Biocide resistance

1. Background

2. Biocides/disinfectants

3. Biocides and resistance

mechanisms

4. QACs and microbial resistance

5. Biguanides and microbial

resistance

6. Nanoparticles and microbial

resistance

7. Cross-resistance of biocides

and antibiotics

8. Techniques to investigate

biocide resistance

9. Clinical trials

10. Expert opinion

Review

Biocides -- resistance,cross-resistance mechanismsand assessmentDivya Prakash Gnanadhas, Sandhya Amol Marathe &Dipshikha Chakravortty†

Department of Microbiology and Cell Biology, Centre for Infectious Disease Research and Biosafety

Laboratories, Indian Institute of Science, Bangalore, India

Importance of the field: Antibiotic resistance in bacterial pathogens has

increased worldwide leading to treatment failures. Concerns have been raised

about the use of biocides as a contributing factor to the risk of antimicrobial

resistance (AMR) development. In vitro studies demonstrating increase in

resistance have often been cited as evidence for increased risks. It is therefore

important to understand the mechanisms of resistance employed by bacteria

toward biocides used in consumer products and their potential to impart

cross-resistance to therapeutic antibiotics.

Areas covered: In this review, the mechanisms of resistance and cross-resistance

reported in the literature toward biocides commonly used in consumer

products are summarized. The physiological and molecular techniques used in

describing and examining these mechanisms are reviewed and application of

these techniques for systematic assessment of biocides for their potential to

develop resistance and/or cross-resistance is discussed.

Expert opinion: The guidelines in the usage of biocides in household or indus-

trial purpose should be monitored and regulated to avoid the emergence of

any MDR strains. The genetic and molecular methods to monitor the

resistance development to biocides should be developed and included in

preclinical and clinical studies.

Keywords: biguanides, biocide resistance, biocides, cross-resistance, disinfectant, quaternary

ammonium compound, resistance detection

Expert Opin. Investig. Drugs [Early Online]

1. Background

The development and dissemination of bacterial resistance to antimicrobials is amajor global public health issue. In the last decade, antibiotic resistance in bacterialpathogens has increased worldwide leading to treatment failures both in human andanimal infectious diseases. Due to this increasing prevalence, there is an increasingneed to control these microorganisms to prevent infections and contain outbreaksof disease, through effective cleaning and disinfection, which relies heavily on theuse of biocides.

Biocides are widely used in different areas, including wood preservation, watertreatment, paint industry and medical field. Disinfection and medical usage togethercontribute the major part of biocide usage [1]. Based on the level of inactivation, theyare classified into low-level (to inactivate vegetative bacteria, some fungi and virusesnamely, ethyl or isopropyl alcohol, quaternary ammonium detergent solution,sodium hypochlorite), intermediate (apart from bacterial spores, most of the bacteria,fungi and viruses -- namely higher concentration of low level biocides) and high leveldisinfectants (e.g., glutaraldehyde, hydrogen peroxide, ortho-phthalaldehyde, peraceticacid) which can inactivate all microorganisms [2].

10.1517/13543784.2013.748035 © 2013 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 1All rights reserved: reproduction in whole or in part not permitted

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The use of biocides is not limited to medical field; they arealso used in consumer products including cosmetics (alcoholsand aldehydes), disinfectants (benzalkonium chloride [BC]),surface cleaning (triclosan), laundry products and pet care.Several biocides are also used in consumer-level products toavoid growth or infection by any microorganism. Triclosanis being used in paint, textiles and plastic products. Anti-microbial agents also play a key role in consumer productsby controlling or preventing the growth of microorganismsin the products and in environments where these productsare used [2].The main cause of antibiotic resistance is attributed to the

suboptimal use and misuse of antibiotics in human medicineand animal husbandry. However, in the recent years, concernshave arisen about the use of biocides in products such astextiles, plastics, household products, cosmetics, processedfoods, etc., as a contributing factor to the risk of antimicrobialresistance (AMR) development in humans and in the environ-ment. These concerns are primarily based on isolation of resis-tant or more tolerant forms of bacteria from in vitro studiesfollowing exposure to suboptimal or sublethal levels ofbiocides [3]. Some antimicrobials are able to induce resistancemechanisms that confer cross-resistance to antibiotics due tocommon resistance mechanisms [4,5] (e.g., efflux pumps) andpresence of resistance genes for antimicrobials and antibioticson the some mobile genetic elements [6]. Though limited innumber, the majority of in situ studies indicate there is littleor no change on susceptibility of bacterial community tobiocides following the use of antibacterial product [6]. It is stillthe case that resistance to disinfectants, in particular, isbelieved to be a relatively rare event because most disinfectantsare often complexes of antimicrobials or adjuncts thatinactivate multiple cell targets.To address the concerns over biocides promoting antibiotic

resistance development, it is important to understand resis-tance mechanisms linked to biocide use and their potentialto confer cross-resistance to other antimicrobials includingantibiotics. In this review, we discuss about the mechanismsof biocide resistance and cross-resistance and the techniquesthat can be adopted to monitor the emergence of resistant

strain with a major focus on the two main biocide groups,namely, quaternary ammonium compounds (QACs) andbiguanides. Rapid diagnostic methods are needed to guiderisk assessments and selection of appropriate biocide to mini-mize the development and spread of antimicrobial-resistantbacteria. There are several advanced techniques reported inthe scientific literature, ranging from physiological methodsto molecular tools which can provide enhanced understandingof the bacterial mechanisms underlying phenotypic changes inbacteria upon exposure to biocides. In this article, we reviewthe reported resistance mechanisms and outline the principlefeatures of physiological and molecular assays in the detectionof resistance mechanisms and discuss specific applications ofthese techniques in the detection of resistance development.

2. Biocides/disinfectants

According to Centers for Disease Control and Prevention,disinfectant is a chemical agent used on inanimate objects todestroy virtually all recognized pathogenic microorganisms,but not necessarily all microbial forms [2,6]. The Environ-mental Protection Agency has grouped disinfectants intogroups as ‘limited’, ‘general’ or ‘hospital’ disinfectant. Severalcategories of disinfectants are being used for differentapplications since very long time [6].

QACs and biguanides are among the biocides that arecommonly used worldwide. Their use also extends in variousapplications like, in paper production, oil and gas field appli-cations, animal sanitation, aquaculture and wood preserva-tion. The mechanisms of action of these compounds andthe resistance mechanisms against them have been studied ingreat detail. Recently, several reports related to the biocideresistance and cross-resistance to antibiotics have beendocumented. These reports alert us about the hazards of usageof biocides without considering their indirect effects onmicrobes.

2.1 Classes of biocidesBiocides can be classified into several groups based either onthe functional group it possess or the target on which theyact. They are classified into at least 22 different categoriesbased on functional chemical groups (Figure 1) [7]. However,based on the target of action, biocides can be broadly classi-fied into four groups, those that exert their action on theproteins, membrane, nucleic acids and cell wall (Table 1).For example, chlorine and oxygen-releasing chemicals act onthe enzymes in membrane as well as cytoplasm and oxidizethem. Alcohol and chlorhexidine can denature the proteins.

QACs, biguanides and phenolic compounds disrupt thecell wall and membrane. The mechanism of QACs is basedon the physical disruption and partial solubilization of mem-brane and cell wall, whereas polymeric biguanides enter intothe cell by destabilizing divalent cations associated with thecell membrane and disrupting the lipopolysaccharide [8,9].The molecular targets of few of the commonly used

Article highlights.

. Classification of biocides, mechanism of action and theemergence of resistance to biocides are provided.

. Unlike antibiotics, the usage of biocides is not regulatedas biocides are commonly used in household andindustrial purpose.

. The resistance mechanism to biocides and antibiotics isvery much similar and can lead to cross-resistance andthus the emergence of multidrug resistance.

. The development of genetic and molecular methods todetect the emergence of resistance is important tocontrol the resistance development.

This box summarizes key points contained in the article.

D. P. Gnanadhas et al.

2 Expert Opin. Investig. Drugs [Early Online]

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biguanides, QACs and phenolics are listed in Table 2. Basedon their action, biocides can also be classified as oxidative,thiol interactive, membrane active, etc.

3. Biocides and resistance mechanisms

Minimum inhibitory concentrations (MICs) defined as thelowest antimicrobial concentration that inhibit the growthof microorganism after overnight or appropriate incubationperiod is considered as a gold standard, for ascertaining thesusceptibility of microorganism to the antimicrobial and forthe prescription of antimicrobial dose or regime. Breakpoints(determined through MIC) are the antimicrobial concentra-tion values that are used in defining whether a particularmicroorganism is susceptible, intermediate or resistant tothe drug. As biocides are not used for treatment, the termbreakpoint is not generally used for classification of the micro-organism as susceptible or resistant. Instead, the term epide-miological cut-off is used for classifying the microorganismas susceptible, intermediate or resistant [10,11]. Based on thisdata the antimicrobial dose is decided [12]. However,MICs are determined under in vitro conditions without con-sideration of drug pharmacokinetics [13]. Lately pharma-cokinetic or pharmacodynamics breakpoints that take into

consideration the drug pharmacokinetic and clinical data areused to determine the antibacterial efficacy of the anti-microbial [13]. As biocides are purposed to kill the microor-ganisms rapidly, instead of MICs microbicidal concentration(MBC), minimal concentration that kills almost all cells(> 99.9%) after constant incubation period (generally 24 h)is used in determining the susceptibility of microorganismsagainst biocide [12]. There have been reports where nocorrelation was observed between MIC and the resistancephenomena [14-16]. If the microorganism is resistant to thebiocide due to its natural properties, it is called insusceptible,whereas if they are less or not affected by concentration ofbiocide effective against susceptible strains, they are calledtolerant or resistant [2]. The term ‘resistance’ should be usedwhile discussing the killing phenomena and ‘tolerance’should be used while discussing adaptation to inhibitoryconcentration [17].

Inappropriate usage of antimicrobials can lead to the resis-tance development. Unlike antibiotics, the target sites ofbiocides are not very specific and hence can lead to nonspecificresistance mechanisms as well. It is highly unlikely for thebacteria to get resistance against a biocide, since biocides caninteract covalently with the bacterial targets. The resistancemechanism of bacteria to biocides can be intrinsic or acquired

Triclosan

Dim

ethoxane, bronidox

Carbanilides, salicylides

Copper, silver, mercury,

titanium compoundsHydrogen peroxide, peracetic acid

Ethyl, methyl, isopropyl, bronopol

4-Aminoquinaldinium,

8-hydroxyquinoline derivative

Ethylene and propyle

ne oxide,

ozone

Lim

onen

e

Tria

zine

s, o

xazo

les,

cap

tan

BIT

, MIT

, CM

IT

Alkaline brom

ine derivativesC

hlorine releasing compounds,

chloroform

Iodine compounds

Acridines, quinones

Glutaraldehyde, formaldehyde

Quaternary ammonium compounds,

anionic, nonionic and

amphoteric agents

Chlorhexidine, alexidine, polymeric

biguanidesPropamidine, d

ibromopropamidine

Form

ic, p

ropi

onic,

sal

icylic

, par

aben

,

sulp

hite

s, v

anilic

acid

est

ers

Cre

sols

, hal

o-ni

troph

enol

s, b

is-p

heno

ls

Dia

zolid

inyl

ure

a, a

ntoi

n

Derivatives of imidazoleBIOCIDES

Phenols

Organic and inorganic acids: esters and salts

Aromatic diamidines

Biguanides

Surface active agents

Aldehydes

Antimicrobial dyes

Halogens

Chlorine compounds

Bromine

Isothiazolones

Derivatives of hexamine

Terpenes

Vapour-phase disinfectants

Quinoline and isoquinoline derivatives

Alcohols

Peroxygens

Heavy-metal derivatives

Anilides

Derivatives of 1,3-dioxane

Phenylethers

Figure 1. Classes of biocides based on the functional groups.

Biocides -- resistance, cross-resistance mechanisms and assessment

Expert Opin. Investig. Drugs [Early Online] 3

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due to mutations in the genetic material or uptake of newgenetic material through horizontal gene transfer (Figure 2).

3.1 Phenotypic changesPhenotypic changes of bacteria might be responsible for thealtered susceptibility against biocides. The MICs are deter-mined in laboratory conditions but the physiological natureof cells in clinical situations are different, like in nutrient-depleted conditions cells can grow as a biofilms, endosporesor intracellularly in macrophages they are resistant to numer-ous physical and chemical agents [18,19]. For example, theresistance (tolerance) of Pseudomonas aeruginosa and Staphylo-coccus epidermidis against tobramycin and BC increased up to100-fold when they were present as biofilms [20,21]. Chlorh-exidine that is widely used in hospitals as a disinfectant caninhibit Serratia strains at 1,024 µg/mL; however, the resistantorganism isolated from hospital environment was able to sur-vive in concentrations of up to 20,000 µg/mL [22]. Formationof biofilms also confers resistance to biocides [23,24]. The ageof the biofilm and disinfectant resistance is not related butthe concentration and the contact time to disinfectants arethe major factors for conferring the resistance [25].Apart from acquired resistance which requires physical pres-

ence of biocide, microorganisms do exhibit natural (intrinsic)resistance pertaining to their physical characteristic like theenvelope architecture. The envelope architecture of Gram-positive and Gram-negative bacteria is highly distinct andmight contribute to the susceptibility of bacteria to biocides.Gram-positive bacteria have a thick cell wall and no outermembrane, whereas Gram-negative bacteria have a thinnercell wall with outer membrane layer. The level of biocide sus-ceptibility among Gram-positive and Gram-negative bacteriais controversial. It has been demonstrated that Gram-positivebacteria are less susceptible than Gram-negative bacteria toBC and ortho-phthalaldehyde [26]. Contrary to this, it wasshown that Gram-negative bacteria are less susceptible tocertain biocides due to the presence of outer membrane andouter membrane proteins (OMPs) that hinders the diffusion

and uptake of biocides, respectively [27]. Bridier et al. reasonthis discrepancy to the methodology used for the estimationof susceptibility against biocide (MICs vs MBC). P. aeruginosa,Providencia stuartii and Proteus spp. which are Gram-negativeshow more insensitivity to biocides due to the impaireduptake [28].

3.2 Modulation of efflux pumpsPresence of efflux pumps is one of the major mechanisms forbiocide resistance in Gram-negative bacteria where broadspecificity efflux pumps are overexpressed [29]. Chromosom-ally encoded acrAB efflux pump provides intrinsic resistanceto multiple drugs in Gram-negative bacteria [30]. This effluxpump, in Escherichia coli, has been shown to export severalantibiotics (tetracycline, ciprofloxacin, fluoroquinoloneb-lactams) as well as other chemicals (ethidium bromide,acriflavine, phenylethylalcohol, sodium dodecyl sulfate, anddeoxycholate) [31-33]. Similarly genome of P. aeruginosa enco-des different multidrug efflux systems like theMexAB,MexCDand MexEF which are able to efflux out variety of anti-biotics [34]. Gram-positive bacteria are also known to possesssome efflux pumps. In Staphylococcus aureus, the multidrugefflux pumps QacA to QacG lead to biocide resistance [35].Plasmid-borne qacH and qacJ decreases the susceptibility ofStaphylococcus haemolyticus to biocides like QACs and bigua-nides [36]. A putative small multidrug resistance transportergene (qacZ) present in Enterococcus faecalis makes the bacteriatolerant to QACs [37]. Chromosomally encoded NorA, NorBand NorC efflux pumps are negatively regulated by mgrA, aglobal regulator in S. aureus. Over expression of these effluxpumps and mutation in mgrA contributes to quinolone resis-tance [38]. Correlation between the efflux pumps genes (qacA,qacB and smr) and decreased sensitivity to chlorhexidinegluconate was recently observed in MRSA strains isolatedin Canada [39]. Phenylalanine-arginine b-naphthylamideand 1-(1-naphthylmethyl)-piperazine are the putative effluxpump inhibitors (EPIs) which reverse the resistance tothe biocides like triclosan, BC, chlorhexidine diacetate,

Table 1. Classes of biocide based on target of action.

Biocides that act

on membrane

Biocides that act

on proteins

Biocides that act

on nucleic acid

Biocides that act on cell wall

QACs [14,133,134]

Biguanides [14,133,134]

Phenols [14,133,134]

Phenylethers [14,133]

Acids [14]

Terpenes [6]

Alcohols [14,133,134]

Anilides [134]

Peroxygens [134]

Parabens [14]

Isothiazolones [14]

Anionic surfactant [14]

Alcohols [134]

PhenolsPhenylethers [133]

Aldehydes [14,134]

Heavy-metal derivatives [133]

Isothiazolones [133]

Acids (parabens) [133]Peroxygens [14]

Chlorine compounds [14]

Biguanides [134]

Vapor-phase disinfectant [134]

Alcohols [133]

Acids (parabens) [133]Antimicrobial dyes [133]

Acridines [14]

Biguanides [133]

Aldehydes [134]

Diamidines [135]

Chlorine compounds [134]

Heavy-metal derivatives [133,134]

Peroxygens [134]

Halogens [134]

Vapor-phase disinfectant [134]

AlcoholsPhenols [136]

Aldehydes [134,136]

Chlorine releasing compounds [136]

Heavy-metal derivatives (mercurials) [136]



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cetylpyridinium chloride (CPC) and trisodium phosphate.There is a decrease in MIC, when the efflux system is blocked.This demonstrates the influence of efflux pumps on naturalresistance [40]. Contrary to this, when porins, required toinflux hydrophilic nutrients in case of Mycobacteriumsmegmatis, were blocked, it led to increased resistance towardmany biocides [7]. ATP-binding cassette (ABC) and majorfacilitators (MFs) super family efflux pumps are the majorcontributors of multidrug resistance (MDR) in yeasts [41]. InCandida albicans, over expression of multidrug efflux pumpMDR1 confers resistance to the fluconazole, an antifungaldrug [42].

3.3 Inactivation of biocidesApart from cell wall modification and efflux effects, inactiva-tion of biocides is another intrinsic resistance property ofmicrobes against biocides. Even though there are limitedchances of inactivation of the biocide, Pseudomonas was foundto inactivate phenols, some aldehydes and triclosan [43-45].

Triclosan (0.4 µg/mL) was inactivated within 96 h byPseudomonas putida [45]. Pseudomonas fluorescens isolatedfrom a sewage treatment plant was able to degrade didecyldi-methylammonium chloride to decyldimethylamine andsubsequently, dimethylamine as intermediates. QACs weredegraded by these strains by N-dealkylation process and hencethey showed higher resistance [46].

3.4 Target alterationApart from target inactivation, target alteration is another wayby which microorganisms gain resistance. Target alteration ormodification due to mutation in the genome can also producechanges in the biocide susceptibility. In E. coli, triclosan inhibitsthe bacterial fatty acid synthesis by affecting FabI (enoyl-acylcarrier protein reductase). However, the altered target site[missense mutation in FabI (G93V)] confers resistance to thebacteria [47]. Similarly in Mycobacterium tuberculosis the analogof fabI (inhA) is the target for isoniazid and ethionamide wherea point mutation in inhA gene confers resistance to drugs [48].

Table 2. Molecular targets of few selected biocide.

Biocide/class of biocides Microorganism Target site/mode of action (MoA)

BiguanidesTarget site: Cytoplasmic inner membrane; low concentrations affect membrane integrity; high concentrations cause congealing ofcytoplasmChlorhexidine Vegetative bacteria [137] Membrane-active agent leading to release of periplasmic enzymes,

causing protoplast andspheroplast lysis; high concentrations cause precipitation of proteinsand nucleic acids

Yeast [138,139] Morphological modifications in cell wall, loss of cytoplasmiccomponents, nucleoprotein coagulation at high concentration,protoplast lysis, interaction with cellular components

Bacterial spores [140-144] Nonsporicidal at ambient temperature but have sporicidal effect at hightemperature, inhibits spore outgrowth but not germination

Virus [145,146] Antiviral action against lipid enveloped viruses, activity against ehnucleic acid core and outer coat of virus

PHMB Bacteria [70,147] Nonspecific damage of cytoplasmic membrane and disrupt cell envelop,leakage of low molecular weight components, inhibition of membranebound enzymes and precipitation of cytoplasmic content andinteraction with nucleic acid

Amoeba [148] Separation of the plasma membrane of intracystic amoeba fromendocystic wall and shrinkage of intracystic amoeba, aggregation ofphospholipids, cytoplasm and nucleus and deformation of organelles

Yeast and Fungi [149] Interacts with cell membraneQACTarget site: Cytoplasmic inner membrane; generalized membrane damage involving phospholipid bilayers, high concentrations targetscarboxylic groups and causes general coagulation

Bacteria [150-152] More effective against Gram-positive than Gram-negative. Enters insidethe Gram-positive bacteria by binding to cell wall proteins and thusdestroys the membrane. In P. aeruginosa, strips off the outermembrane

BC Bacterial spores [153] Inhibits outgrowth but not germinationYeast and fungi [154] Cell wall permeabilization and loss of K [+] ions

PhenolicsTarget site: Proteins, cell membrane and cell wallTriclosan Bacteria [6,155,156] Acts on membrane, inhibits fatty acid synthesis (FabI enzyme) and enoyl

ACP reductase, rapid release of cellular components. At higherconcentration, induces lysis



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Mutation in inhA that confers isoniazid resistance does notconfer triclosan resistance and vice versa [48].Exposure of Listeria monocytogenes to low levels of BC

resulted in altered membrane lipid composition which in turnled to the decreased membrane fluidity and alterations in phys-iochemical properties of the cell surface [49]. Changes in theOMP and modification in lipid membrane permeability hasbeen speculated to confer phenol resistance in E. coli [50]. Bio-cide resistance can be observed more in Gram-negative bacteriathan Gram-positive as Gram-negative bacteria have the abilityto modify the outer membrane. This can lead to increased resis-tance against biocides as compared to Gram-positive bacteriawhich lacks the outer membrane [27]. In yeast, cell wall integritygenes and protein kinase C regulatory mechanism are involvedin polyhexamethylene biguanide (PHMB) resistance [51].The complex nature of the Mycobacterial cell wall makes it

less susceptible to many of the biocides. Mycobacteria possesswater-filled porins which are involved in nutrient transportin the bacteria. The reduced susceptibility in the absence ofporins (MspA and MspA like porins) indicates that the porins

are required for the transport and the actions of various classesof biocides [7] including isothiazolinones, methylenbisoxa-zolidine and the lipophilic biocides PHMB and octenidinedihydrochloride.

3.5 Horizontal gene transferTransfer of genetic elements and the emergence of resistantbacteria are well-known phenomena from 1960s. Plasmidswhich can confer the biocide resistance specifically related toheavy metals were found in several bacteria [52,53]. Theplasmid-mediated mechanism for reduced susceptibility tobiocides has been reported in S. aureus [54-56]. Of late, triclo-san resistance in S. aureus due to acquisition of sh-fabI alleleby horizontal transfer from S. haemolyticus was identified.The sh-fabI islet was detected in staphylococci plasmid (heter-odiploidy condition) indicating active transfer of resistantelement probably due to the positive selection exerted bytriclosan [57]. Recently it was shown that in aquaculture envi-ronments, bacteria harbor integrating conjugative elements(mobile genetic elements) that can provide resistance to

Change in membrane lipid composition

Modification of biocides Mutation in the enzymesof fatty acid biosynthesis

pathway

Plasmid

Acquisition of drugresistant genes byhorizontal transfer

Deletion/mutation in the genes

Modification of OMPs

OMP OMP

Impaired diffusion of hydrophobic biocides in Gram-negative bacteriadue to the presence of outer membrane

Degradation of biocides

Presence/modification of cell wall(Gram-positive bacteria)

Effl

uxpu

mp

Bio

cide

s

Degradationof biocides

Porins

Modified enzymecan act in the

presence of biocide

Inhibition ofbiocide transportdue to modified

structure

Figure 2. Various mechanisms of resistance against biocides in bacteria. Bacteria are inherently resistant to biocide (intrinsic

resistance) or can gain resistance to different biocides (acquired resistance) via different mechanism. Intrinsic resistance is

achieved through presence of cell wall, efflux system, etc. The resistance can also be achieved through mutation in genes that

are responsible for the formation of cell wall, membrane lipid, porins or OMPs. Acquisition of mobile genetic elements like

plasmids through horizontal transfer is another mechanism by which the bacteria gain resistance. Certain genes that encode

for protein that can modify or degrade the biocide can be formed either through alteration in preexisting genes or through

genes acquired through horizontal transfer.



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rifampicin, heavy metals and QACs [58]. Mobile geneticelements that code for enzymes like o-phosphotransferases,o-adenyltransferases and n-acetyltransferases capable ofdegrading biocides were also reported [59]. Bacterial carry theqacE or qacED1 gene in integrons are not necessarily moreresistant to QACs than antibiotic-sensitive strains [60].

4. QACs and microbial resistance

The general structure of QACs is NR1R2R3R+4 X

-, where Rcan be hydrogen atoms or alkyl groups and X is an anion.QACs are used for different applications. Some of the mainapplications are in medical industries, water treatment, agroindustries as well as cosmetics. They are also used in mouth-wash to prevent dental biofilms. Most widely used QACsare BC, stearalkonium chloride, cetrimonium chloride/bromide (cetrimide) and CPC. BC is extensively used in eyedrops, decongestion nose drops, facial moisturizers, facialcleansers, acne treatment formulations, sun protection creamsand lotions, baby lotions, moisturizers, pain relief and handsanitizers. The cationic nature of QAC is the central principleof its antimicrobial properties. QACs can affect lipids andproteins and hence cause disruption of cell membrane andleakage of cellular contents.

QAC resistance can be due to the natural property of anorganism resulting in decreased susceptibility (intrinsic mech-anism) or due to the acquired resistance, resulting from themutation in cellular genes, the acquisition of foreign resis-tance genes or the combination. The major cause of AMR isdue to the horizontal gene transfer rather than mutations [6].

Increased resistance to QACs was observed in P. aeruginosawhen subjected stepwise to increasing concentrations of poly-myxin [61]. Similarly, when the bacteria were treated with lowlevel of QACs, it resulted in different fatty acid profile therebyincreasing the resistance toward QAC [62]. The change in thefatty acid profile is different for different QACs. For example,in case of dodecyldimethyl ammonium bromide resistantPseudomonas, the modifications mainly affected lauric,b-hydroxylauric and palmitic acids [63,] whereas benzyldime-thyltetradecylammonium chloride resistant strains showedchanges in some other fatty acids (b-hydroxycapric, palmitoleicacid [64,65]) apart from the three fatty acids mentioned above. Itwas also shown that qacZ gene that codes for a putative smallmultidrug-resistant transporter provides increased tolerance toBC and not for ethidium bromide in E. faecalis [37].

5. Biguanides and microbial resistance

Chlorhexidine is an important biguanide which is being usedas antiseptic, disinfectant, preservative and pharmaceuticalagent [6]. Chlorhexidine induced precipitation of cytoplasmicproteins and nucleic acids at the bactericidal concentrations ofthe biguanide. However, at lower concentrations it acts as amembrane active agent and induces the release of K+ ions [66].The resistance mechanisms of bacteria against biguanides are

not completely understood. It is proposed that the changes inthe lipid profile may contribute for the resistance. The phos-pholipids may play an important role in biguanides’ resistancesince the phospholipid depleted bacterial cultures showed lesssensitivity to chlorhexidine [66]. Nevertheless, few controversiesarose as no change in the lipid profile of chlorhexidine-sensitiveand resistant strains could be found [66]. Chlorhexidine resis-tance is developed when Proteus mirabilis is exposed to lowlevels of the biguanide and the resistant strains showed reducedsusceptibility to other biocides including cetrimide and BC [67].The MICs of chlorhexidine for methicillin-resistant S. aureuswas found to be 1.5- to 3-fold greater than for methicillinsensitive S. aureus. However, there was no direct correlationobserved between methicillin resistance and chlorhexidineresistance [14]. Exposure of Acinetobacter baylyi to sublethalconcentrations of chlorhexidine induces resistance for lethalconcentrations of the biguanide as well as the resistance tooxidative stress [68].

PHMB is a biguanide which has been used in water treat-ment, as a disinfectant in contact lenses, in poultry industriesto prevent Salmonella infection, Acanthamoeba and fungal treat-ments [51]. PHMB-impregnated gauze dressing was assessed forthe growth of bacteria in vitro. It showed better efficiency forkilling of both Gram-positive and Gram-negative bacteria [69].

In Saccharomyces cerevisiae, PHMB treated upregulatedgenes are involved in cell wall synthesis in case of resistantstrain but not in the sensitive strain. The upregulation ofsome other genes encoding Msn2/4 transcription factorsupon PHMB treatment indicates that the stress responsegenes are also involved in the resistance mechanism [51].

The whole genome transcriptional profiling of PHMB-treated cells demonstrated the involvement of set of geneswhich are responsible for the metabolism of nucleic acidsand DNA repair. These genes contribute to the tolerance ofE. coli to PHMB [70].

6. Nanoparticles and microbial resistance

Recently silver nanoparticles have been used as an antimicrobialagent in different commercial applications including water puri-fication filters [71] and wound dressing [72,73]. It has been claimedthat bacteria cannot develop resistance against silver and silvernanoparticles and hence silver nanoparticles are one of the poten-tial candidates against multidrug-resistant bacteria/patho-gen [74,75]. But there are few reports suggesting the resistancedevelopment against silver [76,77]. Though efflux pump-mediatedresistance for silver was not reported, there are few reportsshowing the plasmid-mediated resistance to silver inPseudomonasstutzeri, Enterobacteriaceae and Citrobacter species [53].

7. Cross-resistance of biocides and antibiotics

Various cases of antibiotic resistance were observed from1960, which is considered as a major threat to clinical prac-tice. Inappropriate prescription and use of antibiotics



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is considered as a major cause for antibiotic resistance [21].Prolonged exposure to sublethal concentrations of biocidescan lead to the overexpression of efflux pumps and hencethe increased resistance in bacteria [21]. MIC of the biocide2-phenoxyethanol was increased when P. aeruginosa was sub-jected to the biocide and the cross-resistance was observedwith dissimilar biocides but not with antibiotics [78]. Differentbacteria resistant to biocides and cross-resistant to antibioticsare listed in Table 3.Triclosan resistance in P. aeruginosa can be due to the target

alteration. It was observed that the resistance mechanism canbe due to the expression of MexAB-OprM efflux system.Upon exposure to triclosan, multidrug-resistant bacteriaemerged due to the mutation in nfxG gene which regulatesthe MexCD-OprM efflux system. These strains showed lowersusceptibility to many drugs and antibiotics, where the MICof ciprofloxacin was increased up to 94-fold [79]. Stenotro-phomonas maltophilia harbors SmeDEF, a multidrug effluxpump, which is activated by triclosan. Triclosan binds toSmeT, a smeDEF repressor and releases it from its operatorinducing the expression of smeDEF and reducing thesusceptibility of S. maltophilia to antibiotics [80].Exposure of subinhibitory concentrations of QACs to

E. coli led to its increased resistance to antibiotics as well asphenolic compounds [81,82]. This was due to enhanced effluxsystem which also conferred cross-resistance [82].The resistance and cross-resistance emergence is specific for

different species as well as different strains of same bacterialspecies [83]. The ability of Salmonella, which is a food-bornepathogen, was assessed for the resistance and cross-resistancemechanism to biocides and antibiotics. Interestingly, Salmo-nella enterica serovar Virchow, resistant to BC showed higherresistance to chlorhexidine, but chlorhexidine-resistant straindid not show any reciprocal cross-resistance to BC [84]. Thisclearly suggests that the resistance and cross-resistancemechanism is specific and not a generic mechanism.

8. Techniques to investigate biocideresistance

There is a need to develop identification techniques to test thepotential of the biocide to confer resistance or cross-resistance.Few such techniques that might help in judging whether thebiocide can confer resistance are discussed.

8.1 Molecular methods and technical aspects: from

research to routine diagnosticsThe outer surface of the bacteria (cell wall or outer membrane)is important for the ability of bacteria to confer resistance toantibiotics [85,86]. The OMPs and the lipid bilayer prevent theaccess of the antimicrobials to the bacteria. The diffusion ofthe antibiotic into bacteria is essential for its action. The hydro-phobic antimicrobials can diffuse via the lipid bilayer in Gram-positive bacteria. Gram-negative bacteria demonstrate toleranceto environmental stresses through number of ways. They have a

highly charged lipopolysaccharide (LPS) layer that prevents thediffusion of hydrophobic antimicrobials. The entry of hydro-philic antimicrobial agents is mediated via the porins in theouter membrane of the bacteria [86]. To resist the action ofsuch antimicrobials, bacteria can alter the expression of theOMPs’ [87,88] increasing export of antimicrobial from the cell.It can decrease the expression of importer porins and increasethat of exporter porins [88]. Bacteria are also found to alter theexpression of the OMPs in order to gain resistance againstthe biocides [21,87,89]. They also change the composition andstructure of outer membrane leading to reduced permeabilityand uptake of antimicrobials. There are several techniquesapplied for characterizing changes in the outer membrane pro-file. The outer membrane profile can be altered either throughthe modifications in the OMPs or LPS profile or throughaltering the organization of these components. These changescan be characterized by assessing for changes in the expressionof OMPs, the LPS components and the fatty acids that makeup the membrane and constitute part of LPS. We have dis-cussed few of the techniques used to identify such alterationsin the microorganisms to adapt to the biocide and developcross-resistance against antibiotics.

8.1.1 Expression analysisThe changed expression of OMPs can be detected by PCR,microarray [90-92], SDS-polyacrylamide gel [93,94], or 2D gelanalysis followed by mass spectrometry [50,78,89]. Recently amodified form of 2D gel, 2D differential fluorescence gelelectrophoresis, has been used to identify the differentiallyexpressed genes in the resistant versus sensitive bacterial strain(Salmonella resistance to triclosan) and the differentiallyexpressed proteins identified by mass spectrometry [95]. SDS-polyacrylamide gel has also been used to analyze the changein LPS profile [94]. PCR (real-time or reverse transcription)has been used for the successful detection or overexpressionof efflux genes in the biocide-resistant bacteria. Examples ofsuch genes include, qac genes [96], ade (multidrug effluxpumps) genes in Acinetobacter [96-98], acr and tol systems inenterobacteriaceae and Aeromonas [98], mex in Pseudomonasand Aeromonas [98], sde genes in Serretia [98,99], lmr inLactobacillus, Bacillus sp. etc. [98].

In order to study whether the biocide is inducing any mod-ification in the expression of the genes, other than OMPs,involved in resistance, one can opt for either of the methodssuch as microarray, real-time PCR and proteome analy-sis [100,101]. PCR-RFLP analysis of the known genetic markersconferring antibiotic resistance would help in testing thedevelopment of cross-resistance [102-104]. Whole genome tran-scriptional profiling was used to assess the novel mechanismof action of PHMB. The role of DNA repair genes wasidentified in the PHMB-tolerance mechanism in E. coli [70].

8.1.2 Uptake/exclusion studiesThe altered expression of exporter porins can also be assessedby drug exclusion studies [89]. Compounds such as ethidium



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bromide, Hoechst and acriflavine are used for suchanalysis [89]. The uptake of the biocide like chlorhexidinecan also be studied using energy-dispersive analysis of X-ray(EDAX). Here the sample is irradiated with electron beamand the X-ray pattern analyzed. As each element gives offX-rays with characteristic energies, EDAX allows assayingthe elemental composition of the sample and hence the pres-ence of biocide like chlorhexidine [88,105]. X-ray microanalysisby EDAX also revealed the distribution of chlorhexidine inthe cells of S. cerevisiae [105] and P. aeruginosa bacteriophageF116 [106].

8.1.3 Changes in surface hydrophobicityAlteration in cell-surface hydrophobicity might limit theuptake or permeability of the biocide. The changed hydro-phobicity can be assessed by bacterial adherence to hydro-carbon (BATH) method where the bacterial suspension ismixed with appropriate hydrocarbon and the partitioning ofbacteria in the hydrocarbon versus aqueous phase are esti-mated to analyze the changed hydrophobicity. The methodwas used by Tattawasart et al. to assess the reason for theincreased resistance of P. stutzeri and P. aeruginosa againstchlorhexidine diacetate and CPC stress [106].

8.1.4 Outer membrane fatty acid profileThe change in the properties of membrane can be assessedby analyzing the lipid or fatty acid profile. For doingso, lipids are extracted from the bacterial surface andanalyzed either by gas-chromatography [65] or thin layerchromatography [49,107]. Mass-spectrometric techniques likematrix-assisted laser desorption/ionization (MALDI) andelectrospray ionization can also be used to analyze the profileof modified lipids [108-111]. Analysis of lipid reveals percent-age of each class of lipids namely saturated versus unsatu-rated, phospholipids versus amino/glycol-lipids, polar andneutral lipids, anionic phospholipids giving an idea whetherthe change can be beneficial in the development of resistance

against the biocide or antibiotics [49]. For example, anincrease in the amount of unsaturated fatty acids in themembrane increases the fluidity rendering the organismresistant to the antimicrobials [112,113]. Reduction in amountof anionic phospholipids or increase in amount of cationicphospholipids can result in reduction of net negative chargeand hence resistant against cationic antimicrobials. Certainbacteria modify the lipid moieties/teichoic acid polymersto increase resistance against cationic antimicrobials [114-118].Addition of groups that render positive charge on the lipidsurface hinders the interaction of cationic antimicrobialswith the bacterial surface. Also, reduction in the phospho-rous group in the lipids helps in reducing the net negativecharge on the bacterial surface making it resistant againstthe action of cationic antimicrobials. Modifications of LPSstructure can give rise to increased resistance against cationicbiocides [119].

8.1.5 Changes in cell-membrane permeabilityBiocides like chlorhexidine, QAC act on outer membrane ofthe bacteria altering the membrane permeability [120]. Tetra-phenylphosphonium ion (TPP+) electrode can be used foranalyzing this changed membrane permeability [121]. In theassay the uptake of TPP+ coupled with the efflux of potas-sium ions indicates that the membrane is permeabilized [121].The entry of TPP+ is generally restricted by the outer mem-brane due to the presence of charged LPS residues [121].However, the alteration of outer membrane by the actionof biocides leads to permeabilization of the membrane andthe diffusion of TPP+ into the cell followed by the effluxof K+ ions [121]. The use of this assay can be extended tocheck the development of resistance in the bacteria againstthe membrane acting (permeabilizing) agents. In order toassess the structural organization of outer membrane,EDAX has proven to be a useful tool [122]. The X-raydiffraction pattern is used to analyze the structure of themembrane [123,124].

Table 3. Biocide and antibiotic cross-resistance.

S. No. Organism Biocide resistance Altered resistance

to antibiotics

Mechanism

1 P. aeruginosa Triclosan Ciprofloxacin Mutation in nfxG gene [79]

2 E. coli BC, didecyl dimethylammonium chloride,dioctyl dimethylammonium chloride

Ceftazidime, cefotaxime,chloramphenicol, florfenicol

Enhanced efflux system [81]

3 Salmonella Triclosan Chloramphenicol, erythromycin,imipenem, tetracycline

Active efflux pumps [84]

4 Mycobacterium Triclosan Isoniazid inhA mutations [157]

5 S. aureus Triclosan Ciprofloxacin Alteration in cell membranestructure and function [158]

6 Campylobacter jejuniand Campylobacter coli

Triclosan, BC Erythromycin and Ciprofloxacin Efflux pumps [40]

7 Citrobacter freundii Triclosan Erythromycin Outer membraneadaptation [159]



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8.1.6 Use of microscopic techniquesTransmission and scanning electronmicroscopy has been used tounderstand the ultrastructural differences between the sensitiveand resistant isolates ofP. stutzeri [122]. The resistance cells stainedintensely with the negative stains (analyzed by transmission elec-tron microscopy) and possessed rough surface with externalamorphous material (analyzed by transmission and scanningelectron microscopy). The amorphous material was claimed tobe exopolysaccharide in nature. The susceptibility of biofilms tothe biocides can be assessed by confocal laser scanning micros-copy (CLSM) by visualizing the spatiotemporal pattern of thebiofilms [125]. Here the bacteria in the biofilms are stained withthe fluorogenic dye such that the damage to the bacterialmembrane (by the biocides) leads to leakage of the dye that ismonitored real-time by CLSM [125]. The patterns of loss of fluo-rescence can help predict the specificity of the biocide action andits limitation, if any, in killing the bacteria in the biofilm [125].

8.1.7 Nucleotide sequencingThough there are many reports for identifying the antibioticresistance mechanism using nucleotide sequencing and wholegenome sequencing [126,] few researches have used these tech-niques to identify biocide resistance mechanism. Nucleotidesequencing revealed that mutation in the sdeS gene promotesexpression of the SdeAB efflux pump genes and hencemultidrug resistance in Serratia marcescens [127].Flow cytometric-based assays can be used to identify the set

of bacteria that are resistant to the biocide [128]. Fluorescence-activated cell sorting can be further used to sort these resistantcells and then characterize the resistance phenomena usingeither of the abovementioned techniques.

9. Clinical trials

Several new biocides are being tried for different applicationsand many of them are in clinical trials (Table 4). The analysis

of resistance mechanism and the resistance development arenot part of the clinical trials as of now. The European law isnow requiring testing of resistance development of com-pounds and/or products for the licensing of a product and/or compound [129]. These available techniques should beinvolved to check the resistance development in future sothat we can avoid any adverse effects due to the introductionof biocides in different applications including household,hospitals and industries.

10. Expert opinion

Emergence of multidrug-resistant strains is a major concernworldwide. The efforts are implemented to control suchemergence like the use of disinfectant/biocides in hospitals,household settings, public places, food industries, etc. How-ever, their indiscriminate or inappropriate use has led to thegrowth of biocide resistance and cross-resistance to the antibi-otics. This has been attributed to the overlapping mechanismof action of biocides and antibiotics. However, controversialresults have been reported that deals with the developmentof cross-resistance to antibiotics in the strains that are resistantto biocides. For example, cross-resistance to antibiotics wasnot observed, when P. aeruginosa were exposed to residuallevels of chlorhexidine but the strains showed increased resis-tance to BC [130]. Nevertheless, few reports suggest thatbiocides are found to exert selective pressure on the microor-ganism to retain the genes (mobile genetic elements) acquiredthrough horizontal gene transfer. It has been shown that pres-ence of triclosan led to increased expression of an efflux pumpin S. maltophilia through the release of the repressor from theoperator. This highlights the perversions associated with theuse of biocides. However, this problem can be taken care ofby using formulations with multiple biocides having differenttargets or formulations consisting of a biocide and an activator(for porins) or inhibitor (for efflux pumps) of the pathway or

Table 4. List of biocides considered for clinical trials.

Biocide/disinfectant Applications Clinical trial phase

Silver nanoparticles Impregnated in central venous catheter (CVC) forclinical applications [160]

Nanotoxicity assessment [161]

IV(I & II)

Chlorhexidine 2% chlorhexidine efficacy as a biocide [162]

Femoral nerve catheters [163]

Preoperative antisepsis [164]

Oral care in ICU patients [165]

IIIII

PHMB In acute traumatic wounds [166]

Foam dressing for chronic wounds [167]

IVII

Octenidine Dihydrochloride Catheter-associated infection [168] IVTriclosan Effects on microbial flora [169]

Effect after molar surgery [170]

Surgical sutures in coronary artery bypass surgery [171]

Efficacy of salivary bacteria post-brushing [172]

IIIVIII

BC Vaginal contraceptive gel [173]Effect on Langerhans cells in the eye [174]

IIII



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process through which biocide exert an effect. Another solu-tion to prevent the emergence of MDR strains is to developnew antimicrobials like metal nanoparticles. Silver ionshave antimicrobial properties on both Gram-positive andGram-negative bacteria. There are many reports documentingbacterial resistance against silver. But there are no reports per-taining to the resistance development against silver nanopar-ticles in bacteria. Biocides are known to alter the propertiesof the microorganism. This can have severe consequences asit might alter the virulence properties of the microorganisms.For example, alteration in lipid profile can increase the adhe-sion of the bacteria to the solid surface increasing its persis-tence in the environment or contributing to its biofilmforming ability. Expression of some virulence genes ofL. monocytogenes increased when the pathogen was exposed

to sublethal concentration of QACs. However, the virulenceof L. monocytogenes was reduced, when peroxy and chlorinecompounds were used [131]. Also, when S. aureus was exposedto sublethal dose of triclosan, biofilm formation and fewvirulence factors were attenuated [132]. Hence, the need of anhour is to formulate the correct combination of biocides atcorrect concentration (after estimation for particular species/strain) and constantly monitor the emergence of resistantstrains with appropriate technique. The breakpoints for eachbiocide have to be cross-verified and redefined if necessary.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.

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AffiliationDivya Prakash Gnanadhas,

Sandhya Amol Marathe &

Dipshikha Chakravortty†

†Author for correspondence

Department of Microbiology and Cell Biology,

Centre for Infectious Disease Research and

Biosafety Laboratories,

Indian Institute of Science,

Bangalore 560012, India

Tel: +0091 80 2293 2842;

Fax: +0091 80 2360 2697;

E-mail: [email protected]



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