae growth and enhanced the binding capacity of penicillin to Klebsiella pneumoniae.

To understand the mechanism of zinc finger nuclease in inhibiting Klebsiella pneumoniae growth, we analyzed the relationship among β-lactamase, PBPs and zinc finger nuclease. First, we detected expression levels of β-lactamase and PBPs in Klebsiella pneumoniae after incubation with zinc finger nuclease for 24 h. Fig. 4A shows that zinc finger nuclease treatment upregulated PBP expression and downregulated β-lactamase production. Knockdown of gene-coding PBPs abrogated the inhibitory effects on zinc finger nuclease for PD-K.p (Fig. 4B). Importantly, our data showed that the inhibitory effects of zinc finger nuclease were more superior to PBPs at the same concentration (10 mg/ml) in regards to PD-K.p growth (Fig. 4C). Notably, expression levels of two members (BlaR1 and Blal) of the β-lactamase signaling pathway in PD-K.p were decreased after zinc finger nuclease treatment (Fig. 4D). To discover the action site of zinc finger nuclease, we transfected plasmids of rpGFP-ZFN into Klebsiella pneumoniae with rpGFP as control. As shown in Fig. 4E, strong fluorescence was observed in surrounding Klebsiella pneumoniae cells delivering rpGFP-ZFN plasmid, while fluorescence was found in the cytoplasm carrying rpGFP, indicating that the action site of zinc finger nuclease is associated with the membrane of Klebsiella pneumoniae. Fig. 4F further confirmed that the action site of zinc finger nuclease is located in the outer membrane of Klebsiella pneumoniae. These results indicate that zinc finger nuclease could regulate PBPs or zinc finger nuclease expression, suggesting that it may represent a potential agent for antibiotic resistance.