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ScienceDirect Quaternary ammonium disinfectants: microbial adaptation, degradation and ecology Ulas Tezel1 and Spyros G Pavlostathis2 Disinfectants play an important role in maintaining acceptable health standards by significantly reducing microbial loads as well as reducing, if not eliminating, pathogens. This review focuses on quaternary ammonium compounds (QACs), a widely used class of organic disinfectants. Specifically, it reviews the occurrence, microbial adaptation, and degradation of QACs, focusing on recent reports on the ecology of QACdegraders, the pathways and mechanisms of microbial adaptation which lead to resistance to QACs, as well as to antibiotics. With the help of culture-dependent and nonculturedependent tools, as well as advanced analytical techniques, a better understanding of the fate and effect of QACs and their biotransformation products is emerging. Understanding the underlying mechanisms and conditions that result in QAC resistance and biodegradation will be instrumental in the prudent use of existing QAC formulations and foster the development of safer disinfectants. Development and implementation of (bio)technologies for the elimination of QACs from treated wastewater effluents will lessen adverse impacts to both humans and the environment. Addresses 1 The Institute of Environmental Sciences, Bogazici University, Bebek, Istanbul 34342, Turkey 2 School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0512, USA Corresponding author: Pavlostathis, Spyros G ([email protected])

Current Opinion in Biotechnology 2015, 33:296–304 This review comes from a themed issue on Environmental biotechnology Edited by Spiros N Agathos and Nico Boon

sub-inhibitory) concentrations of biocides. Recent studies suggest that exposure to sub-inhibitory biocide concentrations facilitates the evolution of resistance to the biocide, and may also lead to co-resistance and crossresistance to other antimicrobial agents such as antibiotics [1,2]. Quaternary ammonium compounds (QACs) are cationic surfactants introduced in the late 1930s along with the first antibiotics, sulfonamides. They are classified as ‘high production volume’ chemicals [3]. The chemical structure of QACs depends on the four aliphatic or aromatic moieties attached to the central nitrogen atom (R1R2N+R3R4). QACs are mainly used in disinfectant and antiseptic formulations utilized in homes, human and animal healthcare facilities, agriculture and industry. They are effective against a variety of bacteria, fungi and viruses at very low concentrations. When QACs are used as disinfectants, the applied concentration is typically between 400 and 500 ppm and almost always below 1000 ppm (e.g., 0.1% w/v in Lysol1) [3]. Domestic, hospital and industrial use of QACs results in QACsbearing waste/wastewater. Because typical wastewater treatment plants are designed to remove major, easily degradable organics, most trace contaminants, including QACs, pass through wastewater treatment plants and are released into the environment. About 75% of QACs utilized annually are released into wastewater treatment systems, whereas the rest are discharged directly into the environment. The mean concentration of QACs in domestic wastewater, treated effluent wastewater, sewage sludge and surface water is reported to be around 0.5 mg/ L, 0.05 mg/L, 5000 mg/kg dry weight, and 0.04 mg/L, respectively, which is significantly below applied concentrations [3–5].

http://dx.doi.org/10.1016/j.copbio.2015.03.018 0958-1669/# 2015 Elsevier Ltd. All rights reserved.

Introduction Disinfectants are extensively used and their formulations contain active ingredients at levels well above the minimum inhibitory concentration (MIC) of targeted microorganisms. Inappropriate application of disinfectants, dilution in the environment after discharge and biodegradation result in biocide concentration gradients. Thus, microorganisms are frequently exposed to non-lethal (i.e., Current Opinion in Biotechnology 2015, 33:296–304

Because QACs are biodegradable under aerobic conditions, their concentrations in indoor and outdoor environments continuously fluctuate. As a result, microorganisms are exposed to QACs dynamically over a wide range of concentrations (i.e., non-inhibitory, sub-inhibitory, overinhibitory concentrations). In general, environmental concentrations of QACs are well below the MIC values. Sewage, biological wastewater treatment units, surface waters and sediments are environments where QACs are present at sub-inhibitory concentrations. When the high microbial diversity in these environments is coupled to sub-inhibitory QAC concentrations, such environments become selective, resulting in the emergence and dissemination of QAC resistance among different bacterial www.sciencedirect.com

Quaternary ammonium disinfectants Tezel and Pavlostathis 297

genera, which may also include clinically important pathogens. Because many of the QAC resistance pathways and mechanisms are similar to those involved in antibiotic resistance, understanding QAC resistance and dissemination is very important in the context of the global antibiotic resistance problem. Although some suggest that there is no clear relationship between antibiotic resistance and exposure of microorganisms to QACs [6], many studies have shown that, for instance, exposure to QACs results in dissemination of integrons (i.e., promoterless mobile recombinational elements) which harbor resistance genes [7,8]. Evidence that soil bacteria and human pathogens share similar resistomes within integrons [9] suggests that there is a link between antibiotic resistance in nature and clinical settings, which is favored by exposure to QACs. Many reviews exist on the fate and effect of QACs in the environment, QAC-related antimicrobial resistance, and implications of QAC resistance to human health [3,10,11]. In this review, we discuss the ecology of QACs-exposed microbial communities, as well as the pathways and mechanisms of microbial adaptation to sub-inhibitory QAC concentrations following the context of a recent review by Andersson and Huges by linking the similarities of antibiotic and disinfectant resistance [12]. In addition, we consider the role of QAC biodegradation on the evolution and dissemination of QAC resistance by creating microenvironments with sub-inhibitory QAC concentrations. Moreover, technologies utilizing novel QAC-degraders for the alleviation of QAC contamination are discussed.

Pathways and mechanisms of QAC resistance at sub-inhibitory concentrations The mode of action of QACs above MIC in bacteria is disruption of the cell membrane’s physical and ionic stability [13]. For example, benzalkonium chlorides (BACs) bind to the cell membrane of Pseudomonas fluorescens by ionic and hydrophobic interactions, bringing about changes of membrane properties and function, followed by cellular disruption, loss of membrane integrity, ultimately resulting in leakage of essential intracellular constituents [14,15]. Above MIC, bacteria with durable cell membrane are selected and proliferate. On the other hand, the mode of action of QACs at sub-MICs is complicated and always includes multiple processes such as loss of membrane osmoregulation, inhibition of respiratory enzymes, the dissipation of proton motive force and oxidative stress, which triggers SOS response, inducing error-prone DNA replication leading to mutations and gene transfers [16,17]. Adaptation to QACs at sub-MICs is achieved by modification of the outer membrane, cell membrane, density and structure of porins, regulatory hyperexpression of efflux pumps, and acquisition of QAC-specific efflux pumps through mobile recombinational elements, such as plasmids and integrons www.sciencedirect.com

upon oxidative stress or (followed by) stress-induced mutagenesis [3]. Major pathways in the evolution of resistance at subinhibitory antibiotic concentrations have been recently reviewed [12,18]. Bacteria follow similar pathways while adapting to QACs. Exposure to QACs at sub-MIC creates (oxidative) stress. For instance, exposure of E. coli to cetyltrimethylammonium bromide resulted in intracellular production of superoxide and hydrogen peroxide [19]. Bacteria compensate for oxidative stress by SOS-response and induction of stress-response sigma factors rpoS, promoting cell survival by DNA repair, while nucleotide polymorphism may occur, resulting in mutations. Oxidative stress responses also boost gene transfer and recombination events via prophages, transposons, integrons and integrative-conjugative elements (ICE) (Figure 1). As a result, resistant sub-populations evolve and dominate in a microbial community upon exposure to any antimicrobial agent [20]. Major mechanisms of adaptation to QACs include modification of cell membrane structure and composition, enhanced biofilm formation, acquisition of efflux genes, overexpression of efflux pump systems, and biodegradation. Generally, multiple mechanisms co-develop during adaptation of bacteria to QACs [21]. Exposure to QACs at sub-MICs enhances biofilm formation [22]. QAC resistant strains of bacteria form biofilms faster and these species are less susceptible to QACs than planktonic species [23]. Presence of multiple species in biofilms increases QAC resistance [24,25]. Expression of certain genes enhanced biofilm formation by QAC resistant Listeria monocytogenes [26,27]. Several mutations result in selection of bacteria with reduced cell permeability. Resistant cells have modified cell membrane fatty acids, phospholipids, and outer membrane lipopolysaccharides [28], resulting in a more anionic and hydrophobic cell surface, thus restricting easy passage of the QACs through the cell surface. Other cell modifications in response to exposure to QACs are density reduction and composition change of the porins [29,30], as well as change of the outer membrane protein composition [31]. Efflux-mediated QAC resistance has received significant interest because it has a genetic origin, confers co-resistance to antibiotics and is transferable among species through horizontal gene transfer. Multidrug efflux pumps mediate the transfer of biocides from the inside to the outside of the cell through an energy or proton-dependent mechanism. Efflux determinants, which confer resistance to QACs, are given in Table 1. QAC resistance via efflux pumps follows two mechanisms. First, QAC resistance is induced by overexpression of efflux pumps upon exposure to QAC or as a result of QAC-induced stress. Such stress either triggers a regulatory system that controls the Current Opinion in Biotechnology 2015, 33:296–304

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Figure 1

Acquisition of efflux genes

Gene cassettes

Integron recombination tI

E cΔ

In

qa

lI

Su

HGT and recombination

ICE induction

Biodegradation

Enhanced biofilm formation

Transposition

RpoS induction

Cl – N+

G TT

Error-prone replication

Error-prone DNA polymerase sRNA

Inhibition of mismatch repair

A TT

Modification of cell membrane

Mutations

Sub-MIC QAC concentration

SOS response

Prophage induction

Over-expression of efflux Replicative DNA polymerase

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Pathways and mechanisms of QAC resistance (adapted from Ref. [12]).

expression of an efflux determinant or causes a mutation resulting in the overexpression of the efflux determinant or increase in its extrusion efficiency [32]. These efflux pumps are generally chromosomally encoded and act against a wide array of antimicrobial agents. Overexpression of these efflux pumps results in a two-fold to eightfold increased tolerance of the adapted bacteria to QACs and other substrates of these pumps [1,33–36]. For instance, the study by Mc Cay et al. [1] showed that

Pseudomonas aeruginosa, variants of which are pathogenic, grown in continuous culture at subinhibitory concentrations of BAC resulted in increased resistance to both BAC and ciprofloxacin; its antibiotic resistance was attributed to amino acid substitution (Val-51AAla) in nfxB, the Mex efflux system regulator gene. The second mechanism of QAC resistance is through acquisition of genes for specialized QAC efflux pumps,

Table 1 Efflux determinants conferring QAC resistance (adapted from Ref. [61]) Efflux family

Efflux proteins extruding QACs

Typical antibiotic substrates

Resistance nodulation division (RND)

YhiUV-TolC, AcrAB-TolC, MexAB-OprM, CmeABC, CmeDEF, SdeXY, OqxAB

Major facilitator superfamily (MFS)

QacA, QacB, NorA, NorB, MdeA, EmeA, MdfA

Multidrug and toxic compound extrusion (MATE)

MepA, NorM, PmpM

Aminoglycosides, b-lactams, Chloramphenicol, Erythromycin, Fluoroquinolones, Novobiocin, Rifampin, Tetracyclines, Trimethoprim Aminoglycosides, Chloramphenicol, Erythromycin, Fluoroquinolones, Lincosamides, Novobiocin, Rifampin, Tetracyclines Aminoglycosides, Fluoroquinolones

Small multidrug resistance (SMR)

QacE, QacED1, QacF, QacG, QacH, QacI, QacJ, smr, EmrE, SugE

Aminoglycosides, Chloramphenicol, Erythromycin, Tetracyclines

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which belong to the SMR family. Among them, EmrE, smr and SugE are multidrug efflux pumps [37], whereas QacE, QacDE, QacF, QacG, QacH, QacI, QacJ and QacZ are QAC-specific efflux determinants [38]. The genes of these efflux proteins are mainly found in mobile genetic elements such as transposons, ICEs, plasmids and integrons. As a result, they can be horizontally transferred between bacteria of the same or different genera. Such genes are abundant in the environment. Acquisition of QAC efflux genes by bacteria involves integration of genes to integrons and plasmids or recombination of efflux gene containing ICE and transposons into the recipient genome. Oxidative stress response plays a crucial role in both QAC resistance mechanisms. Integrons play a significant role in the acquisition and mobilization of QAC resistance genes [39]; they are found in many environmental bacterial species, particularly in those exposed to QACs and/or antibiotic residues [40,41]. QAC contamination is also responsible for the stabilization of integrons and their gene cassettes [42]. A direct link between SOS response and the expression of integron integrases was demonstrated. SOS regulation enhances cassette swapping and capture under stressful conditions, such as during QAC exposure [43]. Plasmids also play a significant role in harboring and disseminating genes related to resistance to QACs and other biocides. Plasmids of the incompatibility (Inc) group IncP-1, also called IncP, as extrachromosomal genetic elements can be transferred and replicated virtually in all Gram-negative bacteria. IncP-1group plasmids are broadly distributed in environments with sub-inhibitory QAC concentrations, such as soils and wastewater treatment plants. IncP plasmids commonly harbor QAC resistance genes along with many other resistance genes [44–47]. In addition, plasmid-associated QAC resistance genes were transferred between non-pathogenic and pathogenic bacteria exposed to QACs, a process that also leads to the co-selection of resistance to other contaminants [48]. Using the SOS-response, ICEs are transferred from one bacterial chromosome to another. ICEs containing multiple QAC resistance genes were identified in strains of Vibrio scophthalmi, V. splendidus, V. alginolyticus, Shewanella haliotis, and Enterovibrio nigricans isolated from fish grown in marine aquaculture systems [49]. Exposure to biocides below inhibitory concentrations also facilitates the conjugation of transposons [50]. Novel transposons carrying QAC resistance genes have been identified in both Gramnegative and positive bacteria [51,52].

QAC biodegradation: ecology, mechanism and genetics Exposure of a microbial community to QACs results in the development of resistance, as well as selection and www.sciencedirect.com

proliferation of resistant bacteria. A very recent study showed that when a microbial community, fed with an organic carbon source was exposed continuously to QACs, its diversity decreased substantially and was dominated by Proteobacteria, mainly composed of Pseudomonads. In addition, a versatile repertoire of antimicrobial resistance genes, such as efflux pumps (e.g., RND family and SMR family) and cell envelope modification systems (e.g., aminoarabinose lipid A modification) were amplified during a long-term exposure to BACs [53,54]. Aerobic biodegradation of QACs has been attributed mainly to bacterial species in the genera of Xanthomonas, Aeromonas, and Pseudomonas [3]. More recently, several bacteria have been identified that are capable of QAC degradation, such as Pseudomonas putida A ATCC 12633 [55], Pseudomonas nitroreducens B and DB [56], Pseudomonas sp. BIOMIG1 (Ertekin and Tezel, 114th General Meeting of American Society for Microbiology, Abstract no: Q352) and Stenotrophomonas sp. B27 (unpublished). In addition, non-culture-based techniques, such as cloning, metagenomics and pyrosequencing have been used to elucidate the composition of QAC degrading, enriched microbial communities. A microbial community originating from a river sediment, utilizing BACs as the sole carbon and energy source had a dramatically decreased phylogenetic diversity as compared to the control (without BAC exposure), and was dominated by Pseudomonas species (>50% of the total) [53,54,57]. Metagenomic analysis revealed that community adaptation to BACs occurred primarily via selective enrichment of BAC-degrading Pseudomonas species, particularly P. nitroreducens, and secondarily via amino acid substitutions and horizontal transfer of selected genes, including a gene encoding a PAS/PAC sensor protein and ring-hydroxylating dioxygenase genes [54,56]. In addition, multi-drug efflux pump genes such as sugE, PmpM, mexAB-oprM and mexEF-oprN were enriched in the BAC-degrading community [53]. Based on the phylogenetic tree prepared using the 16S rDNA sequences of QAC degrading isolates and predominant species in QAC degrading microbial communities, Pseudomonas spp., particularly the P. putida and P. aeruginosa group, are key species in QAC degradation (Figure 2a). In addition Stenetrophomonas spp. (g-Proteobacteria), and Achromobacter spp. (a-Proteobacteria) are frequently identified in communities degrading QACs, indicating that they play a role in the biodegradation of QACs [54]. Three aerobic QAC biotransformation pathways, which differ in the location of the initial reaction, have been observed (Figure 3): (a) hydroxylation of the terminal C of the alkyl chain (v-hydroxylation), followed by multiple b-oxidation cycles, progressing toward the hydrophilic moiety; (b) hydroxylation of the C adjacent to the central Current Opinion in Biotechnology 2015, 33:296–304

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Figure 2

(a)

(b) P. fluorescens [D84013] P. orientalis [AF064457] 47 P. aureofaciens [D84008] 67 100 P. chlororaphis [D84011] 100 P. caricapapayae [D84010] 84 P. syringae [D84026] P. sp. 7–6 [AB278070] 87 P. putida [D84020] 86 86 P. putida ATCC 12633 [AF094736] P. oryzihabitans [D84004] 48 P. putida strain B2 100 98 clone BAC5 [HE650699] P. sp BIOMIG1 91 39 P. nitroreducens B [KC415111] 50 P. nitroreducens BD [KC415112] 97 clone BAC54 [HE650698] 74 P. nitroreducens strain B1 98 P. nitroreducens [D84021] 100 79 P. citronellolis [AB021396] 74 P. aeruginosa [Z76651] P. balearica [U26418] 89 P. stutzeri [U26262] P. denitrificans [AB021419] 100 P. pertucinogena [AB021380] Stenotrophomonas sp. strain B27 52 100 BIOMIG OTU 7 93 Stenotrophomonas maltophilia [JN656280] BIOMIG OTU 1 100 Achromobacter sp. LMG 3458 [HG324053] E. coli K–12 [NR_102804]

Pseudomonas putida S16 AOx [YP_004703499]

100

100

0.06

100

Pseudomonas sp. HZN6 POAOx [AFD54463] 97

64

100

Pseudomonas nitroreducens strain B AOx [KJ911918]

Pseudomonas sp. HZN6 NAOx [AGH68979]

Arthrobacter sp. PAO19 AOx [WP_022875508] 90

Pseudomonas putida BIRD-1 AOx (E4RE15_PSEPB] 100

Pseudomonas putida BIRD-1 AOx (E4R7Q2_PSEPB]

Pseudomonas putida ATCC 12633 TTABMO [AFA52008] 0.6 Current Opinion in Biotechnology

Phylogenetic classification of: (a) QAC-degrading bacterial isolates (in red) and nucleotide sequences of species present in QAC-degrading communities identified in community clone libraries based on 16S rDNA or by pyrosequencing (in blue); (b) QAC dealkylating genes (in red).

N (a-hydroxylation), followed by central fission, resulting in the separation of the hydrophobic from the hydrophilic moiety; and (c) hydroxylation of the methyl-C attached to the central N, followed by fission of the methyl group [3]. In spite of the fact that QACs have a relatively high adsorption capacity resulting in their transfer to anoxic/anaerobic environments, such as anaerobic digesters and aquatic sediments, limited information exists relatively to the fate of QACs under such conditions. The transformation of BACs under anoxic, nitrate reducing conditions was recently reported to be initiated by means of an abiotic nitrite nucleophilic substitution reaction (modified Hofmann reaction) producing alkyl dimethyl amines (tertiary amines) [3,58]. Under anaerobic conditions, there is no evidence of mineralization of QACs that contain alkyl or benzyl groups [3]. In spite of the fact that the energetic burden of the abovediscussed QAC biotransformation pathways (initial reaction) is the same, pathway-b starting with the cleavage of the Calkyl–N bond is the most predominant. Although, pathway-a was the first identified QAC biotransformation pathway, it could not be demonstrated in later studies for Current Opinion in Biotechnology 2015, 33:296–304

similar QACs. Moreover, the product of pathway-b, a tertiary amine, is less toxic than the products of pathway-a and pathway-c [57]. The combination of these two observations suggests that pathway-b is naturally selected as a mechanism for QAC biotransformation to cope with the toxic effects of QACs and to eliminate the detrimental consequences of the other biotransformation pathways. As a result, QAC biotransformation may have evolved as a QAC-resistance mechanism. None of the isolates described above, except Pseudomonas sp. BIOMIG1, was able to grow on the non-alkyl containing amines, such as trimethyl amine and dimethyl amine, after dealkylation. Activation of the C–H bond in the QAC alkyl group was thought to commence with NADHdependent hydroxylation by a monooxygenase in the presence of oxygen [3]. Recently, two enzymes responsible for QAC dealkylation have been identified. Tetradecyl trimethyl ammonium bromide monooxygenase (TTABMO) identified in P. putida ATCC 12633 is a typical flavoprotein that utilizes NADPH and FAD as cofactor [55]. On the other hand, the enzyme responsible for dealkylating BACs by Pseudomonas nitroreducens was identified as a FAD-using amine oxidase (AOx-BAC) www.sciencedirect.com

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Figure 3

R

X– N+

X– N+

R

O2

R

n-3

NADH 2

O

n-3

NAD

NADH 2

+ 2 H 2O

+

HO

HO

O2

X– N+

OH n-5

NAD

(a) ω-hydroxylation of terminal C of alkyl group

R

X– N+

R

O2

n-3

NADH 2

X– N+

n-3

NAD

O2

OH

NADH 2

R

X– N H+

O

+

+ n-3

2 H 2O

OH

NAD

(b) α-hydroxylation of C adjacent to N of alkyl group

O2 R n-3

HO X–

N+

R NADH 2

NAD

X– N+

n-3

O2 R NADH 2

NAD

H + X– N + HO

O

+ 2 H 2O

n-3

(c) α-hydroxylation of C of methyl group Current Opinion in Biotechnology

Aerobic QAC biotransformation pathways.

[56]. AOx-BAC is phylogenetically more closely related to P. putida amine oxidase and Pseudomonas sp. pseudooxynicotine amine oxidase than TTABMO (Figure 2b), which suggests that there may be multiple enzymes responsible for the initial steps in QAC biotransformation. With the exception of certain dimeric gemini QACs, almost all QACs are biodegradable under aerobic conditions [59]. QAC degraders play an important role in mitigating QAC contamination in the environment. However, proliferation of QAC degraders and their transfer in indoor environments are not desired because it would decrease their antimicrobial efficacy, resulting in public health problems. Efforts should be devoted to develop technologies based on immobilized QAC degraders or their enzymes for the treatment of QAC-bearing wastewater. For instance, P. putida ATCC 12633 immobilized using alginate entrapment was able to remove 80% of QACs within 48 hours at a concentration range between 35 and 315 mg/L [60]. To this end, QAC degraders have to be physiologically and metabolically well characterized and factors affecting QAC biodegradation identified. In case QAC detoxifying enzymes are used, strategies to enhance enzyme functionality by optimizing cofactor requirements using directed enzyme evolution approaches will need to be evaluated.

Conclusion and outlook QACs are effective disinfectants that have contributed to maintaining acceptable health standards by ensuring a www.sciencedirect.com

significantly reduced microbial load, and thus the likelihood that pathogenic bacteria are also reduced, if not eliminated. Because QACs are extensively used in domestic, agricultural, industrial and clinical applications, humans and microorganisms are in constant contact with them. Misuse, environmental dilution and biodegradation of QACs often create environments with low QAC concentrations. At sub-inhibitory QAC concentrations, QAC resistance emerges mainly as a bacterial response to QAC-induced oxidative stress, often causing mutations or facilitating gene transfer, ultimately leading to evolution and selection of QAC-resistant bacteria. Wastewater, wastewater treatment plants, wastewater sludge, sludge applied soil, surface waters, and aquatic sediments are environments where QAC resistance evolves and proliferates. QAC resistant bacteria may be transferred from outdoor environments to indoors such as homes and healthcare facilities. QAC resistance genes, especially efflux genes, are frequently found in bacterial isolates obtained from human/animal healthcare facilities. These genes either confer resistance to multiple biocides and antibiotics (co-resistance) or mobile genetic elements that they are attached to carry resistance genes to other biocides and antibiotics. Therefore, QAC resistant bacteria, if they are pathogenic, may pose a serious threat to human health. In addition, QAC resistant bacteria may decrease the efficacy of QAC disinfectants by creating microenvironments habitable by QAC susceptible bacteria, as for example in the case of biofilms. Moreover, Current Opinion in Biotechnology 2015, 33:296–304

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QAC-degrading bacteria create QAC gradients in which susceptible species survive or even develop QAC resistance and proliferate. The connection between QAC degradation and resistance is not well understood. Is QAC degradation a blessing or a curse? Given that QACs degrade, at least under aerobic conditions, QACs could be considered as relatively low risk disinfectants. However, QAC biodegradation creates sub-inhibitory concentrations where otherwise susceptible species may develop QAC resistance through various pathways and mechanisms as described above. Many species are phylogenetically closely related to QAC-degraders. Upon chronic exposure to QACs, these species could become resistant, but unfortunately some of them are pathogenic (e.g., P. aeruginosa) [1,30]. If efficient QAC degraders are used in treatment technologies and then integrated into wastewater treatment plants, release of QACs into the environment may be avoided or at least minimized. As a result, QAC resistance and QAC-induced antibiotic resistance might be reduced. In conclusion, QACs are widely used in many disinfectant formulations and will continue to be used in the future. The underlying mechanisms and conditions that result in QAC resistance and biodegradation, especially in the environment, must be better understood for the prudent use of existing QAC formulations, as well as the development of safer disinfectants. Development and implementation of (bio)technologies for the elimination of QACs from treated wastewater effluents, along with many emerging microcontaminants, will greatly reduce adverse impacts to both humans and the environment.

Acknowledgements U. Tezel acknowledges the EU Research Executive Agency and the Scientific and Technological Research Council of Turkey (TUBITAK) for funding QAC related research through project no. PCIG09-GA-2011-293665 and 113Y528, respectively. S.G. Pavlostathis was supported for research related to this work by the U.S. National Science Foundation under Grant CBET 0967130.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 

Mc Cay PH, Ocampo-Sosa AA, Fleming GTA: Effect of subinhibitory concentrations of benzalkonium chloride on the competitiveness of Pseudomonas aeruginosa grown in continuous culture. Microbiol-SGM 2010, 156:30-38. This study demonstrated increased resistance to both BAC and antibiotics of a potentially pathogenic bacterial species grown in continuous culture at subinhibitory BAC concentrations; resistance was attributed to genetic modifications.

2.

3.

Oh SD: Metagenomic and metatranscriptomic investigation of microorganisms exposed to benzalkonium chloride disinfectants. Atlanta, GA, USA: Georgia Institute of Technology; 2010:. PhD Thesis. Tezel U, Pavlostathis SG: Role of quaternary ammonium compounds on antimicrobial resistance in the environment. In

Current Opinion in Biotechnology 2015, 33:296–304

Antimicrobial Resistance in the Environment. Edited by Keen PL, Montforts MHMM. Wiley-Blackwell; 2012:349-387. 4.

Li XL, Brownawell BJ: Quaternary ammonium compounds in urban estuarine sediment environments — a class of contaminants in need of increased attention? Environ Sci Technol 2010, 44:7561-7568.

5.

Ruan T, Song S, Wang T, Liu R, Lin Y, Jiang G: Identification and composition of emerging quaternary ammonium compounds in municipal sewage sludge in China. Environ Sci Technol 2014, 48:4289-4297.

6.

Gerba CP: Quaternary ammonium biocides: efficacy in application. Appl Environ Microbiol 2015, 81:464-469.

7.

Gillings MR: Integrons: past, present, and future. Microbiol Mol Biol Rev 2014, 78:257-277.

8.

Gillings MR, Gaze WH, Pruden A, Smalla K, Tiedje JM, Zhu Y-G: Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution. ISME J 2014:1-11.

9.

Forsberg KJ, Reyes A, Bin W, Selleck EM, Sommer MOA, Dantas G: The shared antibiotic resistome of soil bacteria and human pathogens. Science 2012, 337:1107-1111.

10. Hegstad K, Langsrud S, Lunestad BT, Scheie AA, Sunde M, Yazdankhah SP: Does the wide use of quaternary ammonium compounds enhance the selection and spread of antimicrobial resistance and thus threaten our health? Microb Drug Resist 2010, 16:91-104. 11. Buffet-Bataillon S, Tattevin P, Bonnaure-Mallet M, JolivetGougeon A: Emergence of resistance to antibacterial agents: the role of quaternary ammonium compounds — a critical review. Int J Antimicrob Agents 2012, 39:381-389. 12. Andersson DI, Hughes D: Microbiological effects of sublethal  levels of antibiotics. Nat Rev Microbiol 2014, 12:465-478. A comprehensive review on pathways and mechanisms of antibiotic resistance at sub-MIC. Review defines sub-MIC environments and how resistant microorganisms evolve and proliferate in such environments. Biochemistry of mutations and gene transfer upon antibiotic exposure is discussed. 13. Wessels S, Ingmer H: Modes of action of three disinfectant active substances: a review. Regul Toxicol Pharm 2013, 67:456467. 14. Ferreira C, Pereira AM, Pereira MC, Melo LF, Simoes M: Physiological changes induced by the quaternary ammonium compound benzyldimethyldodecylammonium chloride on Pseudomonas fluorescens. J Antimicrob Chemother 2011, 66:1036-1043. 15. Morente EO, Fernandez-Fuentes MA, Burgos MJG, Abriouel H, Pulido RP, Galvez A: Biocide tolerance in bacteria. Int J Food Microbiol 2013, 162:13-25. 16. Blazquez J, Couce A, Rodriguez-Beltran J, Rodriguez-Rojas A: Antimicrobials as promoters of genetic variation. Curr Opin Microbiol 2012, 15:561-569. 17. Ceragioli M, Mols M, Moezelaar R, Ghelardi E, Senesi S, Abee T: Comparative transcriptomic and phenotypic analysis of the responses of Bacillus cereus to various disinfectant treatments. Appl Environ Microbiol 2010, 76:3352-3360. 18. Andersson DI, Hughes D: Evolution of antibiotic resistance at non-lethal drug concentrations. Drug Resist Update 2012, 15:162-172. 19. Nakata K, Tsuchido T, Matsumura Y: Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in Escherichia coli cells. J Appl Microbiol 2011, 110:568-579. 20. Hughes D, Andersson DI: Selection of resistance at lethal and non-lethal antibiotic concentrations. Curr Opin Microbiol 2012, 15:555-560. 21. Moen B, Rudi K, Bore E, Langsrud S: Subminimal inhibitory concentrations of the disinfectant benzalkonium chloride select for a tolerant subpopulation of Escherichia coli with inheritable characteristics. Int J Mol Sci 2012, 13:4101-4123. www.sciencedirect.com

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22. Pagedar A, Singh J, Batish VK: Adaptation to benzalkonium chloride and ciprofloxacin affects biofilm formation potential, efflux pump and haemolysin activity of Escherichia coli of dairy origin. J Dairy Res 2012, 79:383-389.

38. Braga TM, Marujo PE, Pomba C, Lopes MFS: Involvement, and dissemination, of the enterococcal small multidrug resistance transporter QacZ in resistance to quaternary ammonium compounds. J Antimicrob Chemother 2011, 66:283-286.

23. Nakamura H, Takakura KI, Sone Y, Itano Y, Nishikawa Y: Biofilm formation and resistance to benzalkonium chloride in Listeria monocytogenes isolated from a fish processing plant. J Food Protect 2013, 76:1179-1186.

39. Cambray G, Guerout AM, Mazel D: Integrons. Ann Rev Gen 2010, 44:141-166.

24. Giaouris E, Chorianopoulos N, Doulgeraki A, Nychas GJ: Co culture with Listeria monocytogenes within a dual-species biofilm community strongly increases resistance of Pseudomonas putida to benzalkonium chloride. PLoS One 2013, 8:e77276. This study tested the durability of biofilm composed of dual species on benzalkonium chloride (BAC) disinfection and resistance of each biofilm species to BAC after biofilm was continuously exposed to BACs. Biofilms were developed on stainless steel coupons, exposed to BACs and species isolated from biofilm were tested for BAC MICs. 25. van der Veen S, Abee T: Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid. Int J Food Microbiol 2011, 144:421-431. 26. van der Veen S, Abee T: HrcA and DnaK are important for static and continuous-flow biofilm formation and disinfectant resistance in Listeria monocytogenes. Microbiology-SGM 2010, 156:3782-3790. 27. van der Veen S, Abee T: Importance of SigB for Listeria monocytogenes static and continuous-flow biofilm formation and disinfectant resistance. Appl Environ Microbiol 2010, 76:7854-7860. 28. Fox EM, Leonard N, Jordan K: Physiological and transcriptional characterization of persistent and nonpersistent Listeria monocytogenes isolates. Appl Environ Microbiol 2011, 77:65596569. 29. Machado I, Coquet L, Jouenne T, Pereira MO: Proteomic  approach to Pseudomonas aeruginosa adaptive resistance to benzalkonium chloride. J Proteomics 2013, 89:273-279. Outer membrane proteins of Pseudomonas aeruginosa adapted to 2 mM BAC by stepwise exposure to lower concentrations were extracted and analyzed with 2D gel electrophoresis. Proteins with different abundance in adapted cells compared to wild-type cells were identified using mass spectroscopy. 30. Tabata A, Nagamune H, Maeda T, Murakami K, Miyake Y, Kourai H: Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR. Antimicrob Agents Chemother 2003, 47:2093-2099. 31. Mavri A, Mozina SS: Development of antimicrobial resistance in Campylobacter jejuni and Campylobacter coli adapted to biocides. Int J Food Microbiol 2013, 160:304-312. 32. Grkovic S, Brown MH, Skurray RA: Regulation of bacterial drug export systems. Microbiol Mol Biol Rev 2002, 66:671-701. 33. Guo W, Cui SH, Xu X, Wang HY: Resistant mechanism study of benzalkonium chloride selected Salmonella typhimurium mutants. Microb Drug Resist 2014, 20:11-16. 34. Buffet-Bataillon S, Le Jeune A, Le Gall-David S, BonnaureMallet M, Jolivet-Gougeon A: Molecular mechanisms of higher MICs of antibiotics and quaternary ammonium compounds for Escherichia coli isolated from bacteraemia. J Antimicrob Chemother 2012, 67:2837-2842. 35. Holdsworth SR, Law CJ: The major facilitator superfamily transporter MdtM contributes to the intrinsic resistance of Escherichia coli to quaternary ammonium compounds. J Antimicrob Chemother 2013, 68:831-839.

40. Gaze WH, Zhang L, Abdouslam NA, Hawkey PM, Calvo-Bado L, Royle J, Brown H, Davis S, Kay P, Boxall AB, Wellington EM: Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment. ISME J 2011, 5:1253-1261. 41. Gaze WH, Abdouslam N, Hawkey PM, Wellington EMH: Incidence of class 1 integrons in a quaternary ammonium compoundpolluted environment. Antimicrob Agents Chemother 2005, 49:1802-1807. 42. Gillings MR, Xuejun D, Hardwick SA, Holley MP, Stokes HW: Gene cassettes encoding resistance to quaternary ammonium compounds: a role in the origin of clinical class 1 integrons? ISME J 2009, 3:209-215. 43. Cambray G, Sanchez-Alberola N, Campoy S, Guerin E, Da Re S, Gonzalez-Zorn B, Ploy MC, Barbe J, Mazel D, Erill I: Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons. Mob DNA 2011, 2:6. 44. Popowska M, Krawczyk-Balska A: Broad-host-range IncP-1 plasmids and their resistance potential. Front Microbiol 2013, 4:44. 45. Dutta V, Elhanafi D, Kathariou S: Conservation and distribution of the benzalkonium chloride resistance cassette BcrABC in Listeria monocytogenes. Appl Environ Microbiol 2013, 79:60676074. 46. Elhanafi D, Dutta V, Kathariou S: Genetic characterization of plasmid-associated benzalkonium chloride resistance determinants in a Listeria monocytogenes strain from the 1998–1999 outbreak. Appl Environ Microbiol 2010, 76:8231-8238. 47. Marti E, Balcazar JL: Multidrug resistance-encoding plasmid from Aeromonas sp. strain P2G1. Clin Microbiol Infect 2012, 18:E366-E368. 48. Katharios-Lanwermeyer S, Rakic-Martinez M, Elhanafi D, Ratani S, Tiedje JM, Kathariou S: Coselection of cadmium and benzalkonium chloride resistance in conjugative transfers from nonpathogenic Listeria spp. to other Listeriae. Appl Environ Microbiol 2012, 78:7549-7556. 49. Rodriguez-Blanco A, Lemos ML, Osorio CR: Integrating conjugative elements as vectors of antibiotic, mercury, and quaternary ammonium compound resistance in marine aquaculture environments. Antimicrob Agents Chemother 2012, 56:2619-2626. 50. Seier-Petersen MA, Jasni A, Aarestrup FM, Vigre H, Mullany P, Roberts AP, Agers Y: Effect of subinhibitory concentrations of four commonly used biocides on the conjugative transfer of Tn916 in Bacillus subtilis. J Antimicrob Chemother 2014, 69:343348. 51. Muller A, Rychli K, Muhterem-Uyar M, Zaiser A, Stessl B,  Guinane CM, Cotter PD, Wagner M, Schmitz-Esser S: Tn6188 — a novel transposon in Listeria monocytogenes responsible for tolerance to benzalkonium chloride. PLoS One 2013, 8:e76835. A novel transposon containing qacH gene was identified in Listeria monocytogenes chromosome using shotgun sequencing. MIC assays performed with strains having and not having transposon and qacH gene as well as qacH expression assays showed that transposon associated qacH gene confers resistance to BACs in Listeria monocytogenes.

36. Morita Y, Tomida J, Kawamura Y: Responses of Pseudomonas aeruginosa to antimicrobials. Front Microbiol 2014, 4:422.

52. Ciric L, Mullany P, Roberts AP: Antibiotic and antiseptic resistance genes are linked on a novel mobile genetic element: Tn6087. J Antimicrob Chemother 2011, 66:2235-2239.

37. He GX, Zhang C, Crow RR, Thorpe C, Chen HZ, Kumar S, Tsuchiya T, Varela MF: SugE, a new member of the SMR family of transporters, contributes to antimicrobial resistance in Enterobacter cloacae. Antimicrob Agents Chemother 2011, 55:3954-3957.

53. Tandukar M, Oh S, Tezel U, Konstantinidis KT, Pavlostathis SG: Long-term exposure to benzalkonium chloride disinfectants results in change of microbial community structure and increased antimicrobial resistance. Environ Sci Technol 2013, 47:9730-9738.

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54. Oh S, Tandukar M, Pavlostathis SG, Chain PS, Konstantinidis KT: Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics. Environ Microbiol 2013, 15:2850-2864. 55. Liffourrena AS, Lucchesi GI: Identification, cloning and  biochemical characterization of Pseudomonas putida A (ATCC 12633) monooxygenase enzyme necessary for the metabolism of tetradecyltrimethylammonium bromide. Appl Biochem Biotechnol 2014, 173:552-561. The gene responsible for dealkylation of tetradecyltrimethylammonium bromide was identified and its product, a flavoprotein, was purified from Pseudomonas putida for the first time. 56. Oh S, Kurt Z, Tsementzi D, Weigand MR, Kim M, Hatt JK, Tandukar M,  Pavlostathis SG, Spain JC, Konstantinidis KT: Microbial community degradation of widely used quaternary ammonium disinfectants. Appl Environ Microbiol 2014, 80:5892-5900. Metatranscriptomic profile of a BAC-degrading microbial community was obtained during BAC degradation. Candidate genes responsible for BAC degradation were determined and cloned in E. coli. A novel amine oxidase gene responsible for transformation of BAC to benzyl dimethyl amine was identified.

Current Opinion in Biotechnology 2015, 33:296–304

57. Tezel U, Tandukar M, Martinez RJ, Sobecky PA, Pavlostathis SG: Aerobic biotransformation of ntetradecylbenzyldimethylammonium chloride by an enriched Pseudomonas spp. community. Environ Sci Technol 2012, 46:8714-8722. 58. Tezel U, Pavlostathis SG: Transformation of benzalkonium chloride under nitrate reducing conditions. Environ Sci Technol 2009, 43:1342-1348. 59. Brycki B, Waligorska M, Szulc A: The biodegradation of monomeric and dimeric alkylammonium surfactants. J Hazard Mater 2014, 280:797-815. 60. Bergero MF, Lucchesi GI: Immobilization of Pseudomonas putida A (ATCC 12633) cells: a promising tool for effective degradation of quaternary ammonium compounds in industrial effluents. Int Biodeter Biodegrad 2015, 100:38-43. 61. Poole K: Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 2005, 56:20-51.

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