Bacterial Resistance to Biocides

Jean-Yves Maillard

Chemical biocides are heavily used as preservatives, disinfectants, or antiseptics¹ in an increasing number of industrial, health care, and domiciliary applications.² Biocides have a long usage history for preventing or controlling infection or controlling spoilage of water and foodstuff. Today, it is impossible to put a number to the quantities of chemical biocides that are being used on daily basis worldwide. It is, however, clear that their usage has dramatically increased during the last 15 years mainly for health care applications and in consumer products. Such increase in usage is causing concerns with issues such as environmental toxicity and emerging resistance in bacteria.² The number of active substances (ie, biocides) that can be used for different applications in Europe has been dramatically reduced.³,⁴ The list of authorized biocides on the European market is currently being reviewed and amended, forcing manufacturers to change the composition of established formulations.³,⁴ The revised European Biocidal Product Regulation⁴ mentioned the need to demonstrate that a product will not induce bacterial resistance. The US Food and Drug Administration⁵ recently enforced a rule that restricts the use of certain biocides, such as triclosan and triclocarban that have been linked to antimicrobial resistance (AMR) in bacteria, in antimicrobial soaps and similar products.

It is appropriate to question whether the widespread use of biocidal products today is justified. The increase usage of biocides not only in consumer products but also in health care may be due, at least in part, not only to a better education and awareness of consumers about microbial infections and contamination⁶ but also to market pressures and opportunities. Important media coverage of poor hygiene and cleanliness in health care facilities also contributed to the demand for better, often biocide-based, solutions. A recent contributor for an increase usage of biocidal products has been the rise in AMR (specifically chemotherapeutic antibiotic) in bacteria. The AMR is a global issue with serious economical and societal consequences.⁷ Recommendations to tackle AMR include better hygiene and control of bacterial pathogens from surfaces in health care and veterinary settings.⁷ Here, the conundrum is that although the use of biocidal products is important to control microbial pathogens, inappropriate applications might lead to an exacerbation of AMR.

One difficult issue about AMR is the definition of what resistance means particularly when biocides are concerned. The definition is usually linked to protocols that measure a change in susceptibility profile.⁸,⁹,¹⁰,¹¹,¹²,¹³ It is unfortunate that in many publications, the term resistance is only linked to an increase in the biocide minimum inhibitory concentration (MIC); MICs are often lower than the in-use concentration of a biocide or product, and the in-use concentration may still be effective.⁸,¹⁴ Where MIC and minimum bactericidal concentration (MBC) may become more relevant is when the during-use concentration is considered. The during-use concentration is the lowest concentration of a biocide or product attained following product application; it encompasses, for example, residual concentration and dilution during use.⁸,¹⁵,¹⁶ The use of the term reduced susceptibility when MIC or MBC are measured is more appropriate and is often expressed as a fold reduction in MIC or MBC. The practical significance of a reduced susceptibility remains, however, to be defined. Generally, the scientific community and regulators have no consensus on the definition of resistance,²,⁸ owing the diversity of terms used, including resistance, tolerance, decreased susceptibility, reduced susceptibility, insusceptibility, and acquired reduced susceptibility. Today, with the diversity of products containing biocide(s) at various concentrations, a bacterium surviving in a product should be defined as resistant whatever the concentration of biocide is in that product.

This book chapter is exploring not only the evidence of bacterial resistance to biocides and products and the mechanisms of bacterial resistance but also the interaction
between biocides and bacteria that would lead to the expression of global resistance mechanisms, putting in perspective the during-use concentration of a product and its effect.

EVIDENCE OF BACTERIAL SURVIVAL IN BIOCIDAL PRODUCTS AND BIOCIDES

There are plenty of examples in the literature of bacteria-contaminating products containing a biocide that lead to an outbreak or pseudo-outbreak (Table 4.1). Bacteria-contaminating biocidal products have been described as early as 1958, with some references mentioning incident dating from the 1940s, and product contamination are still being reported today (see Table 4.1). Bacterial resistance to biocides is therefore not a new phenomenon.²,⁸,⁹ Bacterial survival in a biocidal product can be driven by

Primarily a low concentration attained during product preparation and use (including phase separation for emulsion where the antimicrobial is retained in the oil phase)
The use of contaminated diluent during product preparation
Improper storage of biocidal products
The use of contaminated dispensers holding biocidal products
The use of contaminated disinfectant cloth to disinfect a sterile product

The nature of the biocides plays a role, and most examples provided in Table 4.1 concerned cationic biocides, mainly chlorhexidine and benzalkonium chloride solutions. If the nature of the biocide plays a role and less reactive membrane active agents may be a concern, bacterial resistance to highly reacting alkylating and oxidizing agents can also occur.²,⁶ For example, failure of high-level disinfection during endoscope reprocessing has led to the isolation of resistant bacteria,⁷⁸,⁷⁹,⁸⁰,⁸¹,⁸²,⁸³ some of which have been suspected to be associated with outbreaks and pseudooutbreaks.⁶⁷,⁸⁴,⁸⁵,⁸⁶ The nature of the bacterial contaminant is also important to consider. Bacterial contaminants of biocidal products are predominantly gram-negative bacteria, particularly pseudomonads or Burkholderia species (see Table 4.1), reflecting the versatility of these bacterial genera.⁸⁷ For povidone-iodine, bacterial contaminants such as Burkholderia cepacia and pseudomonads are often considered intrinsically resistant to certain concentrations.⁷¹,⁷²,⁷³,⁷⁴,⁷⁶ Likewise, contaminations of alcohol solutions with bacterial endospores of Bacillus cereus¹⁸,¹⁹ reflect the innate resistance nature of the spores.

Biocides are overall very diverse² and often used in combination in biocidal products. Component of formulations that provide other functions, such as chelation and wettability, and pH may contribute to the overall antimicrobial activity of the product. Yet the efficacy of the full formulations is rarely investigated.⁶,⁸,⁸⁸,⁸⁹ The use of biocidal products in the community or in health care settings on emerging bacterial resistance has not been widely studied, but the few published studies provide some interesting information. One of the main issues of such studies is the definition of bacterial resistance and by what measures it is defined. Thus, the comparison of the outcomes of these studies is difficult.² Investigations from Cole et al⁹⁰ failed to show any cross-resistance between the use of biocides and resistance to chemotherapeutic antibiotics. Likewise, two in situ studies did not observe any cross-resistance between triclosan or triclocarban used in over-the-counter antibacterial liquid hand and body cleansers and antibacterial bar soaps,⁹¹ or triclosan used in consumer products,⁹² and antibiotics. However, Carson et al⁹³ identified a correlation between elevated MIC to quaternary ammonium compounds (QACs) used in home consumer products and bacterial resistance to antibiotics.

The bulk of information regarding bacterial resistance to biocides remains from studies in vitro, which often lack consistency in their approach.²,⁸ Overall, in vitro investigations looking at a change in bacterial susceptibility when exposed to a biocide can be divided into three categories:

Bacterial exposure to increasing concentration of a biocide in defined conditions of time, growth media, and temperature. Such protocols, often referred to as stepwise training of bacteria to biocide exposure, do not necessarily reflect conditions of exposure found in practice.⁹⁴,⁹⁵,⁹⁶,⁹⁷,⁹⁸,⁹⁹,¹⁰⁰,¹⁰¹,¹⁰²,¹⁰³,¹⁰⁴,¹⁰⁵
The use of environmental isolates from health care, biocide manufacturing sites, slaughterhouses, etc, where biocidal products are commonly used. Bacterial isolates often show a reduced biocide susceptibility profile or resistance to specific biocides.⁷⁸,⁸⁰,⁸⁴,⁹⁶,¹⁰⁶,¹⁰⁷,¹⁰⁸,¹⁰⁹,¹¹⁰,¹¹¹,¹¹²,¹¹³,¹¹⁴,¹¹⁵,¹¹⁶
Studies investigating biocidal product contamination that lead to an infectious outbreaks or pseudooutbreaks (see Table 4.1)

The use of in vitro studies to understand bacterial behavior and survival when exposed to a specific biocide may suffer from unrealistic exposure such as excessive low concentrations of a biocide, unrealistic exposure to increasing concentrations, long contact time, absence of organic load, etc.⁶,⁹⁸ Biocides, however, are extensively used in a wide range of products for very diverse applications where inappropriate dilution (see Table 4.1) or exposure to a low concentration during use may occur.² Furthermore, some of these in vitro studies, such as stepwise training, contributed to a better knowledge of bacterial adaptation, albeit slow, to biocides, mainly through the expression of specific mechanisms such as changes in membrane permeability or increasing efflux capability.⁹⁴,⁹⁵,⁹⁶,⁹⁷,⁹⁸,⁹⁹,¹⁰⁰,¹¹⁷

TABLE 4.1 Outbreaks and pseudo-outbreaks due to contaminated biocidal product^a

Biocide	Contaminant(s)	Site(s) of Microbes	Mechanism of Contamination/Source	Year (Reference)
Alcohols
Alcohols	Bacillus cereus	Automated radiometric blood culture system	Intrinsic contamination (spores)	1983 (Berger¹⁷)
	B cereus	Blood (pseudobacteremia), pleural fluid	Intrinsic contamination (spores)	1999 (Hsueh et al¹⁸)
	Burkholderia cepacia	Blood (catheter related)	Contaminated tap water used to dilute alcohol for skin antisepsis	2004 (Nasser et al¹⁹)
Cationic biocides
Chlorhexidine	Pseudomonas species	Not stated	Refilling contaminated bottles, washing used bottles using cold tap water, contaminated washing apparatus; low concentration (0.05%)	1967 (Burdon and Whitby²⁰)
	B cepacia	Blood, urinary, wounds	Not determined	1971 (Speller et al²¹)
	Flavobacterium meningosepticum	Blood, CSF, wounds, skin	Not determined but possibly due to contaminated water and/or topping off of stock solution or low concentration (1:1000-1:5000)	1976 (Coyle-Gilchrist et al²²)
Chlorhexidine	Pseudomonas species, Serratia marcescens, Flavobacterium species	Not stated	Not determined, but authors speculate due to overdilution or refilling of contaminated bottles	1981 (Marrie and Costerton²³)
	Pseudomonas aeruginosa	Wounds	Tap water used to dilute stock solutions; low concentration (0.05%)	1982 (Anyiwo et al²⁴)
	B cepacia	Blood, wounds, urine, mouth, vagina	Metal pipe and rubber tubing in pharmacy through which deionized water passed during dilution of chlorhexidine; low concentration	1982 (Sobel et al²⁵)
	Ralstonia pickettii	Blood	Contaminated bidistilled water used to dilute chlorhexidine; low concentration (0.05%)	1983 (Kahan et al²⁶)
	R pickettii	Blood (pseudobacteremia)	Distilled water used to dilute chlorhexidine; low concentration (0.05%)	1985 (Verschraegen et al²⁷)
	R pickettii	Blood	Contaminated deionized water; low concentration (0.05%)	1987 (Poty et al²⁸)
	Achromobacter xylosoxidans	Blood, wounds	Atomizer (low concentration, 600 mg/L)	1998 (Vu-Thien et al²⁹)
	S marcescens	Blood, urine, wounds, sputum, others	Not determined, but use of non-sterile water for dilution to 2% and distribution in reusable nonsterile containers	1998 (Vigeant et al³⁰)
	R pickettii	Blood (pseudobacteremia)	Distilled water used to dilute chlorhexidine; low concentration (0.05%)	2000 (Maroye et al³¹)
Chlorhexidine	A xylosoxidans	Blood	Atomizer contamination	2005 (Tena et al³²)
	Burkholderia cenocepacia	Various patient specimen, blood, sputum, drainage, catheter	Chlorhexidine solution diluted with contaminated water	2013 (Lee et al³³)
	B cepacia	Blood	Intrinsic contamination, contaminated 0.5% chlorhexidine	2015 (Ko et al³⁴)
	S marcescens	Blood	Intrinsic contamination, 2% aqueous chlorhexidine antiseptic	2017 (de Frutos et al³⁵)
Chlorhexidine plus cetrimide	Pseudomonas multivorans	Wounds	Tap water used to prepare stock solutions; low concentrations (0.05% chlorhexidine and 0.5% cetrimide)	1970 (Bassett et al³⁶)
Chlorhexidine plus cetrimide	Stenotrophomonas maltophilia	Urine, umbilical swabs, catheter tips, others	Deionized water used to prepare solutions; failure to disinfect contaminated bottles between use	1976 (Wishart and Riley³⁷)
Hexidine	S marcescens	Blood	Application of a contaminated solution to disinfect tunnelled catheters	2016 (Merino et al³⁸)
Benzalkonium chloride	Pseudomonas species	Blood	Storage of benzalkonium chloride (0.1%) with cotton/gauze	1958 (Plotkin and Austrian³⁹)
Benzalkonium chloride	P aeruginosa	Blood	Diluted solution of benzalkonium chloride	1959 (Shickman et al⁴⁰)
Benzalkonium chloride	Enterobacter aerogenes	Blood, sinus tract	Storage of benzalkonium chloride (0.13%) with cotton/gauze	1960 (Malizia et al⁴¹)
	Pseudomonas-Achromobacteriaceae group	Blood, urine	Storage of benzalkonium chloride (0.1%) with cotton/gauze; dilution with nonsterile water	1961 (Lee and Fialkow⁴²)
	E aerogenes	Blood, sinus tract	Storage of benzalkonium chloride (0.1%) with cotton/gauze; dilution with nonsterile water	1961 (Lee and Fialkow⁴²)
	Pseudomonas kingii	Urine	Contamination (intrinsic) of antiseptic	1969 (Centers for Disease Control and Prevention⁴³)
	Pseudomonas EO-1	Urine	Contaminated (intrinsic) cleansing-germicide solution	1970 (Hardy et al⁴⁴)
	B cepacia, Enterobacter species	Blood (pseudobacteremia)	Storage of benzalkonium chloride with cotton/gauze, improper dilution, storage bottles infrequently sterilized	1976 (Kaslow et al⁴⁵)
	B cepacia	Bacteremia	Storage of benzalkonium chloride with rayon balls; failure to disinfect squeeze bottles	1976 (Frank and Schaffner⁴⁶)
	S marcescens	Intravenous catheters (dogs and cats), other sites	Storage of benzalkonium chloride (0.025%) with cotton/gauze	1981 (Fox et al⁴⁷)
	S marcescens	CSF	Contamination (extrinsic) of stock bottle	1984 (Sautter et al⁴⁸)
Benzalkonium chloride	S marcescens	Joint	Storage of benzalkonium chloride with cotton/gauze	1987 (Nakashima et al⁴⁹)
	S marcescens	Not specified	Multiple-dose medication vials contaminated with benzalkonium chloride-soaked cotton ball during disinfection	1987 (Nakashima et al⁵⁰)
	Mycobacterium chelonae	Skin abscesses	Storage of benzalkonium chloride with cotton/gauze; improper dilution	1990 (Georgia Division of Public Health⁵¹)
	P aeruginosa	Corticosteroid injection multidose vial	Inoculation with pseudomonads via needle puncture after vial septa were wiped with contaminated disinfectant	1999 (Olson et al⁵²)
	Mycobacterium abscessus	Joint	Storage of benzalkonium chloride with cotton/gauze; dilution with probable contaminated tap water	2003 (Tiwari et al⁵³)
	B cepacia	Blood, catheter	1:1000 aqueous benzalkonium chloride solution	2008 (Lee et al⁵⁴)
Benzalkonium chloride/picloxydine	B cepacia	Blood, urine, wound, sputum	Water used to dilute the antiseptic	1976 (Guinness and Levey⁵⁵)
Benzalkonium chloride/picloxydine	B cepacia	Blood	Water used to dilute the antiseptic	1976 (Morris et al⁵⁶)
Benzethonium chloride	Pseudomonas species	Blood (pseudobacteremia)	Contaminated (intrinsic solution; 0.2%)	1976 (Dixon et al⁵⁷)
Didecyldimethylammonium chloride	Pseudomonas fluorescens, A xylosoxidans	Blood	Contaminated dispenser—product used to decontaminate blood culture bottles	2007 (Siebor et al⁵⁸)
Didecyl diammonium chloride	Achromobacter species	Not specified	Hospital filtered tap water contaminating disinfectant atomizers and patients’ rooms	2015 (Hugon et al⁵⁹)
QAC (not defined)	S marcescens	Not specified	Failure of hospital personnel to clean the disinfectant spray bottles before refilling	1980 (Ehrenkranz et al⁶⁰)
QAC (not defined)	A xylosoxidans	Blood, CSF, respiratory therapy devices	Detergent-disinfectant solution as the source of contamination	2002 (Lehours et al⁶¹)
QAC (not defined)	B cepacia	Blood	Use of a contaminated disinfectant during quality controls in a university blood bank—QAC had been used in order to disinfect the rubber stopper of the blood culture bottle	2005 (Ebner et al⁶²)
Alkyldiaminoethylglycine hydrochloride solution	B cepacia, P fluorescens, Alcaligenes xylosoxidans, P aeruginosa, Pseudomonas putida	Not specified	Unwoven rayon cloths	2012 (Oie et al⁶³)
Glucoprotamin (surfactant)	A xylosoxidans	Blood	Suspected antiseptic reusable tissue dispensers	2016 (Günther et al⁶⁴)
Alkylating biocides
Formaldehyde	P aeruginosa	Blood	Reused formaldehyde solution (low concentration present 0.0014% and 0.005%)	1992 (Vanholder et al⁶⁵)
Formaldehyde (with glyoxal and glutaral)	Klebsiella oxytoca	Blood	Intrinsic contamination to 0.25% formaldehyde	2000 (Reiss et al⁶⁶)
Glutaraldehyde	M chelonae	Automated endoscope washer disinfector	Biofilm formation	2001 (Kressel and Kidd⁶⁷)
Glutaraldehyde	Methylobacterium mesophilicum	Automated endoscope washer disinfector	Biofilm formation	2001 (Kressel and Kidd⁶⁷)
Phenolics
Chloroxylenol	S marcescens	Multiple sites	Contaminated (extrinsic) 1% chloroxylenol soap; sink	1997 (Archibald et al⁶⁸)
Triclosan	S marcescens	Conjunctiva	Intrinsic contamination	1995 (McNaughton et al⁶⁹)
Iodine
Povidone-iodine	B cepacia	Blood (pseudobacteremia)	Intrinsic contamination 10% povidone-iodine (probable B cepacia proliferating on the deionizing resin in the water system)	1981 (Berkelman et al⁷⁰)
	B cepacia	Blood (pseudobacteremia)	Intrinsic contamination	1981 (Craven et al⁷¹)
	B cepacia	Blood (pseudobacteremia), peritoneal fluid	Intrinsic contamination	1989 (Centers for Disease Control and Prevention⁷²)
	B cepacia	Blood (pseudobacteremia), peritoneal fluid	Intrinsic contamination	1991 (Jarvis⁷³)
	B cepacia	Blood (pseudobacteremia), peritoneal fluid	Intrinsic contamination	1992 (Panlilio et al⁷⁴)
	P putida	Blood, catheter tips	Not determined	2004 (Bouallègue et al⁷⁵)
Poloxamer-iodine	P aeruginosa	Peritoneal fluid, wound	Intrinsic contamination	1982 (Parrott et al⁷⁶)
Abbreviations: CSF, cerebrospinal fluid; QAC, quaternary ammonium compound. ^a From Weber et al.⁷⁷ Reproduced with permission from American Society for Microbiology.

The choice of the bacterial isolate to study is important. A recent study, looking at hundreds of isolates of Staphylococcus aureus, reported clear differences in the genetic mutations occurring between isolates exposed to triclosan.¹¹⁸ The investigation of environmental isolates is probably more realistic than the use of adapted standard culture collection strains to understand the ability of bacteria to adapt. Studies of environmental bacterial isolates that have been regularly exposed to biocides do not always show a decreased biocide susceptibility when compared to standard collection strains.¹¹⁴,¹¹⁵,¹¹⁸,¹¹⁹

There is a clear body of evidence that bacteria have a phenomenal ability to survive biocide exposure. Bacterial survival in biocidal products, at time at the in-use
concentration, has been well reported (see Table 4.1). The artificial development of bacterial resistance, however, can be difficult to achieve using realistic in vitro protocols, reflecting, for example, high biocide concentrations or short contact time.⁶,⁹⁸ Nevertheless, it is now clear that bacteria can use an accumulation of mechanisms, ensuring their survival in biocides and certain biocidal products.

MECHANISMS OF BACTERIAL RESISTANCE TO ANTIMICROBIAL BIOCIDES

Bacteria have a number of mechanisms that can be expressed to respond to an external stress and enable their survival. Overall, these mechanisms aim to reduce the damaging concentration of a stressor, such as a biocide, and allow repair to the bacterial cell. A short exposure to a biocide can lead to damages that are reversible (ie, damages can be effectively repaired), whereas a longer exposure often produces irreversible damages that will lead to cell death (Figure 4.1). Maintenance of the internal cytoplasmic pH appears to be key in the bacterial ability to survive.¹²⁰,¹²¹ Although this is quite a simplistic notion, reversible/irreversible damages will depend on the nature of the biocide, concentrations used, and time of exposure. Hence, highly reactive biocides such as alkylating and oxidizing agents are often considered to be more efficient in killing bacteria when compared to cationic biocides.⁸ With this in mind, examples of bacterial survival in a biocidal product often (but not exclusively) concern less reactive biocide chemistries such as cationics and phenolics, but resistance to alkylating agents such as glutaraldehyde have been reported (see Table 4.1).

FIGURE 4.1 Levels of biocide interactions with a bacterial cell. Abbreviation: PMF, proton motive force.

Considering the importance of biocide concentration and exposure in the survival of bacteria, factors that negatively affect biocide/bacteria interactions need to be considered.¹,⁸ These factors can be divided into

Factors inherent to the biocide including concentration, formulation, mechanism(s) of action
Factors inherent to the bacteria including the type (ie, mycobacteria, gram-negative or gram-positive bacteria, bacterial endospores), metabolism (including presence of a biofilm), specific resistance mechanism (eg, overexpression of efflux pumps)
Factors inherent to product usage (ie, the during-use parameters), decreased concentration (ie, following dilution of stock solution or abundant rinsing with water, residual concentration), type and amount of organic load (soiling), effective exposure time, material/surface that is disinfected

The most important parameters to consider are those affecting products during use. These have been rarely considered during in vitro testing,¹⁵,¹⁶ but these will clearly affect bacteria survival during disinfection/antisepsis and
account for bacterial survival in products (see Table 4.1). The concentration of a biocide in a product is key for its efficacy or for allowing bacterial survival.¹²² Where the biocide concentration is close to the MBC, the product may allow bacterial survival and be prone to bacterial contamination.¹²³ During the use of a product, it is often difficult to predict what will be the target microorganisms to kill. In health care settings, outbreaks of Clostridium difficile will dictate the use of sporicides, which are considered the most effective disinfectant products.¹²⁴,¹²⁵ However, despite claims from manufacturers, not all biocide chemistries are sporicidal and only oxidizing agents and alkylating agents have been shown to have efficacy against bacterial endospores.¹²⁴,¹²⁵,¹²⁶ Different microorganisms are recognized to have different susceptibility to biocides.⁸ The main reason for being less susceptible to biocides is their intrinsic or natural properties, which are mainly structural including different cell envelope (eg, different membrane lipid composition, different outer membrane proteins [OMP]) or additional components (eg, efflux, glycocalyx). Bacterial endospores have all together a unique structure and are considered to be highly resistant to biocides (see following discussion). As such, they are often used as biological indicators for testing high-level disinfection of medical devices. Hence, a sporicidal product should be effective in killing vegetative microorganisms.⁸ Apart from their intrinsic properties, a bacterium can acquire resistance to a biocide through gene transfer and mutations.¹²⁷ Whether the change in biocide susceptibility profile is transient or permanent, the mechanisms involved are often similar and initially result from the selective pressure exerted by the biocide or biocidal product in the first place.¹⁵,¹²⁸

Mechanisms Leading to a Decrease in the Concentration of Biocides in Bacteria

Bacteria have several mechanisms at their disposal that enable to decrease a stressor (or biocide) concentration that would be detrimental to them. Bacterial adaptation to a stressor has been suggested from experiments in which bacterial growth curve in the presence of biocides resulted in an extended lag phase¹²⁹ before a normal exponential growth resumed. We now know that bacterial exposure to a biocide will trigger a stress response and lead to the expression of a number of mechanisms enabling survival (see following discussion). The mechanisms involved in bacterial response to a biocide exposure are principally global mechanisms such as changes in membrane composition and expression of efflux pumps, although specific mechanisms such as enzymatic expression and mutations have been reported. These mechanisms often work together to enable bacterial survival.⁶,⁹,¹³⁰,¹³¹,¹³²,¹³³ The use of protocols that informed on gene expressions following a biocide exposure has shown that bacterial metabolism can be altered, leading to marked differences in decreased susceptibility to a specific biocide between susceptible and resistant isolates.¹³³,¹³⁴,¹³⁵

Reducing Biocide Penetration

The impact of bacterial cell structure in decreasing the penetration of a biocide has been well described in the literature in vegetative bacteria, notably gram-negative bacteria and mycobacteria.¹³⁶,¹³⁷ It is also a fundamental property of the bacterial endospore.¹³⁸ The lipopolysaccharide (LPS) layer in gram-negative bacteria has long been established as a barrier to biocide penetration, notably with cationic biocides such as biguanides and QAC. Evidence of the role of LPS comes from the use of permeabilizing agents such as ethylenediaminetetraacetic acid.¹³⁶,¹³⁹,¹⁴⁰ The role of the bacterial outer structure in reducing the effect of biocide exposure has also been demonstrated with the study of protoplasts.¹⁴¹ Overall, the role of membrane-associated proteins in bacteria decreased susceptibility to a QAC and some antibiotics has been documented¹⁴² together with the impact of reducing the expression of membrane porins.⁹⁷,¹³⁰,¹⁴³,¹⁴⁴,¹⁴⁵,¹⁴⁶

In mycobacteria, the mycolate layer associated with the arabinogalactan/arabinomannan cell wall and overall lipid-rich outer cell wall is responsible for preventing biocide and antibiotic penetration.¹⁰⁶,¹³⁷,¹⁴⁷,¹⁴⁸,¹⁴⁹,¹⁵⁰ The reduced presentation of surface-associated porins has also been shown to play a role in reducing the activity of glutaraldehyde and ortho-phthalaldehyde.¹⁵¹

Changes to the bacterial cell envelope in response to biocide exposure have also been documented in a number of studies. Changes in membrane lipid composition,¹³⁰,¹⁵²,¹⁵³,¹⁵⁴,¹⁵⁵,¹⁵⁶,¹⁵⁷ membrane proteins,⁹⁷,¹³⁰,¹⁵⁸,¹⁵⁹,¹⁶⁰,¹⁶¹ membrane potential,¹⁶² and membrane fluidity¹⁰⁴,¹⁰⁵ have all been associated with decreased susceptibility to some biocides.

Efflux Pumps

The expression of efflux pumps allows bacteria to decrease the concentration of stressors that would eventually reach the cytoplasm. Five main efflux pumps have been described in bacteria: the drug/metabolite transporter superfamily, the major facilitator superfamily, the adenosine triphosphate (ATP)-binding cassette family, the resistance-nodulation-division (RND) family, and the multidrug and toxic compound extrusion family.¹⁶³,¹⁶⁴,¹⁶⁵,¹⁶⁶,¹⁶⁷ Carriage of efflux pump genes in environmental and hospital isolates has been particularly well documented in the last few years (Table 4.2). The correlation between decreased biocide susceptibility, antibiotic resistance, and efflux pump carriage in gram-positive and gram-negative bacteria food isolates has been reported in a number of studies.¹⁷²,¹⁷³

The role of efflux pumps in decreased bacterial susceptibility to QAC¹⁶⁶,¹⁷¹,¹⁷⁴,¹⁷⁵,¹⁷⁶,¹⁷⁷,¹⁷⁸,¹⁷⁹,¹⁸⁰,¹⁸¹,¹⁸²,¹⁸³,¹⁸⁴,¹⁸⁵,¹⁸⁶ and triclosan⁹⁴,¹³³,¹⁸¹,¹⁸⁷,¹⁸⁸,¹⁸⁹,¹⁹⁰,¹⁹¹,¹⁹²,¹⁹³ has been well reported. Some publications reported that
the efflux pump gene expression is dependent on the concentration of the biocide.¹⁷¹,¹⁸²,¹⁸³

TABLE 4.2 Examples of studies reporting carriage of efflux pump genes in environmental, food, and hospital isolates

Efflux Gene (% Carriage in Isolate)	Bacteria (Number of isolates)	Origin of Isolates	Biocides	Reference
qacA/B (83.0%) smr (77.4%) norA (49.0%) norB (28.8%)	High-level mupirocin-resistant, MRSA (53)	Health care	Chlorhexidine	Liu et al¹¹⁰
qacA/B (80%)	Staphylococcus epidermidis (25)	Health care	Chlorhexidine	Hijazi et al¹¹¹
sepA (95.3%) mepA (89.4%) norA (86.4%) lmrS (60.8%) qacAB (40.5%) smr (3.7%)	MRSA (82) MSSA (219)	Health care	Chlorhexidine	Conceição et al¹¹²
acrB (96.29%) mdfA (85.18%) oxqA (37.03%) qacA/B (11.11%) qacE (7.40%)	Escherichia coli (27)	Food	Hexadecylpyridinium chloride (QAC)	Burgos et al¹¹⁵
qacA/B (83%) Smr (1.6%)	MRSA (60)	Health care	Benzalkonium chloride Benzethonium chloride Chlorhexidine	Shamsudin et al¹⁶⁸
acrB (100%) AcrAB-TolC system (100%)	Gram-negative (29)	Food	Cetrimide Hexadecylpyridinium chloride Chlorhexidine Triclosan	Fuentes et al¹⁶⁹
qacA (26% for HMRSA, 67% for VISA) qaC (5% for HMRSA, 4% for MSSA, 17% for VISA)	HMRSA (38) Community-acquired MRSA (25) VISA (6) MSSA (25)	Health care	QAC Chlorhexidine	Smith et al¹⁷⁰
mdrL (33%) lde (42%)	Listeria monocytogenes (45)	Food	Benzalkonium chloride	Conficoni et al¹⁷¹
Abbreviations: HMRSA, hospital-acquired methicillin-resistant Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; QAC, quaternary ammonium compound; VISA, vancomycin-insensitive Staphylococcus aureus.

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