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Article Identification of Pathogenic Factors in pneumoniae Using Impedimetric Sensor Equipped with Biomimetic Surfaces

Duyen Thi Ngoc Huynh, Ah-Young Kim and Young-Rok Kim *

Department of Food Science and Biotechnology & Graduate School of Biotechnology, College of Sciences, Kyung Hee University, Yongin 17104, Korea; [email protected] (D.T.N.H.); [email protected] (A.-Y.K.) * Correspondence: [email protected]; Tel.: +82-31-201-3830

Academic Editors: Jean-Louis Marty, Silvana Andreescu and Akhtar Hayat Received: 11 April 2017; Accepted: 9 June 2017; Published: 15 June 2017

Abstract: K. pneumoniae is an opportunistic that causes nosocomial , such as, , urinary tract infections, septic , and gastro intestinal disease. (LPS), capsular polysaccharide, and fimbriae are recognized major factors of K. pneumoniae and play key roles during early stages of infections. In this study, we functionalized the surface of gold electrode with mannose and mucin to monitor the -associated virulence factors of K. pneumoniae. The binding characteristics of K. pneumoniae 2242 wild type and of its isogenic mutants lacking outer-core LPS (∆wabG) or fimbriae (∆fimA) were investigated using Faradaic impedance spectra. The results obtained showed fimbriae are responsible for K. pneumoniae adhesion to the mannose of glycoprotein on the surfaces of epithelial cells, whereas outer-core LPS and capsular polysaccharide are associated with specific binding to mucous. These results concurred with those of a conventional in vitro assay using human ileocecal epithelial (HCT-8 cells) and a human bladder epithelial cell (T-24), indicating that the devised method could be useful for investigating virulence-associated interactions of pathogenic with specific host cells and organs.

Keywords: impedimetric sensor; virulence factors; ; lipopolysaccharide; fimbriae

1. Introduction K. pneumoniae is a well-known opportunistic pathogen and responsible for ~10% of nosocomial bacterial infections, which include , pneumonia, urinary tract infections, and hepatic [1–3]. The bacterium can infect almost every part of the human body, although the urinary and respiratory tracts are most commonly affected [4]. The pathogenic factors of K. pneumoniae have been determined to be capsular polysaccharide (CPS), lipopolysaccharide (LPS), and fimbriae [5]. LPS is a major component of the outer membrane of Gram-negative bacteria, and consists of , core oligosaccharide, and a long chain polysaccharide (O-antigen). LPS is an important pathogenic determinant in K. pneumoniae, which causes pneumonia and bacteremia in man. O-antigen is responsible for bacterial resistance to complement-mediated killing. On the other hand, CPS is considered the most important virulence factor of K. pneumoniae. It covers the bacterial surface and is responsible for its resistance to host phagocytes and serum and promotes inflammation and sepsis [5–7]. The colonization of mucous membranes by bacteria is the result of adhesion between the and the host’s mucous layer [5,8]. The adhesion of K. pneumoniae to mammalian tissue is mediated by two types of bacterial pili, type 1 and type 3 [9,10]. Type 1 fimbriae play an important role in bacterial adhesion to the D-mannose moiety on mammalian cell surfaces due to the specific affinity of fimbrial protein at the tips of fimbriae [11,12]. Type 3 fimbriae are characterized by their affinities for a range of mammalian cells, which include bladder epithelial cells, uroepithelial cells, and endothelial cells [13,14]. The factors

Sensors 2017, 17, 1406; doi:10.3390/s17061406 www.mdpi.com/journal/sensors Sensors 2017, 17, 1406 2 of 13 present on the surfaces of epithelial cells are recognized by and include certain proteins, glycolipids, and carbohydrates [15]. In particular, carbohydrate-protein recognition plays a key role in most pathogenic processes, including bacterial infections, differentiation, and inflammation, and is involved with specific carbohydrate sequences on host cell surfaces [16]. Herein, we describe an impedimetric sensor equipped with a functionalized electrode, which mimics the surfaces of epithelial cells and the luminal surfaces of epithelial organs, and its use to identify the virulence factors of K. pneumoniae associated with the adhesion process during the early stage of . Outer-core LPS and fimbriae of K. pneumoniae are major virulence factors and are known to be responsible for its adhesion to the mucous layer and epithelial cells, respectively [17,18]. The mucins, which are major components of mucous layers, compose a family of glycoproteins that cover the luminal surfaces of epithelial organs [19]. On the other hand, the mannose moiety of N-linked glycoprotein on the outer surfaces of human epithelial cell membranes has been reported to be associated with the specific interaction with fimbriae of K. pneumoniae [20]. We functionalized the surface of a gold electrode with mucin and mannose, and examined its ability to monitor the presence of outer-core LPS and fimbriae of K. pneumoniae. In this study, we employed K. pneumoniae 2242 wild type and isogenic mutants devoid of outer-core LPS and fimbriae. The changes in Faradaic impedance spectra derived from direct interaction between the bacterium and the model surface were analyzed to monitor the presence of virulence factors. Conventional in vitro assays using human ileocecal epithelial cells (HCT-8 cells) and human bladder epithelial cells (T-24 cells) were performed to validate the accuracy of the developed impedimetric sensing system.

2. Materials and Methods

2.1. Materials Mucin (from porcine stomach, Type III), 11-mercapto-1-undecanol, epichlorohydrin, diethylene glycol dimethyl ether, potassium chloride, potassium hexacyanoferrate (II) trihydrate, potassium ferricyanide (III), sodium phosphate, and D-mannose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Absolute ethyl alcohol, sodium hydroxide, acetone, sulfuric acid, sodium acetate, acetic acid, and hydrogen peroxide were from DaeJung Chemicals & Metals Co., Ltd. (Gyeonggi-do, Korea). Carboxyfluorescein diacetate (CFDA) was obtained from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA), and potassium phosphate from Pure Chemicals Co., Ltd. (Kyoto, Japan). Lysogeny broth (LB) broth and agar powder were purchased from BD (BD Difco, Lawrence, KS, USA).

2.2. Bacterial Strains, and Growth Conditions Klebsiella pneumoniae KCTC 2242 (2242) was obtained from the Korean Collection for Type Cultures (Daejeon, Korea). K. pneumoniae KCTC 2242 ∆wabG mutant strain and K. pneumoniae KCTC 2242 ∆fimA mutant strain were constructed by Jung et al. [21] and Huynh et al. [17]. For complementation studies, the wabG and fimA were cloned into pBBR1MCS5 vector (CABRI Consortium, Genova, Italy) as follows. The two genes were amplified from K. pneumoniae KCTC 2242 by PCR using the following primer sets: for the wabG ; 50 AAA AAG CTT ATG AGT AAA TTC AGG CTG GC 30 and 50 AAA GGA TCC TCA GTG GTG GTG GTG GTG GTG TTA ATT CAC CAG ATC 30; and for the fimA gene; 50 AAA AAG CTT ATG AAA ATC AAA ACA CTG GC 30 and 50 AAA GGA TCC TCA GTG GTG GTG GTG GTG GTG TTA CTC GTA CTG CAC 30. Amplified wabG and fimA genes were purified and digested with HindIII and BamH I. pBBR1MCS5 vector was also digested with these restriction enzymes. Digested wabG and fimA genes were ligated into pBBR1MCS5 vector using T4 ligase, and the resulting constructs, pBBR1MCS5::wabG and pBBR1MCS5::fimA vectors, were transferred into K. pneumoniae KCTC 2242 ∆wabG mutant and K. pneumoniae KCTC 2242 ∆fimA mutant, respectively, by electroporation. The complement strains, K. pneumoniae KCTC 2242 ∆wabG containing pBBR1MCS5::wabG and K. pneumoniae KCTC 2242 ∆fimA containing pBBR1MCS5::fimA were selected in LB agar media containing , and subsequently confirmed by colony PCR using the primer Sensors 2017, 17, 1406 3 of 13 sets described above. All bacterial strains were incubated at 37 ◦C in LB broth or LB agar plates containing 10 µg/mL gentamicin. For binding assays, fresh-cultured bacteria were washed three times with PBS buffer (pH 7.4) and then serially diluted in fresh PBS buffer. A conventional plate counting method was used to confirm the number of microorganisms in each sample.

2.3. Cell Lines and Culture Conditions The human ileocecal epithelial cell line HCT-8 and the human bladder epithelial cell line T-24 were provided by the Korean Cell Line Bank (Seoul, Korea). Cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Welgene, Gyeongsan, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 mg/mL). For epithelial cell adherence assays, HCT-8 and T-24 cells were seeded at ~104 cells/well in a 96-well plate (polystyrene; ◦ clear flat bottoms; Corning, NY, USA) and grown in 5% CO2 for 24 h at 37 C.

2.4. Mucin and Mannose Binding Assays

Functionalizations of 96-well plates with mucin or D-mannose were performed as previously described [22,23]. For the mucin binding assay, mucin was dissolved in an acetate buffer (pH 5.0) to a final concentration of 100 µg/mL, and a 96-well plate was incubated with 100 µL/well of a mucin solution at 37 ◦C for 24 h. Unbound mucin was removed by washing three times with PBS (pH 7.0). For the mannose binding assay, D-mannose was first dissolved in 0.1 M NaOH to a final concentration of 2% (w/v) and 100 µL of the solution was added to the wells of a 96-well plate. After incubating at room temperature for 20 h, the plate was washed three times with PBS (pH 7.0) to remove unbound D-mannose. Overnight cultures of K. pneumoniae were harvested by centrifugation, washed three times with PBS, and resuspended in 0.1 M phosphate buffer (pH 8.5). Carboxylfluorescein diacetate (CFDA) (final concentration 2 µg/mL) was added to the bacterial suspension, which was then incubated at 37 ◦C for 30 min. Labeled bacteria were washed three times with PBS to remove unbound fluorophore, and resuspended in PBS to a final concentration of 105, 106, 107 or 108 colony forming unit (CFU)/mL. These bacterial suspensions (100 µL) were introduced to wells and incubated at 37 ◦C for 1 h to assess adhesion. After washing microtiter wells three times with PBS, degrees of bacterial binding to mucin or mannose surfaces was determined by fluorescence spectrophotometry (Tecan, Infinite 200, Grodig, Austria) using excitation and emission wavelengths of 485 and 535 nm, respectively.

2.5. Functionalization of a Gold Electrode with Mucin and Mannose A gold electrode was prepared on glass wafers using an UHV E-Beam Evaporator (ULTECH, Daegu, Korea), as we previously described [24]. Piranha solution (sulfuric acid:hydrogen peroxide, 3:1 (v/v)) was used to remove organic residues from 4-inch glass wafers. After cleaning, glass wafers were pre-coated with a 300 Å-thick titanium layer to promote the adhesion of Au to a glass substrate, and then the chip with titanium layer was subsequently coated with a 1000 Å layer of high purity gold using the same electron beam evaporator. The gold-coated glass wafers produced was cut into 10 mm × 20 mm pieces with a wafer dicing machine (DISCO, Tokyo, Japan). The surfaces of gold electrode were then thoroughly washed with acetone and ethanol. The functionalization of gold electrode with mucin or mannose were carried out as follows. For mucin functionalization, a freshly prepared gold electrode was immersed in an 80% ethanolic solution containing 5 mM 11-mercapto-1-undecanol for 3 h at room temperature. The resulting gold electrode, now possessing surface hydroxyl groups, was then washed with ethanol, dried under a gentle stream of nitrogen, and immersed in a solution of 0.6 M epichlorohydrin containing 1:1 mixture of 0.4 M diethylene glycol dimethyl ether and sodium hydroxide for 4 h at 25 ◦C. After through sequential washing with water, ethanol, and water, the surface of the electrode was treated with a 0.1 M NaOH containing 300 mg/mL of mannose at 4 ◦C for 20 h. To functionalize the surface of gold electrodes with mucin, chips were immersed in acetate buffer (pH 5.0) containing 5 mg/mL of mucin at 4 ◦C for 20 h. The electrodes Sensors 2017, 17, 1406 4 of 13 were thoroughly washed with distilled water to remove remaining chemicals and stored in distilled water until required.

2.6. Electrical Measurement of Bacterial Adhesion to a Model Surface A gold electrode functionalized with mucin or mannose was fixed in custom-built Teflon jigs and fastened with four clips to prevent leakage. The microfluidic jig had two openings, which served as the test solution inlet and outlet, and a platinum electrode immersed into the solution in the reaction chamber. A 0.5 mL sample was introduced into the chamber formed on the surface of gold electrode at a flow rate of 50 µL/min using a syringe pump (Model 11 plus, Harvard Apparatus, South Natick, MA, USA) and 1/16” ID Ethylene-tetrafluorethylene (ETFE) tubing (Upchurch Scientific, Oak Harbor, WA, USA). The surface of gold electrode was then washed with 0.5 mL of PBS buffer (pH 7.4) to remove 3−/4− unbound bacteria. PBS buffer (pH 7.4) containing the redox probe [Fe(CN)6] (at a concentration of 2 mM in the reaction chamber) were introduced before taking measurements. Impedance spectrum data were obtained using an electrochemical analyzer VersaSTAT3 (Princeton Applied Research, Oak Ridge, TN, USA) equipped with V3-Studio software (Princeton Applied Research). The impedance 3−/4− data were collected in the frequency range 0.1 to 1 kHz at the formal potential of the [Fe(CN)6] redox couple. The magnitude of impedance at each frequency point was analyzed by normalized impedance changes with respect to the control (PBS buffer). Normalized impedance change (NIC) values were calculated using the following formula:

Zsample − Zcontrol NIC = (1) Zcontrol where Zcontrol is the impedance of the control and Zsample that of the sample containing bacteria. All experiments were repeated three times and averages and standard deviations were calculated.

2.7. Competition Assay The specific affinity of fimbriae toward mannose was tested by monitoring the binding of K. pneumoniae KCTC 2242 to mannose surfaces in the presence of free mannose at concentrations of 0.05, 0.25, or 0.5 mM in PBS (pH 7.4). Bacterial cultures grown overnight were centrifuged at 2000 g for 1 min at room temperature, and after washing bacterial pellets three times with PBS, 107 CFU/mL bacterial suspensions were pre-incubated with mannose solutions of respective concentrations for 30 min at room temperature. These solutions were then introduced individually into the fluidic chamber containing the mannose-coated electrode. All experiments were performed three times.

2.8. Statistical Analysis One-way analysis of variance (ANOVA) with Duncan post hoc test was used to determine the significances of differences between the wild-type and mutant types. p Values of < 0.05 were considered to denote statistical significance.

3. Results and Discussion

3.1. Characterization of Model Surface The adhesion of pathogenic bacteria to human epithelial cells is the first step of infections that could advance to a series of systematic diseases. Therefore, the ability of a bacterium to bind to specific epithelial cells is currently regarded as the key factor for its pathogenicity. However, several reports indicate the ability of K. pneumoniae to adhere to cultured epithelial cells is not always associated with bacterial infectivity or virulence, for example, an in vitro study showed no correlation with in vivo results in a mouse model [25]. The reason for this lack of correlation may be that epithelial cells in lung, intestinal tract, and bladder are typically covered with a mucous layer, which serves as physical barrier between the extracellular milieu and the plasma membrane. Furthermore, this mucous Sensors 2017, 17, 1406 5 of 13 layer also facilitates the mucocilliary clearance of potential infectious bacteria from organ surfaces. Accordingly, in vitro studies should be accompanied by in vivo studies to determine the virulence factors or pathogenicity of given bacteria, as these are associated with binding-mediated infection. In the present study, we developed an electrochemical sensing system that can determine the virulence factors associated with binding-mediated infection using a modified model surface that mimics the surfaces of epithelial cells and mucus. We modified the surfaces of gold electrodes with D-mannose or mucin, which mimic the outer layer of epithelial cells and the surrounding mucus layer, respectively. Figure1 presents a schematic of the electrode functionalization with functional groups. A gold electrode on a glass substrate was modified with a monolayer of mercaptoundecanol by self-assembly [26], and this layer was derivatized using epichlorohydrin under basic conditions and subsequent mucin or D-mannose coupling achieved by epoxide ring opening reaction [27]. The surface-functionalized electrode was fixed in microfluidic jig (Figure2). In this study, we used two-electrode system to simplify the cell setup. The results were not much different from those obtained from three-electrode system. Surface properties of gold electrode at each step were monitored by taking impedimetric measurements in the presence of 3−/4− a [Fe(CN)6] redox probe. The changes in the electrochemical properties of the redox probe are detailed in the Randle equivalent circuit shown in Figure2. This equivalent circuit consists of the ohmic resistance of the electrolyte, Rs, the Warburg impedance Zw, resulting from the diffusion of ions from bulk electrolyte to the electrode, the double layer capacitance, Cdl, and the interfacial electron transfer resistance, Ret. Rs and Zw are not affected by physicochemical transformations at the electrode surface because they represent bulk properties of the electrolyte solution and diffusion features of the redox probe in solution. On the other hand, Ret and Cdl are determined by dielectric and insulating properties at the electrode/solution interface [28]. The number of bacteria attached to the surface of electrode has been reported to be related with double-layer capacitance [29]. In other words, attached bacteria or any type of attached mass to an electrode would cause a decrease in Cdl by blocking the electrode surface. Figure3 shows the Faradaic impedance spectra, presented as Nyquist plots of the gold electrode, over the course of surface modification. Impedance was measured at frequencies ranging from 0.1 to 1 kHz 3−/4− at the formal potential of the [Fe(CN)6] redox probe. The electron transfer resistance of the gold electrode increased substantially after modifying the surface with a monolayer of mercapto undecanol, which was presumed to be due to the formation of a partitioning or insulating layer of mercapto undecanol on the electrode. The electron transfer resistance of the redox probe further increased after subsequent treatments with epichlorohydrin and mucin. Figure4 shows a Bode plot and a Nyquist diagram for the Faradaic impedance measurement of a mannose-coated gold electrode in the presence of K. pneumoniae 2242 concentrations ranging from 105 to 108 CFU/mL. Impedance changes caused by bacterial attachment to an electrode are typically analyzed using Bode plots [30]. Greatest impedance differences were observed at 0.1 Hz with respect to the control. The number of bacteria attached to the mannose-coated electrode is correlated with double-layer capacitance, which dominates impedance at low frequency, whereas the dielectric capacitance dominates impedance at high frequency. Sensors 2017, 17, 1406 6 of 13 Sensors 2017, 17, 1406 6 of 13 Sensors 2017, 17, 1406 6 of 13

Figure 1. Schematic of the surface modification of the gold electrode with mannose (a) or mucin (b). Figure 1. Schematic of of the the surface surface modification modification of of the the gold gold electrode electrode with with mannose mannose ( (aa)) or or mucin mucin ( (bb).). An epoxy monolayer was formed by the self-assembly of 11-mercapto-1-undecanol on the surface of An epoxy monolayer was formed by the self-assembly of 11-mercapto-1-undecanol on the surface of the gold electrode and this was then reacted with epichlorohydrin. Mannose and mucin were then thethe gold gold electrode and this was then reacted with epichlorohydrin. Mannose Mannose and and mucin mucin were were then coupled to the surface by epoxy ring opening reactions. (R1 = β(1,6)-GlcNAc, R2 = ββ(1,3)-GlcNAc). coupledcoupled to the surface by epoxy ring opening reactions. (R 1 == β(1,6)-GlcNAc,(1,6)-GlcNAc, R R2 2== β(1,3)-GlcNAc).(1,3)-GlcNAc).

Figure 2. Experiment setup showing the sensing chamber composed of a surface functionalized gold Figure 2. 2. ExperimentExperiment setup setup showing showing the the sensing sensing chamber chamber composed composed of a ofsurface a surface functionalized functionalized gold electrode and a Teflon jig (a). AC power was connected to a potentiostat that supplied the required electrodegold electrode and a andTeflon a Teflonjig (a). jigAC ( apower). AC was power connected was connected to a potentiostat to a potentiostat that supplied that suppliedthe required the current between the functionalized electrode and Ag/AgCl electrode located on top of the fluidic currentrequired between current the between functionalized the functionalized electrode and electrode Ag/AgCl and electrode Ag/AgCl located electrode on locatedtop of the on fluidic top of chamber; (b) shows a circuit diagram of the set up used for electrochemical impedance spectroscopy chamber;the fluidic (b chamber;) shows a (circuitb) shows diagram a circuit of the diagram set up ofused the for set electrochemical up used for electrochemical impedance spectroscopy impedance measurements in the presence of a redox probe (b). The circuit elements shown are; solution resistance measurementsspectroscopy measurements in the presence inof thea redox presence probe of(b a). The redox circuit probe elements (b). The shown circuit are; elements solution shown resistance are; (Rs), interfacial electron transfer resistance (Ret), Warbug’s impedance (Zw), and double layer (solutionRs), interfacial resistance electron (Rs), interfacialtransfer resistance electron transfer (Ret), Warbug’s resistance (impedanceRet), Warbug’s (Zw), impedance and double (Zw ),layer and capacitance (Cdl). capacitancedouble layer (C capacitancedl). (Cdl).

Sensors 2017, 17, 1406 7 of 13 Sensors 2017, 17, 1406 7 of 13

Sensors 2017, 17, 1406 7 of 13

Figure 3. Nyquist diagram (Zim vs. Zre) for the Faradaic impedance measurement of a sensing chip in Figure 3. Nyquist diagram (Zim vs. Zre) for the Faradaic impedance measurement of a sensing chip in PBS (pH 7.4) containing 2 mM [Fe(CN)6]3−/4− over the course of the surface functionalization reaction. Figure 3. Nyquist diagram (Zim vs. Zre)3 for−/4 the− Faradaic impedance measurement of a sensing chip in PBS (pHA gold 7.4) electrode containing (a) 2was mM treated [Fe(CN) with6] 11-mercapto-1-undecanolover the course of (b the), epichlorohydrin surface functionalization (c), and mucin reaction. PBS (pH 7.4) containing 2 mM [Fe(CN)6]3−/4− over the course of the surface functionalization reaction. A gold(d). electrode Impedance (a) spectra was treated were measured with 11-mercapto-1-undecanol within the frequency ranging (b), epichlorohydrinfrom 0.1 to 1000 Hz (c at), the and formal mucin (d). A gold electrode (a) was treated with 11-mercapto-1-undecanol (b), epichlorohydrin (c), and mucin Impedancepotential spectra of the [Fe(CN) were measured6]3−/4− redox within couple. the The frequency amplitude rangingof the alternative from 0.1 voltage to 1000 was Hz 5 atmV. the The formal (d). Impedance spectra 3were−/4− measured within the frequency ranging from 0.1 to 1000 Hz at the formal potentialinset ofshows the an [Fe(CN) enlarged6] plot forredox a bare couple. gold electrode. The amplitude of the alternative voltage was 5 mV. potential of the [Fe(CN)6]3−/4− redox couple. The amplitude of the alternative voltage was 5 mV. The The insetinset shows showsan an enlargedenlarged plot plot for for a bare a bare gold gold electrode. electrode.

Figure 4. Bode plot (a) and Nyquist diagram (b) of the Faradaic impedance measurements of a mannoseFigure 4. modified Bode plot electrode (a) and in Nyquist the presence diagram of different(b) of the concentrations Faradaic impedance of K. pneumoniae measurements KCTC of 2242 a Figure 4. Bode plot (a) and Nyquist diagram (b) of the Faradaic impedance measurements of a mannose 5 8 (frommannose 10 to modified 10 CFU/mL). electrode Impedance in the presence spectra of were different measured concentrations within the of frequency K. pneumoniae ranging KCTC from 2242 0.1 5 modified electrode5 in8 the presence of different concentrations of K. pneumoniae KCTC 2242 (from 10 to to(from 1 kHz 10 at to the 10 potential CFU/mL). of Impedance the [Fe(CN) spectra6]3−/4− redoxwere measuredprobe. The within amplitude the frequency of the alternating ranging from voltage 0.1 8 10 CFU/mL). Impedance spectra were measured3−/4− within the frequency ranging from 0.1 to 1 kHz at wasto 15 kHzmV. at the potential of the [Fe(CN)6] redox probe. The amplitude of the alternating voltage 3−/4− the potentialwas 5 mV. of the [Fe(CN)6] redox probe. The amplitude of the alternating voltage was 5 mV. 3.2. Adhesion Properties of K. pneumoniae on the Mannose-Modified Surface 3.2. Adhesion3.2. Adhesion Properties Properties of K. of pneumoniaeK. pneumoniae on on the the Mannose-ModifiedMannose-Modified Surface Surface The fimbriae of K. pneumoniae have been reported to be an important adhesion factor as they Theattach fimbriae Theto a fimbriaespecific of K. carbohydrate of pneumoniae K. pneumoniae moietyhave have on been been the reportedhostreported cell’s to surface to be be an an [17,31].import importantant The adhesion fimbriae adhesion factor formfactor aas specific they as they attach to a specific carbohydrate moiety on the host cell’s surface [17,31]. The fimbriae form a specific attachinteraction to a specific with carbohydrate mannosyl residues moiety of N-linked on the host glycoprotein cell’s surface on the [17 outer,31]. layer The of fimbriae target cells, form which a specific suggestsinteraction that with the mannosylfimbriae of residues K. pneumoniae of N-linked play glycoprotein a critical role on in the early outer stage layer of of infection target cells, [32]. which In this interactionsuggests with that mannosyl the fimbriae residues of K. pneumoniae of N-linked play glycoprotein a critical role in on early the outerstage of layer infection of target [32]. In cells, this which study, we designed a model surface functionalized with mannose on a gold electrode to monitor the suggestsstudy, that we the designed fimbriae a model of K. surface pneumoniae functionalizedplay a criticalwith mannose role in on early a gold stage electrode of infection to monitor [32 the]. In this presence of K. pneumoniae fimbriae using electrochemical impedimetric spectroscopy. The binding study,presence we designed of K. pneumoniae a model surface fimbriae functionalized using electrochemical with mannose impedimetric on a spectroscopy. gold electrode The to binding monitor the ability of K. pneumoniae 2242 and of its isogenic mutant, K. pneumoniae 2242 ∆fimA, which is devoid of ability of K. pneumoniae 2242 and of its isogenic mutant, K. pneumoniae 2242 ∆fimA, which is devoid of presencefimbriae of K. were pneumoniae also examinedfimbriae using using the electrode electrochemical setup. We impedimetricalso tested the binding spectroscopy. characteristics The binding of fimbriae were also examined using the electrode setup. We also tested the binding characteristics of abilityouter of K. core pneumoniae LPS-truncated2242 mutant and of itsstrain, isogenic K. pneumoniae mutant, K.2242 pneumoniae ∆wabG. The2242 wabG∆fimA, gene whichencodes is for devoid outer core LPS-truncated mutant strain, K. pneumoniae 2242 ∆wabG. The wabG gene encodes for of fimbriaeglucosyltransferase, were also examined which is responsible using the electrodefor the attachment setup. We of α also-L-glycero- testedD the-manno-heptopyranose binding characteristics glucosyltransferase, which is responsible for the attachment of α-L-glycero-D-manno-heptopyranose of outerII tocore the О LPS-truncated-3 position of α mutant-D-galactopyranosyluronic strain, K. pneumoniae acid. 2242The outer∆wabG core. The LPSwabG of K. genepneumoniae encodes is for II to the О-3 position of α-D-galactopyranosyluronic acid. The outer core LPS of K. pneumoniae is glucosyltransferase,involvedinvolved in in the the formation whichformation is responsibleofof aa capsulecapsule layerlayer for the onattachment the surfacesurface ofof of thetheα- L bacterium,bacterium,-glycero- Dand and-manno-heptopyranose thus, thus, the the ∆ wabG∆wabG II to themutantmutant O-3 lost lost position the the ability ability of αto to-D form form-galactopyranosyluronic aa capsulecapsule layer on the surfacesurface acid. [21].[21]. The The The outer absence absence core of LPSof capsule capsule of K. formation formation pneumoniae is involvedbyby K. K. inpneumoniae pneumoniae the formation ∆ ∆wabGwabG mutant ofmutant a capsule strain strain waswas layer confirmedconfirmed on the by surfaceby microscomicrosco ofpic thepic analysis analysis bacterium, after after staining andstaining thus, cells cells thewith with∆ wabG crystal violet and ink (Figure S1). The binding characteristics of K. pneumoniae 2242 and of its mutantcrystal lost theviolet ability and India to form ink (Figure a capsule S1). layerThe binding on the characteristics surface [21]. of The K. pneumoniae absence of 2242 capsule and of formation its isogenic mutants to the mannose-coated electrode were analyzed based on NIC value at 0.1 Hz, which by K. pneumoniaeisogenic mutants∆wabG to themutant mannose-coated strain was electrode confirmed were analyzed by microscopic based on NIC analysis value at after0.1 Hz, staining which cells reflectsreflects the the number number of of bacteria bacteria boundbound toto thethe surface of thethe functionalizedfunctionalized electrode electrode (Figure (Figure 5a). 5a). The The with crystal violet and India ink (Figure S1). The binding characteristics of K. pneumoniae 2242 and of its isogenic mutants to the mannose-coated electrode were analyzed based on NIC value at 0.1 Hz, which reflects the number of bacteria bound to the surface of the functionalized electrode (Figure5a). The NIC value was found to increase in proportion to the concentration of test bacteria. The binding Sensors 2017, 17, 1406 8 of 13

NIC value was found to increase in proportion to the concentration of test bacteria. The binding pattern of of K.K. pneumoniae pneumoniae wildwild type type to to the the mannose-coated mannose-coated surfac surfacee was was not not very very different different from from that that of K.of pneumoniaeK. pneumoniae ∆wabG∆wabG, but, but the thebinding binding ability ability of K. of pneumoniaeK. pneumoniae 22422242 ∆fimA∆fimA to theto mannose the mannose surface surface was significantlywas significantly reduced reduced at all at bacterial all bacterial concentrations concentrations tested, tested, indicating indicating that that fimbriae fimbriae were were critically critically responsible for for the attachment of of K. pneumoniae toto mannose surface. surface. To To confirm confirm that that the the loss loss of the ability of bacteria to bind to the mannose surfac surfacee was due to the absence of fimbriae,fimbriae, we examined the binding property ofof thethe complementarycomplementary strain,strain, K.K. pneumoniaepneumoniae ∆∆fimAfimA/pBBR1MCS5-/pBBR1MCS5-fimAfimA,, to to the mannose surface. The The binding binding ability ability of of K.K. pneumoniae pneumoniae ∆∆fimAfimA waswas fully fully restored restored to to the the level of the wild type by introducing the fimAfimA gene to the mutant strain. This results shows that fimbriae fimbriae were responsible for for the attachment of of bacteria bacteria to to the the mannose surface. surface. In In other other words, words, the the binding binding of of K.K. pneumoniae toto human human epithelial cells cells is is at at least in part derived from an interaction between bacterial fimbriaefimbriae and the mannose moiety in the outer surfaces of epithelial cell.

Figure 5. ImpedanceImpedance measurements measurements ( (aa)) and and conventional conventional fluorescence fluorescence measurements obtained using thethe 96-well 96-well plate plate method method ( b) for for monitoring monitoring the adhesion adhesion of of K. pneumoniae KCTC 2242 and its isogenic isogenic mutants to the mannose modifiedmodified electrode. Mean Mean va valueslues with with different superscripts (a,b) at each bacterial concentration indicate significant significant differenc differenceses between the wild type and its isogenic mutant strains ( p < 0.05). 0.05).

Impedimetric results were compared with thos thosee obtained using a conventional assay conducted using a 96-well plate coated with α-D-mannose. CFDA-labeled K. pneumoniae wild type and its using a 96-well plate coated with α-D-mannose. CFDA-labeled K. pneumoniae wild type and its isogenic isogenicmutants mutants were allowed were toallowed bind to to the bind surface to the of surf mannose-coatedace of mannose-coated 96-well plates 96-well for plates 1 h. After for 1 washingh. After washingout unbound out unbound bacteria bacteria from plates from with plates PBS, with amounts PBS, amounts of bacteria of bacteria bound bound to mannose to mannose surfaces surfaces were wereassessed assessed by measuring by measuring the fluorescence the fluorescence emitted fromemitted CFDA-labeled from CFDA-labeled bacteria using bacteria a fluorescence using a fluorescencereader. The binding reader. patternsThe binding of the patterns wild type ofand the itswild isogenic type and mutants its isogenic were almost mutants identical wereto almost those identicalobtained to from those impedimetric obtained from measurements impedimetric (Figure measurements5b). (Figure 5b). Assuming that the binding of K. pneumoniae to the surface surface of epithelial epithelial cell is due to a a specific specific interactioninteraction betweenbetween fimbriae fimbriae and and mannose mannose moiety moiety of the of cell the surface, cell surface, the binding the binding ability of ability the bacterium of the bacteriumshould be should affected be by affected the presence by the presence of free mannose of free mannose molecules molecules because because the free the mannose free mannose would wouldcompete compete with immobilized with immobilized mannose mannose on the surface on the for surface binding for sites binding on fimbriae. sites onThus, fimbriae. we carried Thus, outwe carrieda competition out a competition assay to confirm assay this to hypothesisconfirm this and hy thepothesis accuracy and ofthe developed accuracy impedanceof developed sensor. impedance As was sensor.expected, As binding was expected, of K. pneumoniae bindingto mannoseof K. pneumoniae surfaces was to significantlymannose surfaces and concentration-dependently was significantly and concentration-dependentlyinhibited by the presence of inhibited free mannose. by the The presence binding of patterns free mannose. of K. pneumoniae The binding2242 patterns wild type of and K. pneumoniae 2242 wild type and its isogenic mutants to the model surface in the absence and presence its isogenic mutants to the model surface in the absence and presence of varying concentration of free of varying concentration of free mannose were measured using the impedance sensor, and the results mannose were measured using the impedance sensor, and the results obtained were compared with those obtained were compared with those obtained using the conventional 96-well plate method (Figure obtained using the conventional 96-well plate method (Figure6a,b). The results obtained using the two 6a,b). The results obtained using the two methods demonstrated almost the same patterns indicating methods demonstrated almost the same patterns indicating that the impedance sensor provides accurate that the impedance sensor provides accurate and reliable measurements of bacterial interactions with and reliable measurements of bacterial interactions with specific surfaces. specific surfaces. The binding properties of K. pneumoniae and its inhibition by free mannose were further validated The binding properties of K. pneumoniae and its inhibition by free mannose were further by an in vitro assay using human ileocecal epithelial cells (HCT-8 cells) and bladder epithelial cells validated by an in vitro assay using human ileocecal epithelial cells (HCT-8 cells) and bladder epithelial cells (T-24 cells). Binding patterns and degrees of inhibition in the presence of free mannose

Sensors 2017, 17, 1406 9 of 13

(T-24 cells). Binding patterns and degrees of inhibition in the presence of free mannose were consistent with those obtained using the impedance sensor (Figure6c,d). The binding of K. pneumoniae to the mannose-coatedSensors 2017, 17 surface, 1406 or to epithelial cells was only affected by the absence of fimbriae9 of 13 in ∆fimA mutant strain. Furthermore, the binding abilities of K. pneumoniae ∆fimA to the mannose surface and to epithelialwere cells consistent were fully with restored those obtained to the using level ofthe wild impedance type when sensor the (FigurefimA 6c,d).gene The was binding complemented of K. by pneumoniae to the mannose-coated surface or to epithelial cells was only affected by the absence of fimA introducingfimbriae pBBR1MCS5- in ∆fimA mutant. strain. The presence Furthermore, or absence the binding of outer abilities core of LPS K. pneumoniae and capsular ∆fimA polysaccharide to the did notmannose affect bacterial surface and binding to epithelial to the cells mannose were fully surface restored or toto epithelialthe level of cells.wild type Interestingly, when the fimA binding of K. pneumoniaegene was2242 complemented∆wabG mutant by introducing to mannose pBBR1MCS5- surfacefimA and. The to epithelialpresence or cells absence was of even outer highercore than that of itsLPS parental and capsular strain. polysaccharide This implies did that not the affect mutant bacterial strain, binding which to lackedthe mannose both outersurface core or to LPS and capsularepithelial polysaccharide, cells. Interestingly, is able to binding associate of K. with pneumoniae epithelial 2242 cells ∆wabG more mutant than toits mannose parental surface strain, and which is to epithelial cells was even higher than that of its parental strain. This implies that the mutant strain, consistent with a previous report that capsular polysaccharide reduces bacterial adherence to epithelial which lacked both outer core LPS and capsular polysaccharide, is able to associate with epithelial cells in vitrocells [more8,18 ,33than]. Inits∆ wabGparentalmutant, strain, twowhich restraints is consistent limit adhesionwith a previous to epithelial report cells.that capsular The first is that the thickpolysaccharide layer of capsular reduces polysaccharide bacterial adherence around to epithelial bacteria cells partially in vitro [8,18,33]. masks the In ∆ adhesionwabG mutant, component two in fimbriaerestraints that otherwise limit adhesion interacts to epithelial with the cells. mannose The first moiety is that ofthe epithelial thick layer cells. of capsular The second polysaccharide is that capsular polysaccharidesaround bacteria interfere partially with masks the biosynthesisthe adhesion component of fimbrial in fimbriae subunits that into otherwise mature interacts fimbriae with on the bacterial surfacesmannose [34]. It ismoiety noteworthy of epithelial that thecells. enhanced The second binding is that abilitycapsular of polysaccharidesK. pneumoniae 2242interfere∆wabG withto the epithelial biosynthesis of fimbrial subunits into mature fimbriae on bacterial surfaces [34]. It is noteworthy that cells was also clearly monitored by impedimetric measurements of the mannose-coated model surface the enhanced binding ability of K. pneumoniae 2242 ∆wabG to epithelial cells was also clearly (Figure6monitored). by impedimetric measurements of the mannose-coated model surface (Figure 6).

Figure 6. Competition assay results showing the role of mannose on fimbriae-mediated adhesion of Figure 6.K. Competitionpneumoniae 2242 assay wild type results and its showing isogenic mutants the role to of the mannose epithelial oncells. fimbriae-mediated The results obtained using adhesion of K. pneumoniaethe impedance2242 wild sensor type (a) andwere its compatible isogenic with mutants those toobtained the epithelial from the cells. conventional The results 96-well obtained using theplate/fluorescence impedance sensor method (a ()b were) and with compatible in vitro assay with results those obtained obtained using from HCT-8 the human conventional ileocecal 96-well plate/fluorescenceepithelial cells method (c) and T-24 (b) andbladder with epithelialin vitro cellsassay (d). resultsThe ∆fimA obtained mutant showed using HCT-8 lowest binding human to ileocecal epithelialthe cells mannose (c) and surface T-24 bladderand its epithelialbinding was cells unaffected (d). The ∆byfimA the mutantpresence showed of free lowestmannose. binding The to the concentration of bacteria used for all the test was 107 CFU/mL. Mean values with different mannose surface and its binding was unaffected by the presence of free mannose. The concentration of superscripts (a,b,c) at each mannose concentration indicate significant differences between the wild 7 bacteriatype used and for its all isogenic the test mutant was strains 10 CFU/mL. (p < 0.05). Mean values with different superscripts (a,b,c) at each mannose concentration indicate significant differences between the wild type and its isogenic mutant strains (p < 0.05). Sensors 2017, 17, 1406 10 of 13

3.3. Adhesion Properties of K. pneumoniae on Mucin Modified Surfaces The ability of bacteria to adhere to specific epithelial cells is generally regarded as a potential pathogenicSensors 2017, 17 factor, 1406 during the initial stage of infection. However, it has been reported that the ability10 of 13 of K. pneumoniae to adhere to cultured epithelial cells is not necessarily associated with bacterial infectivity, because the results obtained in an in vivo study using mouse model showed no correlation 3.3. Adhesion Properties of K. pneumoniae on Mucin Modified Surfaces with those of an in vitro study [35]. We suggest that the reason for this results was that most epithelial cells Thein intestine, ability oflung, bacteria and bladder to adhere are to not specific direct epithelially exposed cells to lumen is generally but typically regarded are ascovered a potential with apathogenic thick layer factor of mucous. during theThe initial mucins stage are of a infection. family of However, glycoprotein it has and been a reportedmajor component that the ability of the of mucousK. pneumoniae that coverto adhere the luminal to cultured surfaces epithelial of epithelial cells is not organs necessarily and serves associated as physical with bacterial barrier infectivity, between thebecause extracellular the results milieu obtained and inplasma an in membrane. vivo study usingThus, mouse we also model functionalized showed no the correlation surface of with the thosegold electrodeof an in vitro withstudy mucin [35 to]. Wemimic suggest actual that luminal the reason surfac fores thisof epithelial results was organs that mostand to epithelial investigate cells the in adhesionintestine, lung,properties and bladder of K. pneumoniae are not directly wild exposedtype and to its lumen isogenic but typicallymutants arelacking covered outer with core a thickLPS (layer∆wabG of) mucous.or fimbrial The protein mucins (∆ arefimA a family). The surface of glycoprotein of gold andelectrode a major was component functionalized of the with mucous porcine that gastriccover the mucin luminal which surfaces is simila of epithelialr to human organs colonic and mucin. serves K. as pneumoniae physical barrier 2242 betweenwild type the or extracellular its isogenic mutantmilieu andstrains plasma were membrane.introduced to Thus, the chamber we also functionalizedequipped with thea mucin-coated surface of the electrode gold electrode and allowed with tomucin adhere to mimic to its actualsurface. luminal After washing surfaces ofthe epithelial surface organswith PBS, and amou to investigatents of bound the adhesion bacteria propertieson mucin surfacesof K. pneumoniae were measuredwild type using and the itsimpedance isogenic sensor. mutants Increases lacking in outer NIC values core LPS indicated (∆wabG an) orassociation fimbrial betweenprotein ( ∆bacteriafimA). The and surface the artificial of gold mucous electrode layer was on functionalized the gold electrode, with porcine and are gastric proportional mucin whichto the numberis similar of to bound human bacteria colonic (Figure mucin. 7).K. It pneumoniae is interesting2242 to wildnote typethat adhesion or its isogenic of bacteria mutant to strainsthe artificial were mucousintroduced layer to was the chamberunaffected equipped by the absence with a or mucin-coated presence of fimbriae electrode but and was allowed affected to by adhere outer tocore its LPSsurface. and Aftercapsular washing polysaccharide. the surface Adhesion with PBS, level amounts to the of artificial boundbacteria mucous on layer mucin was surfaces significantly were lowermeasured for K. using pneumoniae the impedance ∆wabG mutant sensor. than Increases for the in wild NIC type. values In addition, indicated the an associationbinding ability between of K. pneumoniaebacteria and ∆ thewabG artificial mutant mucous to the mucous layer on layer the goldwas electrode,fully restored and by are complementing proportional to ∆ thewabG number mutant of strainbound with bacteria pBBR1MCS5- (Figure7).wabG It is , interesting which to note implied that adhesionthat the association of bacteria between to the artificial bacteria mucous and the mucouslayer was layer unaffected was mediated by the absencethrough orouter-core presence LPS of fimbriae or capsular but polysacchari was affectedde by on outer the coreouter LPS surface and ofcapsular the bacterium. polysaccharide. Adhesion level to the artificial mucous layer was significantly lower for K. pneumoniaeThe association∆wabG betweenmutant than K. pneumoniae for the wild and type. the In mucous addition, layer the bindingwas also ability investigated of K. pneumoniae using a conventional∆wabG mutant 96-well to the mucousplate coated layer with was mucin, fully restored and the by results complementing obtained were∆wabG comparedmutant strainwith those with obtainedpBBR1MCS5- usingwabG the plasmid,impedance which sensor. implied We thatfound the the association results were between almost bacteria identical, and thewhich mucous indicated layer wasthe impedance mediated through measurements outer-core offer LPS a orreliable capsular alte polysaccharidernative to the conventional on the outer surfacestaining of method. the bacterium.

Figure 7. Impedance measurements ( a) and conventional measurements obtained using the 96-well plate/fluorescenceplate/fluorescence methodmethod (b(b)) for for the the adhesion adhesion of K.of pneumoniae K. pneumoniaeKCTC KCTC 2242 and2242 of and its isogenic of its isogenic mutants mutantsto mucin to modified mucin modified surfaces. surfaces. For impedance For impeda measurements,nce measurements, normalized normalized impedance impedance change (NIC) change at (NIC)0.1 Hz at was 0.1 used Hz towas measure used theto adhesionmeasure ofthe bacteria adhesion to mucin of bacteria surfaces. to Increasesmucin surfaces. in impedance Increases signals in impedancewere proportional signals towere number proportional of bacteria to nu inmber the range of bacteria from 10 in5 tothe 10 range8 CFU/mL from 10K.5 pneumoniaeto 108 CFU/mLKCTC K. pneumoniae2242 ∆wabG KCTCshowed 2242 the ∆ lowestwabG showed level of interactionthe lowest withlevel mucin of interaction surfaces. with Mean mucin values surfaces. with different Mean valuessuperscripts with different (a,b) at each superscripts bacterial concentration(a,b) at each ba indicatecterial concentration significant differences indicate significant between the differences wild type betweenand its isogenic the wild mutant type and strains its isogenic (p < 0.05). mutant strains (p < 0.05).

The association between K. pneumoniae and the mucous layer was also investigated using a conventional 96-well plate coated with mucin, and the results obtained were compared with those Sensors 2017, 17, 1406 11 of 13 obtained using the impedance sensor. We found the results were almost identical, which indicated the impedance measurements offer a reliable alternative to the conventional staining method.

4. Conclusions A simple and sensitive impedance sensor was developed to monitor the major pathogenic factors of K. pneumoniae. The ability of K. pneumoniae to adhere to host cells is governed by fimbriae, outer core LPS, and capsular polysaccharide, which have specific affinities for the D-mannose-containing trisaccharides of the host glycoprotein and the mucous layer that covers epithelial organs. We designed an electrode functionalized with mannose or mucin to mimic epithelial cell surfaces and the luminal surfaces of epithelial organs. The binding characteristics of K. pneumoniae 2242 and its isogenic mutants, ∆fimA and ∆wabG, which lacked fimbriae and outer-core LPS, respectively were examined using an impedance sensor equipped with a functionalized electrode. Based on the results obtained, we conclude that type 1 fimbriae are responsible for the recognition of and binding to mannose containing trisaccharides of host glycoprotein, whereas outer-core LPS and capsular polysaccharide of bacteria are associated with bacterial interactions with mucins. Wild type K. pneumoniae 2242 demonstrated strong interactions with mannose and mucin-coated surfaces based on impedance measurements. However, the binding ability of the bacterium to mannose surfaces was significantly diminished when the fimA gene was knocked out. Binding of the bacterium to mucin surfaces was also significantly hampered by removing the ∆wabG gene, which is responsible for the synthesis of outer-core LPS. Furthermore, based on impedance measurements, the binding abilities of these knockout variants were fully restored by complementing the mutant strains with the fimA or wabG gene, respectively, which suggested these genes are responsible for specific interactions. In addition, impedance results were compared with those obtained using the conventional 96-well plate method and in vitro assays using HCT-8 human ileocecal epithelial cells or T-24 human bladder epithelial cells. We found all impedance measurements were consistent with conventional and in vitro assay results, which indicates that the devised impedance sensor implemented using a functionalized model surface could replace conventional methods. We suggest this impedance sensing system offers a promising alternative means of monitoring or investigating the virulence factors of pathogenic bacteria associated with detrimental interactions with specific tissue or organs at early stages of infections.

Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/17/6/1406/s1, Figure S1: Visualization of capsule in K. pneumoniae KCTC 2242 and its isogenic mutants. Acknowledgments: This research was supported by the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2012-0009575) and the Cooperative Research Program for Agricultural Science & Technology Development (PJ01199303), Rural Development Administration, Korea. Author Contributions: Y.-R.K. designed the experiments. D.T.N.H. and A.-Y.K. performed the sensor construction, testing and validation study. D.T.N.H. constructed the plasmid harboring fimA and wabG gene for complementary study and performed conventional binding assays including in vitro experiments. Y.-R.K. and D.T.N.H. analyzed data and wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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