Polyhexamethylene guanidine hydrochloride

shows bactericidal advantages over

chlorhexidine digluconate against ESKAPE

bacteria

Zhongxin Zhou1

Dafu Wei2

Yanhua Lu1∗

1State Key Laboratory of Bioreactor Engineering, School of Bioengineering,East China University of Science and Technology, Shanghai, People’sRepublic of China2Key Laboratory for Ultrafine Materials, Ministry of Education, School ofMaterials Science and Engineering, East China University of Science andTechnology, Shanghai, People’s Republic of China

Abstract

More information regarding the bactericidal properties ofpolyhexamethylene guanidine hydrochloride (PHMG) againstclinically important antibiotic-resistant ESKAPE (Enterococcusfaecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, andEnterobacter species) pathogens needs to be provided for itsuses in infection control. The bactericidal properties of PHMGand chlorhexidine digluconate (CHG) were compared basedon their minimum inhibitory concentrations (MICs), minimumbactericidal concentrations, and time-course–killing curvesagainst clinically important antibiotic-susceptible andantibiotic-resistant ESKAPE pathogens. Results showed thatPHMG exhibited significantly higher bactericidal activitiesagainst methicillin-resistant Staphylococcus aureus,

carbapenem-resistant Klebsiella pneumoniae, andceftazidime-resistant Enterobacter spp. than CHG. A slightbactericidal advantage over CHG was obtained againstvancomycin-resistant Enterococcus faecium, ciprofloxacin-and levofloxacin-resistant Acinetobacter spp., andmultidrug-resistant Pseudomonas aeruginosa. In previousreports, PHMG had higher antimicrobial activity againstalmost all tested Gram-negative bacteria and severalGram-positive bacteria than CHG using MIC test. Thesestudies support the further development of covalently boundPHMG in sterile-surface materials and the incorporation ofPHMG in novel disinfectant formulas. C© 2014 International Union ofBiochemistry and Molecular Biology, Inc. Volume 00, Number 00, Pages1–7, 2014

Keywords: polyhexamethylene guanidine hydrochloride, chlorhexidinedigluconate, ESKAPE, bactericidal activities

Abbreviations: CFU, colony forming units; CHG, chlorhexidinedigluconate; CoNS, coagulase-negative staphylococci; ESKAPE,Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species;MBC, minimum bactericidal concentrations; MIC, minimum inhibitoryconcentration; MRSA, methicillin-resistant Staphylococcus aureus; PBS,phosphate buffer solution; PHMG, polyhexamethylene guanidinehydrochloride.∗Address for correspondence: Yanhua Lu, PhD, State Key Laboratory ofBioreactor Engineering, School of Bioengineering, East China University ofScience and Technology, 130 Meilong Road, Shanghai 200237, People’sRepublic of China. Tel.: +86-21-64251185; Fax: +86-21-64251185; e-mail:luyanhua@ecust.edu.cn.Received 7 March 2014; accepted 26 May 2014DOI: 10.1002/bab.1255Published online in Wiley Online Library(wileyonlinelibrary.com)

1. IntroductionThe Infectious Diseases Society of America (IDSA) states thatthere are few candidate drugs moving forward that will treatinfections caused by antibiotic-resistant bacterial infections andin particular the “ESKAPE” pathogens (Enterococcus faecium,Staphylococcus aureus, Klebsiella pneumoniae, Acinetobac-ter baumannii, Pseudomonas aeruginosa, and Enterobacterspecies) [1–3]. In 2010, the IDSA supported a program calledthe 10 × 20 initiative to develop 10 new systemic antibacterialagents by 2020 in which the resistant ESKAPE pathogens wereprimary targets [2].

Recent important developments are worth considering inthe field of chemically synthesized guanidine-based cationicantimicrobial polymers effective against some antibiotic-resistant bacteria [4–8]. Previous studies have shown thatthese compounds had excellent antibacterial [9, 10], an-tifungal [11, 12], and antiviral activities [13, 14]. For

1

Biotechnology andApplied Biochemistry

example, an interesting recent report indicated that guani-dinium side-chain-functionalized polycarbodiimides offeredsignificant antibacterial activity against methicillin-resistantS. aureus (MRSA) [5]. Neomycin B and kanamycin A con-jugated with multiple guanidinium groups restored theantimethicillin-resistant Staphylococcus epidermidis activityof kanamycin A and the anti-MRSA activity of both [4]. Guani-dinylation of the polycationic headgroup in neomycin B-derivedcationic lipids enhanced the antibacterial activity againstneomycin B-, kanamycin A-, and gentamicin-resistant P. aerug-inosa [4]. Synthesized para-guanidinoethylcalix[4]arene andchlorhexidine digluconate (CHG), both of which containmultipleguanidinium groups, also have very good activity against MRSAand the coagulase-negative staphylococci (CoNS) vancomycin-resistant E. faecium [6, 7]. Antibiotics used in combinationwith the synthetic polycationic polymer polyethylenimine sig-nificantly reduced their minimum inhibitory concentrations(MICs) by 1.5- to 56-fold against a resistant clinical isolate ofP. aeruginosa [8].

As a guanidine-based cationic antimicrobial polymer, poly-hexamethylene guanidine hydrochloride (PHMG) has receivedincreasing attention in recent years for two reasons. First, itcould be developed as a highly effective disinfectant in com-bination with other compounds. Second, it could be bound tosubstrate material to create covalently bound, nonleachingantimicrobial surfaces. A recent successful trial was performedon functional additives prepared by covalently bound PHMG tochitosan [15] or potato starch [16] that were then adsorbed ontocellulose fibers to improve the antimicrobial activity of paperproducts. PHMG is one major component of Akacid plus R©, anew member of the guanidine family of disinfectants [17, 18].In 2011 and 2012, PHMG’s ability to fight bacterial spores[19] and fungi [20, 21] was also reported. As for PHMG com-bating antibiotic-resistant bacteria, Oule et al. [22] reportedthat a PHMG-based disinfectant can fight against MRSA andnosocomial infections.

These reports suggest that PHMG has great potential for thedevelopment of covalently bound, nonleaching antimicrobialmaterials [23] and highly effective disinfectants [19]. Moreinformation about its antimicrobial properties against clinicalstrains and especially against clinically important antibiotic-resistant ESKAPE pathogens is required for its further use inthe control of infection.

CHG, another nonpolymeric member of the guanidinefamily of disinfectants, possesses broad bactericidal activityagainst many Gram-negative and Gram-positive bacteria, andit has been used in commercial products for several decades inthe hospital, agricultural, and domestic environments [17, 24].

However, there are no systematic investigations compar-ing differences in the bactericidal properties of PHMG andCHG, especially for combating clinically important, antibiotic-resistant ESKAPE bacteria. Furthermore, disinfectants aretypically used in commercial products that contain the activeingredients in different concentrations, making it difficult tocompare the antiseptic properties of the active substance itself

and decide which would be the appropriate choice for specificapplications.

In this study, the bactericidal activities of PHMG and CHGwere compared based on their MICs, minimum bactericidalconcentrations (MBCs), and time-course–killing curves againstclinically important antibiotic-resistant ESKAPE pathogens.

2. Materials and Methods2.1. Chemical substancesPHMG was synthesized by condensation polymerization of hex-amethylene diamine and guanidine hydrochloride according toa previously reported procedure [25]. The number-averagedmolecular weight of prepared PHMG was 481 g/mol, and itsweight-averaged molecular weight/number-averaged molec-ular weight was 1.2, as estimated by electrospray ionizationtime-of-flight mass spectrometry [26]. Hexamethylene diamineand guanidine hydrochloride were purchased from SinopharmChemical Reagents Co. (Shanghai, People’s Republic of China).

A stock solution of CHG 20% was purchased from Sigma–Aldrich Co. LLC (Shanghai, People’s Republic of China). Thestock solution was diluted with sterile 10 mM phosphate buffersolution (PBS, pH 7.4) with a final pH of 7.4 before use. Allother reagents were of analytical purity.

2.2. Bacterial strainsClinically isolated, antibiotic-susceptible and antibiotic-resistant ESKAPE pathogens were provided by the HuashanHospital, Shanghai Medical College, Fudan University (Shang-hai, People’s Republic of China). These clinical ESKAPE strainswere isolated from individual patients from the Huashan Hos-pital in 2009 as previously reported [27] and kept frozen at–80 ◦C prior to use. ESKAPE strains were identified usingthe Vitek automated identification system (BioMerieux, Marcyl’Etoile, France) and were confirmed using the API-GN system(BioMerieux). Strain resistances were tested using the Kirby-Bauer disk diffusion method, and results were defined usingClinical and Laboratory Standards Institute (CLSI) standards.

2.3. Antibacterial testing2.3.1. Minimum inhibitory concentrationThe MIC of PHMG and CHG was determined using the standardmethod of Mueller-Hinton broth microdilution as described inthe CLSI with minor modifications [28]. Briefly, PHMG or CHGwas dissolved in 10 mM PBS (pH 7.4) with a final pH of 7.4 toprepare stock solutions. The corresponding stock solution wassubjected to twofold serial dilution (0.25–564 μg/mL) with 10mM PBS (pH 7.4). Bacteria were cultured overnight at 37 ◦C onan agar plate with 5% sheep blood, and cultures were furthersuspended, centrifuged, and resuspended with 10 mM PBS (pH7.4) for three cycles. Then, a prepared cell suspension with anoptical density of 0.35 at 600 nm was diluted to approximately5 × 105 colony-forming units (CFU) per milliliter in twofoldcation-adjusted Mueller-Hinton broth (Oxoid, Basingstoke,Hampshire, England). On the basis of agar plate counts,Escherichia coli cell suspensions with an optical density of

2 Bactericidal Activities of Guanidine Polymer

0.35 at 600 nm corresponded to 2.5 × 108 CFU/mL. MICendpoints were read as the lowest antimicrobial concentrationthat completely inhibited macroscopically visible growth of theinoculum [17]. Quality control was ensured by the concurrenttesting of ATCC strains. The MIC results were expressed as themean of three parallel measurements. Data were analyzed witha chi-square test. The difference between the MIC values of thedifferent polymers was considered significant when P < 0.05.

2.3.2. Minimum bactericidal concentrationMBC testing was performed using microtiter plates set up forMIC determinations as indicated [29]. Aliquots (10 μL) takenfrom eachwell up to and including theMIC endpoint were trans-ferred and spot plated onto the appropriate agar and incubatedovernight. MBCs were expressed as the lowest concentrationof biocide at which growth was not observed after 5 days ofincubation. The MBC results were expressed as the mean ofthree parallel measurements. Data were analyzed with thechi-square test. The difference between the MBC values of thedifferent polymers was considered significant when P < 0.05.

2.3.3. Determination of bactericidal activity bytime–killing curves

Killing curves for PHMG and CHG were performed onclinically isolated, antibiotic-resistant ESKAPE strains,including vancomycin-resistant E. faecium, MRSA,carbapenem-resistant K. pneumoniae, ciprofloxacin-and levofloxacin-resistant Acinetobacter spp., multidrug-resistant P. aeruginosa, and ceftazidime-resistant En-terobacter spp. The preparation of PHMG and CHGsolutions and bacterial cell suspensions was thesame as for antimicrobial testing. Bacterial cells at6.25 × 106 CFU/mL were incubated with various concen-trations of PHMG or CHG for different times at 25 ◦C. Aliquots(10 μL) of the various cultures were withdrawn at differentintervals, immediately followed by a 1:100 dilution in 10 mMPBS, which was used to arrest treatment and reduce its drawout, and then a serial 10-fold dilution was applied. Cell suspen-sion (200 μL) from each dilution was spread on Mueller-Hintonbroth agar plates and incubated at 37 ◦C for 24 H, and thenumber of CFU from surviving bacteria was counted. Controlswere performed in the presence of 10 mM PBS without anyantimicrobial agent. All assays were repeated three times.

3. Results and Discussion3.1. Comparisons of the antimicrobial activity of

PHMG and CHG based on MIC test againstclinically important ESKAPE strains

Currently, the majority of hospital infections are caused byESKAPE strains, which effectively “escape” the effects ofantibacterial drugs [3]. The MICs of PHMG and CHG againstantibiotic-susceptible and antibiotic-resistant clinical strainsare provided in Table 1. As for the validity of the data in ourassay system, our MIC values were comparable to results fromprevious reports because CHG had the same MIC range against

all tested ESKAPE clinical strains, as has been previouslyreported [17, 30].

Table 1 shows that, for ESKAPE strains, PHMG was moreeffective against Staphylococcus spp., K. pneumoniae, andEnterobacter spp., with MIC90 values of 4–8 μg/mL, andE. faecium, Acinetobacter spp., and P. aeruginosa, with a MIC90value of 32 μg/mL. CHG was active against Staphylococcus spp.,with MIC90 values of 8–16 μg/mL, but lower than that againstK. pneumoniae, E. faecium, Enterobacter spp., Acinetobacterspp., and P. aeruginosa, with a MIC90 value of 32 μg/mL.

For the above-mentioned strains S. aureus, K. pneumo-niae, and Enterobacter spp., PHMG had low MIC90 values of4–8 μg/mL, whereas CHG had the significantly higher MIC90values of 8–64 μg/mL. Therefore, based on the lower MIC90value and MIC range, PHMG had the significantly higherantimicrobial activity than CHG against S. aureus, K. pneumo-niae, and Enterobacter spp. for both antibiotic-susceptible andantibiotic-resistant phenotypes.

For vancomycin-susceptible and vancomycin-resistant E.faecium, MIC90 values and the high limit of the MIC range werethe same for PHMG and CHG. However, the MIC50 value andthe low limit of the MIC range for PHMG were smaller than thatfor CHG. Therefore, PHMG still showed a slight advantage overCHG against vancomycin-susceptible and vancomycin-resistantE. faecium.

For wide-type and ciprofloxacin- and levofloxacin-resistantAcinetobacter spp., the MIC50, MIC90, and MIC range for PHMGwere exactly the same as for CHG, respectively. For wide-typeand multidrug-resistant P. aeruginosa, both compounds alsohad almost the same MIC50, MIC90, and MIC range, respec-tively. Irrespective of the antibiotic-susceptible and antibiotic-resistant phenotypes, PHMG and GHC had similar antimicrobialactivities against Acinetobacter spp. and P. aeruginosa.

Our previous study demonstrated that PHMG had the bestactivity against methicillin-susceptible S. aureus, MRSA, andCoNS, with an MIC range of 1–8 μg/mL, which was lowerthan the 4–32 μg/mL range of CHG [31]. CHG had the bestactivity against β-hemolytic streptococci, with a MIC valueof 2 μg/mL [31]. MRSA and β-hemolytic streptococci are theprincipal Gram-positive pathogens responsible for complicatedskin infections [32]. Infections caused by CoNS are particularlyfrequent in patients [33]. In 2008, Oule et al. [22] reported thatPHMG could be used as a safe disinfectant to fight against MRSAbased on testing its activity against four quality-control strains.These cumulative data support PHMG’s application as a noveldisinfectant and antimicrobial material against nosocomialinfections, especially MRSA and methicillin-resistant CoNS.

A literature search showed that PHMG exhibited higheror very similar antimicrobial activities in MIC test against al-most all tested Gram-negative bacteria (with the exception ofSerratia marcescens) and some Gram-positive bacteria whencompared to CHG [31]. PHMG had a significantly lower MIC90than CHG against Klebsiella spp., Proteus mirabilis, Citrobacterspp., Enterobacter spp., indole-positive Proteae, S. aureus,and CoNS for antibiotic-susceptible and antibiotic-resistant

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TABLE 1In vitro minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PHMG and CHG against

antibiotic-susceptible and antibiotic-resistant ESKAPE clinical strains

MIC (μg/mL) MBC (μg/mL)

Organism (number of strains tested) PHMG HG PHMG CHG

Staphylococcus aureus

Methicillin susceptible (20) MIC50 2 8 MBC50 2 16

MIC90 4 8 MBC90 4 16

Range 1–4a 8 Range 1–4a 8–32

Methicillin resistant (20) MIC50 2 8 MBC50 2 16

MIC90 8 16 MBC90 8 32

Range 2–8a 8–32 Range 2–8a 16–64

Klebsiella pneumoniae

Wide type (20) MIC50 8 16 MBC50 8 16

MIC90 8 32 MBC90 8 32

Range 4–8a 16–32 Range 4–8a 16–32

Carbapenem resistant (10) MIC50 8 16 MBC50 8 16

MIC90 8 32 MBC90 8 32

Range 4–8a 16–32 Range 4–8a 16–32

Enterobacter spp.

Wide type (15) MC50 4 32 MBC50 4 32

MC90 8 32 MBC90 8 32

Range 4–8a 8–64 Range 4–8a 8–64

Ceftazidime resistant (10) MC50 8 16 MBC50 8 16

MC90 8 32 MBC90 8 32

Range 8a 8–32 Range 8a 8–32

Enterococcus faecium

Vancomycin susceptible (15) MC50 4 8 MBC50 4 8

MIC90 32 32 MBC90 32 32

Range 2—32 8–32 Range 2–32 8–32

Vancomycin resistant (10) MIC50 8 16 MBC50 8 16

MIC90 32 32 MBC90 32 32

Range 4—32 16–32 Range 4–32 16–32

Acinetobacter spp.

Wide type (10) MIC50 16 16 MBC50 16 16

MIC90 32 32 MBC90 32 32

Range 8–32 16–32 Range 8–32 16–32

(Continued)

4 Bactericidal Activities of Guanidine Polymer

TABLE 1Continued

MIC (μg/mL) MBC (μg/mL)

Organism (number of strains tested) PHMG HG PHMG CHG

Ciprofloxacin and levofloxacin resistant (10) MIC50 16 16 MBC50 16 16

MIC90 32 32 MBC90 32 32

Range 8–16a 16–32 Range 8–16a 16–32

Pseudomonas aeruginosa

Wide type (15) MIC50 16 16 MBC50 16 16

MIC90 32 32 MBC90 32 32

Range 16–32 8–32 Range 16–32 8–32

Multidrug resistant (10) MIC50 16 16 MBC50 16 16

MIC90 32 32 MBC90 32 32

Range 16–32 8–32 Range 16–32 8–32aSignificantly different (P < 0.05) when the MIC or MBC range of PHMG was expressed as mean ± SD of MIC or MBC values of different isolatescompared to that of CHG.

MIC50/90, MIC for 50% and 90% isolates of the organism, respectively.

MBC50/90, MBC for 50% and 90% isolates of the organism, respectively.

phenotypes, based on the chi-square test (P < 0.05). For E.coli, P. aeruginosa, Acinetobacter spp., Stenotrophomonasmaltophilia, and E. faecium, PHMG and CHG had verysimilar MIC90 values. However, for Gram-positive Streptococ-cus pneumoniae, β-hemolytic streptococci, and Enterococcusfaecalis, PHMG had significantly higher MIC90 values thanCHG. Clinical microbiologists increasingly agree that antibiotic-resistant Gram-negative bacteria, rather than Gram-positivebacteria, currently pose the greatest risk to public health[34]. The superior activity of PHMG relative to CHG againstGram-negative bacteria supports the further development ofPHMG to combat against antibiotic-resistant Gram-negativebacteria.

3.2. Comparisons of the bactericidal activity of PHMGand CHG based on the MBC test against clinicallyimportant ESKAPE strains

To evaluate the bactericidal properties of PHMG and CHGagainst clinically important ESKAPE pathogens, their MBCswere assayed. As shown in Table 1, MBC values for bothPHMG and CHG were observed at 1× MIC against six clinicallyimportant ESKAPE species, with both susceptible and resistantphenotypes. There was little or no difference between the MBCsand MICs of PHMG and CHG, which is consistent with previousreports on this cationic antimicrobial agent [29].

PHMG had a lower MBC50 and MBC90 than CHG against S.aureus, K. pneumoniae, and Enterobacter spp., irrespective ofthe antibiotic- resistant phenotype. For wild-type and antibiotic-

resistant E. faecium, Acinetobacter spp., and P. aeruginosa,PHMG and CHG had the same MBC90 value.

3.3. Comparisons of the bactericidal activity of PHMGand CHG based on time-killing kinetics againstclinically important, antibiotic-resistant ESKAPEstrains

For further comparison, the killing kinetics of PHMG and CHGwere compared. Time-killing experiments were completedagainst one antibiotic-resistant strain of each clinically impor-tant ESKAPE bacterial species. Killing curves for six antibiotic-resistant ESKAPE strains are shown in Fig. 1. PHMG at twice theMIC eradicated MRSA, carbapenem-resistant K. pneumoniae,ceftazidime-resistant Enterobacter spp., vancomycin-resistantE. faecium, ciprofloxacin- and levofloxacin-resistant Acineto-bacter spp., and multidrug-resistant P. aeruginosa within 1.5–2.5 H. However, the same concentration of CHG took more timeto eradicate the corresponding antibiotic-resistant ESKAPEstrain. These results suggest that PHMG had a stronger bacteri-cidal activity against these antibiotic-resistant ESKAPE strains.

From 2008 to 2012, guanidine-based, cationic, antimi-crobial polymers and some amphiphilic, cationic compoundscontaining multiguanidine group were shown to have high ac-tivity against antibiotic-resistant bacteria [4–8]. These reportsdid not draw the attention of academics, however. Our activitydata show that PHMG possesses superior bactericidal activityrelative to CHG against six clinically important antibiotic-resistant ESKAPE strains. Our cumulative results revealed that

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FIG. 1Time–kill curves for PHMG and CHG on sixrepresentative antibiotic-resistant isolates oftested ESKAPE clinical strains at 25 ◦C.(A) Methicillin-resistant Staphylococcus aureus;(B) carbapenem-resistant Klebsiella pneumoniae;(C) vancomycin-resistant Enterococcus faecium;(D) ceftazidime-resistant Enterobacter spp.;(E) ciprofloxacin- and levofloxacin-resistantAcinetobacter baumannii; and(F) multidrug-resistant Pseudomonas aeruginosa.

PHMG and other cationic, guanidine-based polymers shouldbe explored further as powerful chemotherapeutic agents orpermanent sterile-surface materials created by covalent bond-ing with matrix material, and thus combating clinically impor-tant antibiotic-resistant bacteria.

It has been demonstrated that dose-dependent membranedisruption is the main bactericidal mechanism of cationicguanidine-based antimicrobial agent [25]. For the reason thatPHMG shows advantage over CHG against above-mentionedbacteria, it is possible that the flexible straight-line alkyl chainof PHMG has better partition ability into the hydrophobicregions of the phospholipids membrane, which could damagethe phospholipid bilayer and kill the bacteria, than the rigid

benzene ring of CHG. The different structures of the cellmembranes of bacterial genera resulted in the differences ofantimicrobial activities of PHMG or CHG against the above-mentioned bacterial genera.

For the application aspects, Oule et al. [22] reported thatPHMG could be used as a safe disinfectant to fight againstMRSA. PHMG is one major component of Akacid plus R©, anew member of the guanidine family of disinfectants [17, 18].Our experimental tests showed that PHMG exhibited advan-tage over CHG against MRSA, methicillin-resistant CoNS, andabove-mentioned six clinically important antibiotic-resistantESKAPE strains. Synthesized guanidine-based cationic antimi-crobial polymers were recently reported to have good activitiesagainst some antibiotic-resistant bacteria [4–8]. These cumu-lative data support PHMG’s further developments as a noveldisinfectant to combat against nosocomial infections causedby antibiotic-resistant bacteria. In addition, PHMG can bechemically bound to or coated on various materials to prepareantimicrobial materials, for example, antimicrobial paper. Ithas been reported that functional additives prepared by co-valently bonding PHMG to chitosan [15], potato starch [16],and beeswaxes [35] improve the antimicrobial activity of paperproducts.

6 Bactericidal Activities of Guanidine Polymer

4. ConclusionsThe present study emphasized on the superior antimicrobialproperties of PHMG against clinically important antibiotic-resistant ESKAPE strains and Gram-negative bacteria. Forclinically important antibiotic-resistant ESKAPE pathogens,antimicrobial and bactericidal tests showed that PHMG hada significantly higher bactericidal activity than CHG againstMRSA, carbapenem-resistant K. pneumoniae, and ceftazidime-resistant Enterobacter spp. For vancomycin-resistant E. fae-cium, ciprofloxacin- and levofloxacin-resistant Acinetobacterspp., and multidrug-resistant P. aeruginosa, PHMG showed aslight bactericidal advantage over CHG. Together with previousreports, cumulative data showed that PHMG exhibited higherantimicrobial activity against almost all tested Gram-negativebacteria (E. coli, Klebsiella spp., P. mirabilis, Citrobacterspp., Enterobacter spp., indole-positive Proteae, P. aeruginosa,Acinetobacter spp., and S. maltophilia) and some Gram-positivebacteria (S. aureus, CoNS, and E. faecium) that are commonin nosocomial infections. These results are encouraging forthe use and further development of PHMG in permanent,covalently bound sterile-surface materials and as a highlyeffective disinfectant for combating antibiotic-resistant strainsand Gram-negative bacteria in the control of hospital infection.

5. AcknowledgementsThe authors are grateful for the financial support by theNational Natural Science Foundation of China (No. 21204020),the China Postdoctoral Science Foundation (Projectno. 2013M530184), and the Fundamental Research Fundsfor the Central Universities.

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